plant cell biology for masters
TRANSCRIPT
Plant Cell Biology for Masters
http://plantcellbiology.masters.grkraj.org/Plant_Cell_Biology_Table_Of_Contents.html
i. Plant Cell Structureii. Plant Cell Division
iii. Plant Cell Apoptosis iv. Plant Cell Geneticsv. Plant Cell Physiology
vi. Plant Cell Biochemistry and Metabolism vii. Plant Growth and Development
i. Plant Development Introductionii. Plant Hormones introduction
iii. Auxinsiv. Gibberellinsv. Cytokinins
vi. Abscissinsvii. Plant Cell Vacuoles
viii. Ethyleneix. Morphactins and Othersx. Application of Plant Hormones
xi. Physiology Of Floweringxii. Physiology Of Vernalization
xiii. Physiology Of Dormancyxiv. Physiology Of Plant Movementsxv. Circadian Rhythm and Biological Clock
viii. Plant Cell Gene Engineeringix. Plant Molecular Biology
Plant Development-Introduction
Concepts of Growth and Differentiation
In living organisms’ production of a zygote to produce a zygote are two events separated by
space and time. The zygote in an embryo sac divides and redivides to produce a mass of
cells. Concomitant with the cell divisions polarity of future shoot apex and root apex is also
determined and established which is accompanied with physiological and structural changes.
After a period of active growth, the embryo suspends its active state and becomes dormant.
At this stage, the embryo which is enclosed in a seed coat is shed from the mother plant.
Then after a period of rest, which may be short or long, if conditions are favorable, the seed
germinates and develops into a full fledged plant. Such plants inturn, after going through a
period of vegetative development produce reproductive structures in which once again a
zygote develops due to the act of fertilization. And the cycle is repeated. Between these two
stages the time required for the development varies from as short as 24 hours, to several
years. This is however determined by the genetic potentiality of the given plant, but
environmental factors have a strong and stimulating influence on the plant genome for its
expression of manifestation.
A careful analysis of various events in the development of higher plants reveals that there are
two important processes that go hand in hand. One is growth and the other is differentiation;
together it is called development. Growth is generally defined as an irreversible increase in
size/volume accompanied with an increase in the protoplasmic mass. Differentiation, on the
other hand, is a process in which new structures and new functions generate from the
preexisting cellular components. For that matter, these two events are not mutually
exclusive.
Increase in the number of cells accompanied with the increase in volume has an apparent
effect on the increase in the size of the plant body. The same can be determined by either
counting the number of cells or measuring the volume or both. But differentiation involves
the molecular changes in undifferentiated cells, leading to the formation of new structures,
like tissues, leaves, buds, branches, flowers and fruits. So the development can be considered
as the summation of all physiological activities.
Measurement of growth:
Normally growth is measured in terms of increase in the number of cells, increase in height or
weight with time. These can be determined by known physical methods. Even the number of
leaves, branches, roots, flowers and fruits produced can be measured by simple methods.
The developments of later said structures are the products differentiation which can be
further subjected to quantitative or qualitative studies.
The growth of a seedling, in terms of increase in length or weight, can be plotted as the
function of time which may be in hours or days. The graphical expression, which results in a
curve called growth curve. Different plants, for those matter different organs of the same
plant, exhibit different kinds of curves. For example, a tree plant increases in height and
weight with time. Similarly, the number of leaves, flowers and fruits produced show different
pattern, which is quite distinct from that of growth pattern of a single leaf, flower or fruit. The
plants exhibit indefinite growth and determinate growth pattern. But in both one can observe
the growth pattern which passes through three different phases one leading to the other.
They are lag phase, log phase and steady or constant phase.
It is possible to utilize the above said growth curves in determining overall growth rate or the
growth rate of individual phases. This can be done by taking a tangent angle (90) degree
derived from two points on the curve, which provides the information about the increase in
length or weight in a given period of time. By integrating them together, one can obtain the
growth rate. The same value i.e. growth rate (increase in length or wt.xTime) can be plotted
against a larger time scale. This plot gives a general pattern of growth rate at different
phases of growth of the whole plant. The finer analysis of these growth patterns can be
employed to determine the efficiency of plants in terms of its increased or deceased dry
matter, photosynthetic efficiency, flowering, fruit production, etc.
In fact, using the above methods, it is also possible to determine relative growth rate, not
assimilation rate and the ratio of leaf area to dry weight or leaf area ratio. The above
methods provide an invaluable data in determining the genetic potentiality of plants in
response to different environmental conditions. The efficiency of the plants in terms of input
and output can also be determined.
Growth Rate and Phases of Growth:
Different plants exhibit different growth rate; this is the law of nature. Generally, most of the
plants initially show a period of slow growth. This is called lag phase. At this stage most of
5he available reserve food material is metabolized to generate energy rich compounds and
various organic compounds necessary for building the structural components of cells. In this
period, most of the reserve food material is drawn into metabolic pool.
Further development depends upon the ability of the plants to synthesize its own food
material, which is achieved by the development of leaves. Once sufficient number of leaves
and roots are produced, the plant becomes self sufficient and then the plant grows at a faster
pace. In fact, the increment of growth doubles with time. This phase of growth is called log
period or exponential period of growth.
The term exponential is derived from the fact, the increase in the number of cells or increase
in height or weight, when measured at equal periods of time always doubles, etc., 1, 2, 4, and
8, 16, 32, 64 and so on. Such a relationship between the number of cells produced in a given
time is expressed n the form of an equation i.e. N=4t, where t is the time interval and N is the
number of cells or weight or length. If the growth pattern obeys the said equation, then it is
called exponential growth. If fact, it is at this period of time, the plant reaches the maximum
differentiation and development, in terms of production of leaves, buds, branches, etc., and
the plant gains maximum height and weight.
The magnitude of exponential growth in this period however, varies from plant to plant. For
example, Calamus gigantica (bamboo) grows at the rate of 2 ft./day, Asparagus at 1 ft/day,
inflorescence axis in Agave 6 inches/day, Basella bacella 2 inches a day. On the contrary
Cycas, a gymnosperm, grows at the rate of 1.2 to 2 inches/year. The above examples clearly
indicate that the ability of growth of each plant is ultimately determined by its genetic
potentiality.
Once plants reach their maximum growth point, they start producing flowers. This of course
varies from species to species. With this, the growth rate of plants falls sharply, but the other
non flowering branches continue to grow but slowly. Thus a steady rate of growth is
maintained. Such a phase is called steady phase. But I may annuals, with flowering and
fruiting, the growth comes to stand still and ultimately the plant dies. Nevertheless, the
perennial plants (herbs, shrubs or trees) exhibit further seasonal vegetative growth and
seasonal flowering/fruiting periods, where the growth curve is different from that of annuals.
Such a behavioral pattern in exhibited by evergreen plants as well ad deciduous plants.
While the above observations are true for the entire plant, the growth rate of leaves, flowers
and fruits exhibit a different pattern. They too have a lag phase, a log phase and constant
phase where the growth of the said organs is virtually stopped. All structures or organs which
have determinate growth show this pattern. Furthermore, the magnitude of growth of
different organs varies from species to species.
Mechanism of Growth and Differentiation:
It is very important to understand the regions of growth that are involved in the development
of the plant body. This can be taken as the basis for understanding the mechanism of growth
and differentiation. The apices of shoots and roots provide valuable information about the
structures involved in growth and differentiation of cells during development of plant
structures. Refer to the topic cell differentiation as the function gene expression to
understand the molecular basis of differentiation.
Both root apex and shoot apexes are made up of three important regions i.e. the region of
meristems, the region of elongation and the region of differentiation. The meristematic
region is restricted to the apexes, where the cell will be in an active state of cell division. In
the region of elongation, cells are in the process of physical elongation, thus less region
exhibits an apparent growth. Just behind this region, the elongated cells or the cells that are
still in the process of elongation show marked differentiation in their cellular structures and
functions. In this region, one can see different types of cells and tissues like pith, xylem,
phloem, endodermis, cortex etc. The analysis of each of these structures and their functions
will give a comprehensive picture of how cells grow, differentiate and produce specific organs.
Meristems: The higher plants, in their embryonic state itself, acquire polarity for their future
development. The embryo possesses plumule at one end and radicle at the other end of the
plant axis. The plumule develops into shoot system and radicle into root system. But shoot
apex and root apexes contain a single or a group of meristematic cells. Each cell in this
region is endowed with a potentiality of active cell division and this potentiality is retained
throughout the lifetime of the plant. Such meristematic cells are rich in cytoplasm, compactly
arranged with a number of intercellular protoplasmic strands as interconnecting structures.
Vacuoles in these cells are nearly absent. Nucleus is active. Cells exhibit maximum
respiratory activity to generate energy rich compounds. Other metabolic pathways are very
active in generating various compounds to build up various cellular components and
structures. The nuclear DNA undergoes periodic replication and various sets of genes are
expressed in producing required TRNAs, RRNAs and RNAs. Once these RNAs reach the
cytoplasm, they are utilized in synthesizing a host of proteins of which some are used in the
organization of different cellular structures like membranes of ER, plasma lemma,
mitochondria, plastids, nucleus, lysosomes, golgi bodies, cytoskeletal structures,
microtrabaculae, chromatin, ribosomes, etc. and other proteins act as enzymes and
regulatory factors for various metabolic processes. These activities in meristatic cells are
maintained as well as sustained till
the death f the plant. Except under dormant conditions where all the cellular activities are
temporarily suspended, they are fully active through out the life of the plant.
Cell Division and Plane of orientation:
Once the cellular components are made and the protoplasmic mass reaches a particular
proportion in terms of mass, the cells initiate mitotic divisions. This results in the production
of daughter cells. Some of the daughter cells which are away from the mother meristem cell
act as derivatives. T he most crucial part of mitotic divisions is the plane of division. This is
however determined by the plane of orientation of mitotic apparatus in the dividing cells. As
shown in the fig upper cells undergo another period of preparation for another cycle of cell
division to produce another set of derivative cells and the process goes on and on thus the
terminal cells fund in the apical dome continue to divide and redivides ceaselessly to
generate new cell derivates. The regulatory factors that control cell divisions continue to be
produced in mitotically active meristematic cells there by the said cells keep on their mitotic
activity unhindered. On he contrary, the cell derivatives produced by the upper meristematic
cells, because of their positional effect, nutritional and hormonal influences produce certain
regulatory factors some of which block gene expression required for continuous mitotic
activity and others stimulate the cells to undergo certain transformation leading to
differentiation.
Mutation studies in yeast cells clearly suggest that mitotic cycle is controlled by at least 36-38
mutational sites an each one of them are distinct and specific in their activity. Some of the
genes, if activated, may block the continuous mitotic activity. On the other hand, if some
specific regulatory genes undergo mutation, it induces uncontrolled mitotic activity which
may lead to cancerous growth. However in higher plants to keep pace with the increase in
the number of meristematic cells, the tunica layer of cells which cover the apical dome divide
and redivides radially. It is interesting to note that the outer tunica layer of cells or epidermal
cells are also meristematic but their plane of cell division is always radial. The intrinsic factors
that are responsible for such a behavior of these cells are perplexing and fascinating. Nothing
is known about this phenomenon.
Transitional Zone:
The basal cells though derived from the terminal meristematic cell(s) exhibit different pattern
of growth. This is however, determined by the underlying mature or partially matures cells
which supply nutrients in the form of mineral elements, organic compounds and plant
hormones. The gradient and magnitude of various chemical components supplied from the
lower cells have profound influence on the cells derived from meristematic cells. The
positional effect of transitional zone cells, compounded by the hormonal and nutrient
gradients the cells found in the transitional may undergo one or more cell divisions or they
may directly undergo transformation leading to differentiation.
Onset of cell elongation and cell differentiation:
The cell derivatives thus produce, utilizing nutritional ingredients and phytohormones,
undergo dramatic transformations. Not all the cells behave in the same way. Some elongate
considerably and undergo transformation to produce different cell types, such as xylem
elements, phloem elements, and endodermal cells and so on. Others without much
elongation develop into cortical parenchyma, pith parenchyma and pericyclic cells. In stem
apex, just below the meristematic zone cells, just below the tunica layer, some of the cells
organize into leaf primordia, which actually cover the entire meristematic dome.
In roots, epidermal cells in the region differentiation elongate and develop into root hairs.
However, among the vascular elements the first to be differentiated is phloem and then xylem
elements develop. Though most of the cells are derived from the same group of meristems
because of intrinsic factors, and programming the derivatives undergo transformation and
develop into various cell types perform different functions.
Phloem as a Model for Understanding the process of differentiation:
Whether it is stem apex or root apex the first cell type to be differentiated are sieve tubes.
The cells that are destined to become sieve elements are in line with the sieve cells of
vascular bundles found below. The destined cell receives auxins from the cells found above
and nutritional factors and sucrose from the lower vascular elements. Here sucrose appears
to play a significant role in the formation of sieve cell. In response to hormones and
nutritional factors, the said cell first undergoes an unequal cell division in the vertical plane.
The smaller cell produced undergoes one or two divisions and develop into companion cells.
But the larger cell undergoes remarkable transformation. First the cell elongates and
expands. Concomitantly the nucleus disintegrates into small chromatin bits. It s at this
juncture, the chromatin becomes active and produces substantial amount of mRNAs required
for the formation of P-proteins and other factors. The mRNAs thus produced retain their
translational activity for a period of 4-5 months. Once the p-proteins and its associated
elements organize into longitudinally oriented tubular structures the tonoplast membrane
disappears rendering the peripheral protoplasm and vacuolar materials, indistinguishable.
The functional activity of such sieve tubes and companion cells are sustained for quite a long
time.
Differentiation of xylem elements:
Similar to the development of sieve elements, some of the cells found by the side and just
below the newly formed sieve tubes undergo differentiation and development into xylem
structu5es. Experiments involving the induction of vascular elements in callus clearly show
that the application of sucrose favor the development of sieve tubes and indole acetic acid on
the other hand, stimulates the formation of xylem elements. So supply of IAA to these cells is
crucial in the development of the xylem elements.
Responding to IAA and other nutritional factors, the cells that are destined to become xylem
elements enlarge in length as well as in breadth. At the same time, the entire cellular
machinery gears up to produce cell wall materials in massive amounts. Golgi bodies become
very active, the vesicle loaded with cell wall components and enzymes are transported across
the cell and the same are unloaded outside the plasma membrane. Synthesis and deposition
of various components of secondary wall takes place on a large scale. Meanwhile
endoplasmic reticulum starts associating with the plasma membrane at different sites. These
regions become free from cell wall deposition and the same develop into pit canals. But in
other regions, cell wall is deposited in a characteristic pattern in different cells of different
xylem elements. During the development of xylem the protoplasm is completely used and
finally it disappears; it is an apoptotic phenomenon, a programmed cell death. At the
molecular level, it is clear that differential expression of genes is mainly responsible for the
production of massive amounts of MRNAs required for the formation of enzymes needed for
cell wall components. Even the deposition of cell wall material is regulated. Thus
undifferentiated cells undergo differentiation into xylem structures to perform their specific
functions.
Molecular Paradox in Morphogenesis
During morphogenesis, the derivatives of meristems are subjected to nutritional and
hormonal pressures. As a result cellular programs of these cells get modified due to the
activity of regulatory factors. A set of genes which were active during early stages of mitotic
cycle, at least, some of them get repressed, thus they switch off all the programmes for cycle
events. At the same time, a new set of genes are activated which inturn exhibit cascade
effect. In the sense, synthesis of a new group of mRNAs and their translation products
activate another set of genes and such sequential gene inactivation and activation results in
the transformation of undifferentiated cells into new cell types which by virtue of possessing a
characteristic structure and functional potential perform specific functions; thus the division
of labour sets into the system. There must be a master switch (s) in the form of regulatory
factors that act at every stage of differentiation and growth. This can be similar to that of
Homeobox or Hox gene clusters of animal system. Existence of such gene clusters have been
discerned but not fully evaluated.
The regulation of gene expression may operate at the transcriptional level, translational level
or at post translational level, but each of these events have their own feed back mechanisms.
Besides nutritional factors and environmental factors phytohormones have a profound
influence on gene regulation. Unlike animal systems plants have just a set of hormones like
auxins, gibberellins, cytokinins, abscissins and ethylene. Among them the first three act as
growth promoters and the others act as growth inhibitors. The site of synthesis, the time of
synthesis, the concentration of each of these hormones at any given site and time, nflu8ence
the molecular expression according to their specific effects. Though individual hormones
elicit certain specific responses, their activity is greatly influenced by the presence of other
hormone. One hormone in one tissue type may promote or inhibit the activity of the other
hormone. Thus by modulating the levels of growth hormones, they induce different
morphological structures. This is possibly achieved through a very complex gene regulation
mechanism about which we know nothing.
Molecular aspects of IBA induced new root formation:
Adventitious root initiation in response to auxins like IBA has been used in understanding the
molecular events that lead to redifferentiation in plants. In response to IBA, pericyclic cells
found in the hypocotyl tissues of phaseolus vulgaris undergo transformation and reorganize
into root primordial cells. The entire process takes place within 24-36 hours after the
treatment with the hormone.
Molecular events: Within 15-30 minutes of auxin treatment, even in the presence of
actinomycin the rate of protein synthesis in the hypocotyls increases without any concomitant
increase in mRNA synthesis, which suggests that early effect, is on IBA translational
machinery. This view has been experimentally borne out by invitro translational studies.
After another 60-90 minutes transcription and translational process further increase by 4-5
fold over control tissues. Among many changes observed in the pattern of proteins
synthesized, the increased synthesis of 55-58 K Dalton proteins and another high molecular
weight protein is very significant. Using SDS page, invitro translational system and
immunoprecipitation methods the 55-58K protein has been identified as Tubulin.
Tubulin is known to be an important component of microtubule needed for mitotic apparatus
and cytoskeleton structures. Furthermore tubulin polymerization into microtubules also
increases in the presence of IBA and cytoplasmic factors. The above observations suggest
that the IBA induced microtubules formation has some role in dedifferentiating the cells. In
order to ascertain this view when the IBA pretreated hypocotyls are exposed to colchicine or
cytochalasin B for a period of 36 hours, IBA mediated new root formation is completely
inhibited, but after IBA mediated new root formation is completely inhibited, but after 36
hours of IBA pretreatment colchicine has no effect. The above results indicate that auxin
mediated increase in tubulin synthesis, tubulin polymerization into microtubules, the
organization and orientation of mitotic apparatus ultimately determines the plane of division.
Besides another important observation is the transformation of parenchymatous cells into
mitotically active cells. Thus IBA induces cell transformation and also determines the plane of
division in parenchymatous tissue which ultimately determines the organization and
development of root primordia.
Interestingly, the IBA mediated root initiation can be inhibited by higher concentration of
cytokinins. Anatomical studies show that in the hypocotyls, treated with both auxin and
cytokinins, the pericyclic cells show greater mitotic activity but root primordial organization is
totally absent. But in the segments treated with cytokinin alone pericyclic cells do not show
any changes.
In order to find out the interesting interaction between IBA and cytokinins and their effect on
molecular events studies on the rate f protein synthesis and RNA synthesis show that the
rate of protein synthesis increases as early as 15-30 minutes as cytokinin treated segments
but the increase in RNA synthesis is delayed by 4-6 hours. Similarly in the hypocotyl which
are exposed to both IBA and cytokinins, the rate of protein synthesis increases at 15-30
minutes, but the IBA mediated RNA synthesis is inhibited by cytokinin for a period of 4-6
hours. Later RNA synthesis and protein synthesis increase substantially in both the cases.
Particularly in the stems treated with IBA+cytokinin, the increase is highest.
Analysis of in vivo protein pattern and invitro protein pattern reveals that though cytokinin
increases the level of tubulin synthesis as in the case of IBA. It inhibits the synthesis of high
mol.wt. Protein induced by IBA. The above features suggest that cytokinins inhibit IBA
mediated root initiation by modulating the microtubular organization by way of inhibiting high
moo. wt. proteins that are needed for root primordial organization. Probably this is the only
example in plant system which explains the molecular events that lead to the initiation of root
formation and the inhibition of it in response to combination of phytohormonal treatment. A
model has been given to follow the events for new root formation in response to IBA and BAP
is given.
Fucus as a Model to Understand Structural Changes during Morphogenesis:
From the earlier discussions it is clear that the temporal and quantitative regulation of gene
activity through transcription and translation ultimately determines structural changes during
differentiation. Though not much is known about the molecular events that lead to ultra
structural changes during development, it is well known that the fertilized egg in the embryo
sac always undergoes a polar division as if it is predetermined and produces cells which
develop into radicle and plumule. Such behavior of cells during the early part of its
development is known to be a function of intracellular compartmentalization regulated by
certain intrinsic interactions of nucleocytoplasmic components.
Development of umbrella shaped reproductive gametangial structures in Acetabularia,
development of hold fasts in cladophora and rhizoidal formation in the zygotic development of
Fucus, are some of the examples which have been used in understanding ultra structural
changes during organogenesis.
The developing zygote is Fucus first produces a rhizoidal process a rhizoidal process at the
region of contact with the substratum. If such spherical zygotes are subjected to unilateral
illumination from one side, rhizoidal structures develop at the opposite pole. If the direction
of illumination on the zygote is changed at short intervals of time, the end at which the rhizoid
develops is determined by the last treatment. The time required for such polarity fixation is
about 10-16 hours of treatment, beyond that the polarity of rhizoidal development remains
unchanged.
If such programmed cells are studied under electron microscope, it is revealing to find a large
number of osmophilic bodies some unknown granular materials, dense fibrillar structures,
ribosomes and mitochondria congregated at the region of rhizoidal formation. Interestingly,
the perinuclear region facing the rhizoidal pole shows polarized finger shaped projections
indicating the flow of informational materials from the nucleus in the direction of rhizoidal
pole.
The above observations suggest that the duration between the time of induction by light and
the time at which rhizoids develop is the most crucial period at which various molecular and
structural events fix the polarity. In fact, these events are the basis for differentiation.
Furthermore, if zygotes, which re subjected photo inductive treatment, are treated with
inhibitors of protein synthesis like CHI for above 9-10 hours, rhizoidal formation is not
inhibited. This clearly suggests that the denovo protein synthesis is not required for polarity
fixation. The polarity fixation is achieved with whatever structures or components available in
the cell. Instead if such zygote is provided with cytochalasin B or colchicine or both for the
same period of time, i.e. 9-10 hours, the polarity of rhizoidal formation is totally inhibited and
it remains labile. Cytochalasin B and colchicine are known for disrupting the formation of
microfilaments and microtubules respectively. Transmission electron microscopic studies of
polarized zygotes of Pelvetia fastigata reveal cortical clearing. in the region of rhizoidal
formation. Bundles of microfilaments and microtubules are found oriented in line with
rhizoidal pole. The above studies clearly suggest that the polarity fixation is due to the
organization and orientation of microtubules and microfilaments. The intrinsic components
that lead to the organization of such cytoskeleton are not yet clear. Furthermore, the plane of
cell division is also controlled by the orientation of mitotic apparatus.
It is very important to know more about microtubules and micro filaments and their role in
organogenesis (refer chapter proteins). The assembly of tubulin units into microtubules and
their orientation within the cell is determined by the position of nucleating centers and the
availability of polymerization factors like Tau, Maps, and GTP & ATP. In a developing system,
the assembly and orientation of microtubules and microfilaments are under close spatial and
temporal regulation. In recent years, the role of microtubules and microfilaments in hormone
induced growth and development is attracting greater attention.
Morphogenesis involves growth and differentiation. Growth generally refers to the enlargement of the cells or the production of a group of cells of similar kind. In contrast to growth, differentiation involves the development of a cell or an organ which is structurally and functionally different from the cell it originates. In the development of a plant both growth and differentiation, go hand in hand. Plants like animals start their development from an unicellular structure, which by a series of pre-determined cell divisions produces a variety of cell structures and organs. Except for a few cell types such s sieve tubes among phloem elements, almost all living cells of all kinds are totipotent and if proper nutritional conditions are given they are capable of giving rise to a complete plant. This developmental potential is inherent in their genetic make-up. In recent years, however, it is becoming clear that the differential expression of the genetic potential is controlled by plant growth substances that are produced in the system itself. It is their interaction with the genetic apparatus which results in the full expression of the phenotype of the plant.
Supplement material from my thesis
PLANT GROWTH REGULATING SUBSTANCES: HISTORICAL ACCOUNT:
The existence of a plant growth substance was first perceived by Charles Darwin [1],
who in his own elegant way, described how plants perceive the stimulus of light and respond
to phototropic growth. Since then, botanists have made great strides in unraveling the
mystery of the diffusible substance, which is responsible for growth. Kogl and Haagen Smit
isolated and identified the growth promoting substance and coined the name “hetero auxin”
or as it is known today, indole-3-ylacetic acid (IAA) [2,3]. This gave a great impetus to the
plant scientists all over the world, and the search culminated in a series of discoveries of plant
growth substances, such as Gibberellin B (GAB) by Yabuta and Sumuki (1938), Cytokinin by
Miller and Skoog (1955), and Abscisic acid (ABA) by Robinson and Wareing (1964).
Simultaneously a host of synthetic growth promoting as well as growth inhibiting substances
were also made available to plant scientists. This led to intensive investigations on the effect
of the various plant hormones on plants. Various hormones, elicited a wide array of
responses in different kinds of tissues. It was soon realized that the physiological response to
any given growth substance depends, in the first instance, on the type of cell receiving the
stimulus. For example, a developing leaf cell, in a barley plant, will respond to GA3 by
elongating, whereas an aleurone cell, from the same plant, will respond by producing a-
amylase. The specificity of response is built into the cell by its previous developmental
programme. It is curious to note that the highly specific animal hormones affecting specific
cells have no counterpart in plants. In general, it can be stated that the auxin is known as
growth promoting substance, but the same substance is also capable of eliciting responses
such as, apical dominance, root initiation, prevention of leaf abscission, fruit setting and so
on. While Gibberellic acid is known to be effective in promoting growth, induction of a-
amylase, bolting and flowering, Cytokinin is considered as the hormone that controls
cytokinesis, a part of cell division. Abscisic acid is known to act as a growth inhibitor,
controlling processes such as bud dormancy and seed dormancy. Ethylene, recognized as a
hormone recently, shows bizarre effects. In the development of the plant body, it is the
interaction between the various hormones that controls and regulates the process of
morphogenesis.
HORMONAL INTERPLAY
Although Haberlandt (1913) considered the tissue and organ culture in sterile
conditions as a theoretical possibility, it was realized as a practical proposition for research by
two independent workers, Kottle (1992) in Germany and Robbins (1922) in America. The
advent of tissue culture techniques opened up a new vista in the studies of hormonal
interactions in morphogenesis.
When plant growth substances are supplied to plant tissues exogenously, each of them
elicits unique responses. However, many responses are overlapping. But if two or more
different hormones are supplied in known concentrations, in many cases synergistic or
antagonistic responses are exhibited and this effect is referred to as hormonal interaction.
Studies on tobacco pith callus tissue have revealed that the relative concentrations of
hormones control the expression of callus either into roots or shoots. Low, high and
intermediate ratios between auxin and cytokinin induce roots, shoots and callus respectively
[4]. Auxin-cytokinin-induced shot formation in the callus is inhibited by GA3; however
GA3 enhances the growth of the callus. The inhibitory effect of GA3 can be alleviated by the
addition of ABA, which by itself is a weak inhibitor of callus growth [ 5,6 ].
The form of shoots developed from callus is dependent on GA3-cytokinin ratio. The
higher ratios induce the formation of tall, spindly shoots with narrow leaves, whereas lower
ratios produce dwarf shoots with rounded leaves [7].
Specific hormones are found to influence the relative levels of other hormones and this is very well substantiated in the culture system, where auxin-cytokinin induced callus growth is reduced by certain culture conditions, which also inhibit the biosynthesis of GA [6]. Thus, the interactions of hormones appear to be very complex.
This complexity is further compounded by the recent observations, in which, it has
been shown that each hormone can influence the biosynthesis, degradation, conjugation or
transport of one or the other hormones. The end ogenous level of IAA in a plant tissue is
enhanced by the exogenous application of GA or Kinetin. But, ethylene, which is known to be
induced by excess auxin, reduces the level of auxin itself. Again, auxin levels can be
increased by the addition of cytokinin. This complex relationship appears to be very
fascinating, because of the findings in which cytokinins are known to bring down the levels of
ABA, through the increased biosynthesis of GA. But ethylene promotes the synthesis of ABA,
which in turn enhances the levels of ethylene. The close relationship between ethylene and
auxin provides an excellent feed back control mechanism. Letham et al. (1978) have
illustrated the complex interaction between plant growth substances as indicated in the
diagram (Fig. 1) [8]. Further investigations into hormonal interactions at molecular level, will
have far reaching impact in understanding morphogenesis at the molecular and sub cellular
levels.
ULTRASTRUCTURAL CHANGES DURING DIFFERENTATION: MICROTUBULES,
MICROFILAMENTS IN CELL POLARITY AND ORGANOGENESIS:
The temporal and quantitative changes in the regulation of gene activity forms the
basis for the ultra structural changes determining the process of cell development and
Fig. 1: Hormonal Interactions.
Fig.1: Hormonal interactions which affect hormonal levels in plant tissues. Open
arrow from A B indicates hormone A causes increase in the levels of B. Closed arrow from
A B indicates that hormone A decreases the levels of B. differentiation.
The molecular events leading to such ultra structural organization are not very clear. For
example, it is not clear how and why a fertilized egg in the embryo sac, always undergoes a
polarized division in such a way, that one of the two cells, equal or unequal, develops into
radicle and the other into plumule. This polar behavior of the cells at the earliest part of their
development is the function of intracellular compartmentalization, regulated and governed by
certain intrinsic interactions of nucleo cytoplasmic components, whose expression in turn is
controlled by the inherent genome.
Polar growth like cap (reproductive organ) formation in Acetabularia, rhizoidal
development in Fucus, hold-fast development in Cladophora are some of the best studied
systems in the plant kingdom. In the case of zygote development in Fucus, if the spherical
zygote is subjected to unidirectional light treatment, the rhizoid develops at the shaded side
of the cell. If such a programmed zygote is subjected to electron microscopic investigation, it
is observed that in the region of rhizoid formation, large number of osmophilic bodies and
undefined extracellular materials accumulate. The peri nuclear region showed highly
polarized finger shaped projections towards the site of rhizoid initiation. Along with these
charges, heavy concentration of mitochondria, ribosomes and dense fibrillar vesicles are also
found. These structural changes represent an expression of a fixed axis for rhizoidal growth
[9]. The time taken for such polarity fixation has been determined to be 10-16 hrs. During
this period, if the cells are treated with cycloheximide (CHI) for 9-15 hrs after fertilization,
they still show a fixed polar axis. However, if cytochalasin-B, which is known to disrupt the
microfilaments, is present during polarity fixation, the polar axis remains labile and axis
establishment is prevented [10].
The role of cytochalasin-B and colchicines, in lactin Con-A induced “cap” formation in
animal cells, has been reviewed by Edelman (1976) and Nicolson (1976) [11,12]. The authors
have explained how the microtubules and microfilaments control the mobility, stabilization
and topographical distribution of cell membrane and surface components during lectin
induced “cap” formation. The cross linking of membrane receptors embedded in the plasma
membrane, to a variety of fluorescent labeled polyvalent ligands such as antibodies or lectins
lead to form patches and clusters which ultimately culminates in the “cap” formation at one
end of the cell. Similar to “cap” formation in animal cells, “cortical clearing” in the region of
rhizoidal emergence is observed in zygotes of the plant Pelvetia fastigata [13]. In the same
system, Peng etal. (1976) have shown the deposition of new membrane patches (30 um)
covering the rhizoidal region at the time of cortical clearing [14]. These studies clearly
suggest that microtubules and microfilaments play a significant role in cell polarity, which is a
prerequisite for organogenesis. Ever since, the discovery of microtubular components in few
plant cells by Lederbetter and Porter (1963) [15] the presence of such structures, in most of
the plants, has been established.
Basic components of the microtubules are not known to be tubulin monomers, which
exist in the form of heterodimers, consisting of α and β subunits, of molecular weight
110,000- 120,000 daltons [16]. Several proteins are associated with microtubules, of which
the proteins known as TAU and microtubule associated proteins (MAPS) appear to be
important, for they take part in assembly and disassembly of microtubules [17,18,19]. In
addition to the above components, the presence of nucleating centers or organizing centers
within the cells, account for an ordered or controlled assembly of microtubules [20,21]. In a
developing system, microtubule assembly is under close spatial and temporal regulation. The
nucleating centers function in controlling initiation, orientation, directionality and
patternization of microtubular polymerization. Recently attempts have been made to isolate
microtubules and study their organization in plants [22,23]. The cross reaction of antibodies,
raised against porcine brain tubulin, with the plant protein, strongly suggests the similarity of
plant tubulin with animal tubulin, at least at the antigenic level. Considerable attention has
been given to relate the orientation of microtubules during plant hormone induced growth.
Hormone induced growth in Vigna angularis, hypocotyl of Lactuca sativa and coleoptile
segments of Triticum vulgare, is inhibited by colchicines, urea and vineblastin sulfate [24-
26] and this inhibitory effect is attributed to microtubular disruption [27]. Similarly, the
inhibition of protoplasmic streaming in Avena, and growth inhibition in Zea mays root by
cytochalasin-B are again attributed to the role of microfilaments and microtubules [28,29].
Electron microscopic studies on the deposition of cellulose in cell walls during secondary
growth and movement of secretory vesicles are shown to be directed by microtubules [30].
However, the available information on plant hormone induced changes in the microtubular
assembly, synthesis and their role in morphogenesis is scanty.
PLANT HORMONES AND NUCLEIC ACID METABOLISM
Plant growth regulating substances, very often, exhibit dramatic effects on growth and
development in plants. The molecular mechanisms which control such processes involve
changes in nucleic acid and protein metabolism. DNA replication, transcription and
translation are the three major molecular events involved in growth and differentiation. An
understanding, as to how plant hormones control such molecular mechanisms, as to how
plant hormones control such molecular mechanisms, is very pertinent to interpret
morphogenesis at the molecular level. In spite of the enormity and complexity of the problem
involved, considerable Progress has been made in this field.
DNA METABOLISM
A majority of investigations reveal that hormone induced cell elongation does not require DNA
synthesis [31,32]. However, Wardell (1975) has reported that auxin, in flowering tobacco plants,
enhances DNA synthesis within an hour of treatment. On the contrary, in pea root cortical cell cultures
[33], Libbenga and Torrey (1973) have shown that enhanced DNA synthesis is possible, only in the
presence of auxin and kinetin together, but not with auxin alone [33a].
In long term experiments, most of the growth promoting substances enhances DNA
synthesis which is always accompanied by cell division [34]. In cucumber hypocotyls, GA3 –
promoted growth is fluoro-deoxyuridine (FUDR) sensitive. This increase in DNA synthesis, in
response to GA3 is due to the increased chloroplast proliferation, which is prevented by
chloramphenicol. The above experiments suggest that GA3 has some preferential effect on
chloroplast biogenesis [35, 36].
On the other hand, hormones such as ethylene and Abscissic acid inhibit DNA
synthesis. This correlates well with their growth inhibitory effects in vivo [37-39]. However, it
is not clear whether this effect is direct or indirect.
Although, cytokinin is known to induce cell divisions, it does not affect DNA synthesis by
itself as a prelude to cytokinesis. However, it promotes DNA synthesis in the presence of
auxin [40]. Hence, it is suggested that auxin has a permissive role in DNA synthesis, while
kinetin stimulates it. This is further exemplified in an experiment, where hormone depleted
and aged tobacco pith explants, could be induced to undergo mitosis without the
accompanying DNA replication by the addition of cytokinin. This suggests that cytokinin
perhaps regulates cell division, by controlling the synthesis of specific proteins for mitosis
rather than acting directly on DNA replication [41,42].
DNA SEQUENCE COMPLEXITY
In higher plants, it is estimated that the amount of DNA present per cell ranges from
1pg – 100 pg and this amount of DNA codes for more that 106 different proteins. The rough
estimates indicate that there are 50,000 or more structural genes which are expressed at one
time or the other during the life of the plant [43]. The number of structural genes operating in
different plant structures like root, stem, leaves and flowers may vary. On the basis of
hybridization studies between polysomal RNA and 3H-labelled single copy DNA, it has been
estimated that there are about 2700 diverse structural gene transcripts in the tobacco leaf
cells [44]. DNA, in higher plants, consists of three base sequence classes i.e., highly
repeated, moderately repeated and unique. Unique sequences are believed to code for mRNA
sequences [45]. These sequences can be detected, characterized and quantified by studying
the reassociation kinetics of denaturated and sheared DAN. Highly repeated sequences
reassociate rapidly at low cot values ( cot = concentration of DNA in mole nucleotide per liter
X time in seconds ) and unique sequences reassociate slowly at high cot values [45]. Studies
on changes in DNA sequence complexities in response to phytohormones in plants are few.
Nevertheless, reannealing studies of DNA isolated from the control and auxin treated
artichoke tubers, suggest that auxin causes an apparent loss in the rapidly reannealing
fraction of DNA, but leads to some dramatic changes in the cot values for other fractions
[46]. On the contrary, GA3 has no effect on the DNA isolated from the roots of Cucumber but
DNA from the treated shoots and leaves of the same plants, exhibit a nine-fold increase in the
intermediate reassociation fractions. According to Britten and Davidson (1969) such
sequences may have a regulatory role [47]. In cymbidium protocorm tissue, while auxin
causes an increase in AT-rich DNA, GA3 promotes an increase in GC-rich DNA [48]. Similar
studies have been carried out in various plants such as wheat embryos, pea epicotyls etc.
[49]
However, the above studies do not indicate whether, phytohormone induced changes in
DNA population is due to primary or secondary effects of the hormones.
PHYTOHORMONES AND TRANSCRIPTION
Protein synthesis is a crucial event in growth and differentiation, and therefore the
effect of phytohormones on transcription and processing of different kinds of RNA would
determine the process of morphogenesis.
AUXIN-MEDIATOR/ACCEPTOR PROTEINS AND RNA SYNTHESIS
The mechanistic aspects of auxin function appear to be very complex. Previous
investigations have suggested that there may be one or two cellular sites at which auxin may
act and elicit its responses on growth and differentiation. One such site is found to be located
in the plasma-membrane and the other may be a non-plasma membrane site located either in
the cytoplasm or nucleus.
Auxin-mediated early cell enlargement is known to function through the activation of
membrane bound factors [50-52]. The early growth is insensitive to both cycloheximide and
actinomycin D; hence neither RNA nor protein synthesis is required for this function.
However, the second phase of growth requires both RNA and protein synthesis. The auxin-
induced mechanism of early growth is speculated as due to the cell-wall loosening effect
caused by the secreted protons, which are pumped out by the auxin-activated membrane
bound factors [53]. The existence of soluble auxin-binding proteins of molecular weight
3,15,000 and 10,000 daltons have been reported in dwarf bean seedlings and soybean
cotyledons respectively [54,55]. It is not clear, whether these auxin binding proteins have
any role in transcription. Hardin et al. (1972) [56-58] have demonstrated that in soybean
hypocotyls and onion stems, auxin treatment results in the release of a plasma membrane
factor into the cytosol, which stimulates -Amanitin sensitive RNA polymerase activity.
However Mathyse and Philips (1969) have shown the existence of auxin-receptor complex in
the nuclei and this factor is responsible for the enhanced RNA synthesis. Auxin-receptor
protein has been isolated from coconut nuclei and shown to have a molecular weight of
10,000 daltons. It binds to the auxin non-covalently at a molar ratio of 2:1. In the presence of
transcribing system, consisting of -amanitin sensitive RNA polymerase (presumably RNA
polymerase II), native DNA, and an initiation factor, the auxin binding factors stimulate RNA
synthesis 2-3 fold, only in the presence of auxin. Although, these factors are not well
characterized, this constitutes a good evidence for the existence of auxin-receptor proteins.
Using changes in melting point profiles and bathochromic shifts in DNA and chromatin,
Fellenberg (1971) and Bamberger (1971) have claimed that auxin interacts with DNA and
chromatin directly, there by facilitating transcription [62,63]. However, these observations
turned out to be due to changes in the PH of the medium, rather than to the direct effects
[64].
While the studies on auxin-histone interactions have not yielded any significant information,
non-histone proteins are now considered to be the most likely candidates influencing specific
gene transcription. Non-histone proteins are heterogenous and include a number of enzymes
and structural proteins. They are species-specific and tissue-specific. Their phosphorylation
pattern changes with the different states of gene activity [65,66]. Such an involvement is
very well exemplified in the studies involving progesterone and estrogen receptor complexes
with the chromatin of target cells in different animal systems.
In plants, the existence of zone-specific non-histone proteins is known in soybean
hypocotyls [68]. Recently, 2,4-dichlorophenoxy acetic acid (2,4-D) induced changes in the
phosphorylation of nuclear proteins has been shown in soybean, but the correlation between
auxin induced RNA synthesis and the phosphorylation pattern ahs not been established
unequivocally [69].
Tissere et al. (1975) have isolated factors from the acidic fraction of the chromatin
proteins from the lentil roots, referred to as , , and factors. On auxin treatment,
the , and factors remain unchanged but the factor increases two-fold. While the factor
regulates RNA polymerase I , affects polymerase I and II. Hence these factors are known to
regulate the activity of RNA polymerases without enhancing the synthesis of the enzyme [70].
Generally, auxins induce RNA synthesis. Silber and Skoog (1953) for the first time
correlated auxin-induced cell enlargement with enhanced RNA synthesis [71]. Since then,
considerable work has been carried out on the auxin induced changes in the levels of various
RNA species.
Auxin has been reported to preferentially increase rRNA content in pea epicotyls and
soybean hypocotyls [72]. But, it has also been shown that the enhancement in RNA synthesis
occurs well after auxin has effected growth. It has been found that 5-fluorouracil [5-FU] does
not prevent cell enlargement in soybean hypocotyls and artichoke tuber, but auxin induced
growth in oat coleoptile and soybean hypocotyls is inhibited by actinomycin D. The above
observations suggest that auxin induced growth in the earlier stages does not require RNA
synthesis, but requires the synthesis of messenger RNA later [73,74].
Investigations, using polysome content as a measure of quantification of mRNA
synthesis have shown that auxin induced polysome levels are actinomycin D sensitive, but 5-
FU insensitive. This indicates that the synthesis of new ribosomes is not necessary for the
increase in the levels of polysome [75-78]. Verma et al. (1975) using in vitro cell free
protein synthesizing system and cellulase specific antibody, have shown that the auxin
induces the synthesis of cellulase-specific RNA [79]. However, increase in other m RNA
populations is not ruled out.
It is not clear, whether, the auxin induced messenger RNA is synthesized as a precursor
heterogenous RNA (hnRNA) in plants. In short-term labeling experiments (45 minutes), auxin
enhances the labeled precursor incorporation, preferentially into the nuclear fraction. Long
term labeling experiments, however indicate that incorporation into both nuclear and
cytosolic fractions is enhanced [80]. Recent studies with Petroselium sativum (parsley),
have demonstrated that most of the poly (A)-RNA is synthesized as hnRNA. This is established
by DNA-RNA hybridization-kinetic studies [81].
Attempts have also been made to use Escherichia coli RNA polymerase core enzyme,
to measure transcription of DNA/chromatin, as an index of differential gene expression
chromatin, isolated from auxin treated soybean seedlings has been found to code for
synthesis of many fractions of RNA species, of which the synthesis of TB-RNA, several
fractions of hetero disperse RNA AND 4S-5S RNA are much more than the control. This
chromatin preparation enhances labeled amino acid incorporation into proteins by 2-3 fold in
coupled transcription-translation assays [82,83]. Similar studies involving the plant tissues
have clearly indicated that auxin can influence polymerase activity, as well as template
activity.
GA-BINDING/MEDIATOR PROTEINS AND RNA SYNTHESIS
The presence of GA3 – binding protein in pea stems, epicotyls and other plants has been
reported. The approximate molecular weight of the GA3 – binding proteins, extracted from
dwarf epicotyls of pea plant, has been determined to be 5, 00,000 and 60,000. Both the
fractions bind GA3 noncovalently and easily exchange with non-labeled GA3.
Not much is known about the effects of GA3 binding proteins on transcriptional events.
Isolated pea nuclei do not respond to exogenously added GA3 but, if the nuclei are isolated
after GA3 treatment, transcription is enhanced [86]. Attempts to localize GA3 – binding sites,
in cells by autoradiography or by cell fractionation, have yielded limited results [87]. GA7 has
been shown to interact with DNA containing higher AT than GC content. It is also reported
that GA7 interacts With DNA isolated from a number of plant sources, as well as some phages,
but it does not interact with either animal or bacterial DNA. This specificity of GA7 interaction
has not been explained. In the presence of DNA Ligase, GA7 causes the formation of
covalently linked loops in cucumber DNA, but cytokinins and auxins do not have any effect.
Recently, it has been reported that at low concentrations of ethidium bromide, GA 4, GA7, or
GA3, show synergistic effects on elongation in cucumber hypocotyls, perhaps by exposing
binding sites in DNA and thus making gibberellic acid more effective.
Studies on the effect of GA3 on transcription of either isolated chromatin or isolated
nuclei from pear or cucumber show, that the increase in RAN synthesis is due to increased
RNA polymerase, as well as due to the increased template availability [90]. A wide variety of
tissues such as barley leaf segments, clover seedlings, lentil epicotyls, potato buds, cucumber
hypocotyls and sugar beet have been used to study GA3’s effect on RNA synthesis. A
significant amount of information on the effect of GA3 on RNA synthesis comes from the
studies with barley aleurone cells. GA3 causes a selective increase in m RNA for -amylase,
which on release to the endosperm facilitates the mobilization of carbohydrate source for the
growing embryo. If actinomycin-D is added along with GA3, the increase in -amylase
synthesis is inhibited [91]. The labeling of poly (A)–RNA, specific for -amylase, shows an
increase after GA3 treatment with a lag period of 3-4 hrs. This correlates well with the lag
period of -amylase synthesis observed in vivo. However, Higgins et al. (1976) and Carlson
(1977) have suggested that GA3 activates the preexisting -amylase m RNA by post
transcriptional process into an active translatable form of m RNA but, Rodaway et al. (1978)
have considered this possibility as very unlikely similar effects of GA3 on poly (A)-RNA
synthesis have been established in etiolated maize seedlings, but their translational products
have not been characterized.
CYTOKININ-RECEPTOR PROTEIN AND RNA SYNTHESIS
The presence of cytokinin-receptor protein has been indicated by the work of
Mathyse et al. (1969) [97]. The cytokinin-protein complex enhances RNA
synthesis in vitro in presence of pea bud chromatin or DNA as template and e. coli or calf
thymus, kinetin-receptor does not stimulate RNA synthesis. Similar studies using nuclei-rich
preparations from soybean, tobacco callus and pea buds, have shown that the cytokinins
enhance [14 C] –ATP incorporation into TCA precipitable and KOH digestible RNA fractions by
40-100% within 5-10 minutes after treatment. To the phytohormone-starved tobacco pith
callus, the addition of cytokinin alone does not cause any major charges in RNA metabolism,
but cytokinin with auxin treatment enhances [32 p] – orthophosphate incorporation into
polysomal RNA within 3 hrs of treatment [98,99]. On the contrary, in the hypocotyls
of soybean, even at early stages after treatment, the auxin induced incorporation of [14 C] –
uridine into RNA is inhibited by kinetin. After 6 hrs of incubation with cytokinin and auxin
together, the inhibition of labeled precursor incorporation into rRNA and tRNA is 90% and TB-
RNA 30-50%. However, the labeling into D-RNA is not affected [ 100-103 ].
Cytokinin-mediated increase in the specific isoacceptor leucyl-tRNA species has been detected in
cotyledons and hypocotyls of beans [ 104, 105 ]. This could be the result of and effect of direct
synthesis or on post-transcriptional modifications. In general, it seems that the occurrence of
cytokinins in tRNA is not longer viewed as necessary for eliciting the cytokinin response [106-108].
ETHYLENE-RECEPTOR AND RNA METABOLISM
Although no coherent information is available regarding ethylene binding to receptor
proteins, transcriptional studies with isolated chromatin from ethylene treated tissue, show
qualitative changes in the RNA species synthesized. There is a change in the nearest
neighbor frequency of the RNA synthesized, thus indicating transcription of new template in
response to ethylene [37].
Generally ethylene is known to induce abscission and fruit ripening in plants. During
abscission, ethylene enhances the synthesis of cellulase and pectinase, which are responsible
for the breakdown of the cell wall and pectin [109, 110]. In cotton, Coleus and bean plants,
ethylene induced abscission can be prevented by actinomycin D. Furthermore, using 5-FU as
an inhibitor of rRNA synthesis, investigations have demonstrated that the ethylene mediated
abscission involves induction of specific mRNA, but the products have not yet characterized.
Similar results have been obtained in the ethylene induced ripening
of Pyrus mallus fruits [111]. In ripening tomato fruits, ethylene brings about changes in the
relative amounts of iso-accepting species of leucyl-, lysyl-, methionyl- and tyrosinyl-tRNAs.
ABA-RECEPTOR AND RNA SYNTHESIS
ABA affects a number of physiological processes, such as dormancy, leaf senescence, abscission,
seed germination and flowering, and also antagonizes the effects of other plant hormones, in a number
of plant tissues. It is not clear, whether such physiological responses to ABA are brought about through
mediator molecules. However, various reports indicate that the effect of ABA is on RNA – turnover and
translational process.
ABA has been shown to inhabit the transcriptional capacity of isolated chromatin [112]. This
inhibitory effect is ascribed to the effect of ABA on polymerase activity [113,114 ].
In Fraxinus embryos [115], ABA inhibits labeled precursor incorporation into RNA, and reduces
polysome formation, but protein synthesis is not affected. The above results have been interpreted to
mean that ABA prevents the synthesis of mRNA and other RNA species, thereby affecting growth.
ABA, in pear embryo and lentil roots, causes a preferential increase in UMP rich RNA and a
substantial decrease in GMP, but there is not change in AMP, CMP contents of RNA (115 a). This
observation is significant in view of the recent observation, that translation control RNA (tcRNA), which
controls translation of certain mRNAs has very high (50%) content of UMP nucleotides [116]. Additional
support to the above observation comes from the findings that ABA treated germinating seedlings
contain UMP rich (40-50%) RNA species [117]. It is not clear whether this high level of UMP rich RNA is
due to new synthesis or preferential degradation of the other RNA species.
ABA also inhibits GA3 - promoted -amylase synthesis, in barley aleurone cells. Some
investigators have shown that ABA inhibits [14 C] – uridine incorporation into polydisperse RNA [92,
118]. There are also reports that ABA does not inhibit the synthesis of any species of RNA [119]. In
this context, the observation about certain short chain fatty acids (of C5 and C9 length) at a
concentration of 10-4 M, inhibiting GA3 – induced amylolysis in barley endosperm is interesting. Such
short chain nonionic fatty acids have been detected in many germinating seeds such as oat and
fenugreek [120].
PHYTOHORMONES AND POST – TRANSCRIPTIONAL EVENTS
Perichromatin and Precursor RNAs:
Most of the RNA species, transcribed within the nucleus, are in the form of larger molecules and
these are processed and transported to the cytoplasm across the nuclear membrane pore complex
[121]. The existence of extra chromosomal and extra nucleolar ribonucleoprotein structures called
perichromatin granules (presumably precursors of mRNAs) in rat liver nuclei and plant cells, has been
reported as early as 1969 [122]. The transport of such particles across the membrane pore complex
has been speculated. Active movement of such chromatoid body (containing RNA) across the pore
complex, during rat spermatogenesis is every well documented [123]. In plants, synthesis of large
precursor RNAs (hnRNA) and their processing into the mature forms have been reported [43,124,125].
POLY (A) AND “CAP”
During the processing of hnRNA, additional structures such as the “cap” at the 5’ end poly (A)
chain at the 3’ end are tagged to the mRNA. Addition of poly (A) segments of 50-200 nucleotides, at
the 3’ end of the hnRNA or mRNA is catalyzed by poly (A) polymerase [126]. The existence of such
polyadenylated mRNA has been demonstrated in the nuclei and cytoplasm of plant cells [93,127,125].
However, not all mRNAs of RuDP carboxylase in chloroplasts lack such mRNA (?), it is one such example
[128].
Capping at the 5’ end of the mRNA involves the addition of 7-methylguanosine, which is linked by
a 5’-opopopo5’ triphosphate bridge to a 2’-0-methylated Adenine [129,130]. The “cap” structure is
implicated in influencing the processing of pre-mRNA, initiation of translation , transport of mRNA and
stability of the same [131, 132]. However, “cap” is not required for the translation of all mRNAs [133].
Recent reports suggest that the prokaryotic mRNAs, which are “capped” in vitro, translate very
efficiently in cell-free eukaryotic protein synthesizing system [134]. Sonenberg et al. (1979) have
isolated a “cap” binding protein whose molecular weight is 24,000. This protein stimulates translation
of capped mRNAs in an in vitro system derived from HeLa cell extracts, under conditions that do not
increase translation of non-capped mRNAs from satellite necrosis virus [134a]. In plants, there are not
many reports about “cap” structures in mRNAs. However, addition of 7-methyl guanosine 5’-
monophosphate (m 7G5P) to the French bean seed mRNA – programmed cell free protein synthesizing
system derived from wheat germ, inhibits [35 S] - methionine incorporation into proteins by 90%,
thereby suggesting that the mRNA isolated from French be an cotyledons are “capped” [135]. The role
of plant growth substances in the “capping” process is not clear.
The non-coding sequences found between the first AUG codon and the “cap” at the 5’ end and
the last terminator codon and the first adenine nucleotide of the poly(A) segment at the 3’ end are
suggested to play an important role in initiation and termination of protein synthesis [136]. Such
information is not available with plant mRNAs.
INFORMOSOMES
The messenger RNA is transported from the nucleus to cytoplasm as m RNA protein complex (m
RNP). These m RNP particles may release the m RNA for polysome formation or the m RNPs may
remain in an inactive form. The latter particles are referred to as informosomes. Such informosomes
are found in a number of embryonic tissues [93,137,138] and other non-embryonic structures such as
stems, tubers, etiolated leaves, dry mosses, spores of ferns, Acetabularia and Dictyostelium . Such
inactive m RNPs have also been reported in animal cells [145-147].
The m RNPs that are found in most of the organisms have poly(A) tail at 3’ end [148]. The
presence of such m RNPs ensure the preservation of m RNAs during periods of dormancy and certain
adverse environmental conditions such as drought, lack of optimal photoperiodic stimulus and lack of
light of actinic wavelength.
ACTIVATION AND INACTIVATION OF m RNAs
The activation of preexisting dormant m RNPs to external stimuli has been reported in a variety
of systems such as germinating seeds [138,159] rejuvenating moss[141], etiolated leaves exposed to
light [140], ferritin synthesis in rat liver [146] and fertilization in sea urchin eggs [160]. Specific effects
of plant hormones on this process have also been documented. These examples include GA3 effect
in barley aleurone cells and spores of Anemia phyllitidis [93,149], IAA effect in pea epicotyls [79] and
cytokinin effect inGlycine max [102,103]. In all the above mentioned cases, when external stimuli are
provided, the dormant m RNPs get activated and the rate of protein synthesis is enhanced, even in the
presence of actinomycin-D, or 5-FU, thereby indicating that the enhanced protein synthesis is
independent of new RNA synthesis. It is not clear, whether these external stimuli or added
phytohormones act directly on m RNPs or function through other mediating-factors, which may either
activate ribosomal machinery or bring about the conformational changes in the secondary structure of
m RNPs.
TRANSFER RNA:
Transfer RNA (tRNA) is also synthesized as a large precursor and then processed into smaller
products [131]. Transport, slicing and secondary modifications such as methylation, thiolation of pre-
tRNAs are some of the important controlling processes, which make available, the functional tRNAs for
protein synthesis. Addition of CCA at 3’ terminal of t RNA and activation of amino-acyl tRNAs and
activation of amino-acyl t RAN synthetases are other crucial steps that regulate the translation
processes in Pisum sativumseedlings, GA3 is known to enhance aminoacyl t RNA synthetases by 5-8
fold [149]. In early 1960s, auxin was suspected of form a complex with t RNA thus regulating t RAN
function [150]. However, ti is now considered that this may not be the case [151,152].
As the genetic code is degenerate, it is thought, that multiple iso-accepting species of tRNAs may
exert a regulatory role in the translation of mRNAs [153] and the relative changes in the population of
such iso-accepting species of t RANs [154] and the relative changes in the population of such iso-
accepting species of tRNA during different stages of development, has been recorded in plants
[155,156]. The presence of cytokinins-effect may be mediated through tRNAs [106]. The occurrence of
the highly active cytokinin6 – de (6-3 methyl-2-butenyl amino purine) adjacent to the A base of the
anticodon triplet has been demonstrated in yeast serine- tRNA I, II and E. coli- tRNA [157]. Such
cytokinin containing tRNAs have been identified in various plants and it is felt, that cytokinins may
exert their effect on growth through regulating the translational process. However, recent studies
[158] show that there is no correlation between the effect of hormone-induced growth and the
presence of cytokinin in tRNA species. Nevertheless, there are evidences to show that cytokinins can
affect the tRNA activities either by regulating the levels of tRNAs or by secondary modification of tRNA
by methylation or by changing amino acyl-synthetase activity [108].
CONTROL OF PROTEIN SYNTHESIS BY PHYTOHORMONES
In radish cotyledons, cytokinin-enhanced protein synthesis does not require mRNA
synthesis de novo [161]. Similarly cytokinin enhances protein synthesis in soybean callus, as early as
30 minutes after treatment [102]. Klambt (1976) [99] has demonstrated that N5 (2- isopentyl)adenine
stimulates cell-free protein synthesis slightly in systems derived from tobacco pith and corn extracts.
Such stimulation is suggested as due to selective binding of cytokines to a specific protein of the
ribosome. Fox et al. (1975) [162] have identified one low and one high affinity cytokinin binding site
on wheat germ ribosomes. The binding affinity is lost on boiling the 0.5M KCL wash or on treatment
with trypsin, suggesting that this high affinity receptor is a ribosomal protein. Such cytokinin-binding
protein from tobacco callus is associated with 4OS ribosomes [163]. In animal systems [164] increased
or reduced phosphorylation of ribosomal protein controls the rate of protein synthesis. Cytokinin is also
thought to enhance protein synthesis through dephosphorylation of ribosomal proteins. Tapfer et al.
(1975) [165] have found that, when cytokinin requiring soybean callus is transferred to a cytokinin
deficient medium, the decreased rate of protein synthesis is accompanied by increased
phosphorylation of some ribosomal proteins.
In soybean hypocotyls, a parallel situation is found where Auxin causes increased
protein synthesis in a cell-free protein synthesizing system [166]. Travis et al . (1976), have
proposed, that the polysome formation is dependent on ribosome activation as well as mRNA
supply. In the same system they have also observed the incorporation of labeled amino acids
into 3 ribosomal proteins, one of which shows several fold higher incorporation that that of
the control. However, ribosomal dissociation studies, clearly indicate that these auxin
induced proteins are associated with 60S ribosomal sub-units. Evidence for hormone-induced
protein synthesis, independent of mRNA synthesis, is also found in Pea epicotyls for [99].
Here, auxin has been found to activate the mRNA specific for cellulase at very early periods,
when there is no increased mRNA synthesis. The poly (A) – RNA isolated from treated
segments, on translation in vitro, shows enhanced synthesis of cellulase. The poly(A) – RNA
from control plants hardly codes for any cellulase. The above results suggest that the auxin
perhaps activates the preexisting, dormant mRNAs into active translatable form of mRNA.
Thus, the evidences presented above indicate that most of the growth promoting
hormones increases protein synthesis through increased mRNA synthesis as well as through
activation of dormant mRNAs or the ribosomal system. Such dual action of phytohormones is
well documented (165 and 166) ABA, in general, appears to exert an inhibitory effect on the
synthetic processes. Although, ABA inhibits light-promoted unrolling and greening of etiolated
leaves in wheat, it does not prevent light induced polysome formation [167]. In etiolated
leaves of barley seedlings, ABA not only reduces the amount of polysome formation in the
dark, but also inhibits light-induced increase in polysome size [168]. In barley aleurone cells,
GA3 – induced -amylase synthesis is inhibited by ABA and this inhibition is prevented by the
addition of cordycepin. This observation indicates that ABA has both transcriptional as well as
translational control [169]. In this context, increase in UMP-rich RNA component, in response
to ABA treatment, is highly significant. Heywood et al. (116) have isolated a translational
control RNA of mol.wt. 10,000 daltons, which is rich in UMP. This translational control RNA is
believed to play a significant role in translational control where it inhibits translation of
mRNAs, probably by pairing with poly-AMP segments of mRNAs [116 &170]. This observation
correlates well with the effects of ABA on growth inhibition in plants.
PHYTOHORMONES AND RNA – PROTEIN TURNOVER
The regulation of RNA and protein turnover is another important facet of cellular
differentiation. Phytohormones are known to regulate such events. ABA, when applied to
excised barley leaves, enhances chromatin bound nuclease and deoxy-ribonuclease (DNase)
activities [180]. In excised Avena leaves, it affects one out of the four nuclease isoenzymes
[171]. Similarly in lentils (Lens culnaris), ABA reduces the levels of RNA and also inhibits
growth [172]. However, in maize coleoptiles, ABA suppresses the incorporation of radioactive
precursors into RNA without affecting the levels of RNase for 8 hours [173].
Auxin is also implicated in suppressing the RNase activity in number of cases
[174,175]. However, no direct relationship between nuclease levels and RNA stabilization has
been established. In pea epicotyls, IAA elicits a large increase in membrane-bound RNase,
which in turn is suppressed by kinetin [176]. Similarly, a correlation has been established
between nucleases and auxin treatment in wheat root callus cultures [177].
In senescing leaf tissues, increased activities of RNase and DNase are correlated well
with the decreased content of RNA and yellowing of the leaf. Cytokinins suppress such
activities and delay senescence, which is popularly known as Richmond and Land effect [178-
180]. In the absence of a “water stress” on leaves RNase activity is suppressed by kinetin but
the same hormone under “water stress” condition enhances RNase activity [181].
In mustard and corn leaves, during sene scence kinetin and 6-benzylaminopurine retard
the loss of radioactive label from prelabelled proteins [182, 183]. In wheat leaves, cytokinin
suppresses proteolysis of RuDP-carboxylase and this is prevented by CHI, suggesting that the
proteolytic enzymes are being synthesized de novo during senescence [184].
During differentiation in Dictyostelium (from plasmodial stage to sporangium) [144],
blue-green algae (vegetative to heterocyst) [185], and soybean callus cells (non-dividing to
dividing state) [103], appearance and disappearance of some specific proteins have been
noticed. This selective appearance and disappearance of certain proteins during
developmental process may involve differential expression of genes. Such transitory proteins
may play a very important role in morphogenesis.
ADVENTITIOUS ROOT INITIATION AS A MORPHOGENETIC SYSTEM
Introduction:
In the present investigation, hormone induced root initiation in the hypocotyls tissue
of Phaseolus vulgaris Linn. Is used as a model system, to study the molecular events
involved in the differentiation process. A brief review of the literature on adventitious root
formation is therefore presented here.
The root that develops from any part of the plant body other than the radicle is called
an adventitious root. Duhmel du Monceau (1758) for the first time explained adventitious
root formation in stem as due to the downward movement of sap. Sachs (1880) postulated
the existence of a highly potent root forming substance originating from leaves [186]. Van
Tieghen and Douliot (1888) observed adventitious root formation in 67 dicotyledonous and 21
monocotyledonous families [187]. In 1933, Bouilleune and went propounded a hypothesis
that “Rhizocauline”, a specific factor, is responsible for root initiation [188]. Went and
Thimann (1937) observed for the first time that auxin, in addition to its growth-promoting
effect, also induces root initiation in stem outings. Thus, a practical use was also found for
the auxin [189].
Anatomical studies:
Previous anatomical studies reveal that during root initiation, meristematic activity is found in
the region of endodermis, pericycle, especially over the vascular bundles [190-192]. However, there
are many examples of adventitious roots originating from phloem parenchyma or phloem ray cells. It is
also not uncommon to find roots arising from tissues such as primary xylem parenchyma, callus etc.
[193]. The common observation of adventitious roots arising near vascular bundles, prompted Priestly
and Swingle (1929) to suggest that nutrient supply may be a factor determining the site of root
formation (194).
In recent years, pea epicotyls (Pisum sativum) and hypocotyls of Glycine
max (soybean) have been used as systems for studying the effect of plant growth
substances in the regulation of cambial activity and organization of root primordial in plants.
Excised or intact hypocotyls, epicotyls segment and callus in response to auxin treatment,
undergo dedifferentiation leading to the formation of root primordial. Preliminary anatomical
studies have shown that parenchymatous cells, in the cortex of 24 hr auxin treated soybean
hypocotyls and pea epicotyls, appear to be swollen and exhibit few cell divisions
[72,195,196]. After 48 hr the number of cell divisions increase and disintegration of many
cortical cells sets in, leaving lacunae. Root primordials originate from the cambial cells near
vascular tissues and become recognizable after 3 days of hormone treatment. The primordial
cells continue to divide and occupy the lacunae. In 4-5 days the new roots break through the
epidermal layers of the segments.
Factors other than phytohormones:
Many environmental factors such as water supply, temperature, sunlight, acidity of the soil, etc.
are known to affect root initiation. Young leaves promote root initiation but old leaves are found to
inhibit them. Roots themselves inhibit new root formation, perhaps as a result of the production of
some inhibitors [197].
The role of organic micronutrients is new root formation is significant. Adenine,
arginine, aspargine and uracil promote root initiation [197] but guanine inhibits root formation
in Phaseolus mungo [198]. Among other organic micronutrients, nicotinamide, niacin,
thiamin (vitamin B1), vitamin K an vitamin H and pyridoxyl enhance root formation only in the
presence of auxin [197].
It is also interesting to note, that some cortisols stimulate adventitious root formation in
the intact hypocotyls segments of mung beans [199]. The cortisol sensitive phase during root
initiation is between 2nd and 4th day of treatment. However, cortisols in the presence of
auxins, stimulate root initiation, but inhibit growth of the roots [200]. Recently it has been
reported that vitamin D and its analogues by themselves promote root formation slightly, but
in the presence of IBA, IAA or NAA, root production is greatly enhanced [201].
Gentics of root formation:
Zobel (1975) has described mutants in tomato with respect to root formation. These mutants are
referred to as rosette (ro), lateral-les (dgt) and dwarf (drf), of which r has been established as
adventitious rootless mutant [202]. The diploid chromosome number of these plants is 24 (2n = 24).
Breeding experiments and genetic analysis have revealed that ro gene is located on the second
chromosome and follows simple Mendelian inheritance. Hence, it is suggested that a single pair of
alleles controls adventitious root formation.
Plant hormone interactions:
The most effective adventitious root forming substances known are IBA and NAA [188].
Staurt (1938) demonstrated that excised stem cuttings of Phaseolus mobilize carbohydrate
reserves as soluble sugars within 24 hr of auxin treatment [203]. Altman and Wareing (1975)
obtained similar results using [14 C]-CO2 in Phaseolus vulgaris cuttings [204]. However, the
application of exogenous sugars could not replace the requirement of auxins for new root
formation [205].
Ethylene stimulates root initiation in many leafy stem cuttings [206], but there are also
cases where ethylene has no promotive effects [207,208]. Batten et al. (1978) have claimed
that there is no correlation between the effectiveness in rooting and induction of ethylene
production by a variety of auxins. Application of ethylene has no effect on root initiation and
it proceeds normally even in the presence of 7% carbon dioxide or in hypobaric conditions
[209]. In this context, it is pertinent to note, that in the hypocotyls of mung bean, IAA induces
a specific protein, which in turn inhibits IAA induced ethylene production [210].
While ABA exhibits both stimulatory and inhibitory effects on new root formation
[211,212], cytokinins and GA3 in general are inhibitory [213-215]. However, it has been
suggested that the early stages of root formation are most sensitive to cytokinin and
GA3inhibition, but under certain conditions GA3 promotes root formation in Citrus embryoids
[216]. Verga et al. (1974) have found that pretreatment of leaves with GA3, before excision,
enhances adventitious root initiation. This effect is believed to be due to GA3induced
enhancement of auxin synthesis [217].
In callus cultures, in general, root formation is dependent on the ratio between auxin and cytokinin concentration. While higher ratios promote root formation, a lower ratio inhibits this process but induces shoot primordial. However, at intermediate ratios, both roots and shoots are produced. Hence, it is deduced that the relative concentration of auxin to cytokinin is very critical in the differentiation and development of organs such as roots and shoots [4] Fig.2.
BIOCHEMICAL EVENTS:
During adventitious root initiation in the epicotyls of pea and hypocotyls of soybean,
increase in DNA, RNA and protein contents has been observed. After 3 days of auxin
treatment, cellulase activity increases 30-40 fold, pectinase 7-8 fold, pectin esterase and 1 -
> 3 Gluconase by 3-fold [195]. Similarly, during rhizogenesis, in detached cotyledons of
mustard (Sinapis alba L.) increase in RNA and protein contents have been noticed [218].
Some evidence has been presented to show that the increase in cellulose activity is totally
abolished, if the segments are treated with actinomycin D or azaguanine or puromycin along
with the auxin. Hence, it is discerned that the continued synthesis of RNA and protein is
essential for the increase in the activity of cellulase [196].
Trewavas (1976) has documented some interesting observations on the changes in
nucleic acid metabolism and protein synthesis, specifically cellulose, during adventitious root
initiation in pea epicotyls and soybean hypocotyls [196] (Fig. 3) In epicotyls of pea plant, over
a 3 day period, auxin causes an increase in DNA and protein contents by 2.5 fold and RNA
content 4.0 fold. In soybean hypocotyls, a 4-5 fold increase in DNA and protein contents and
a 10-foldincrease in RNA content have been observed. Under similar conditions, actinomycin
D and puromycin inhibit the auxin induced increase in DNA, RNA and protein contents.
In the presence of auxin, the ratio of microsomal RNA to soluble RNA is 6.9 compared to
the control value of 4.8. These changes are attributed to auxin induced differential turnover
of RNA. Furthermore, labeling studies have indicated that initial lag period for auxin mediated
increase in RAN synthesis is l hr in pea segments and 3 Hr in soybean. After several hours of
treatment with auxin, increased labeling has been found in rRNA and tRNA [72,151].
Using in vitro transcriptional assay system and DNA-RNA hybridization techniques, O’
Brien et al. (1968) and Verma et al. (1975) have demonstrated qualitative and quantitative
changes in RNA [79,83]. This implies selective unmasking of the genome, which correlates
well with the time point at which cell divisions start.
In a time-course study in pea segments, Davies et al. (1963) have detected a further
shift in the labeling pattern of the polysomal populations. At the end of the third hour the
proportion of polysomes containing 10 ribosomes or more has increased 3 fold. Ribosomal
populations double within 6-10hours. After 10 hr of treatment, the ratio of polysomes to
monosomes reaches a value of 9, compared to 0.5 in control tissue [219].
In soybean hypocotyls a shift from monosomes to polysomes is noted after 3 hr of
auxin treatment and this is sensitive to actinomycin D, but not to 5-FU, 6-methylpurine in
polysome size is independent of ribosomal RNA synthesis, but dependent upon the continued
synthesis of tightly bound (TB) and DNA-like RNA (D-FNA). It has been further demonstrated
that the increase in protein synthesis by individual polysomes in vitro is due to the increased
activation of chain initiation, probably through the synthesis of 3 specific 60s ribosomal
proteins [77,166].
The present investigation has been undertaken to study (1) the early molecular events
leading to dedifferentiation of pericyclic cells into root primordial; (2) the identification of
specific gene products essential for adventitious root initiation; (3) a delineation of sequence
of events at molecular level leading to the emergence of root initials. Adventitious root
initiation in hypocotyls segments of Phaseolus vulgaris Linn. In response to the hormone
IBA treatment has been used as the model system.
Fig.2: Interaction between Auxins and Cytokinins during Organ differentiation in callus
cultures. Open arrows indicate the promotion of organs and closed arrows indicate inhibition.
Fig.3: Time of initiation of metabolic changes induced by IAA after addition to Soybean
hypocotyls or pea epicotyls.
PLANT HORMONES-Introduction
By convention hormone are said to be a substances whose site of synthesis and site of action
are different; the two events are separated by space and time. Hormones are known to elicit
specific responses. Charles Darwin first demonstrated the existence of such substances in
plants. Who in his own inimitable way explained growth of plant tips, respond to light and
exhibits photo induced curvature movements. Since then botanists all over the world made
studies in unraveling the mysteries of diffusible substances called hormones which control
growth and development of the plant body.
DISCOVERY OF PLANT HORMONES
Darwin used canary grass coleoptile tips to demonstrate the sensitiveness of the stem apex to
light mediated curvature movements. Later Boysen Jensen used Avena coleoptile tips to
demonstrate the presence of plant hormones. A gelatin block was placed on the decapitated
coleoptile tip, then the tip was replaced over the gelatin block and the tip was illuminated
from one direction. In response to light, the stem tip bent towards the light source. This was
explained as due to the downward movement of some substance from the tip through gelatin
block down wards. And the substance was considered as the cause for growth curvature.
Paal.A, on he other hand, placed the cut coleoptile tip asymmetrically over the decapitated
coleoptile segments and placed the seedlings in tip dark. After few hours, he observed the
curvature of the stem tip away from the side at which apical tip was placed. Pal’s experiment
further demonstrated the presence of some kind of growth promoting substances in coleoptile
tip, from which the substance was able to diffuse and bring about growth on one side hence
the curvature. What ones people called diffusible substances have be restated as
transportable substances for there are carriers in the cell membranes which do the role.
These are carriers specific.
F.W. Went collected growth promoting substances by placing the coleoptile tips on the square
agar blocks. By placing such loaded agar blocks asymmetrically on the decapitated coleoptile
tips in dark, showed the growth curvature movements. Persuing the above methods he
established quantitative bioassays. The bioassays explain and correlate quantitative
relationship between the amount of hormone applied and the magnitude curvature as a
response.
It is at this juncture of time biologists and chemists started identifying the chemical
component that is responsible for growth promoting activity. To their surprise, they found the
human urine as a rich source for the said hormone. Kogl and Haagen Smit starting with 33
gallons of urine, extracted 40 mg of the active principle in the form of crystalline powder
which showed 50,000 fold grater activity. First they called this substance as Auxin A. Using
the same extraction procedures they isolated another active substance from corn germ oil
and called it as Auxin B. Not satisfied with their purification methods, they used charcoal
adsorption column chromatographic procedures
for isolating a pure form of growth substance. The substances obtained from this method
were called Heteroauxin. Later heteroauxin was identified as Indole Acetic Acid. But this
substance was known as a chemical to them for it was already identified by E & H
Salkowaski. However, Salkowski’s did not know about the property of IAA as growth hormone.
The discovery of Indole acetic acid as the plant growth hormone gave impetus to plant
physiologists. As a result, new hormones were discovered from different plant sources. Their
site of synthesis, chemical structure, site of action and their physiological and morphological
effects have been studied in detail. So far, five phytohormones have been identified from
different parts of the plant body; most importantly all the hormones can be detected in the
same plants. They are Indole Acetic Acid (IAA), Gibberellic acid or Gibberellins (GA), cytokinin,
Abscisic acid (Abscissin or ABA) and Ethylene. Simultaneously a host of synthetic compounds
have been developed which stimulate plant hormones in many respects. The figure below
shows each hormone synthesis pathway.
PHYTOHEROMONES and PLANTS’ RESPONSES
Investigation on the effects of hormones on plants has revealed that the hormones elicit a
wide array of responses in different types of tissues in the same plant body. Physiologists
have realized that the responses to the hormonal treatments depend upon the kind of tissue
and the physiological state of the tissue. For example, in a developing stem segment, in
response to GA3, the internodes elongate considerably but the same hormone in maize grains
elicit the synthesis of alfa amylase enzymes is aleurone cells. Thus the specific response to a
particular hormone depends upon the inbuilt potentiality of the said tissues; this behavior is
because of its previous developmental programmes. It should also be remembered that the
specialized hormones found in animal systems have no counterparts in plants with respect to
the target cells and specific functions. The auxin which induces the growth in one part of the
plant body, fails to bring about the same effect in the other part, but it may have different
effects like apical dominance, new root formation or parthenocarpy, etc, at different site.
While GA is known to bring about the gene activation in aleurone cells leading to the
synthesis of alfa amylase, the same hormone acts on rosette shaped Hyoscyamus plant and
induces bolting and flowering. But in pea plants, it overcomes genetic dwarfism. The above
observations suggest that each and every phytohormone elicit more than one response in the
same plant body but at different sites. Furthermore as all hormones are synthesized at
different sites within the same plant body the said hormones interact with each other and
control the growth and development.
HORMONAL INTERPLAY
Although Haberlandt considered the tissue and organ culture under sterile conditions as a
theoretical possibility, Kattle in Germany and Robbin in USA have succeeded in developing
methods to culture tissues in a defined media. With the advent of tissue culture techniques,
studies on experimental morphogenesis progressed in leaps and bounds in the past 45 years
or so.
Using tissue culture methods, it is possible to treat the tissue with one or the other hormone
at will. Use of phytohormones in tissue cultures, have revealed that though a single
hormones has a specific effect, two or more hormones together at different concentrations,
elicit different responses in tissue from. In the presence of two different hormones, the
effects may be promotive, synergistic or antagonistic where one may modify the activity of
the other. Such reactions are referred to as Hormonal interplay.
Callus tissue can be grown from tobacco pith cells and the same can be maintained in a
defined nutrient medium in the presence of both IAA and cytokinins at particular
concentrations. The same callus can be induced for organogenesis by changing the relative
concentrations of IAA to that of cytokinins. In the presence of higher amounts of auxin to
cytokinins, callus generates roots. On the contrary if the concentration of cytokinin is higher
in relation to auxins, the callus cells produce shoots only. If the concentration of the two said
hormones is balanced, the callus induces the formation of both roots and shoots.
The above mentioned observations suggest that the hormonal interplay has a significant role
in organogenesis. Similarly, GA3 promotes callus growth but the same hormone inhibits
auxin-cytokinin induced shoot formation. The above said hormonal effects are not just direct
effects, but they also influence the levels of other hormones either by activating the synthesis
of a particular hormone or by inhibiting the synthesis of it.
Various studies on hormonal effects show that the endogenous levels of auxin in plant tissues
is elevated by the applications of GA, Cytokinins or both. While auxin induces the synthesis of
ethylene which in turn induces the formation of ABA, which on the contrary enhances the
levels of ethylene? Thus, they show both cooperative and promotive effects on each other.
It is also known that ethylene and ABA together bring down the levels of the auxin. This
effect can be overcome by the addition of cytokinins, for cytokinins are capable of bringing
down the levels of ABA through the increased levels of GA biosynthesis. The close
relationship and interplay between GA, auxins, cytokinins, ABA and ethylene, exhibits an
excellent feedback control mechanism. However, understanding of hormonal interaction at
the level of gene expression and their product is very important in interpreting the hormonal
interplay and effects.
Plant Hormones -AUXINS
Distribution
Though auxin is synthesized in the plant apices of shoots and roots, it is transported towards
their respective basal parts. Quantitative estimation of auxins found in the segments of
seedlings, by spectrophotometric analysis; show that stem apex possesses 1.5 to 2.0 fold
higher amounts than found in the root apices. The lowest concentration is found in the region
of the stem where cotyledons are attached. Even leaves contain some amount of Auxins.
Looking at the structure, functional responses, plants are no different from animals’ system
neuronal network. Holistically one can imagine plants having a system are no different from
animal systems.
Auxins exist either in bound form or in a free state. The non diffusible form is considered as
bound form and it is active, but the freely diffusible form is referred to as an inactive form.
The bound state of the auxin has been explained as due to the binding of receptor proteins or
binding proteins to auxins. Such proteins are found in the plasma membranes, cytoplasm and
chromosomes. The mol. wt. of auxin binding proteins has been determined as 10,000 and 31,
5000 Daltons. Such proteins have been isolated from the free nuclei of coconut liquid
endosperm. These proteins in the presence of IAA are found to be active in inducing gene
expression. In living cells, the
relative concentrations of bound form and free forms of auxin is not constant but depends
upon the environmental conditions or the functional state of cells.
TRANSPORTATION
Auxin is transported from the site of synthesis to the site of action, which is not far away.
Nevertheless auxin is also translocated to other regions of the plant body. The transportation
of auxin is polar, i.e., from apex to the base, which is called basipetal movement, but
acropetal movement i.e. from the base to apex has also been observed but the amount
transported is almost negligible. The ratio between basipetal and acropetal movement is
approximately 3:1.
Furthermore, the transportation is more or less an active process. The long distance
transportation appears to take place through sieve tubes and it is a facilitated mechanism.
The rate of auxin movement is about 6.4-20 mm/hr, which is many times faster than the rate
of diffusion. It is now clear that the transportation is through carriers. Auxin exists in ionized
form called A (-) and uncharged form called AH. Transportation across the membranes is
through carriers and in ioized form. But transportation in free space is diffusion and it is in AH
form. One of the proteins that is responsible for efflux transport of auxins is PIN; there are
several forms of these proteins.
SITE OF SYNTHESIS:
The natural auxin found in plants is called Indole Acetic Acid (IAA) and it is the first of the
phytohormones to be discovered. Indole acetic acid is mostly synthesized in the stem and
root apexes. Using radioactive 14C as the tracer, it has been demonstrated that the
meristematic cells, just above differentiating into vascular tissues, are found to be active in
synthesizing IAA. Shoot apexes synthesize more auxin than the root species.
BIOSYNTHESIS OF INDOLE ACETIC ACID
Tryptophan has been found to be the precursor for IAA synthesis. In some cases Indole
acetonitril has also been found to be used in the synthesis of IAA. The enzymes responsible
for the biosynthesis of IAA have been identified. There are two pathways which converge in
the production of IAA. To start with, tryptophan is converted to Indole pyruvate by
deamination reaction catalyzed by specific deaminase enzymes. The indole 3D pyruvate is
then subjected to decarboxylation to produce indole acetaldehyde, which is then oxidized by
alde-hydrases to indole 3- acetic acid. On the other hand, tryptophan may be first subjected
to decarboxylation step. The tryptamine synthesized in this reaction is deaminated to indole
3- acetaldehyde then it is oxidized to IAA.
Indole aceto nitrile is also found in plants and the same is used in the synthesis of IAA by
converting IAN to Indole acetamide then to IAA or IAN to IAA directly.
SYNTHETIC AUXINS
Once the native auxin has been identified as indole acetic acid, plant biochemists started
looking for similar compounds in nature as well as in the laboratory. As a result, a host of
synthetic auxins have been discovered and their effects as growth promoting hormones have
been characterized, ex. Indole propionic acid Indole butyric acid Naphthalene acetic acid,
phenyl acetic acid, 2,4 Dichlorophenoxy acetic acid are just few of the known synthetic
auxins.
The hormonal activity of IAA and the other synthetic compounds has been attributed to the
presence of a ring system as the nucleus, a side chain possessing a carboxyl group and the
presence of at least one carbon atom between the ring and the carboxyl end. The length of
the side chain, the number of substituents in the nucleus and the side chain and the basic
structure of the nuclear ring have been found to exert profound influence in eliciting
physiological responses.
EFFECT ON GENERAL METABOLISM
Isolated tissues, hypocotyls segments, epicotyl segments, leaves, excised roots and even
whole plants have been used to monitor various biochemical and physiological responses to
hormone treatment. Auxin has been found to accelerate cellular metabolism in treated
tissues. Particularly respiratory rate increases by at least by 20%.
The general activation of metabolic processes in terms of turnover is all pervading. Even
mitochondria and plastids show increased activity. Many of the increased metabolic activities
in response to auxin treatment have been attributed to the changes in membrane
permeability and activation of some membrane factors. As a consequence of increased
respiratory activities, photosynthetic activity, amino acid metabolism, nucleic acid synthesis
and protein synthesis and others, cells build up the required materials for their growth.
Auxin binding protein
Plant growth involves interaction between metabolites such as sugars, phytohormones and
their action on gene expression. Auxin as a signaling molecule has various effects depending
upon the tissue where it acts.
EFFECT ON NUCLEIC ACID SYNTHESIS
Auxins show some dramatic effects on growth and development of plants. Though the
immediate effect of auxin is known to be at the plasma membrane and cytosolic level, with
time its effect on gene activation and protein synthesis is over bearing. The effectiveness of
Auxin’s activity is due to the presence of auxin binding proteins. They act as receptors and
after complexing with the auxins, they are rendered highly active.
The effect of auxins induced cell elongation does not require DNA synthesis, but in long term
effect, DNA synthesis is always accompanied with cell division. The interaction between auxin
and cytokinin has been interpreted as at the auxin plays permissive role in DNA synthesis and
cytokinin stimulates it.
Auxins do not induce transcription immediately after application of the hormone. But auxin
induced transcription sustained for 49-50 minutes. But auxin after effect on increased
translational activity has been explained as due to the activation of translational machinery
through the activation of translational factors. However, transcriptional activity in response to
auxin at later stages results in the synthesis of all species of RNAs, such as mRNAs, tRNAs and
rRNAs. Among the population of mRNAs induced by auxins, some specific mRNAs for
cellulase, tubulin and others, are found is higher concentrations.
EFFECT ON PROTEIN SYNTHESIS
As mentioned earlier, auxin, in many systems enhances the rate of protein synthesis very
early without the concomitant increase in RNA synthesis; which suggests that auxins earlier
effect is on translational activity. This may be achieved either through the activation of
inactive mRNPs, or through the activation of translational machinery and translational
factors. Increase in polysome content in response to auxin treatment in many plant cells is
good evidence in support of auxins’ promotive effect on protein synthesis. However, the
increased level of protein synthesis at later stages is actually due to the auxin induced
transcriptional activity, which really sustains the translational activity for a longer period of
time. Though auxin enhances the synthesis of almost all house keeping enzymes, it also
induces the synthesis of specific proteins like cellulase, cellulose synthase, tubulins and other
specific factors required for cell elongation. It should be remembered that the specific effect
of auxin on the expression of a particular protein product depends upon kind of tissue
involved. Auxin induced activation of polymerization of tubulins into microtubules is another
interesting feature of auxins effect.
EFFECT ON CELL ELONGATION
Among all the hormonal effects the effect of auxin on cell elongation has been studied in
detail. For a long time, the exact mechanism of auxin induced cell elongation has remained
unsolved. For historical interests it is essential to understand a few theories which where
proposed in the past to explain the mechanism. In this text recent views have also been
provided.
TURGIDITY THEORY
This is probably the oldest of the theories that have been proposed so far. This theory is
based on the assumption, that auxin stimulates respiratory and other metabolic activities.
As a result, the osmotically inactive components of cells are degraded to osmotically active
molecules. This in turn affects the diffusion pressure deficit of the cell and water potential
gradient is created between the growing cells. So water from neighboring other cells,
particularly xylem diffuses into the activated cell passively. As a consequence of this turgour
pressure that builds up within the cell, the cell is stretched from within, thus the cell
elongates. According to this concept the cell elongation is a passive phenomenon.
It is true that auxins enhances the respiratory activity, but for the cell to elongate, due to the
turgour pressure developed inside, the cell also requires loosening of the cell wall, without
which whatever turgour pressure that develops, cannot force the cell to elongate or expand.
In recent years, with the use of refined techniques, it has been demonstrated that in many
plant systems the auxin induced cell elongation takes place under negative turgour pressure.
Furthermore, respiratory inhibitors like DNP, cyanide inhibit auxin mediated cell elongation,
which suggests that cell elongation is an active process. Because of these the turgour theory
is not favored. One of the proteins involved in cell expansion is expansin
GENE ACTIVATION THEORY
With new discoveries in the field of molecular biology, plant scientists also thought, that auxin
brings about the cell elongation through gene activation. Bonneret.al. suggested that as
auxins are acidic in nature, they can easily bind to basic proteins found associated with the
chromatin material. The binding of auxins to histones, certain segments of DNA’s are freed
from the surrounding histone proteins for transcriptional activity. As a result, the required
mRNAs for cellulase are produced. As cellulase is required for degradation and loosening of
the cell wall, they proposed gene activation. The combined effect of gene activity and auxin
induced turgour pressure has believed to be mechanism of cell elongation. Further elongation
stops at later stages and the synthesis of cell wall materials continues which again is due to
the activation of gene expression.
This theory however falls short of its expectations because transcriptional activity in response
to auxin increases only after 15-30 minutes later, but the apparent growth of cell starts as
early as 10-15 minutes after auxin treatment. So this theory also fails to explain the early
phase of growth, moreover, the binding of auxin to the histone in bringing about gene
expression is no more tenable.
ACID THEORY
Cleland and others have demonstrated that stem segments on exposure to auxin, secrete
protons into external medium and render it acidic. Sharp fall in pH of the medium due to
auxin treatment has been demonstrated by many workers. Proton secretion has been
attributed to the activation of H/ATPase found in the plasma membrane. As the protons
diffuse through the cell wall, the pH in that region falls and acidic form cellulase will be
activated. As a result, the cell wall fibrils are cut and loosened which greatly facilitates the
elongation of cells.
Acid theory explains the early effect of auxin on cell elongation even before the auxin induced
transcription starts. This theory also assumes that the internal pressure responsible for the
elongation as turgour pressure. It is now known that not in all cases turgour pressure has
been demonstrated as the cause for cell elongation. However, this theory explains the early
effect of auxin on cell elongation.
Auxin also induces cellulase enzymes which act on cellulose fibers and loosen the cell wall
and facilitate the expansion due to increase in turgid pressure.
CYTOSKELETON THEORY
This theory is the most recent theory and it is based on many experimental evidences. It is
very important to know that auxin induced cell elongation or growth exhibits two phases. The
first phase is initiated as early as 5-15 minutes after treatment. It is rapid and lasts for 30-45
minutes. This phase is insensitive to the inhibitors of transcription and translation. But it is
highly sensitive to colchicine treatment. The above features suggest that the early rapid
growth does not require transcription or translation products. But it requires microtubule
formation because colchicine inhibits polymerization of tubulin monomers into microtubules.
On the other hand, the second phase of growth is slow but steady. It is sensitive to
Actinomycin-D, CHI and also colchicine. It means the second phase requires transcriptional
products, translational products and polymerization of tubulins into microtubules which act as
cytoskeletons.
In the first phase, auxin first enhances the rate of respiration, thereby ATP production
increases. At the same time, it also activates nucleating centers and H/ATPase pump located
in the plasma membrane. Nucleating centers are a group of aggregated tubulin monomers
which on activation start assembling available tubulin monomers from the cytosolic pool.
Thereby, a large number of microtubules grow and elongate from the nucleating centers.
Polymerization of tubules requires GTP and ATPs as energy source. The hydrolysis of them
generates significant amount of H+. Meanwhile, the activated H/ATPase pumps excrete H+
into the exterior of the plasma membrane by active process. As the pH in the cell was
becomes acidic, the acidic form of cellulase enzyme which are already present become active
glucanase activity they hydrolyze the cellulose fibrils, thus the cell walls fibrils loosened. In
this process, certain amount of Ca2+ ions are also removed from the middle wall, which
renders the middle wall more labile and plastic. As more and more microtubules grow and
elongate and build up, they build up a kind of mechanical force within the cell and the cell is
stretched. MTs contribute to the deposition of cell wall materials to be transported and
deposited out side the plasma membranes. The elongation is further facilitated by the
loosened cell wall. Thus the cells grow in length.
The second phase of cell elongation however required more and more of tubulin pool because
most of it is depleted in the first phase. So it also requires fresh synthesis of tubulins,
cellulase enzymes and other required factors for the sustained cell elongation as per the
demand transcriptional activity increases and the RNAs produced are used by the
translational machinery and more of proteins are synthesized. Though there is an increase in
the synthesis of most of the house keeping proteins, the increased synthesis of tubulin,
cellulase and cellulose synthesizing enzymes is significant. Utilizing tubulin monomers
continue to polymerize and more of microtubules generate at the same time loosening of cell
wall continues. There by the second phase of cell elongation proceeds slowly but steadily.
That is why the second phase is so sensitive to the inhibitory actions of actinomycin D, CHI
and colchicine.
This theory appears to explain most of the observed properties of cell elongation.
Furthermore, it also explains how cells elongate even in the absence of sufficient turgour
pressure within the cell.
EFFECT ON NEW ROOT FORMATIONS
Roots developing from any part of the plant body other than the radicle are called
adventitious roots. It is not an uncommon phenomenon to see the plant parts are propagated
by inducing new root formation. But the exogenous supply of auxins to stem and leaf cuttings
readily induces the new root formation, which ensures vegetative propagation. The synthetic
hormones like IBA and NAA are more effective in new root formation than the native IAA.
If defoliated stem cuttings are continuously washed in water to leach out the endogenous
auxins, they do not produce any adventitious roots in a nutrient media. But if such washed
segments are treated Indole Butyric Acid for about 10-20 minutes and placed in a nutrient
medium, stem cuttings produce a large number of roots. These roots normally develop from
the terminally differentiated pericyclic tissue found around phloem tissue. Within 36-72 hours
after treatment some of the founder cells in the said pericyclic region undergo transformation
and organize into root primordia, which later grow through the cortex and emerge out of the
stem.
The transformation and organization of pericyclic cells into root primordia is a phenomenon of
dedifferentiation. Studies on molecular aspects of IBA induced new root formation in the
hypocotyls of phaseolus vulgaris reveal that the new root formation is due to the differential
gene expression. Using techniques like translation of isolated mRNAs from treated and
untreated segments at different time periods in a cell free system and the analysis of invitro
and in vivo protein products by polyacrylamide SDS gel electrophoresis and autoradiography
and immunoprecipitation of labeled proteins by using specific antibodies, show that among
the many proteins synthesized, the synthesis of 55-58 KD proteins and 105 kd proteins in the
IBA treated segments increases significantly. The 55-58% proteins have been identified as α
and β tubulin, which are the precursor for microtubules. Involvement of microtubular
assembly and orientation during the differentiation and organization of pericyclic cells into
root primordia is confirmed by the use of colchicine and cytochalasin B. The said drugs
prevents the root initiation in the IBA treated hypocotyl segments within 36
hours after hormonal treatment, but the drugs have no effect if the hypocotyls that have
already passed through 36 hours after IBA treatment. The above results indicate how auxins
can bring about new root formation by inducing differential gene expression. Some of the
concepts of auxin action on gene expression has been shown below in the form consolidated
figures which are self explaining.
Lateral root develop from preexisting roots and new roots develop from stems. The root
initials start from cambial founder cells. It is important to remember that even plant tissue,
as in the case of animal systems, contain stem cells, called them as founder cells. Proper
signals can induce organ formation from such stem cells. Auxin activates founder cells in
pericycle to enter into G1-S transition. It is now clear that auxin induces certain cyclins and
CDKs (B) types. Auxin induced transcriptional mechanism perhaps go through activators,
Auxin binds to auxin response factors TIR1, which in turn translocates into the nucleus and
bind to auxin response elements of genes. However the degradation ARF/IAA/Auxin protein
complex by SCF –TIR is important for the ARFs to bind to auxin response elements properly. It
is also possible several other factors are involved in induction of lateral roots; probably auxin
binding protein (ABP) is one among them.
Once auxin is transported into cells, it binds to its receptor protein TIR, which inturn binds
ARFs /IAA/Auxin protein complex which are acting as repressors. IAA-TIR binding leads to
activation of SCF mediated degradation of IAA/Aux binding protein which frees ARFs and now
ARFs bind to ARE promoter elements. Aux/IAA binding proteins were acting as repressors.
The auxin-TIR1 now binds to Aux/IAA proteins and using SCF complex ubiquitinate AUX/IIA
proteins and feed to proteosomes for degradation. Thus the ARFs become active and activate
specific genes to which they are bound. This is general mechanism for auxin induced gene
activation.
Auxin binding protein (ABP)
TIR1 protein bound to auxin activates Auxin response factors and thus gene expression.
The above figure proposed by Thomas Guiylfoyle provides the input how auxin mediated
specific genes are activated. This proposal is considered to be the most famous among plant
molecular biologists.
EFFECT ON APICAL DOMINANCE
Plants like pines and other conifers exhibit a growth pattern, which is quite distinct from a
banyan tree or a tamarind tree. The conical growth of the pine plant is due to the
predominant growth activity of the apical meristems where the growths of lateral buds are
more or less suppressed. On the other hand, the growth pattern in banyan tree is diffused,
where lateral branches grow as vigorously as the apical branches. The suppressive effect of
apical buds on the growth of lateral buds is often called apical dominance. Such a
phenomenon is not just restricted to only conifers, but it is also found in other plants.
Apical dominance has been explained as due to the action of auxin present in the main apex.
This view is amply supported by an experiment, where if the stem tip is cut off, the axillary
buds found below sprout immediately. Instead, if an agar block containing auxin placed over
the decapitated stem, the axillary buds remain suppressed. It is clear from the above
experiment, that auxin present in main apex some how inhibits the growth of the axillary
bud. The severity of apical dominance is greater on the axillary buds present nearer to the
main apex. Nevertheless, apical dominance exerted by the auxins can be overcome by the
application of cytokinins to axillary buds. This is because cytokinin induces cytokinesis.
Probably, the mitotic block that is operating in axillary buds may be due to the inadequate
supply of cytokinins. But how the supply of cytokinin to axillary buds is made inadequate by
the apical bud is not clear.
The idea of nutritional inadequacy to the axillary buds due to the stronger influence of apical
meristems has been considered by many scientists. It is assumed that because of apical
dominance, most of the nutrition is drawn towards the apex than to the axillary bud. This
explanation for apical dominance has no conclusive evidences to prove the claim. On the
other hand, studies have shown that higher concentrative of IAA induces the synthesis of
ethylene. As apical buds contain more of auxins, it may induce the synthesis more of
ethylene which in turn may inhibit the normal growth of the axillary bud. But by removing the
apical bud, the concentration of auxin drops, so also ethylene hence axillary buds sprout.
Effect on Phototropism
The growth curvature in stem apex in response to light is called phototropic movement. Such
growth curvature in shoot tips can be induced even in the absence of light by placing auxin
containing agar blocks asymmetrically on decapitated stem tips. This indicates that unequal
concentration of auxin is responsible for unequal growth there by the curvature. But in light
induced curvatures, how does light brings about unequal concentration of auxin and which
part of the light spectrum is responsible for the curvature are the few questions that needs
explanation. Using monochromatic light, it has been determined that the most effective
spectrum is 445 mm. But the answer to the first question is still elusive and various theories
have been proposed from time to time to explain this phenomenon.
Lateral Transport Theory
This theory was proposed by Cholodney & F.W. Went. Accordingly, the concentration of auxin
in the stem apex is uniform all-round. Once the light rays fall on the stem from one side, it
induces the movement of auxin from the illuminated region towards the darker region. By
such movement, auxins accumulate in greater amounts in the darker region. The motive
force for the movement of the auxin in response to light is attributed to the differential
electrical charges on the said surfaces. Then he tips were illuminated from one side and the
basipetally diffused auxin was collected and the amount of auxin found in the agar blocks was
determined. According to the authors in the control tips they detected more of auxin in the
far side the surface that was in the dark than from the surface that was illuminated. But in
the tip with a mica plate the amount of auxin found in both the blocks was same. So it was
deduced that the difference in the amount of the auxin detected in the blocks on the darker
side is actually was due to lateral movement. Accordingly resultant differential concentration
is responsible for differential growth, hence the phototropic curvature. The growth is
dependent auxin concentration.
Recombinant GUS gene expression of a gene that is responsive to auxin shows the
distribution of Auxin in response to light
Their claim was disputed by other workers, who used radioactive C-labeled auxin for exact
quantitative determination of the transported auxin from one region to another. They did not
find any substantial lateral transportation. So the lateral movement theory has remained
unconvinced.
Inhibition of basipetal Transport Theory:
Gordon and others proposed that the unequal distribution of auxins in response to light
treatment is not due to lateral movement, but due to the inhibition of basipetal movement
from the illuminated side, which causes unequal concentration of auxins and growth
curvature is due to it. Slow growth of the stems in day times and steady growth of the plants
in intense sun light has been attributed to this effect. However this theory has not been
tested with covincing experiments.
Photo inactivation Theory:
When blue light at 445 nm has been detected as the action spectrum for the induction of
phototropic curvature, plant physiologists started looking for pigments that absorb blue light
at 445 nm. This logic was based on the fact that known photo responses like photosynthesis
and photoperiodic responses are due to specific pigments. The search for such pigments
revealed the presence of B carotenes and riboflavin in the stem tips. As some plants, which
respond to light induced phototropic movement, did not possess B carotenes it was deduced
that riboflavin as the causative pigment. The photo inactivation was explained on the
assumption that riboflavin after absorbing light gets activated and the same inactivates the
auxin directly or destroys the auxin through certain IAA oxidase. This result in unequal
concentration of auxin in the stem tip which in turn is believed to be responsible for the
phototropic curvature again this theory has never been proved unequivocally.
Present concept:
In the past, many experimental results were based on very crude extraction methods. Such
experimental results are not very convincing today. Use of radioactive isotopes i.e. (14C) IAA,
solvent extraction methods combined with GLC analysis for qualitative and quantitative
estimations of auxins have cast grave doubts about previous theories. In the light of recent
work, it has been suggested that light has profound effect on inducing the synthesis or
releasing the growth inhibitors like ABA. The release of ABA in the region where it is
illuminated causes inhibition of growth in the said region. The effect of ABA on osmotic
changes by creating effluxes of ions is well known. Moreover, it is now known that auxins
could be made inactive or active by auxin binding proteins. If binding protein complexes with
IAA, it is rendered physiologically active, if it is free, it remains inactive. So light induced
changes in the concentration of ABA and other auxin binding factors are believed to bring
about variations in the endogenous levels of active auxins. Such changes are ultimately
responsible for phototropic growth curvatures,
For a long time the above concepts were accepted as facts, however, finding of phototropin, a
blue light absorbing protein, has role in phototropic curvature is also an accepted fact.
Phototropin exists in two forms; both are flavin binding proteins of mol.wt 110 Kda and
124Kda. Interestingly these proteins contain at least 11 kinase (PAS) domains; these domains
are celled LOVE domains (Relation to Light, Oxygen and Voltage). They also contain different
domains such as BTB and D1-to D15 domains. Phototropins act as light receptors and
absorption leads to autophosphorylation at serine/threonine sites and they are involved in
phosphorylation of many other membrane bound proteins. Membrane bound proteins have
many roles in transportation of ions, transportation of Auxin/auxin receptor.
However the exact mechanism by which they bring about photrophic curvature movement is
yet to be discerned.
Conformational changes of Love domain proteins called Phototropin in the presence of light
and in the absence of light.
Plants regulate their growth directions in response to light direction, and its response is called
phototropism. Phototropism is a model research to understand the regulatory mechanisms of auxin
metabolisms in response to environmental stimuli, and we are studying on this topic by a molecular genetic
approach using Arabidopsis mutants. We identified a signal transducer RPT2 and a novel blue-light
photoreceptor phot2, both of which are required for the phototropic responses of Arabidopsis. We indicated
that phot1 and phot2 show partially overlapping functions in two different responses, phototropic response
and chloroplast relocation, in a fluence rate-dependent manner, as blue light receptors. To reveal the
phototropin-signaling pathways, We studied on the regulation of cytosolic Ca2+ concentrations by
phototropins, involvements of RPT2 and another phototropin-signaling factor NPH3 in chloroplast relocation
and stomatal opening, and functional sharing between phototropins and other blue-light receptors
cryptochromes. Now, We are studying on a dephosphorylation mechanism of NPH3 and transcriptional and
post-transcriptional regulations of RPT2 in response to blue light irradiation.
In addition, we revealed that photoreceptors phytochromes and cryptochromes regulate auxin biosynthesis,
metabolism, and transport in response to light irradiation and indicated that a suppression mechanism of
expression of auxin transporter PGP19 by phytochromes and cryptochromes is one of mechanisms to
enhance the phototropic response by phytochrome and cryptochromes. Other analyses on the light
signaling and auxin mechanisms were also published by collaborations with other research groups.
Light activates three kinds of photoreceptor families, phototropins, phytochromes, and cryptochromes, and
affects the plant growth directions through changes of auxin biosynthesis/metabolism and transport.
Effect on Growth Curvatures:
Growth movement of any part or the organ of the plant body in response to gravitational pull
of the earth is referred to as geotropic movements. The most remarkable feature of the plant
body is that, though both shoot and root system are derived from the same embryonic cells,
they respond differently to the same gravitational stimulus. This property may be due to
unique embryonic developmental programmes dictated by the inbuilt genetic factors, which
probably enforce the respective organism to behave differently to different environmental
factors.
Growth curvature away from the earth’s gravity is called negative geotropism and the growth
of the roots towards earth is called positive geotropism. Most of the roots, with the exception
of pneumatophores and coralloid roots which are negatively geotropic, exhibit positive
geotropism. However, not all stem show absolute negative geotropism. It is a common
observation that some of the underground stems like rhizomes, suckers, etc, grow obliquely in
the soil and such growth movements are called dia-geotropic movements.
Mechanism of geotropic for with Curvatures:
Again, Cholodney and went are the pioneers in explaining geotropic movements. They
proposed that stem apexes and root apexes require different concentration of auxins to bring
about the maximal growth. In the sense, it is said, that the concentration of auxin that is
favorable for the growth of shoot apex is inhibitory to the growth of the root apex. On the
contrary, the concentration, that is optimal for the growth of the root apexes, is not adequate
for the growth of the stem tips. It implies that stem cells require greater amount of auxins for
the in maximal growth and root cells require very low concentration of auxins for their optimal
growth. Based on these premises the mechanisms of geotropic responses have been
explained.
If a straight seedling is placed horizontally on the soil, shoots curl upwards and roots bend
towards the soil. This behavior has been attributed to the gravitational force that acts upon
the respective organs for they are endowed with different programmes, though both have the
same genetic properties. Cholodney and went assumed that the gravitation once acts on
both root and shoot. So auxins having certain mass of their own move downwards and
accumulate in greater amounts in the lower cells which are in contact with the soil.
As higher concentration of auxin is promotive in stem tips the cells grow faster than the
others. So the stem curves and grows upwards. On the contrary, as the higher concentration
of auxin is inhibitory for the root cells, the growth of root cells is inhibited, but the cells
containing less amount of auxins show maximal growth activity. Hence root tips curve
towards soil and grow forwards in to it. Thus stems exhibit negative geotropic growth
movements and roots show positive geotropic movements.
The above said theory enjoyed the general acceptance for a long period of time. But people
realized that gravitational force, by its mass action, has greater effect on amyloplasts than on
auxins found in the plant cells. Because of their greater mass, amyloplasts settle down on the
plasma membranes of the cells as shown in the figure. The contact of the amyloplasts with
plasma membranes acts as on irritant as a result growth of cells on that side of the
membrane is inhibited but the growth on he other side is favored. But this explanation has
never been favored because some plants which are lacking in amyloplasts also exhibit
geotropic responses.
In recent years, research work on geotropic movements has revealed that the site of
geotropic perception is not the root apex, and it requires root cap for its response. If the root
cap is cut off, the decapitated root dies not show any geotropic responses. When the cap is
replaced he roots respond normally for geotropic stimulus. This observation has been
explained as due to the synthesis of ABA in the root caps. The ABA is transported basipetally,
and then it also moves downwards due to the gravitational force. As the lower cells receive
ABA their growth is inhibited. But the upper cells grow normally and bring about geotropic
curvature.
Effect on Parthenocarpy:
Development of fruits without fertilization is known as parthenogenesis and the fruit is said to
be parthenocarpic fruit. Auxins have been found to be effective in inducing parthenocarpic
fruits in some plants. It has been demonstrated that the extracts of pollen grains also induce
the development of parthenocarpic fruits, discharge of pollen tube contents into embryo sac
is believed to be cause for the increase in he content of auxin in embryo sac. Some botanists
suspect that pollen tubes carry some enzymes which
produce
more auxins.
It is important to note the synthesis of more auxins not lead to fertilization. Whether the act
of fertilization has any stimulatory effect on the synthesis of auxin is not clear nonetheless
many plants respond to auxin treatment produce parthenocarpic fruits.
Induction of parthenocarpic fruits by auxins has greater application value in cultivating fruit
yielding plants. The auxin induced fruits, besides seedless, they are larger in size and
sweeter. Commercial production of such fruits brings more income to farmers.
Effect on Abscission
It is common to observe that older leaves, debladed petioles, abortive flowers, and often
fruits, fall off from the plants. In the above said cases a distinct and characteristic layer of
cells develop at the base of the petiole of the leaf or the pedicel of fruit, which acts as a weak
point, hence the said structures fall down. The layer that is responsible for this process is
called abscission layer or abscission zone.
Structure of Abscission Layers
It consists of a number of layers of thin walled cells which are rich in cytoplasm and actively
dividing. Disappearance of middle lamella from the cells of this layer is a characteristic
feature. This is due to the activity of pectinase enzyme. As a result, this part of the stalk
becomes weak and the leaf or the fruit falls down by its own weight.
In many plants, just below the abscission zone towards the stem, a layer of actively dividing
cells develops. This layer is called protective layer. This layer develops after the fall of leaf at
the free surface, but in some cases the protective layers develop even before the fall of the
leaves.
These cells by repeated cell divisions produce many layers of cells, which get suberized and
protect the inner layer of cells from injury or infection. Thus, this layer acts as wound healing
tissue.
Development of Abscission Layer:
Leaves fall with the age and fruits fall down after ripening, but in deciduous plants, onset of
winter acts as the signal for the plants to shed their leaves. Sometimes, unseasoned cold
waves induce premature falling of fruits. In all these cases, the falling of leaves or falling of
fruits is due to the formation of abscission layer at the base of their stalks. The factors that
induce the formation of abscission layers vary. In some cases, ageing acts as an important
causative factor, in others environmental conditions like winter may act as the factor. In the
case of aging the accumulation of senescing factors induce abscission layer formation. One of
the senescing factors is believed to be ABA. Reduction in the quantity of auxin n the distal
parts of the leaf is also known to be another causative factor. In the initial stages of
abscission layer formation, if IAA is applied to distal part of the leaf, the development of the
abscission layer is inhibited. Instead, if auxin is applied at a later stage of abscission the
process I shortened and the leaf falls quickly.
Once, it was thought that ABA is mainly responsible for leaf abscission, but recent
investigations indicate that ethylene is highly effective in inducing abscission layer formation,
but the role of ABA is not totally ruled out. Incorporation of radioactive label during the
abscission layer formation suggests, differential gene expression in the abscission zone
results in the production of pectinases and cellulases, which by their activity breakdown the
middle wall and also some cellulose. At the same time the cells in this zone become
meristematic.
Uses:
The application of auxin to leaves and fruits is now known to prevent the development of
abscission layer. Many synthetic hormones like NAA, IBA, 2,4-D have been found to very
effective in preventing the premature fall of fruits, particularly commercial crops like citrus,
apple, oranges, mangoes, grapes. The use of such hormones not only prevents the
premature falling of fruits, but also increases the quantum of fruit setting. And fruits thus
produced are larger and sweeter. Thus auxins can be used for commercial gains in the field
of pomiculture.
Auxin and Cytokinins interactions:
In plants each of the phytohormones, though have independent effects, often they affect the
other or they may have synergistic effect. For example Auxin prepares the cell but cytokinins
execute cell division. When auxin induces new root formation at a particular concentration,
cytokinins inhibit auxin induced new root formation.
Plant Hormones-Gibberellins
Gibberillic acid was first discovered in Japan under unusual circumstances. Farmers in Japan,
before the World War, found that their paddy crop plants were afflicted with a strange disease
called Bakane. The diseased plants showed unusual growth where plants were very tall, weak
and sterile. This disease affected 20-30% of the crop plants and the farmers were subjected
to great loss.
Investigation into the cause, Hori a Japanese plant pathologist discovered that this disease
was due to a fungus called Gibberella fujikoroi, but it is now identified as Fusarium
moniliforme. Later Sawada and Kurosowa found that the extracts of this fungus simulated
what the fungus could cause on infection. Then a group of Japanese workers led by Yabuta
and Sumuki obtained an active principle from the extracts of the causative fungus, and later
Yabuta and Hayashi identified the active principle as Gibberellic acid, which is new popularly
called as Gibberellins (GA). Western scientists did not know anything about these discoveries
before the First World War. After the war, they came to know about GA. Then they searched
for this substance in higher plants and found this substance in all known higher plants. In fact
they succeeded in obtaining GA in pure crystalline form and also they established its chemical
structure.
Maryland mammoth
Distribution
Almost every kind of plant, including algae, ferns, fungi, bacteria, is known to contain GA.
Quantitative analysis of various parts of the higher plants shows that young leaves, which are
just unfurling, are rich in GA. Even germinating seedlings contain significant amount of GA.
Further studies revealed that proplastids found in young and developing leaves act as the site
of GA synthesis and it is of great interest to know that their synthesis and release from
plastids is under the subtle control of phytochromes. Though GAs is synthesized in plastids,
they are translocated to different regions of the plant body through sieve tubes by active
translocation mechanism.
The diagram shows the concentrations and spatial distribution of different phyhormones in a plant body under normal conditions.
Structure and Classification of GA
Gibberellins are diterpenoid acids derived from tetra cyclic diterpenoid known as Kaurene.
The basic carbon skeleton of GA is known as Gibbane ring. But today the term Gibbane is in
use. However, the systematic nomenclature of GAs is based on Kaurene and Gibbane
structures.
Modified solvent extraction methods and the use of sophisticated analytical tools like HPLC
etc. have greatly helped plant biochemists in identifying different forms of GAs. So far, 52 to
57 different kinds of Gas have been identified. Some of the gibberellins are known to be
conjugated with glucose as glucose esters.
Chemical analysis of different parts of the plant body for GAs shows that different kinds of
GAS are located in different parts of the plant body; their content and kinds vary at different
stages of development of the plant body. Two or more GAs are found in the same plant but in
different organs. In addition, Gibberellins exist in a dynamic state, in the sense; they undergo
rapid interconversions from one kind to another, which however depends upon the intrinsic or
extrinsic factors. It is also known that some of the gibberellins which are active in one organ
are found to be inactive in another organ and vice versa. The dynamic equilibrium between
different kinds of GAs, their synthesis and their multifaceted effects in plants is really
fascinating.
Biosynthesis:
Biosynthesis of GA takes place in young plastids. Acetate is the precursor for the synthesis of
all kinds of gibberellins.
In a series of multi step reactions acetate is used to produce a diterpene compound called
Kaurene. Specific enzymes responsible for each creates have been more or less identified.
The site of synthesis is proplastids or plastids or both. Plastids are also the site of fatty acid
synthesis in plants.
Kaurene thus synthesized is converted to Gibberellic acid, which is then subjected to
hydroxylation, methylation or glycosylation to produce different forms of Gibberellins. The
remarkable feature of the biosynthetic pathway of GAs is that the same pathway is also used
by plastids to synthesize certain plant pigments, Abscisic acid and some sterols. The needed
enzymes for the biosynthesis of the above said compounds are regulated by phytochromes
and other factors. The plastogenome and nuclear genome play a significant role in regulating
the biosynthesis of Gibberellins and other mentioned compounds, how plastogenome
contributes to the synthesis and its regulation is not known.
Effect of GA on of Plants
1. Effect on General Metabolism:
Effect of various factors on cellular activities.
Gibberellins, as growth promoting hormones, accelerate the rate of cellular metabolic
pathways such as respiration, protein synthesis etc. During the germination of cereal grains,
GA is known to promote mobilizing the food materials for the growing embryo. It plays a
significant role in metabolizing lipids to glucose and then to sucrose through gluconeogenesis
process. The promotive effect of GA on nitrogen metabolism and HMP pathway is very
significant. GA is also known for its effect on membrane transformation through rapid
turnover of lipids. Interestingly, GA also favor C3 pathway in C3 plants, but the same
hormone adversely affects Hatch and Slack pathway in C4 plants. Thus GA exerts a wider
influence on general metabolic pathways.
2. Effect on Transcription and Translation:
The metabolic effects exerted by Gibberellins show variations and it depends upon the
structure, species and developmental stage of the plant body that receives it. The presence
GA binding proteins have been identified from various sources like pisum epicotyls, phase his
etc. Mol. Wt. of such proteins has been determined as 6.0 x 104 to 5.0 x 105 Daltons. GAs
are also known to elicit specific responses through such receptor proteins.
In cucumber hypocotyls, GA3 promotes the replication of DNA in plastids; thereby it exhibits
its specific effect on biogenesis of chloroplasts. Gibberellins are also known to stimulate
transcriptional activity of DNA templates through the binding of GA to AMP rich regions of
DNA. In some cases, GA’ effect on transcriptional activity has been attributed to the
activation of RNA polymerase through their receptor proteins, where the synthesis of all
species of RNAs is enhanced.
The most specific effect of GA on transcription has been observed in the germinating barley
grains. GA3 induces the mRNA synthesis for alfa amylase enzymes in aleurone cells and the
endospermal do not produce mRNA for alfa amylase. Probably this effect of GA is one of the
best exemplified actions of phytohormones on differential gone expression. In spite of having
voluminous data about GA’s effect on different plant species, nothing is clear how different
GAs bring about different transcriptional activities leading to morphological expressions.
Gibberellins are also known to activate dormant mRNPs and the translating machinery. This
view is supported by the fact that GA enhances polysome formation even in the absence of
concomitant synthesis of mRNA. Synthesis of alfa amylase enzyme in barley aleurone layers,
appearance of some new proteins during bolting and flowering are few examples to
demonstrate the effect of Gibberellins on protein synthesis.
Effect on Genetic Dwarfism:
Genetic dwarfism, in many plants including garden peas and some species of French beans, is
controlled by single genes. Such genetic dwarfs respond very favorably to GA treatment and
in response to the hormone they grow as tall as normal tall varieties.
However the phenotypic transformation of genetic dwarfs into tall plants is not
heritable. Quantitative analysis of tall and dwarf peas and bean plants reveal the
presence of high concentrations of GA in tall plants than is dwarf varieties, which
suggests that dwarfism is due to a single gene mutation affecting one of the steps in
GA biosynthesis. Cross breeding experiment do support this view.
The fascinating aspect of Gibberellin’s effect on genetic dwarfs in its specific action is on
internodal growth. The internodes respond favorably than any other parts. The elongation of
internodes is possible by two simultaneous processes i.e. one by meristematic divisions of
intercalary meristems and the second is by the elongation of meristematic cells. The exact
mechanism by which cell elongation is achieved is not clear, nonetheless, GA induced early
growth of internodes can be inhibited by colchicine, but not by the inhibitors of transcription
and translation. This indicates that the cytoskeleton structures like microtubules are involved
in GA mediated growth similar to that of auxin induced cell elongation.
Dwarf rice plants also respond very well to GA treatment. GA induced internode elongation in
rice plants is due to the activation of inter-calary meristems. After few cell divisions, the cell
derivatives elongated 80-1000 times the original size. Elongation, in this case, is due to the
excretion of protons and labializing the cell wall to be plastic. Meanwhile, intracellular
turgidity also builds up due to the action of GA on plasma membranes. The combined effect
of loosening the cell wall, the increased turgour pressure results in the cell elongation.
Effect on Aleurone Layers in Cereal Grains:
Cereal grains of Zea mays, sorghum, Hordium, Oryza etc have a distinct layer around the
endosperm called aleurone layer, the cells of which are rich in protein granules. During
germination, aleurone cells become active and with time, they secrete enzymes into
endospermous tissue, where the reserve starch gets degraded to glucose and the same is
utilized by the growing embryos.
There is a definite relationship between the activity of aleurone cells and developing embryo.
If the embryo is separated from the rest of the grain and incubated separately, aleurone cells
fail to secrete any enzymes. On the other hand if the embryo is also incubated with the rest
of the grain, after a period of time, aleurone cells start secreting alfa amylase. Instead of
incubating embryo with the rest of the grain, if GA3 is added to the incubating medium,
aleurone cells produce alfa amylase enzymes. Thus it is clear that during the early part of
germination the young embryo produces Gibberellins which on reaching the aleurone layer
activates the cells to produce the required enzymes like alfa amylase, protease, phosphoryl
choline glyceride transferase and phosphoryl choline citidyl transferase. The last two
mentioned enzymes are required in lecithin biosynthesis which activates membranes.
Recently it has been reported that GA besides inducing the expression of alfa amylase
inducing enzyme, it also triggers the expression of ribosomal RNA synthesis to a greater
extent.
The synthesis of GA induced enzymes can be inhibited by actinomycin D or cycloheximides,
which are the inhibitors of transcription and translation respectively. This suggests that GA
preferentially activates the gene expression for the above said enzymes.
GA binding protein
Studies on GA induced molecular events clearly suggest than GA at very early stages
activates preexisting mRNPs for alfa amylase but later the hormone activates specific genes
in aleurone cells. However, the increase in the levels of transcription for amylase and
protease reaches maximum at 6-7 hours after hormone treatment. The above results further
supported by the fact, that when GA induced mRNAs from aleurone layer are translated in an
vitro system, among the many proteins produced, the levels of alfa amylase and protease is
found to be high. In actuality, the newly synthesized enzymes are loaded into membrane
vesicles and the same is secreted out of the aleurone cells into endospermous tissue, where
they bring about hydrolytic activity and thus mobilize the food materials for the developing
embryos.
Endosperms cells are dead cells. In recent investigations it has been shown that GA not only
induces the synthesis of mRNAs for alfa amylase but also induces the synthesis of RNA. But
ABA suppresses the GA induced mRNA for alfa amylase. However ABA does not prevent the
synthesis of RNAs.
GA signaling takes through trimeric G receptor protein. This leads to pathways, one-Ca+
dependent pathway and the other Ca+ independent pathway; not much is known about how
GA induces signal transduction.
The Ca+ independent pathway activates phosphorylation of DELLA proteins, which are
repressors of GAMB gene ( GA induced Amylase-beta). Phosphorylation of F-box proteins
(SLN1 and SLR1) leads to ubiquitinated protein degradation. This facilitates the GA-GA
receptor complex bind to GAMYB gene promoter and activates the transcription of the genes.
Translated product GAMY beta protein, a transcriptional regulator protein, enter the nucleus
and binds Amylase gene regulatory elements called GARE (GA response elements) and induce
amylase gene expression. Transcripts on translation produce amylase proteins which are
transferred into ER and loaded into vesicle and stored in cytoplasm. They are released in
response to GA through what is called Ca+ dependent pathway. Note MYB is a family of
genes there cans any where 80-108 in number; they are the most abundant transcription
factors.
Effect on Vernalization:
Not all seeds released from the fruits germinate immediately. Some pass through a period of
dormancy. And those seeds which exhibit temporary suspension of growth activity are called
dormant seeds. In order to make dormant seeds germinate they have to be subjected to
scarification or stratification or some of them have to be subjected cold treatment. Plants or
seeds acquiring the ability to accelerate flowering in response to cold treatment is called
vernalization.
GA is known to overcome cold treatment for plants requiring vernalization, but it can also
break the seed dormancy. Quantitative estimation of GA in vernalized seeds do not show any
increase in GA content in response to cold treatment, however, during germination at normal
temperature, the concentration of GA increases significantly; these studies suggest that cold
treatment per se does not increase GA content, but provides the ability to synthesize more GA
during germination and growth. In some instances, where dormant seeds requiring far-red
treatment to break the dormancy GA acts as the substituent.
Effect on Parthenocarpy:
Where auxins fail, GAs are found to be very effective in inducing Parthenocarpy. In addition
to it, GA induced parthenocarpic fruits are larger in size and sweeter in content. GA is used to
increase the total yield of grape fruits. It is also highly effective in increasing the yield and
sugar content of sugar cane. However, the application of GAs should not be more than what
is required in grape cultivation; otherwise fruits will be damaged .
Gibberellins and Auxin Interactions:
Gibberellins and auxins are found to induce cell elongation, parthenocarpy and metabolic
activities including RNA and protein synthesis. But GA and auxins have their own specific
effects on different tissues of the plant body. While GA promotes internode elongation,
overcomes genetic dwarfism, induces amylase synthesis in aleurone cells and in some cases
it can substitute cold treatment or far red treatment. IAA does not elicit any of these effects.
On the other hand, auxins impose apical dominance, induce adventitious roots and induce
cellulase synthesis. On the other hand GAs don’t elicit any of these responses. However, in
the case of cell elongation though GA and IAA have independent actions in promoting the
growth of etiolated normal pea stem sections, if both the hormones are provided together,
their total effect is just additive but not synergistic. On the contrary, if internodes of dwarf
pea stems are treated with either GA or IAA, the promotive effect on stem segments is very
little, but if both are provided together the stimulation in terms of growth is highly
pronounced and the total effect is synergistic. The above observation indicates that
gibberellins need auxins for synergistic activity. This particular conclusion is further
substantiated by the fact that a decapitated internodal segment does not respond to GA
treatment alone but if the apical meristem or an agar block containing auxin is placed on the
decapitated segment, elongation of the internode is stimulated in the presence of GA. This is
because apical meristems do synthesize auxins, which interact with gibberellins to bring
about the combined effect. Recent studies in in vivo and invitro system strongly suggest that
GA has an important role in promoting the biosynthetic pathway of auxin, thus in the
presence of GA, the levels of auxins increase. Probably GAs enhances the rate of IAA
synthesis.
Microarray of GA induced gene expression
Effects IAA GA
Stem Growth + +
Parthenocarpy + +
Root initiation + -
Callus formation + -
Induction of amylase - +
Bolting and flowering - +
GA and ABA:
Plant hormonal interactions are fascinating for the simple reason that many of them act
antagonistically and some cooperatively and few synergistically. Some of the interactions are
concentration dependent. GA and Auxin induce parthenocarpy; Cytokinins and auxins act
antagonistically in Auxin induced new root formation. Induction of dormancy and breaking
dormancy are two opposite development pathways in seeds. ABA can induce dormancy and
GA can break the dormancy. Cold treatment induces vernalization in some plants in ABA
dependent manner, but vernalization can be broken by GA.
The diagram illustrates how ABA and GA act in opposite ways in response to environmental inputs.
GA and Flowering:
Plants which require longer photoperiodic conditions for flowering are called long day plants
and those plants requiring shorter photoperiodic are called short day plants. Long day plants
do not flower under short day conditions and vice versa. Correct photoperiod treatments
stimulate the synthesis of flowering hormone called florigin which inturn induces flowering by
differential gene expression. However, there are a large number of plants which do not
require such photoperiodic conditions for flowering.
FCA and the control of Arabidopsis flowering time. (A and B) Schematic representation of the principal genetic pathways controlling flowering time in winter annual and rapid cycling
accessions of Arabidopsis. Promotive activities are denoted by arrowheads, repressive activities are denoted by T-bars. The photoperiod (PP) and gibberellin signal transduction (GA)
pathways are shown activating genes with a floral meristem identity function (FMI), while FLC is shown to repress this. FRI, the FCA-containing autonomous pathway (AUT) and vernalization
pathway (VRN) regulate FLC in an antagonistic manner. The most frequently found difference between winter annual (A) and rapid cycling (B) Arabidopsis accessions is allelic variation
at FRI, with most rapid cycling accessions carrying inactive, loss-of-function fri alleles. (C) A comparison of the phenotype of wild-type Ler plants and late flowering fca-1 mutant plants.
Although grown for the same time and under identical conditions, wild-type plants have already flowered while the fca-1 plants have remained in the vegetative state and continued to
produce more leaves as opposed to floral organs. (D) Schematic representation of the alternative processing ofArabidopsis FCA pre-mRNA. Exons are represented as filled boxes and intron
by lines.
Some of the long day plants under non inductive short day conditions exhibit rosette leaves
because of extreme condensation of internodes. If such plants under non inductive conditions
are sprayed with Gibberellins, they get stimulated and the internodes elongate dramatically;
such GA induced elongated stems also initiate flowering, such a phenomenon is known as
Bolting and Flowering. Though bolting and flowering are two different events, GA has been
found to effect the elongation of internodes first during which process flowering substances
are also produced as a result floral buds are initiated.
Furthermore, quantification of GA in untreated plants and plants which are in flowering show
very low amount of GA in the former and higher content in the later. It amounts to the fact
that during proper photoconductive conditions synthesis of Gibberellins increases. However,
GA has no effect on majority of short day plants. Strangely particular variety of short day rice
plants responds very well to GA treatment and produce flowers.
From the above discussions, it is clear that Gibberellins actually over come long day
treatments are photo period requiring plants. The interrelationship between long day
photoperiodic treatments and GA effect has been explained on the basis of the following
facts. Plastids synthesize and store the same under long day photo periodic condition. At the
same time, the phytochrome pigments which are also present within plastids, absorb red light
and transform into far red light absorbing pigment. This form of pigments labelises the
plastid membranes and thus Gibberellins are released from the plastids. Thus the
concentration of GA increases in photo period induced long day plants. In spite of it, the
exact mechanism by which GA brings about differential gene expression during flowering is
not clear. Probably, it may also require other factors for floral induction.
Effect on inflorescence and floral structure:
Though GA is known to induce bolting and flowering in long day plants, the effect of GA on a
short day plants like sorghum bicolor is very interesting. In these plants, GA3 stimulates
flowering even under non inductive conditions. But in combination with far red light
treatment GA’s effect is synergistic. The hormone also brings about marked increase in the
number of spikelets and glumes. Along with these promotive responses, GA also increases
the number of floral structures like ovaries and promotes fertilization, but inhibits stamen
development.
Effects of GAs on Flowering and Flowering Genes”“By Valerie Sponsel, Biology Department, University of Texas, San Antonio, TX, USA, ‘with acknowdgment”
Flowering in Arabidopsis, a plant for which more tools exist to study the molecular genetics of
the process than in any other species, is regulated by four separate pathways: (a)
the photoperiodic (long day) pathway, which operates in the leaves; (b) the
convergentautonomous (leaf number)/vernalization (low temperature) pathway; (c)
the carbohydrate (sucrose) pathway; and (d) the gibberellinpathway. The latter three
pathways all operate in the shoot apical meristem. The four pathways converge on a number
of floral pathway integrators that together regulate floral initiation.
The recent identification of FLOWERING LOCUS T (FT) as the gene expressed in leaves that
encodes a phloem-mobile mRNA or protein that fits the description of a universal flowering
stimulus, florigen, has been an exciting development after the decades-long search for this
elusive flowering signal (Huang et al. 2005; and see Zeevaart 2006, for a review). Additional
work is seeking to further define the integrative networks that exist between the different
pathways. Web Essay 25.2, from which Figure 1 is taken, provides a complete consideration
of this subject. This topic briefly addresses the GA pathway, when it operates, and what is
known about how it is integrated with the other pathways.
Figure 1 Multiple developmental pathways for flowering in Arabidopsis: photoperiodism, the autonomous (leaf number) and vernalization (low temperature) pathways, the energy (sucrose)
pathway, and the gibberellin pathway. The photoperiodic pathway is located in the leaves and involves the production of a transmissible floral stimulus, FT protein. In LDPs such as Arabidopsis, FT protein is produced in the phloem in response to CO protein accumulation under long days. It is then translocated via sieve tubes to the apical meristem. In SDPs such as rice, the transmissible floral
stimulus Hd3a protein accumulates when the repressor protein, Hd1, is not produced under short days, and the Hd3a protein is translocated via the phloem to the apical meristem. In Arabidopsis, FT
binds to FD, and the FT/FD protein complex activates the AP1 and SOC1 genes, which trigger LFY gene expression. LFY and AP1 then trigger the expression of the floral homeotic genes. The
autonomous (leaf number) and vernalization (low temperature) pathways act in the apical meristem to negatively regulate FLC, a negative regulator of SOC1. The sucrose and gibberellin pathways,
also localized to the meristem, promoteSOC1 expression. (After Blázquez 2005.) (Click image to enlarge.)
In Arabidopsis, flowering occurs in long days (LD) even in the GA-deficient ga1-3 mutant,
although it is somewhat delayed compared to wild-type plants. However, this mutant
will not flower in short days (SD) unless treated with GA. Double mutants of ga1-3 and co, (the
wild-type CONSTANS is normally expressed in leaves and is upstream of FT in the LD
pathway) will not normally flower, either in SD or LD. From this and other information it is
proposed that the LD and GA pathways are independent, with the GA pathway being essential
for flowering in noninductive conditions. Input from the LD pathway (transduced through FT)
and from the GA pathway converge on SOC 1 (SUPPRESSOR OF OVEREXPRESSION OF CO 1),
that is expressed in the apical meristem. Thus, GA treatment toga1-3 mutants in SD causes
both enhanced expression of SOC in the apical meristem, and flowering. Over expression
of SOC1 in ga1-3mutants allows them to flower in SD without GA treatment (Moon et al.
2003).
An additional floral pathway integrator is LFY, the product of a floral meristem identity gene
that is expressed in apical meristems.LFY expression, which is high in LD-grown plants, is only
observed in SD-grown ga1-3 mutants that have been treated with GA.
Constitutive LFY expression will also allow SD-grown ga1-3 to flower without GA treatment
(Blazquez et al. 1998). Using deletion analysis, Blazquez and Weigel (2000) identified an 8 bp
motif in the LFY promoter that is necessary for GA responsiveness, but not for response to day
length. This sequence is a potential target for MYB transcription factors, and candidate MYBs
whose expression increases in the shoot apex in response to exogenous GA4, or to elevated
levels of native GA4, have been identified in both Lolium temulentum and Arabidopsis (Gocal
et al. 1999, 2001b). Interestingly, LDs—but not GA treatment—cause a rapid increase in
expression of the L. temulentum ortholog of FT (LtFT), indicating in this plant too that the LD
and GA pathways converge downstream of FT (King et al. 2006).
Plant Hormones-CYTOKININS
Skoog and his students, while working on the callus cultures under in vitro conditions,
Cytokinins were discovered. They found the callus that develops from the stem explants,
containing both pith and vascular elements, develops well. But the explants containing just
pith cells produces callus, but further growth of its stops, even in the presence of optimal
concentration of auxins. This is because the cell in the callus somehow rendered incapable of
cell division. If such callus is supplanted vascular tissues, extract of vascular tissues, coconut
milk or malt extracts, the growth of the callus will be restored and the cells exhibit mitotic
activity. This effect has been attributed to the presence of some active principle in the
supplanted coconut milk. Cytokinins have various functions in association with other
hormones or its accessories.
The active principle responsible for inducing cell division was isolated first from the extracts of
yeasts. Such a substance was called kinetin, later the name was changed and called as
cytokinin; kinetin terminology was misleading for another class of compounds called kinins
which were already known to be found is animal systems. However, the term cytokinin has
been given to all those compounds that are capable of inducing cell division in the presence
of optimal concentration of auxin. Now it is known that a good source for cytokinin is coconut
liquid endosperm and milky endosperm of sweet corns.
Some of the naturally occurring cytokinins are 6-furfuryl-aminopurine, Ribosyl zeatin, Zeatin,
Isopentinyladenine and Dihydrozeatin. Interestingly, the above compounds are also found in
denatured products of nucleic acids. Many synthetic cytokinins are also available in the
market, ex., 6 Benzyl aminopurine, 6 Phenyl aminopurine.
Distribution:
Cytokinins have been detected in a wide variety of plants; from unicellular yeasts, algae to
multi cellular higher plants. Particularly in higher plants, cytokinins are found in root tips,
xylem, young leaves; endosperms of developing fruits, germinating seeds and tumour tissues.
Site of Synthesis:
Most of the cytokinins required for the plant body are synthesized in the root tips, and then
they are translocated to different regions particularly to meristematic and expanding tissues;
transportation is through xylem stream. This observation has been supported by many
studies.
Site of CK synthesis
For example, the amount of cytokinin found in the excised petiole is less than the petiole that
has rooted. If the root tips are cut, the growth of the stem apex is more or less inhibited till
adequate supply of cytokinin is restored by new root formation. Significant amount of
cytokinin is synthesized in required endosperm of palm fruits, kernel of cereal grains and
others.
Almost all naturally occurring cytokinins are the products of purine nucleotide derivatives.
The presence of iospentenyl adenine in some tRNAs has misled people to believe that the
source of cytokinins is tRNAs but it is not the case. Nevertheless the site of synthesis of
cytokinins in young and developing plants is restricted to meristematic regions of the root
tips; where certain enzymes utilize purine and convert them to cytokinins. The presence of
isopentenyl adenine in many tRNA is not due to the incorporation of cytokinins into tRNAs.
However, the exact mechanism and biosynthetic pathway of cytokinins is yet to be
elucidated.
Effects:
General Metabolism:
Cytokinins’ effect on respiration is very interesting. It enhances the rate of respiration in the
callus, but the same hormone when applied to senescing leaves, brings down the rate of
respiration, retards the degradation of chlorophyll and enhances the rate of chlorophyll
synthesis. In addition it also increases the rate of metabolic activities involved in C3
pathway. In certain systems cytokinin induces nitrate reductase activity, but in callus it does
not. The root nodule development in legumes and its metabolic activity is subtly regulated by
the interaction between auxins and cytokinins.
Nucleic acid Synthesis:
Although cytokinin is known to stimulate cell division, it does not induce DNA synthesis as a
prelude to cell division. But in the presence of auxin, it promotes DNA synthesis. So it is
suggested that cytokinin stimulates and auxin promotes DNA synthesis. For example, an
aged and non proliferating callus cells can be stimulated to undergo mitotic divisions by the
application of cytokinin.
Cytokinin binding protein i.e. Receptor proteins have been identified in many plant systems.
The cytokinin protein complex is known to induce transcription activity a pea bud chromatin in
a cell free system. In Soy bean and French been hypocotyls, it transitorily inhibits IBA induced
RNA synthesis for a period of 6-7 hours, but later it stimulates RNA synthesis.
Specific species of tRNAs, containing cytokinin activity, is seen in the cotyledons and
hypocotyl segments of bean plants. But this has been identified as due to post transcriptional
modification of tRNA. Recent investigations have clearly elucidated that the presence of
cytokinin moiety in some tRNA structures is not responsible for eliciting any cytokinin
mediated responses, however it is to be noted that many tRNAs contain isopentenyl adenine
(IPA) in the anticodon loop region
PROTEIN SYNTHESIS:
Quite a number of experiments, involvement in vivo systems or cell free invitro systems, have
demonstrated that cytokinins first stimulate translational activity without any concomitant
increase in the synthesis of mRNAs. This particular effect has been attributed to cytokinin’s
ability to activate pre existing mRNPs and ribosomal proteins needed for chain initiation. Fox
et al, have demonstrated that cytokinins bind to ribosomal surface through a receptor
protein. This protein activates ribosomes by dephosphorylating a specific ribosomal protein.
Furthermore, the increased activity of protein synthesis in response to cytokinin has been
found to be concentration dependent. It is also interesting to note that cytokinin mediated
protein synthesis; either as short term or long term effects, shows the synthesis of new
proteins in treated tissues. At least in the hypocotyls of French bean, cytokinin inhibits some
proteins that are induced by IBA, but cytokinin by itself enhances the rate of tubulin synthesis
similar to that of IBA.
Cell Division:
The name cytokinin is derived from its ability to induce cytokinesis during cell divisions.
Though cytokinins stimulate the process, the permissive effect of it is controlled by auxins.
When cytokinin is provided to a liquid culture medium containing plant cells, the protein
synthetic activity of the cells is greatly stimulated. Moreover, some of the proteins thus
synthesized are new ones. These observations suggest that cytokinins control cytokinesis by
regulating the synthesis of some specific protein factors that are required for cytokinesis.
Cell Enlargement:
Isolated cotyledons of Cucumis, radish and other plants expand dramatically when they are
treated with cytokinin. The expansion of cotyledons is rather more due to cell enlargement
than due to cell divisions. During cytokinin induced cell enlargement, respiratory activity
increases significantly and greater amounts of K+ ions are accumulated in the cells. At the
same time, cells in response to the hormones induce the synthesis of few minor species of
RNAs and some proteins. But the inhibitors of respiration, transcription and translation
completely inhibit cytokinin mediated cell enlargement. Interestingly such cotyledons also
respond to red light treatment and enlarge. However the red light induced enlargement
cannot be reversed by far red light treatment which further suggests that cytokinins bring
about permeability changes within the membranes.
Richmond & Lang’s effect:
Senescense of leaves leads to yellowing and finally leads to the fall from the plant. If a young
excised leaf is kept in water, it slowly changes its color to yellow and dies. If such leaves are
provided with cytokinin, the yellowing is significantly delayed and such an effect is called
Richmond and Lang’s effect; named after the discoverers.
Senescense is a common feature exhibited by parts of the plant which show definite growth
pattern. With age, the structures like leaves, flowers, etc., senesce and die. Among various
factors, decrease n the content of auxin acts as a very important factor in inducing
senescence. In matured leaves, still attached to the plant body with time, senescence sets in
and leads to the degradation of chlorophyll. Catabolic activity increases and the formation of
abscission layer begin. In detached leaves also one finds similar processes. But cytokinins
prevent and prolong the initiation of senescence for a quite a period of time. This effect of
cytokinin has been explained as due to the prevention of degradative catabolic processes by
the way of repression activity of few hydrolysing enzymes like protease, RNAse, DNAse etc.
Furthermore, cytokinin facilitates the chlorophyll synthesis. It also sustains the activity of
carbon fixation, RNA synthesis and protein synthesis. Still the exact mechanism by which
cytokinins prevent aging and senescence is not known.
Effect on Dormancy:
Dormant buds that develop due to certain adverse environmental factors remain inactive for a
long time. If such buds are treated with cytokinins they come out of dormant state and
sprout. This is due to the effect of cytokinin in activating cell division which was prevented by
mitotic blocks present in the dormant besides. Interestingly, cytokinins also overcome auxin
imposed apical dominance and stimulate the growth of the axillary buds, probably by
overcoming the factors such as mitotic blocks.
Interaction of cytokinins with auxins in morphogenesis:
Plant development is a series of biochemical events which ultimately leads to morphogenic
changes. This process is regulated by a number of phytohormones which bring about
differential gene activity leading to the development or specific phenotype. The interaction
between various hormones is very complex and they operate at different levels like
differentials transcription, protein synthesis, enzyme activity, permeability etc. Here the
interaction between cytokinin and auxin id discussed briefly.
The culturing of plant explants with various combinations of cytokinins and Auxins show
the inhibitory effect of cytokinin on auxin induced new root formation.
In tissue culture experiments, the plant explants are supplemented with optimal nutrients
including carbohydrate as the energy source and hormones as growth regulators. Plant
explants, which may be leaf segments stem segments, roots or embryos develop and produce
an undifferentiated tissue called callus is a defined culture medium. This behavior is due to
the presence of balanced concentration of auxin and cytokinin in the nutrient medium. The
callus is like a cancerous tumour, where the cells are undifferentiated and they are under the
spell of uncontrolled mitotic activity. If the concentration of auxin with respect to that of
cytokinin ratio is changed, from the ratio that is favorable for the growth of only callus, the
cells of the callus undergo transformation to produce organs like roots or shoots. If the ratio
between auxin and cytokinin is high, the callus cells undergo transformation and produce
roots. On the other hand, if the ratio between auxin and cytokinin is low the callus cells
initiate shoots. As all cells in the callus are totipotent, depending upon the hormonal
concentration, cellular genetic material is differentially expressed and their products induce
specific morphogenetic events leading to organ formation. In this system it is the
concentration of each of the hormones provide the signal for differentiation of
undifferentiated cells into new organs.
In spite of having sufficient knowledge about the hormonal effects in organogenesis, the
molecular basis of morphogenesis is not very clear; however, one example can be used to
illustrate how auxins and cytokinins interact and modulate molecular events during
morphogenesis. Adventitious root formation in the hypocotyls of phaseolus vulgaris in
response to auxin treatment is a process of redifferentiation and reorganization of pericyclic
cells into root primordia. While IBA induces new root formation, cytokinins inhibit IBA
mediated root initiation. At molecular level both auxin and cytokinins increase the rate of
protein synthesis without increase in transcriptional activity at 30 minutes of treatment. In
the case of auxin treated hypocotyls increase in transcription is detected after 45-60 minutes,
but not in cytokinins treated. In the segment treated with both auxin and cytokinins one does
not observe such changes at short time but observed at longer periods. This observation
comes from quantitating in vivo and in vitro protein synthesis using 14C leucine. Also purified
poly-A is used for quantitation using in vitro translation. The results show increase in
translation at later stages is due to transcriptional activation. General Protein analysis on SDS-
PAGE at 24hrs, 48hr and 72 hrs, shows a remarkable increase in two bands at 55Kda and
58Kda and few other bands of high molecular weight. However increase in the said proteins
was not detected in cytokinin treated segments. The bands were identified as Tubulin alpha
and beta proteins. More over in Auxin treated segment discerned from in vitro translation of
Pol(A) RNA shows one more 115Kds band showed up only in auxin treated but not in any
others. This increase is only transitory from 24hrs to 36hrs. Then the protein band
disappears. The role of this protein is not known.
At molecular level Auxin acts through Auxin response factor for activation of genes. But the
activation of transcription at 36 hrs or more, by cytokinins is yet to be found. However
cytokinins action of activation of genes has been discerned to some extent in Arabidopsis.
Cytokinin acts as signal molecule and it binds to dimeric receptors anchored in plasma
membrane. The receptors are believed to be similar to receptor tyrosine kinases (RTKs), but
their kinase activity is restricted to histidine residues (HPK), so they are called Arabidopsis
histidine protein kinases (HKs). This kinase activates histidine phospho transfer proteins
(AHPs) transducers. These are response regulators which can activate or repress gene
expression. AHK-ps enter the nucleus and interact with ARRs (nuclear response regulators)
and activate transcription. Such components were also observed in maize.
Outline of cytokinins pathway
There are four steps in cytokinins signaling pathway as shown in the obove diagram. AHK
sensing and signaling, AHP nuclear translocation and localization and activation of ARR genes
and a negative feedback loop through Cytokinin inducible ARR gene products.
Arabidopsis encode three cytokinins receptors; cytokine response gene1 (CRE1 also called
AHK4) ,Woodenleg (Wol) and AHK2/3. There are other histidine kinases such as CK11 and
CK12 (AHK5) which are independent of cytokinins but they do respond to cytokinins. Mutants
of CRE1 and Wol show defects in cytokinins mediated shoot formation and defects in root
vasculature.
The ARR (A response regulator proteins) ( such asARR1, ARR2,and ARR10, which are
transcriptional activators carry MYB like DNA binding domains, and also contain glutamine Q’
rich activating domains. ARR p-lation activates transcription of A-type ARRs. Expression of
cyclin D and ARR5 show they are the major sites of cytokinins action in root and shoot
meristems. There are 54 gene encode AHKs, AHPs and ARRs.
Cytokinins requirement for nodulation
In procambial cells (PCs), the coordinated signaling by cytokinin and auxin induces the
expression of genes that are involved in the maintenance and growth of procambial activities
into xylem elements. The auxin-signaling pathway might be involved in gene expression of
auxin-response factors, such as MONOPTEROS (MP), that also function as transcriptional
activators, and their repressors, the AUX/IAA proteins. Cytokinin might be perceived by the
WOL/CRE1/AtHK4 cytokinin receptor, which, in turn, transmits an intracellular signal that is
mediated by a His–Asp phosphorelay mechanism to PC-related histidine-containing
phosphotransfer factors (AHPs) and then to PC-related type-B response regulators (ARRs). The
type-B ARRs might function as transcriptional activators of PC-related genes including the
genes of their repressors, the type-A ARRs. The presence of repressors in auxin- and
cytokinin-signaling pathways might allow cytokinin and auxin signaling to be temporal.
Brassinosteroids (BRs in the figure) are biosynthesized actively in PCs and secreted, but
brassinosteroids do not work as a signal for the maintenance of procambial activities. Instead,
brassinosteroids, in the presence of auxin, might initiate differentiation of procambial cells to
precursors of xylem cells (pXCs) after recognition by a receptor, which might be a
heterodimers, composed of either brassinosteroid-insensitive-1 (BRI1) or one of the BRI1-like
proteins (BRL1–BRL3), plus BRI1-associated receptor kinase-1 (BAK1). The brassinosteroid
signal inactivates the negative regulator BIN2 (brassinosteroid-insensitive-2), which allows the
unphosphorylated form of bri1-EMS-suppressor-1 (BES1) and brassinazole-resistant-1 (BZR1)
to translocate to the nucleus and to promote pXC-related gene expression. Among the most
important pXC-related genes that are induced by brassinosteroids might be the HD-ZIP-III-
homeobox gene family, which might function in further xylem cell differentiation. KANADI and
the microRNAs MIR165 and MIR166 might suppress differentiation of PCs to pXCs. The
suppression by the microRNAs might be caused by the rapid degradation of the HD-ZIP-
III gene mRNA through RNAi machinery.
The above diagram shows how cytokinins play a role in localization of CRF6 in cytosol.
RFs in Arabidopsis
We have taken a genetic approach identifying knockout mutants in each of the CRF genes in order to better understand their function in plant growth and development.One research area we on is determining the b UC Cytokinins induce CRF genes. There are six such CRFs; they are cytokinin response/regulatory factors and transcriptional activators. They play important role in plant development. Mutants in CRF genes produce malfunctions in leaf and cotyledon development as shown in the diagrams above.
This figure provides general information about cytokinin signal pathways. Arabidopsis, each of which into pairs of related
genes that arose as ancient genome duplication in plants. Each pair of
genes has been maintained over time and may have become specified in function.
--.
While only three have taken a genetic approach identifying knockout mutants in each of the CRF genes in order to better understand their
function in plant growth and d
Cytokinin activated gene expression is shown in the form of microarray.
CRFs act in parallel with type-B ARRs to mediate cytokinin regulated gene expression.
(A) Wild-type, arr1,12, crf1,2,5, andcrf2,3,6 seedlings were treated with either 10 μM
BA or a DMSO control for 1 h and gene expression analyzed by using a microarray.
Genes that displayed a ≥2-fold change in response to cytokinin in the wild type are
shown. (B) Venn diagram of the 135 cytokinin-regulated genes affected by
the arr1,12, crf1,2,5, and/or crf2,3,6 mutations. (C) Model of cytokinin signaling. Both
AHPs and CRFs move into the nucleus in response to cytokinin. Once there, the AHPs
phosphorylate the type-B ARRs, which, together with CRFs, mediate cytokinin-regulated
gene expression.
Another diagram shows few more Cytokinin signaling features.
Plant hormones-ABSCISINS
Plant physiologists knew presence of growth inhibiting compounds in plants for sometime.
But the active substance responsible for it was discovered by Ohkuma and Cornforth, later
Ohkuma established its structure in 1965; Cornforth further confirmed this in the same year
by synthesizing the compound in the laboratory. Today, we know that growth inhibiting
substance as Abscisic acid or Abscissin II, which was once called as ‘Dormin’. Besides ABA,
plants also contain other natural growth inhibitors such as Coumerin, Ferulic acid, Para
ascorbic acid, phaseic acid, violoxanthin, etc. In addition, plant chemists have identified some
synthetic growth inhibitors. Ex. 2,3,5 tri iodo-benzoic acid, morphactins, caproic acid, phenyl
propionic acid, Malic hydrazide, etc.
Distribution:
Abscissin has been isolated from a wide variety of plants. The amount of ABA found in plants
varies from species to species and from organ to organ. For example, in the pulp of avocado
fruits the concentration of ABA is 10 mg/kg. And in the dormant buds of cocklebur it is 20
mg/kg. Again depending upon the environmental conditions the concentration of ABA varies
in the same part or the organ of the plant body. In the leaves of phaseolus vulgaris, if the
plant is subjected to water stress within 90 minutes the amount of ABA raises from 15 mg/kg
to 175 mg/kg. Intense light and other dormancy inducing factors are highly effective in
increasing the concentration of ABA is the plant body.
ABA like other phytohormones exists in free state or in bound form. The free state of ABA is
supposed to be an active form. The bound forms of ABA are conjugates of glucose and its
derivatives. Depending upon the influencing factors, they undergo rapid changes from one
form to another to elicit their respective functions. ABA is mostly transported through sieve
tubes in the plant body. But the rate of transportation ranges from 25-45 cm/hr, which is far
below the rate of sucrose transport that takes place in sieve tubes, which suggests that ABA’s
transport is independent of sucrose.
Structure and Biosynthesis:
Abscissin I is a sesquiterpene consisting of 3 isoprenoid units. It has a 6-carbon ring and a 5-
carbon chain ending with a carboxyl group. The ring has a double bonded oxygen group.
However, naturally accuring ABA exists in two stereo isomeric forms like (+) ABA and (-) ABA
but the active form is ABA (-).
The biosynthetic pathway of ABA is almost similar to that of Gibberellins. In fact, mevolonate
acts as the precursor. But some research workers consider that ABA is a break down product
of violoxanthin, however, the detailed studies, using radioactive isotopes as labels, have
revealed that it is derived from simple isoprenoid compounds and not from violoxanthin or
such products.
The site of synthesis of ABA has been identified as plastids, which includes proplastids,
etiolated and also mature chloroplasts. The initial biosynthetic reactions are more or less the
same as that of GA up to Farnesyl pyrophosphate FPP and GGMP. From GGMPP onwards, the
synthesis of GA and ABA go through two different pathways; which is controlled by
phytochrome, water stress and environmental factors. Depending upon the factors, one
pathway is favored over the other. Adverse environmental factors like water stress, salt
stress and severe cold favor the synthesis of ABA. But red light favorable conditions enhance
the synthesis of GAs.
Thus plastids play an important role in regulating the synthesis of anyone of these two
hormones at any given time depending upon phytochrome state or water potential of the cell.
Effects of ABA:
Dormancy:
Abscisic acid is a stress induced hormone. During winter season in temperate and cold
regions, most of the axillary and terminal buds undergo a period of suspended physiological
activities called a period of dormancy. Even seeds belonging to various species exhibit
dormancy; which has been attributed to ABA’s effect on physiological activities of the cells.
Studies on ABA’s effect on transcription and translation indicate, the hormone inhibits RNA
polymerase activity. Its effect on translation has been attributed to its inductive ability to
synthesize a RNA species, which is poly (A) rich a (mol. Wt. of 10,000 Daltons). This poly(A)
RNA is capable of inhibiting the translation of mRNA by hybridizing with 3’ region of mRNA.
Thus it affects both transcription and translation. During the seed development or during
induction of dormancy transcriptional activity has been found to be normal but the mRNA
synthesized is made inactive by ABA by the above said mechanism. Most of the mRNAs
remain as inactive mRNPs or informosomes. Thus ABA imposes dormancy in the said
structures like buds and seeds. ABA is also known to promote dehydration in seeds and
render them dormant. How ABA brings about this effect is not clear, but it is known that ABA
has profound effect on Ca+, K+ and H+ fluxes across the membranes.
ABA inhibits GA mediated Alfa amylase synthesis:
It is very interesting to know that one hormone interacts with another hormone in regulating
the gene expression. Interestingly, both GA and ABA are synthesized in the plastids and they
follow the same pathway in the initial steps. Curiously enough, both the said hormones inhibit
each other’s physiological effects.
While Gibberellins activate aleurone cells to produce alfa amylase and proteases through
differential gene expression, ABA completely inhibits the GA mediated synthesis of amylase
and protease by inhibiting the translation of mRNA through ploy-A rich RNA. By another
mechanism ABA also activates the release of specific short chain fatty acids that inhibits GA3
induced amylosis in barley endosperm by inhibiting transcription and translation of alfa
amylase mRNAs.
Role of GA in countering ABA action during seed gemrination
Effect on Cell Expansion:
Normal plants treated with GA or IAA exhibit pronounced growth by way of activating cell
elongation, but ABA inhibits the cell growth considerably. Considering this, some botanists
think that the genetic dwarfism is probably due to the presence of excess of ABA in their plant
body. But quantitative studies reveal that there is no such correlation between the genetic
dwarfism and ABA content. And genetic dwarfism is rather due to the deficiency of
Gibberellins than to the presence of excess ABA. But the inhibitory effect of ABA has been
attributed to its effects on membrane permeability and ion fluxes. It is well known that ABA
brings about the extrusion of K+ ions from guard cells and causes closing of the stomata.
Thus ABA may act on other cells as in the case of guard cells and inhibit GA or IAA mediated
cell elongation.
Senescence:
Abscisic acid is known to initiate aging in leaves in which process the detached leaves loose
their green color and becomes yellow. During ABA induced senescence, degreening takes
place by the synthesis of an enzyme called chlorophyllase, whose synthesis can be blocked by
transcriptional or translational inhibitors. ABA also induces high rate of respiration for only a
short period of time. The labalizing effect of ABA on lysosomes leads to the release of
enzymes, which start degrading cellular macromolecules. Photosynthesis and other anabolic
processes like protein synthesis, etc., are greatly reduced. The induction of ethylene
synthesis is believed to be activated by ABA in certain cases, where the auxin concentration is
very low. The ABA induced ethylene in turn causes the formation of abscission layers in the
stalks of leaves and fruits.
On the other hand, cytokinins are known to overcome ABA’s effect, by decreasing degradative
processes.
ABA influences the formation of abscission layer. This process has been exploited
commercially in harvesting fruits and cotton balls. By spraying ABA on cotton plants, most of
the stalks of cotton balls develop abscission layers simultaneously and it will greatly help in
mechanical harvesting of cotton balls. Similarly, in fruit orchards, to obtain uniform
harvesting, controlled application of ABA facilitates the harvesting of most of the fruits at one
time.
Effect on Turgour Movement:
In recent years, ABA’s effect on membranes, in changing ion fluxes, is drawing the attention
of all plant physiologists. ABA has a positive effect on guard cells, where the cells loose most
of the K+ ions to subsidiary cells and collapse to induce the closing of the stomata. In fact,
guard cells also contain chloroplasts and it is suspected that ABAs are released from them
under certain conditions like water stress or high temperatures, which in fact induce the
closing of the stomata.
Stomatal movement in closing and opening of guard cell is an excellent system in
understanding interaction of cellar and external factors. Co2 depletion in the atmosphere,
water stress leads to activation of ABA, that is released from chloroplast found in guard cells.
They acxtivate Potassium and H-channels for expulsion og ions and another class of channels
opens for inward movement of Ca+ ions. This leads to the closing of the stomata; this process
is reverse of opening, where K+ ions are translocated in to build up of ion concentration so as
to facilitate the movement of water into guard cells, so the cells turgour pressure increase
and stomata opens.
The figure is not very explanative, though looks good.
Similarly, the turgour movements of motor or bulliform cells in grass leaves, turgour
movements of parenchymatous cells in the pulvinous of Mimosa pudica and other plants
which exhibit nastic movements are believed to be due to the action of ABA. In recent years,
the role of ABA in photo induced growth curvatures has been gaining importance. When stem
tips are unilaterally illuminated from one side, the cells that receive light release some
quantity of ABA, which causes the efflux of K+ ions, which results in the collapsing of cells. At
the same time, they also inhibit cell elongation. But on the other side i.e. darker side cells
grow normally and bring about growth curvatures. It look like ABA by their controlled
synthesis and release bring about innumerable physiological is plants effects.
ABA and Gene Expression:
ABA is also involved in gene expression of ABA response factor (TFs), whose upstream binding
sequences are given in the table. Activation leads to the production of factors that provide
stress tolerance.
ABA is involved in a variety of stress tolerance activities such osmotic stress, cold stress,
superoxide (ROS) stress, salt stress and others. Only diagrams have been provided they are
self explanatory in nature.
Ros response involves dsRNA mediated mi RNA
Plant Cell Vacuoles
Mature plant cells contain a single large central vacuole and it is the most conspicuous
compartment of the cell which occupies nearly 50-70% of the total cell volume. On the
contrary, meristematic cells are lacking in such vacuoles. Plant cell vacuoles are distinct and
characteristic in having a single unit membrane called tonoplast, which separates the
vacuolar content from the rest of the cytoplasmic fluid. The liquid present within the vacuole
in called cell sap this contains a host of inorganic and organic compounds.
Cell derivatives of meristems; first undergo expansion, and then differentiation. During this
stage many smaller vacuoles arise from cytoplasmic membranes of both RER and SER and
fuse with one another to form a large central vacuole.
Tonoplast membranes at their cytoplasmic surfaces are associated with polysomal complexes
engaged in protein synthesis. In the course of time the central vacuole gets loaded with wide
variety chemical components. The most perplexing situation is that during transformation of
mature parenchyma cells into meristematic cells the central vacuole disappears. What
happens to the vacuolar components is not known.
Plant vacuoles can be identified from other membranous vesicles by vital staining
techniques. The neutral red stain being basic in its properties binds components of cell
vacuole. These are highly inducible enzymes. ER is involved in the intracellular
transportation of various components like proteins, lipids, carbohydrates and others via golgi.
Some of ER membranes, containing specific proteins within their lumen, are pinched off into
vesicles which later fuse with each other or get processed via Golgi complex and get
integrated with specific cell organelles. Even cellular vacuoles of various kinds and
dimensions show metabolic activities like amino acid metabolism, fatty acid oxidation.
The loading of variety of components in to cell vacuoles bring about changes in the osmotic
potential of the cell sap. As a consequence of this, water may freely enter into protoplast; this
has been taken as evidence to argue that tugour changes within the cell sap is mainly
responsible for the growth of the negative turgour pressure. But now it is known that the
growth of the cell takes place without development of turgour pressure.
Vacuolar expansion and concentration is another dynamic feature, where vacuoles play a
predominated role in the development of young buds. The swelling of vacuole forces the cell
wall to bulge into a bud. Similarly in the case of stomata, guard cells contain a number of
small contracted vacuoles, but at the time of opening, the same vacuoles fuse and enlarge
into a large central vacuole in the guard cells, thus they bring about the movement of guard
cells. Nonetheless, it is speculated that hormones like Abscisic acid and cytokinin play
important roles in closing and opening of stomata
Vacuolar Contents:
Inorganic substances found in the vacuole show variation from cell type to cell type. For
example, more than 90% of the total cellular Mg2+ ions are found within the vacuole. On the
contrary, the total concentration of calcium ions and copper ions is just 6% and 2%
respectively. But the most common ions like K+ ions are equally distributed between
cytoplasm and vacuoles. In certain cases more than 40% of the total phosphates are found in
vacuoles and most of it is in the form of polyphosphates called volutin threads.
Enzymatic components:
It is really interesting to observe the presence of a wide variety of hydrolysing enzymes within
the vacuoles. The common enzymes found are carboxypeptidase, RNase, DNase,
phosphotases, b-glycosidase, alfa and beta–amylase etc. The concentration and composition
of such enzymes vary from cell to cell and species to species, strangely leaf cells of
Solanaceae members like tomato, potato, contain significant amount of protease inhibitors
within their vacuoles. How such variety of enzymes and other components are retained and
released is a mystery.
Other organic compounds:
Plant cell vacuoles also contain a good number of organic carboxylic acids, amino acids,
amides, mucilage, anthocyanins, flavones, gums, alkaloids, anthocyanin and other pigments
and even tannins. In certain plants like citrus, the vacuoles are filled with highly acidic citrate
compounds whose pH is about 2.5 and curiously enough such acidic pH is prevented from
inactivating cytosolic components by tonoplast membranes and provides a distinct
compartmentalization. In some cases more than 25 per of the total amino acid pool is found
in vacuoles.
Specialized Vacuoles:
Aleurone vacuoles: Certain vacuoles in seeds or grains of various pulses and cereals store
a variety of proteins; such granular components are referred as aleurone grains. Inulin,
legume, vanillin, glycerin, etc. are some of the common proteins found as storage products in
Aleurone vesicles. Such vesicles are derived from RER-SER membrane transitions. Most of
the stored or aggregated nonliving structures are called ergastic substances.
Spherosomes and starch vacuoles: Similar to Aleurone vesicles, lipids and starch are also
stored in special vacuoles called spherosomes and amylosomes. Spherosomes are derived
from RER SER transitional endomembranes. Such membranous vesicles are endowed with
enzymatic system from synthesis interconversion. On the other hand, starch is synthesized in
chloroplasts but transported to be stored in amyloplasts. Such starch containing
membranous organs are found in large numbers in storage tissues like root tubers, stem
tubers, cotyledons and endosperms of seeds and grains. The role of pyrenoids found in lower
algal cells and their role in storing starch is very interesting.
Functions:
Vacuoles are highly dynamic. They store many inorganic and organic compounds including a
host of enzymes. At the same time they are engaged in protein synthesis at the cytosolic
surfaces of tonoplast where they of found as polysomal complex. Some of the ions are
transported across tonoplast membranes against concentration gradient. In crassulacian
plants carboxylic acids are released into cytoplasm at nights. The diurnal behavior of
vacuoles is a unique feature for succulents. The synthesis and aggregation of various
substances like calcium oxalate crystals, raphides and other ergastic is very common for plant
central vacuoles.
Plant Hormones-ETHYLENE
In olden days, villagers, even now, used to accelerate the ripening process of banana, mango
and other fruits, just before they were taken to market places. The method employed by
them was simple. They used to keep the raw and unripened fruits in tightly closed earthern
pots and fill the pot with smoke generated by burning cow dung at the base and then seal it.
After 12 to 24 hours of this treatment, the fruits would appear yellowish and just started for
full ripening and they were ready for marketing. Even today, villagers use this method
without knowing why and how ripening is accelerated by the smoke generated by burning the
cow dung. But plant physiologists discovered ethylene as the gas that induces and augments
fruit ripening. This phenomenon and technology of fruit ripening was known to our village
farmers many many centuries back. Even now renowned plant physiologists don't know this.
All of us know cow dung produces atmospheric polluting gases such as methane and
ethylene. For that matter animal feca and fruit release more pullutants than all the cars (put
together) that emit pullutants. Though scientific studies were initiated as early as 1900s,
understanding the process of fruit ripening and identification of the causative factor was
possible only in 1924. Since then, detailed studies have been made on ethylene and its effect
on plants.
Many plant physiologists call ethylene as a plant hormone in gaseous state. But some do not
agree with this view instead, they consider ethylene as a byproduct of reactions induced by
other phytohormones and not an hormone perse. Two critical enzymes involved are SAM and
ACC synthase. Both gene can be used for antisense technology for prolonging commercial
fruit ripening, which is help full for farmers for their products will have longer shelf life and
this protectiveness ha sno deleterious effect on eaters.
Ethylene is produced in response to exogenous stimuli as shown in the above diagram. In any
of the plant developmental processes plant hormones interact with one another.
Distribution:
Ethylene is found in almost all parts of the plant body. But it is found in greater amounts
particularly in old and yellowing leaves and ripening fruits. This compound being a gaseous
substance diffuses through the intercellular spaces easily and rapidly reaches different
regions of the plant body.
Biosynthesis:
Ethylene consists of two CH2 groups held by a common double bond H2C=CH2. The
synthesis of ethylene is greatly enhanced by higher concentrations of auxin. Even
Gibberellins and cytokinins induce the synthesis of ethylene indirectly. The precursor for
ethylene was once believed to be methionine.
But recent investigations, using radioactive isotopes have shown that the precursor for
ethylene is 1-amino cyclopropane carboxylic acid and not methionine. ACC acts as the direct
and immediate precursor. In fact, higher concentration of auxin induces the synthesis of a
group of ethylene synthetase enzymes. These enzymes require FMN, H2O and Cu2+ as the
cofactors for their activity. The auxin induced enzymatic activity can be inhibited by
actinomycin D and CHI, which suggests that ethylene synthases are inducible enzymes. The
site of synthesis of ethylene has been suspected to be chloroplasts and its release is believed
to be regulated by phytochromes.
Apart from Auxins, factors like wounding, aging, irritation, light, cold temperature and drought
can also induce ethylene synthesis. Most of the above mentioned are stress factors even;
ABA is known to induce ethylene
production.
Effects:
Abscission: Onset of winter, cold treatment, drought and such conditions induce the
formation of abscission layer in the stalks of leaves, flowers and fruits. Eventually the said
structure separates by death of cells from the plant body and withers, it is also called in Greek
language as Apoptosis. The structure and the development of abscission layer has been
explained in the chapter ‘Auxin’. To put it in a nut shell, ethylene induces differential gene
expression in the region, where the abscission zone develops. As a result, pectinase and
cellulase enzymes produced and the same act upon the cell wall and degrade the same. Thus
the abscission layer becomes the weak point and the leaves, fruits, etc., fall down by their
shear weight.
Fruit Ripening: Once the fruit reaches a particular stage of development, the raw, hard,
green colored fruits undergo transformation to produce matured, soft, sweeter and
yellow/red/orange/coloured fruits. The repining process requires period of time ranging from
24 hours to a week or so. And ethylene is known to initiate this phenomenon. Once the
ripening is on, there is way to stop it.
With the maturity of fruits, the synthesis of ethylene is induced and ethylene induces more
ethylene production. During early part of ripening process, ethylene initiates a cascade of
events, which follow one after another and end up in a crescendo of biochemical reactions or
what is called climacteric state at which all the biochemical reactions are at their maximum
efficiency.
Ethylene to be effective in its action requires a copper containing metallo protein. Strangely,
CO2 is known to bind to the same site of the protein at which ethylene binds and thus it
competitively inhibits ethylene action. The active ethylene in its complex form first activates
respiratory process by which reserve food materials and organic acids if found, are subjected
to oxidative and decarboxylation reactions. The increase in respiratory activity is unusually
cyanide insensitive, which means that the electron transport chain used in this process
appears to be different from usual mitochondrial electron transport chain. Studies in this
regard have revealed that the electron transport chain in this process branches of from Cyt.C
and bypasses the cyt.a3 oxidase enzyme which is actually the site of cyanide inhibition. Most
of the respiratory and other metabolic pathways that are stimulated by ethylene lead to the
formation of more and more of sucrose and organic acids.
As the respiratory activity is reaching its climax ethylene simultaneously affects the
membrane permeability and also activates a set of genes resulting in the synthesis of specific
mRNAs. On translation of these mRNAs, specific proteins such as pectinase and cellulases are
produced. The enzymes then act on the middle wall and primary walls to loosen up the cells,
thus render the hard fruit into soft fruit. Ethylene induced fruit ripening can be effectively
inhibited by actinomycin and CHI, which suggests that ethylene induces differential gene
expression.
Ethylene also affects the membrane stability and permeability. As a result, the pigments
found in the tonoplast leak out and most of the membrane structures get disturbed.
Furthermore, ethylene induces the degradation of chlorophyll by chlorophyllase which is again
a product of gene expression. Simultaneously some anthocyanins are also synthesized which
develop attractive coloration to the skin and the flesh of the fruit. Thus ethylene ultimately
makes the fruit into a softer, sweeter and colorful commodity.
Effect on apical dominance;
Plants which show conical growth, ex., conifers, is known to have a strong apical dominance
effect on the lateral buds. This has been attributed to strong influence of IAA present in the
apical meristems found in the main axis. Recent investigations, it has been found that IAA
induced apical dominance is more due to ethylene production than to auxin itself. Apical
meristems of the main axis synthesize auxin and the same is translocated downwards. At the
same time, some amount of IAA produced in very young leaves is also translocated towards
the stem and more of auxin gets accumulated in the nodal regions. As higher concentration
of IAA stimulates the synthesis of ethylene, which on synthesis, diffuses into lateral buds
inhibits the growth. So the apical dominance is actually enforced by IAA through its second
messenger i.e. ethylene. But the apical dominance can be overcome by the application of
cytokinin, which removes the mitotic block imposed by ethylene and activates cell division, so
the lateral buds grow into branches.
Effect on Geotropic Movements:
Geotropic responses are explained as due to the sensitivity of stem tip and root tip to
different concentrations of auxins. But ethylene, a product induced by higher concentration
of auxin brings about a reverse of geotropic curvatures called ageotropic effects, where roots
instead of growing downwards into the soil curl upwards. In fact, ethylene treated roots loose
their sense of directional growth. Sometimes, the effect of ethylene will be similar to the
effects of morphactins. In addition, the other effects induced by ethylene, such as stunting,
stem enlargement and prostrate habit by an impaired response to gravity are called ‘Triple
response’ to ethylene.
Ethereal: In recent years, ethylene derivatives are sold in the market as ethereal or
ethephon, a patented product. This compound is nothing but 2 chloro ethyl phosphonic acid.
When these compounds are applied to plants in solution form, they release ethylene which in
turn brings about its effects. Application value of this compound in agriculture, pomiculture is
very well exploited during harvesting cotton balls and other fruit products. Application of
ethereal induces not only ripening in most of the fruits irrespective of the age and degree of
ripening, it also induces the abscission layer formation uniformly in stalks, which greatly
facilitates harvesting either by mechanical means or manually at any given time.
Ethylene signal transduction pathways:
Ethylene though exists in gaseous form, it diffuses across cells, but when it enters a cell it
binds to specific receptor. The signal transduction process are more like RTK but for they
have histidine kinase activity. Very often in some cases it looks like an RTK pathway. The
ultimate effects are different dpending upon the organ on which it works and the time at
which it works. Belwo only the self explanatory diagrams are give for your reference and
looking for more information.
Plant hormones- MORPHACTINS And Others
Morphactins are a group of substances which act on morphogenesis and modulate the
expression of plants. Chemically, they are the derivates of fluorene compounds. Fluorene by
itself is inactive, but the addition of COOH group in the 9th position makes it active. Majority
of morphactins, as synthetic compounds, have very diverse effects on plant growth and
development. Ex., chloroflurenol, flurenol, methyl benzilate, methyl chloroflurenol, methyl
dichloroflurenol.
Most of these components have transitory effect and they are degraded rapidly in plants. The
half life varies from compound to compound and plant to plant. Moreover, cellular and
environmental factors have greater influence on the stability of them. Sometimes, the
applied morphactins may be modified by glycosylation or demethylation. They are
translocated in plants basipetally as well as acropetally through both sieve tubes and xylem
elements.
The effect of morphactins is very interesting. Particularly in the presence of other natural
hormones, they exhibit both synergistic and antagonistic effects, which is however depends
upon the relative concentrations. Generally, morphactins have adverse effects on plant
morphogenesis. They inhibit seed germination, but sprouting, growth of seedling, internode
elongation etc. In many cases, they depolarize cell division which probably leads to distorted
morphogenesis. Morphactins are very effective in inducing lateral bud development so
tillering will be profuse. Strangely, some morphactins stimulate flowering in certain short day
plants.
In many respects, morphactins resemble ABA in inducing seed dormancy, bud dormancy,
suppressing stem elongation, etc. Most of their effects can be reversed by GA3 treatment.
The overall effect of morphactins appears to be polyvalent antiregulators.
Other known hormones
Other identified plant growth regulators include:
Brassinolides - plant steroids that are chemically similar to animal steroid hormones. First isolated from pollen of the mustard family and extensively studied inArabidopsis. They promote cell elongation and cell division, differentiation of xylem tissues, and inhibit leaf abscission.[17] Plants that are deficient in brassinolides suffer from dwarfism.
Salicylic acid - activates genes in some plants that produce chemicals that aid in the defense against pathogenic invaders. Jasmonates - are produced from fatty acids and seem to promote the production of defense proteins that are used to fend off
invading organisms. They are believed to also have a role in seed germination, and affect the storage of protein in seeds, and seem to affect root growth.
Plant peptide hormones - encompass all small secreted peptides that are involved in cell-to-cell signaling. These small peptide hormones play crucial roles in plant growth and development, including defense mechanisms, the control of cell division and expansion, and pollen self-incompatibility [18].
Polyamines - are strongly basic molecules with low molecular weight that have been found in all organisms studied thus far. They are essential for plant growth and development and affect the process of mitosis and meiosis.
Nitric oxide (NO) - serves as signal in hormonal and defense responses. Strigolactones , implicated in the inhibition of shoot branching.[19]
Jasmonate
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Jasmonic acid
Methyl jasmonate
The jasmonates (JAs) are a group of plant hormones which help regulate plant growth and development. Jasmonates include jasmonic acid and its esters, such asmethyl jasmonate (MeJa). Like the related prostaglandin hormones found in mammals, the jasmonates are cyclopentanone derivatives which are derived biosynthetically from fatty acids. They are biosynthesized from linolenic acid by the octadecanoid pathway.
The level of JA in plants varies as a function of tissue and cell type, developmental stage, and in response to several different environmental stimuli.[1] High levels of JA are also found in flowers and pericarp tissues of developing reproductive structures and in the chloroplasts of illuminated plants;[1] JA levels also increase rapidly in response to mechanical perturbations such as tendril coiling and when plants suffer wounding.[2][3]
Demonstrated roles of JA in planta include:
JA and MeJA inhibit the germination of nondormant seeds and stimulate the germination of dormant seeds[1]
High levels of JA encourage the accumulation of storage proteins; genes encoding vegetative storage proteins are JA responsive and tuberonic acid (a JA derivative) has been proposed to play a role in the formation of tubers[4][5]
JA application can induce chlorosis and inhibition of genes encoding proteins involved in photosynthesis, although the purpose of this response is unknown it is proposed that this response to JA could help reduce the plant's capacity for carbon assimilation under conditions of excess light or carbon[1]
The role of JA accumulation in flowers and fruit is unknown; however, it may be related to fruit ripening (via ethylene), fruit carotenoid composition, and expression of genes encoding seed and vegetative storage proteins[1]
JA plays a role in insect and disease resistance. Many genes during plant defense are induced by JA; JA and ethylene may act together in defense response[6]
The perception of jasmonate is via the ubiquitin system, like auxins. After the conjugation of jasmonate and an amino acid isoleucine, it led to the SCFCOI1 complex degrade the ubiquitin markerd JAZ protein, and then releasing the transcription of other transcription factors
Yi Zhang and John Turner at the University of East Anglia found that when leaves of the model plant Arabidopsis are wounded, cell division in the apical meristem is reduced, growth of the plant is arrested within days, and the new leaves grow to only one-half of their normal size although the size of leaf cells is unaffected.
Unexpectedly, the suppression of cell division in the apical meristem occurs through a signal pathway initiated by the wound hormone, jasmonate, which is synthesised in the damaged mature leaves. Mutant Arabidopsis lines unable to synthesise or to respond to jasmonate are not only larger than normal plants, but their growth is not reduced by the wound stress.
The jasmonates are a group of plant stress hormones that naturally occur in plants following exposure to certain
types of stresses, including pathogen and herbivore attacks. (+/-)-Methyl jasmonate is a mixture of trans (3R,7R and
3S,7S) isomers. Methyl jasmonate induces the synthesis of proteinase inhibitors in plant leaves.1 In cancer cells, it
suppresses proliferation and induces apoptosis.2 More specifically, methyl jasmonate inhibits hexokinase that is
bound to mitochondria.3 As hexokinase is overexpressed in cancer cells and contributes to cancer cell growth and
survival, methyl jasmonate’s disruption of mitochondrial hexokinase activity selectively targets, and kills, cancer
cells. Methyl jasmonate derivatives also have potential as anti-inflammatory agents.4
Brassinosteroids
There are approximately 60 steroidal compounds known as brassinosteroids named after the first one identified, brassinolide, which was found in
mustard pollen. Effects include:
1. Stimulation of stem elongation.
2. Inhibition of root growth and development.
3. Promotion of ethylene biosynthesis and epinasty.
Salicylates
Salicylates have been known to be present in willow bark for quite some time. Salicylic acid is synthesized from the amino acid phenylalanine. Effects include
1. Thermogenisis in Arum flowers.
2. Plant pathogen resistance-stimulates plant pathogenesis protein production.
3. Reported to enhance longevity of flower ( practice of adding a bit to cut flowers).
4. Reported to inhibit ethylene biosynthesis.
5. Reported to inhibit seed germination.
6. Blocks the wound response.
7. Reverses the effects of ABA.
Jasmonates
Jasmonates are represented by Jasmonate and its methyl ester. They were first isolated from the jasmine plant in which the methyl ester is an important product in the perfume industry. Jasmonic acid is synthesized from linolenic acid which is an important fatty acid. Jasmonates have a number of effects such as:
1. Inhibition of many processes such as growth and germination.
2. Promotion of senescence, abscission, tuber formation, fruit ripening, pigment formation, and tendril coiling.
3. They appear to have important roles in plant defense by inducing proteinase synthesis.:defence mechanism against fungi
Practical applications of Plant Hormones:
Agriculturists all over the world have developed certain unusual methods by which they
successfully cultivate the crop plants. It is only in recent years plant physiologists discovered
how plant hormones can be effectives used in agriculture, horticultures, pomiculture and
other related fields. As described earlier, plant hormones have a wide variety of effects and
most of these responses are concentration dependent. Fortunately phyto chemists have also
identified many synthetic hormones, some of which are more potent than natural hormones.
Experimentation and experience have shown that the judicial use of hormones or combination
of hormones can e employed in agriculture and related industries to get the maximum
benefit. Quantity and quality of agricultural products are very important factors in the
agricultural economics. How best the phytohormones can be utilized in this direction requires
imagination and training.
There are many areas in agriculture, horticulture, pomiculture, moriculture, etc., where
phytohormones can be used in successful cultivation to obtain greater yield. The high
percentage of germination of sown seeds in the field has a bearing on the output.
Pretreatment of seeds with IAA, NAA, GA, etc. has been found to be very effective not only in
increased the percentage of germination but also in the total yield of the crop plants.
Suitable concentration and combination have to be determined for each and every crop
plants.
The overall growth of plants, number of tillers and branches that produce from every plant in
the field contribute to the total yield. Use of GA or IAA greatly enhances the growth of plants
and total area of leaf surfaces. Some morphactins can also be used to produce more tillers.
In the case of sugar cane, use of GA has been found to increase the length of the internodes
and also the sugar content.
Plants can be multiplied by vegetative propagation. Many horticultural plants are propagated
by this way. The success of this method depends upon rooting. Hormones like NAA and IBA
are very effective in inducing roots in stem cuttings. These hormones can also be used fro in
grafting propagation. This way most of the plants can be propagated in large numbers and is
quick time.
Use of hormones like IAA, NAA, IBA and Gibberellins ensures fruit setting and many of the
fruits which develop from such hormone treatments are seedless, larger in size and sweeter in
content. In many cases the total yield will be very high. Quantitatively and qualitatively such
products yield more income to the farmer. Grapes, apples, oranges, mangoes and other fruit
yielding crop plants can be treated with some of these phytohormones for the better yield.
Furthermore, these hormones prevent premature falling of fruits, otherwise nearly 50-70% of
the set fruits fail to mature and most of them fall off because of the formation of abscission
layer in their stalks.
Preservation of agricultural products before marketing is another area in which hormones can
be used effectively. Tubers, rhizomes, bulbs and such products sprout while they are stored.
This will affect the farmer in terms of financial gain. Some of the synthetic hormones like
NAA, maleic hydrazide can be used to preserve the said products for quite a period of time,
thus one can improve the keeping quality of agricultural products.
Another judicial use which can be commercially exploited is the use of GA and other
hormones in inducing flowering in unseasonal periods. But one should known which hormone
is effective on which plant; otherwise phytohormones fail to produce the desired results.
Today farming is labour oriented and economically it is becoming impossible. Particularly
removing weeds in the field is a nuisance, costly and time consuming. However specific
synthetic hormones are available in the market to destroy the weeds selectively. 2, 4-D, 2, 4,
5-T can be used in paddy fields to destroy weeds. Similarly, monocot grasses can be
destroyed by using specific weedicide hormones. Sometimes, water hyacinth and such water
plants grow and multiply so fast they spread and establish their population in large tracts of
tanks and streams. This causes considerable damage to water storing capacity of tanks. For
example, 2 methyl 4 chloro 5 isopropyl phenoxyacetic acids can be effectively used to destroy
water hyacinth and save the tanks. Thus one can use different hormones for different
purposes, it can be for good or for bad, and it is like a knife with two cutting edges. It is left to
the man who uses it.
Application of Plant Hormones
Agriculturists all over the world have developed certain unusual methods by which they
successfully cultivate the crop plants. It is only in recent year’s plant physiologists discovered
how plant hormones can be effectives used in agriculture, horticultures, pomiculture and
other related fields. As described earlier, plant hormones have a wide variety of effects and
most of these responses are concentration dependent. Fortunately phyto chemists have also
identified many synthetic hormones, some of which are more potent than natural hormones.
Experimentation and experience have shown that the judicial use of hormones or combination
of hormones can be employed in agriculture and related industries to get the maximum
benefit. Quantity and quality of agricultural products are very important factors in the
agricultural economics. How best the phytohormones can be utilized in this direction requires
imagination and training.
There are many areas in agriculture, horticulture, pomiculture, moriculture, etc., where
phytohormones can be used in successful cultivation to obtain greater yield. The high
percentage of germination of sown seeds in the field has a bearing on the output.
Pretreatment of seeds with IAA, NAA, GA, etc. has been found to be very effective not only in
increased the percentage of germination but also in the total yield of the crop plants.
Suitable concentration and combination have to be determined for each and every crop
plants.
The overall growth of plants, number of tillers and branches that produce from every plant in
the field contribute to the total yield. Use of GA or IAA greatly enhances the growth of plants
and total area of leaf surfaces. Some morphactins can also be used to produce more tillers.
In the case of sugar cane, use of GA has been found to increase the length of the internodes
and also the sugar content.
Plants can be multiplied by vegetative propagation. Many horticultural plants are propagated
by this way. The success of this method depends upon rooting. Hormones like NAA and IBA
are very effective in inducing roots in stem cuttings. These hormones can also be used fro in
grafting propagation. This way most of the plants can be propagated in large numbers and is
quick time.
Use of hormones like IAA, NAA, IBA and Gibberellins ensures fruit setting and many of the
fruits which develop from such hormone treatments are seedless, larger in size and sweeter in
content. In many cases the total yield will be very high. Quantitatively and qualitatively such
products yield more income to the farmer. Grapes, apples, oranges, mangoes and other fruit
yielding crop plants can be treated with some of these phytohormones for the better yield.
Furthermore, these hormones prevent premature falling of fruits, otherwise nearly 50-70% of
the set fruits fail to mature and most of them fall off because of the formation of abscission
layer in their stalks.
Preservation of agricultural products before marketing is another area in which hormones can
be used effectively. Tubers, rhizomes, bulbs and such products sprout while they are stored.
This will affect the farmer in terms of financial gain. Some of the synthetic hormones like
NAA, maleic hydrazide can be used to preserve the said products for quite a period of time,
thus one can improve the keeping quality of agricultural products.
Another judicial use which can be commercially exploited is the use of GA and other
hormones in inducing flowering in unseasonal periods. But one should known which hormone
is effective on which plant; otherwise phytohormones fail to produce the desired results.
Today farming is labor oriented and economically it is becoming impossible. Particularly
removing weeds in the field is a nuisance, costly and time consuming. However specific
synthetic hormones are available in the market to destroy the weeds selectively. 2, 4-D, 2, 4,
5-T can be sued in paddy fields to destroy weeds. Similarly, monocot grasses can be
destroyed by using specific weedicide hormones. Sometimes, water hyacinth and such water
plants grow and multiply so fast they spread and establish their population in large tracts of
tanks and streams. This causes considerable damage to water storing capacity of tanks. For
example, 2 methyl 4 chloro 5 isopropyl phenoxyacetic acids can be effectively used to destroy
water hyacinth and save the tanks. Thus one can use different hormones for different
purposes; it can be far good or far bad. It is left to the man who uses it.
Application of plant hormones:
Experimental morphogenesis
Tissue Culture:
Since the days of Haberlandt attempts to grow plant cells, tissues and organs in an artificial
but defined nutrient medium have met with great success. Various methods have been
established to raise plantlets starting from single cells, pith, leaf, roots, etc. By modulating
the nutrients and hormonal concentration, it is possible to regenerate the entire plant body
from any living cell from any part of the plant body, which suggests that all cells are
totipotent. Hormonal concentrations play a significant role in culturing explants into
undifferentiated callus and callus to differentiate into roots, shoots or the entire plant from eh
callus.
Plantlets from Callus:
Tissue culture techniques have been very well exploited in understanding the role of different
hormones and their interaction in organogenesis. Such systems offer excellent experimental
materials for biochemical or molecular studies. Tissue explants in the presence of a particular
concentration of auxin, proliferate and produce an undifferentiated mass of cells called
callus. However, further growth of the callus depends upon the availability of cytokinin, for
the callus by itself cannot synthesize cytokinins.
The callus cells can be further induced to develop into shoots, roots or both by providing
auxins and cytokinins in a defined ratio. As shown in the figure, at high ratio of auxin to
cytokinin callus produces only shoots, at lower ratio the callus induces only roots, but at an
intermediate ratio both shoots and roots develop. In certain tissues, by manipulating the
concentration of auxin, cytokinins and gibberellins one can induce flowering directly from the
callus.
Tissue culture methods can be employed in propagating somoclonal variations from the same callus.
By inducing numerous plantlets from the single callus, the plantlets can be transplanted to field
conditions and then desirable varieties can be elected in a shortest possible time. This is because cells
in the callus show cot of variation in the chromosomal number. Tissue culture systems also provide an
opportunity to understand the effects of each hormone, nutrients, interactions between the hormones
and their effects on plant systems. Using the same methods, it is also possible to study molecular
events that lead to organogenesis. The potentiality of this technique in the applied fields like
horticulture, agriculture and other related fields is great; actually thee is not limit. These methods can
be employed to create haploid plants from pollen grains, which is very useful in hybridization
techniques.
Somatic Cell Hybridization
Protoplasts: Plant cells from leaves, stem and roots, for that matter from any source, can
be separated from one another by subjecting the tissues to certain cell wall digesting
enzymes like pectinases. The enzyme pectinase digests the middle wall, thereby the cells get
separated. Once the cells are freed from one another, cellulase can be used to remove the
surrounding cell walls. The resultant cells lacking is cell walls are called protoplasts which
contain only plasma lemma as the outer membrane and such cells assume spherical shape.
Cell Hybridization:
Insolated protoplasts can be used for single cell cultures in a defined medium and the calluses
can be obtained from such cells. Further, embryoids or organs can be raised from such callus
tissue. For every plant species the defined nutrient medium has to be determined for
obtaining callus and plantlets.
Protoplasts obtained from two different species can be forced or induced to fuse with one
another by various methods. It can be done by treating the protoplasts with polyethylene
glycol (PEG) or (X) viruses. These agents make the cell membrane labile and favor the
binding of the membranes between two protoplasts. Once the cells bind to each other, the
membranes fuse with one another leading to the fusion of cytoplasm between the cells. If the
protoplasts thus fused belong to two different species variety or genera, the product is called
as somatic cell hybrid.
It is also very important to note that just fusion of two cells by their cytoplasm is not an end.
The fusion of nuclei is also very important. Once the nuclei fuse, the hybrid cell is ready for
propagation. Such cells can be plated onto a defined medium which is suitable for the
development of both the species. In such a medium the hybrid cell divides and redivides to
produce the callus; again by manipulating hormonal combinations and nutrient media, it is
possible to induce plantlets from such callus. The success of these methods requires a large
number of trial and error experimentations, where one has to determine the suitable media
for each species and then one has to obtain another proper medium to make the hybrid cells
to respond. The success of these techniques required efforts and imagination and lastly the
luck.
Introduction of recombinant DNA into protoplasts:
Protoplast culturing techniques can be very well exploited in incorporating exogenously
supplied DNA into protoplasts. When protoplasts are incubated in a known medium
containing DNA, the cells take up DNA slowly. The amount of DNA that is incorporated into
the cells has to be determined. More than that, the entry of DNA into the cell is not enough;
the DNA has to reach the nucleus and it has to be incorporated into the host chromatin. The
DNA that is supplied may belong to different species or it may be recombinant DNA of a
known gene or gene(s). The DNA can also be injected into the nucleus directly by simple
injecting transgenic techniques. If the protoplasts incorporate such supplied DNA into their
nuclei, then the protoplasts can be cultured. One can develop a new species from the said
techniques. However, one has to find out suitable factors for the expression of incorporated
genome. These methods have been successfully employed in many laboratories.
In recent years the technique of cloning of known or desired gene has been perfected. A
cloned gene can be transferred to a bacteria or a phage easily and the same can be made to
express. The same technique can be used to transfer known genes into animal cells by
directly injecting the known genetic material into zygotes and by transplant back into uterus it
possible obtain the offspring with the exogenously supplied DNA incorporated into the
chromatin material unfortunately plant cells are not amenable for such techniques. However,
the plasmids from Agrobacteriam tumificians (which causes can be like tumors are plants)
have been isolated and characterized. Methods have been developed to inactivate the
plasmid into harmless structure, but such plasmids are still active when transferred as
exogenously supplied genetic material. By transferring known genes with using such
plasmids it can be used as an effective vector to transfer genes into protoplasts or to the
living plant itself either by rubbing the plants into wounds or by using a gene gun. By this
was many desirable games can be introduced into different plants.
Application of somatic cell hybridization techniques:
Somatic cell hybridization is a reality, but this is a technology of the twenty first century. The
success of this technology has raised new hopes in mankind. Already plant technologists
have succeeded in fusing different species of tobacco protoplasts and also they have obtained
a complete plant put of such somatic cell hybrids. The fusion of potato and tomato
protoplasts has produced pomato. But this hybrid has yielded plantlets which are capable
producing a tuber like structures in the terminal region than at the base of the plant. What
one wishes is to obtain hybrids which can yield two crops by a single plant.
The most ambitious project that man is thinking of is to develop plant and animal cell hybrid.
In fact, the hybrids between HeLa cell (Human cell line) and an yeast cell has been achieved.
What futurist plant genetic engineers expect is to fuse a plant cell with an animal cell and
make the plants to produce animal tissue. Ex. Plants producing pork or animal cell proteins
can be used as vegetables. Though the wishes are wild the success of such dreams are
becoming a reality.
Similarly introducing of recombinant DNA into protoplasts has generated
great expectations in the field of agriculture. For example, if nitrogen fixing genes are
introduced into cereal crop plant cells, the plants obtained from such experiments do not
need nitrogen fertilizers. It saves the farmer from providing nitrogen fertilizers and saves him
lot of money. Similarly, introduction of some protein genes, which yield proteins of good
nutritional value. It is of immense help in saving man from malnutrition. So the application
value of this technology has no limit. Many industries have been established to develop new
varieties of plants and plant products which have a greater commercial value.
PHYSIOLOGY OF FLOWERING
Plants, to begin with go through a period of vegetative growth. The extent of vegetative
growth is endowed with its genetic potentiality. Accordingly, they may grow into herbs or
shrubs and some may develop into trees or climbers. Generally, every plant after going
through a period of vegetative growth, responding to environmental clues, start producing
floral structures, which may be in the form of characteristic single flowers or inflorescences.
Many plants for that matter, a large number of plant species (higher plants), after a period of
vegetative growth, start flowering irrespective of the season. But some plants flower only in a
particular season of the year. Based on the duration required for the plants to produce
flowers, they have been classified into annuals, biennials and perennials. All plants have to
acquire ripeness to flowering. Annuals complete their vegetative growth and flowering in one
season and then they die. Biennials produce vegetative growth in one season and flower in
the next season. But perennials remain for many years and flower seasonally.
In fact, some trees do not flower till they reach a certain age. For example, coconut and
areca nut plants start producing flowers only when they reach an age of 6-8 years. On the
other hand in the case of bamboo plants, they grow for a number of years, and flower only
once in their life span. As soon as they flower, produce seeds and plants die (monocarpic
plants). Interestingly there are many plants which flower throughout the year, ex.,
Catharathus roseus.
Parts of a flower from outer to the central region
Aestivation of some flowers- refers to the organization of each floral part with respect to each
other, especially holds good for sepals and petals.
Plants growing in different regions of the globe are exposed to different climatic conditions
and different day lengths. In fact they are adapted to such environs in such a way, they
exhibit alternate vegetative and flowering cycles. It means that plants with their inherent
genetic potentiality interact with environmental conditions, accordingly, they respond and
behave. Humans, homo sapiens present day species, just about 25000yrs to 40000yrs old,
copulated with Homo eructus, mostly in Asia and made them extinct; when they evolved and
colonized sites of their own, after observing for many centuries the above said natural
phenomenon, they have devised different methods to cultivate crop plants in different
seasons of the year, so as to get the harvest at the right time of the year. They also
domesticated animals for their use.
The common knowledge of the farmer has been extended and explained by plant
physiologists ; why and how the said plants behave in response to different environmental
conditions.
Plants have all the needed signal transduction pathways to respond to environmental signals.
Such signal pathways has been worked out in Arabidopsis.
Developmental pathway of plants and its structures start from the zygote and end in all the
structures.
Discovery of flowering response:
Though it is a common knowledge that different kinds of plants respond to different seasons
of the year in producing the flower, it was left to G.Gassner & W.W. Garner to explain the
phenomenon by their pioneering scientific studies. Gassner observed that winter variety of
petkus rye plants called Secale cereal, responded favorably to cold treatments. Almost at the
same period of time, Garner and Allard demonstrated how plants produce flower in response
to different lengths of the day and night in a 24 hours day cycle. The above two phenomenon
are popularly called as Vernalization and Photoperiodism respectively. The above studies
have lead to the discovery of how plants rhythmically respond and behave to day and night
duration or to temperature fluctuation in different seasons of the year and they also observed
rhythmical behavior of the plants which is referred to as ‘biological rhythm’. And the
operational time measuring system found with in the plant structures is called ‘Biological
Clock’.
PHOTOPERIODISM (PP):
Earth, because of its revolution on its own axis and rotation around the sun, exhibits a period
of day and night and seasonal changes. The duration of the day and night again shows
variations because of the angle and distance between the earth and the sun at any given time
of the year. Thus plants and animals living on different parts of latitudes or longitudes are
subjected to different periods of photo periods and different temperatures at different seasons
of the year.
If we use three points or places on the globe, located at different positions as the reference
point, to measure the day and night periods, it will be apparent how different are the day
periods and temperatures of such places. Brazil in South America and Congo in Africa exhibit
almost 12 hours of day and 12 hours of night in all the months of a year. But a city like
Philadelphia located in the east coast of USA at latitude of 40 degree N, in the month of
December; it experiences 9 hours of day and 15 hours of night. On the other hand, in the
month of June, the day period is 14 ½ hours and night is 9 ½ hours long. Similarly, cities of
Norway, during December, experience 6 hours of day and 18 hours of night, but in June, it
enjoys 18 hours of day and 6 hours of night. Such day periods also accompany with changes
in extreme temperatures. The above observations suggest that organisms living in these
regions are subjected to seasonal variations of day and night and also to changes in seasonal
temperature fluctuations.
Garner and Allard , while working the department of Agricultural Station, Beltsville, Maryland,
USA, demonstrated remarkable relationship between the effect of the day period and
flowering in a mutant tobacco plant called Maryland Mammoth. They observed that the
mutant failed to produce flowers but tall, so they are called Maryland Mammoth. They also
observed that the same plant started flowering in summer under field conditions. But the
same plant started flowering when transferred to green house where it was subjected to short
day and long night conditions. So the plant was called short day plant. Since then, a large
number of plants have been subjected to various cycles of photoperiod i.e. treatment and
according to their responses, plants have been classified into different groups. The flowering
response in plants to photoperiodic treatment is now called photoperiodism. Light induced
responses in photo morphogenesis are many and intricate, and this can be only represented
in the form of network.
Classification:
Based on the responses to different photoperiods, most of the plants are grouped into 3 major
classes, viz. Short day plants, long day plants and day neutral plants. However, detailed
studies on each of these groups resulted in further classification of them into sub groups like
long short day plants, short long day plants etc. Each of these groups has been further
grouped into qualitative and quantitative varieties based on the specificity of the appropriate
light periods.
Critical Day Period:
It is the duration of the photoperiod or the dark period that ultimately determines whether the
plant has to go through vegetative growth or to produce flowers. Different plants require
different periods of light or dark for 100% flowering. If that period falls short then plants do
not produce 100% flowering. Such requirement of a minimum of photoperiod or dark period
for effective flowering is called critical day period. The length of light and dark period for
different long day and short day plants varies. For example Xanthium requires a critical
length of 15 ½ hours of dark period for its effective flowering. If the dark period is less than
15 ½ hours plants do not induce any flowering, but longer dark periods d not inhibit
flowering. On the other hand, the long day plant, ex. Hyoscyamus niger requires a critical 11
hours of exposure to light. Anything less than that, plants fail to produce flowers. If the
length of the day period for this plant is more than 11 hours, it does not affect the flowering.
Similarly, different plants have different critical day periods and the correct photoperiod has
to be determined individually by subjecting them to photoperiodic treatment.
Ripeness to flowering and site of perception:
Not all photoperiodic plants respond to the light treatment until and unless the plant has
grown to certain vegetative maturity. For example, Wulfia requires at least one leaf:
Xanthium responds well if it has few partially mature leaves. In Zea mays, at least there
should be 5-6 leaves to respond for photo periodic treatment.
Stem apex is the site for development of flower; a dramatic change in the structural and
functional features of the floral structures; they are nothing but modified leaves. In spite of it
is to be noted, the floral structures are same as the vegetative structure but modified; sepals,
petals, stamens and carpels are all derived from leaves. The most intriguing question that
has puzzled scientists for such a long time, how the genes hitherto remain silent, start
expression and develop these structure. The molecular aspects of gene expression that
changes leaves into floral organs are just opening up, yet only in bits.
Looking at the start of the plant body from the zygote a fertilized egg, in course of time
divides and redivides and differentiates and develops tissues and organs. Developmental
process in plants, at molecular level is more or less the same pattern as in animal systems.
Look at the Stem Apex Meristem (SAM) has undifferentiated central dome of progenitor cells
(pleuripotent in nature) covered by a layer of epidermal cells. Signals have to come from
different modes and methods to convert such potent cells to go through developmental
programmes. It is possible one such cell is enough for the development, just like stem cells in
animal systems. Signals are varied such as sunlight, sunlight duration, temperature, and
organic chemicals such as Gibberellins and sucrose are internal signals. Cellular transduction,
in response different signals, is more complex, for the signals arrive as environmental factors
Light (Photoperiodic pathway) temperature (Vernalization pathway) or inbuilt factor
(autonomous pathway). Most of the environmental signals impinge on leaves and resultant
downstream products have to be translocated to the SAM; this can be long distance for the
site of perception and the site of response are separated in time and space (Einstein).
Plants without leaves do not respond to any photoperiodic treatment, which suggests that the
vegetative buds are incapable of perceiving the stimulus. Similarly, plants with only old and
mature leaves not only fail to respond but also they inhibit or nullify the photoperiod effect.
However, partially mature leaf or leaves that are just unfolding, are highly sensitive. A
remarkable feature is that even one such sensitive leaf is enough to respond to proper
photoperiodic induction. It means whatever reactions or a product produced in one leaf is
enough to induce flowering in the entire plant. Let us start with light mediated induction.
Light and Duration mediated:
Photo inductive Cycle:
A plant, if subjected to certain length of day period and night period in 24-36 hours duration,
then it is called one photoperiodic cycle. Such periodic cycle responsible for inducing flower
formation, is referred to as ‘photo inductive cycle’. The required number of photo inductive
cycles varies from species to species; this is because of inherent genetic makeup of the said
plant. For example, Cockleburr needs just one photo inductive cycle but plants like Plantago
lanceolata or Salvia accidentales require at least 17-25 cycles for 100% flowering. If the
provided inductive cycles are less than what is required, the number of flowers produced in
such plants will be less than 100%. This suggests that during the inductive cycles some
flower inducing material gets accumulated and if such a substance produced reaches a
threshold value the flower production is maximal.
It is important to know that the photoperiodic response is ‘all or none’ phenomenon. Once
the provided stimulus produces a proper impact on the genome, the flowering is initiated and
once it is initiated it cannot be reverted to vegetative condition under normal circumstances.
Importance of Dark Period:
Systematic studies, using different lengths of light and dark period, it is noticed that the dark
period is important for short day plants for flowering. Shortening of photoperiod has profound
influence on the quantitative yield of flowers. The importance of photoperiod, i.e. Day period
on short day plants is very interesting. For example Xanthium requires 15 ½ of continuous
dark period and 8 ½ hours of day period for 100% flowering. If such a plant is maintained
with 15 ½ long dark period and the day period is shorted by 2 or 3 hours, the total number of
flowers produced will be significantly lower than the plant that is exposed to 8 ½ hours of day
period. This suggests that the photoperiod affects flowering ability in quantitative terms, it
means that proper dark period is essential for flowering but one cannot expect the plant to
grow and produce flowers in continuous dark. This is because light is required for
photosynthesis for it provides energy rich and components for the development of floral
primordia. If the photosynthate supply is not adequate the total growth of the floral axis is
affected. In fact, in one of the experiments, a short day plant which is kept in continuous dark
conditions is induced to produce flowers by providing sugar solution to the leaves. Similarly if
the CO2 supply is cut off during photosynthetic period, the short day plants in spite of
receiving proper dark and light periods, they do not produce flowers to their maximum
ability. The above observations suggest that photosynthate provides the necessary raw
materials for floral primordia for full expression.
Another important factor that affects the floral induction is the intensity of light. If the light
provided is of low intensity i.e., less than 100 ft candles, flower production is totally inhibited,
though the meristems are organized into floral primordia. But if the intensity of light is
increased above a critical level, the number of flowers produced also increases up to certain
level. This is because the light intensity affects the total yield of photosynthate, so flower
production is also affected by the said factor.
Action Spectrum of Light:
Finer analysis of the active part of white light that is effective is photoperiodic inductions
reveals that the red light at 660 mm and far-red light at 730 mm are the most effective
wavelengths in inducing or inhibiting the flower initiation. It has been established that
continuous far-red irradiation inhibits flowering in long day plants, on the contrary, continuous
red light treatment blocks flowering in short day plants.
The dramatic effects of red light and far red light can be demonstrated on a short day plant
like xanthium. It is known that a short break in continuous dark period with white light in a
short day plant brings about the total inhibition of flowering. If the short break is due to red
light, the inhibition is 100%, but if the break is due to far red light flowering is not inhibited.
Interestingly, if the red light and far red treatment is repeated alternately but ending in Far
Red as short breaks results in the reduction of total number of flowers produced. If the
number of cycles is extended the flowering will be totally inhibited though the last light
treatment is far red light. This is possibly due to the breakdown or exhaustion of some
intrinsic factors generated during short day treatments.
Phytochrome as the Photoreceptor:
The effectiveness of red light and far red light inducing or inhibiting the induction of flowers
strongly suggests the presence of some substance or substances that could absorb light in
the said wavelengths. By absorbing the
Light at particular wavelength, the said substances probably undergo excitation of
chromophore and the protein bound undergoes conformational change.
This protein complex induces signal transducing activities leading to flower induction. Search
for such a compound in plants resulted in the discovery of a blue / yellow colored pigment
called phytochrome.
This complex when get excited with the absorption of light at red wave length, its protein gets
activated and acts as a aspartate kinase and it autophosphorylates itself.
When phytochromes were discovered there was great excitement among plant physiologists.
This led to intensive research work on various aspects of the structure and functions of
phytochromes in plant morphogenesis. Now scientist have extracted and identified five
phytochromes; Phy A to Phy-E. Yet in recent years another pigment was discovered, called
Cryptochrome whose role is not well discerned.
Distribution:
In recent years, the techniques for extraction and quantification of this subtle pigment have
been standardized. The pigment has been found to be located in all possible organs of the
plant body. At the cellular level, it is mainly associated with plasma membrane, cytoplasm
and the membranes of plastids. The presence of this pigment in plastid membranes is
significant, because plastids are also known to perform many other photo biological
processes.
Chemical composition and structure:
The pure form of phytochrome appears blue / yellow colored pigment in solution form.
Phytochrome, in fact, is made up of two moieties; one is a protein and the other is a
chromophoric component. The phytochrome-associated protein has been isolated from maize
seedlings and other sources. The Mol.wt. of phytochrome is 123-125 KD and it is a tetramer.
But chromophore part is made up of linear chain of four pyrole rings. The protein subunits are
firmly bound to the A-pyrole ring via S-bond of the chromophore unit (chromophorobilin).
With the absorption of light at 660 mm or 730 mm, the double bonds found within the
chromophore get disturbed and shifted. These changes inturn bring about conformational
changes in 3-D structure of the pigment and also in protein chains either in in trans form
(activated) or cis form (inactive form). Probably, the above said changes due to absorption of
light transform them into excited form of molecules and they inturn elicit certain physiological
functions in the cells.
The two leaf like structures are dimeric phytochrome apoproteins
Dual forms of phytochromes:
In the earlier days of its discovery, people suspected the presence of two kinds of
phytochrome pigments because they showed different absorption spectrum at 660 mm and
730 mm. But Norris and others, using dual wavelength spectrophotometer, demonstrated
that the same phytochrome pigment exists in two alternate forms. They are called red light
absorbing pigment and far red light absorbing pigment. The Pr form after absorbing red light
gets transformed into Pfr form which by absorbing Far red light gets converted back to PR
form. The Pfr form naturally undergoes decay back to Pr form. Thus phytochrome exhibit dual
forms. The concentration of each form is dependent upon quality of the light source and the
physiological state of the cell.
The PFR form of the pigment formed due to the absorption of red light is not very
stable. It decays back to PR form or it is destroyed by some enzymes in dark
condition, but this process is slow. On the other hand, if the PFR form absorbs
far red light, it gets converted to PR quickly. But the decay of PfR pigment to PR
form on its own takes place in dark or it is converted by certain enzymes and it is
temperature dependent. In the presence of oxygen, the pigment undergoes
irreversible destruction. In spite of their labileness and sensitivity, they remain
quite stable at pH 6 and pH 8. Furthermore, the stability of these forms of
pigments is controlled by the firm binding of protein moiety to the chromophore
part of the pigment.
Activated phytochrome undergoes autophosphorylation at serine/threonine residue, which
then binds to its receptor protein and moves into the nucleus, where it associates with
transcriptional factors and activate their target genes whose products inturn activate other
genes and promote flowering.
Phytochrome in response light undergoes conformational change and moves into the nucleus where it binds to PIF3. The Phy-PIF3 dimers bind to G-box of the response gene and activate the gene expression. Light also triggers phytochrome mediated G-trimeric membrane protein activation, that leads to the production of cGMP and also the release of Ca2+ ions. One of the early gene expressed in response to Phytochrome activation is CCA1.
PHY-Pfr (), not only moves into the nucleus but also interact with membrane trimeric
alpha/beta/gamma G protein, that activates cGMP and release of Ca^2+.
The effect of which is not clear (at least to me). The PKS is serine protein kinase and NDPK2 is Nucleotide biphosphate kinase.
Involvement of Pfr-Phy-A and other in chromatin remodeling is speculated but not discerned.
The effects activated Phy-A is many where it activates many genes and their effect is seen or felt in cytoplasm, this includes flowering time locus FT synthesis.
Functions:
Probably phytochrome is the only pigment that is known to have multifarious activity and
elicit diverse responses. Among the 35 to 45 functions attributed to this pigment-protein
complex, bud dormancy, seed dormancy, flower induction is the well known phenotypic
effects. At the metabolic level, the phytochrome is known to act upon cell respiration,
permeability of membranes, transcription, translation and enzyme activity. Some of these
activities have been very well demonstrated in different plant systems. In this text the
discussion is restricted to its role in flower inductions and dormancy.
Role of phytochromes in flower induction:
Phytochromes being omnipresent in the plant body, they are always subjected to both red
and far red radiations in the day conditions. Accumulation of pR forms and pFR forms of
phytochrome in sufficient amounts in plants is critical and important. The effective
concentration of any of these forms over a threshold values in the perceptive organs like
leaves is absolutely essential to bring about certain biochemical functions which may
ultimately lead to the induction of flowers.
In long day plants, the Pr form of the pigment by absorbing red light throughout the day
transforms the substance to Pfr form and it accumulates in greater amounts. Such Pfr
pigments, when preset in higher concentration above the threshold value, activate the cell
machinery and ultimately induces flowering. However, recent studies indicate that the PfR
form alone is not active, but it also requires another substance called X whose properties are
not well characterized. The PER-X complex is believed to be highly effective in inducing
flower formation in long day plants. The X factor is known to be Phytochrome interacting
factor (PIF3).
PR – > PfR –> PRR.X – Induction of
flowering.
Light activated Phy-A binds to the receptor FHY1-FHL (FHY means elongated hypocotyls) that
enters the nucleus where it activates light response genes including flowering genes.
Accumulation PhyA represses the production of FHY3-FAR1 for they bind to gene loci of the
same. With the dissociation of Phy-A from FHY1-FHL the genes for FHY3 and PAR1 get
activated to produce FHY1-FHL that is found in cytosol after transcription and translation,
which are used for the binding of Activated Phy-A.
On the contrary in short day plants, because of long dark periods, whatever PfR pigments
formed in the day conditions are subjected to decay back to PR form. However, higher levels
of PR pigments are effective in inducing flower formation in short day plants. Conversely,
higher amounts of PR forms inhibit flower initiation in long day plants and PfR forms prevent
initiation of flowers in short day plants. So the kind of pigment or the form of pigment that
has a promotive effect on one kind of plant acts as an inhibitor to the other kind. The dual
form of pigments performing dual role is really intriguing.
The PHY –PIF3 complex binds to LHY/CCA (long hypocotyls and Chlorophyll a/b binding
proteins) region of the said genes and activate its transcription. The CCA has its own target
genes such as TOC which in turn interacts with PHY-PIFr dimer complex. The ultimate effect is
on the synthesis of FT in conjunction with CO. This FT moves into phloem vessels that
translocates to the base of SAM.
Light activated signal transduction pathway (general):
This actually explains why gibberellins are effective on long day plants but not so on short day
plants. Extrapolating this view it is possible to visualize that there are two or more different
genetically regulated regulatory factors acting at two regulatory sites. Added to this, plants
requiring vernalization can be short cut the flowering by GA treatment. But one thing is
certain that one of the factors is GA and other factor, i.e. Anthesins may be anything, possibly
it may be a highly labile protein or it may be one of the mRNPs, RNPs or a protein or signal
factor or a kind of ligand that can induce signal transduction just binding to the receptors on
plasma membranes or cytoplasmic receptor found in of the receiver tissues. The most
enigmatic situation was GA actually synthesized in the apex of the future stem tip or
inflorescence meristems, precursors for GA synthesis are found in proplastids and fully
developed plastids. The photoperiodic stimulation takes place in young leaves; if so
translocation of the phytochrome induced signal has to be transported a long distance to the
non determinant stem meristem the future floral meristem. What is the structural element
that can transport such substances? Is it Xylem or phloem? Xylem elements participation can
be ruled out for the simple reason that the xylem elements transport is mostly from root to all
other regions. But phloem transportation takes place in both directions; starting form vienlets
to veins and to the midrib of the leaf and from there the transportation is bidirectional.
Whether the bidirectional movement takes lace in the same sieve tube-companion dimers or
separate sieve tube elements are involved is little ambiguous.
In addition to photoperiodic and GA pathway, plants use autonomous pathway and
vernalization pathways also, where GA pathway provides a kind of interlink between
photoperiodic and vernalization pathways.
Concept of Florigin:
The PR and PfR forms of pigments are the products of photoperiodic stimulus and they in turn
are responsible for inducing flower formation. So the respective pigments elicit certain
responses in the plants which probably produce some kind of a substance (s) which is/are
responsible for transforming vegetative shoot into reproductive shoot. Production of such
substances by plants has been suspected by many plant physiologists long ago. But so far,
no one has succeeded in isolating or identifying such compounds. In spite of it, the presence
of substance (s) responsible for flower induction has been proved by different methods and by
different investigators.
The grafting experiments conclusively prove the presence of flowering substance(s). If a
short day plant, kept under proper photo inductive conditions, is grafted to another plant or
plants (by serial grafting) which are maintained under non photo inductive conditions,
flowering is induced in all plants. Whether the plants receiving the graft are short day plants
or long day plants, it does not make any difference. This experiment clearly indicates the
production and existence of a flower inducing substance in a photo induced plant made
product in response to stimulus, which is capable of diffusing through the graft to the receiver
plant. What is this substance is it a chemical signal such as cAMPs like or is it a mRNA or a
protein. Any substance that is induced and produced in leaf cells has to be transported to
long distances and should be stable. It is possible that the substance produced should cross
through the cell wall of mesophyll cells into sieve tube cells, and then it has to be transported
to stem apex meristems (SAMs). There again it has to cross cell wall barriers to reach a whole
mass of cells. So this substance should be a small molecule that can be easily transportable
and easily induce signal transduction that is capable of spreading.
Chailkhyan, a noted Russian botanist named such flower inducing elusive hormone as
‘Florigin”. Attempts to isolate such a substance have failed. In fact, people have made
attempts to collect the substance from the donor plant to a receiver plant through a water
jacket, but failed to obtain any stable compound which could induce flowering in other plants.
Probably, the suspected florigin may be an extremely unstable, labile and sensitive
compound, which could not withstand the most simple extraction methods. However recent
experiments involving solvent extraction methods indicate that florigin might be a compound
similar to sterols or mRNA-mRNP complex or a labile protein. But there are may plant
physiologists who suspect the very existence of such a compound because they feel that
some of the known growth promoting hormones by themselves may bring about flower
induction by some complex interactions. Most of the known hormones are small molecules,
easily transportable and bind to receptors and induce signal transduction. Either such
hormones and their binding proteins or the combined complexes may be involved.
However, the transport of such substances has been found to be through sieve tubes, but the
rate of translocation in short day plants and long day plants varies. In short day plants, the
rate of transportation is about 45-50 cm/hr. but in long day plants, it is about 2-2.5 cm/hr.
The rate of translocation of the said flowering substance is found to be 40-100 times slower
than the rate of transportation of sucrose, though the components involved in transportation
are the same. The different rates of transportation observed in short day plants and long day
plants are suspected to be due to the presence of two different substances. It is also known
that sieve tubes translocate different substances at different rates because of specific carriers
involved in the translocation process. The puzzling feature that is not known is that whether
the so called florigin is one compound or a complex of compounds. If it is one compound,
then the flowering substance produced by both the short day plants and the long day plants
should be the same. If there are two compounds, the rates of translocation may differ. But
why should they differ? The probable explanation is that one of the compounds is
constitutively synthesized and such substances reach their destination earlier and the other
compound that is synthesized when it is subjected to photo inductive conditions reaches the
destination later. However, for the inductive action, both compounds are required, but it is
difficult to visualize whether these compounds elicit their action in complexed form or
independently at one or two different sites.
But recent studies reveal that plants produce the elusive florigin, which is synthesized in
leaves and translocated through sieve cell and reach the base of SAM. The complex of
substance now called the actual “Florigin”, is a “Molecule of the century” has been identified
as FT (flowering time protein correctly Flowering locus T), not the FT mRNA suspected earlier.
Actually FT is synthesized in response to constans (CO). It is now believed that it combines
with another protein called FD, together activate AP1, SOC1 in the apical Meristem, they
inturn activate the expression of LFY. Then AP1-LFY triggers the expression of floral homeotic
genes. This just explains light induced components, but the flower initiation is also due to GA,
sucrose, vernalization and in a large number of plants it is autonomous. There is a kind of
confluence of the products of these effects ultimately responsible for triggering the floral
homeotic genes.
Red light activated Phy-A induces certain genes, not very well documented, the ultimate flowering product is FT; so also GA has an effect on inducing FT.
Effect of GA on Flowering:
Fascinating aspect of flower inducing substance (s) is that when the extracts, obtained from
photo induced leaves of Xanthium is applied to lemma plant kept under non inductive
conditions, the extracts induce flowering in lemma plants. However, the induction of
flowering by the extracts should be supplemented with gibberellins, without which the
extracts alone or gibberellins alone has no effect. This experiment suggests that gibberellins
are probably one of the components of elusive florigin (like elusive “Himalayan snow man’),
but the nature of the other component is still a mystery.
It is very well known that gibberellins induces bolting and flowering in rosette leaved long day
plants, but not in short day plants (with some exceptions). In long day plants, GA not only
stimulates the elongation the condensed internodes, but at the same time, it also promotes
the formation of factors needed for flowering. Thus GA treatment substitutes photoperiodic
treatment in long day plants. Added to this complexity, application of high concentrations of
gibberellins and cytokinins to the callus, obtained from Arachis hypogea (peanut plant),
results in the induction of flowers directly from the callus. Paradoxically ABA, a growth
inhibitor, is very effective in inducing flowers in some short day plants like Fragilis, Pharbatis,
etc. But ABA does not induced flowers in xanthium, another short day plant. This particular
case is very interesting, at the same time, it is also intriguing and it further raises doubts
about the existence of florigin per se. But such experimental results are very few and
conditions used for such materials have to be carefully analyzed. Much more perplexing that
is observed in some cases is that the application of cytokinins to the whole plant induces
flowering, when the plant is at a particular stage of development.
Based on gibberellin’s promotive effect on long day plants and its failure on short day plants,
Brain and is colleagues (1958-59) came out with a working model to explain the action of
photoperiods on flower inducing substances. According to this model, during photo inductive
red light treatment a precursor gets converted to Gibberellin or Gibberellin like substance.
The same substance is believed to undergo decaying back to the precursor either
in dark or under far-red light treatment. According to their concept it is assumed that
Gibberellin like substances have to be maintained at high concentrations in long day plants to
be effective in producing the elusive compound called ’florigin’. But in short day plants,
according to Brain, et.al. GA like substances are effective only when their concentration is
very low for higher concentration of GA is inhibitory. That is why when GA is applied to short
day plant there is no effect in terms of flower induction.
But the said scheme of events fails to explain how low levels of GA like substances can
produce sufficient quantities of florigin in short day plant to bring about the induction of
flowers and it is expected that the florigin that is produced in short plants or long day plants
should be the same.
Even today, with all the knowledge of molecular biology of the exact processes involved in
inducing flowers are not known. Still, it is very important to understand the model proposed
by Chailkhyan, a great Russian plant scientist. He toiled his entire life time to understand this
phenomenon and his proposed model is very worth understanding. Cajlachjan, another way
to pronounce his name, has assumed that the florigin formation takes place at two levels but
in two steps. Further, florigin is not one substance but it is a complex of two substances, i.e.,
gibberellins and Anthesins. It is also assumed that long day plants synthesize anthesins
irrespective of photoperiods, which means anthesins are produced constitutively, which
indicates that the genes responsible for the synthesis of the above said compound are
constitutively expressed all the time in long day plants. But the synthesis of GA or GA like
compounds is under the control of long day photoperiodic conditions. In the sense, the pFR
produced in long day plants has an important role in activating the pathway of GA synthetic.
On the other hand, short day plants are believed to synthesize GA constitutively and the
synthesis of anthesins is under the control of short day photoperiods. It means the pR form of
the pigments is effective in inducing the synthesis of anthesin.
Gibberellin pathway and Light quality pathway. Each of them has certain products and they
get integrated at the base of the meristem and determine and differentiate the floral
meristem. Many of the components that are synthesized in floral meristem or axis, very
young leaf primordials ultimately interact and integrate in activating the apical Meristem into
floral meristem.
Light has another discerning, but subtle activity is inducing the synthesis of GA1 and its
derivatives that activates LFY, and hypocotyl elongation and shade avoidance processes.
In spite of numerous experiments and detailed studies well over 50 years or so, the
explanation given to describe the entire mechanism of flower induction is confusing and often
contradictory in nature. Particularly investigations at the molecular level are few and they are
not convincing. Skoog and his students working on a cultivar variety of Nicotiana tabbacum
plant demonstrated that when the total DNA extracted from the leaves of photo induced
plants is applied to the stem tips of tobacco plants kept under non photo inductive conditions,
the receiver plant flowered. It is not clear from their experiments whether on not the DNA
extracted is really pure and free from proteins and other stimulatory small molecules that can
easily diffuse into stem apex.. Yet it indicates that pure DNA perse cannot express until and
unless they are provided with regulatory factors which are mostly proteins. If proteins are
associated with such extracted DNA, what are those proteins? Unfortunately, nothing is
known about them. But it is clear from their experiments that the applied DNA is not free
from proteins and such proteins are highly stable when they are associated with DNA. It
would be interesting to isolate chromosomal proteins from induced plant and provide the
same to non induced plants and see whether the receiver plants produce flowers or not.
A group of Japanese workers demonstrated an increase in the levels of different species of
RNAs and different species of proteins is buds during floral initiation. Unfortunately it is not
clear whether there are any qualitative difference among the mRNAs and proteins produced
during floral initiation. It is very important to understand the molecular events that lead to
the transformation of vegetative buds into floral buds. It beats any one’s imagination why
such work has not been successfully elucidated so far; even though the technology is
available at hand.
Nevertheless, it is now clear that the induction of flowering due to photoperiodic effects is
regulated at two sites separated by space and time. In the first event the site of response to
photoperiodic induction is young leaves. The second site is the terminal or axillary shoot
buds. Which on receiving the flower inducing substance(s) are stimulated to produce floral
axis or floral buds? Shoot buds per se do not respond to photo inductive treatments, but they
respond when leaves are subjected to such treatment. Which means whatever substances
produced in leaves reach the vegetative buds and induct flowering in them?
At the level of leaves, phytochromes able to receive signals form correct photoperiodic
treatments, undergo excitation and induce gene expression in the cells leading to the
synthesis of flower inducing substances. According to the prevailing views, the pFR pigments
produced in long day plants induce the synthesis of GA or Gibberellin like compounds in
proplastids/plastids and the same are released into cytoplasm. In the same long day plants,
whatever small amounts of pR pigment found in the leaf cells might be able to induce gene
expression to produce anthesins all the time irrespective of photoperiodic treatment. On the
contrary in short day plants, the pR form of pigments that accumulated during long dark
periods are suppose to induce anthesins production and the little amount of pFR present
might be involved in producing GA like compounds constitutively all the time. The anthesins
appears to be proteinaceous or protein like compounds.
That is why the rate of translocation of flowering compounds varies. Anthesins appears to be
translocated faster than gibberellins. Whether the genes that respond to two different
photoperiodic treatments are located in the nucleus or plastids or both, is difficult to visualize,
until and unless one obtains nuclear or cytoplasmic mutants for flowering.
Once such components are produced in leaves they are translocated to different site of cell
vegetative buds. It is conceivable that as long day plants produce anthesins and short day
plants produce GA like compounds constitutively; they reach the meristems of the vegetative
buds earlier. On the contrary as GA like substances in long day plants and in short day plants
produced under photo inductive conditions; reach the vegetative mounds later. If one of the
components reaches the vegetative meristems earlier, it cannot bring about the total gene
expression without the presence of the other factor. So both the factors are required in the
vegetative buds for total and comprehensive effect to be effective. Once the vegetative buds
receive both factors (i.e. GA and Anthesins) in required quantities, the florigin complex
activates a battery of genes requiredfor the transformation of vegetative buds into floral
buds.
Whether the florigin components activate one gene or a group of genes, the activation leads
to a cascade effects. It means a specific gene or gene products may activate group or group
of genes, which inturn may activate another set of genes. The products of these genes
ultimately bring about the transformation of vegetative meristems into floral meristems and
commit them to develop into floral structures. It is important to remember that the
production of stalk, bracts, sepals, petals, stamens and pistils is not a single event nor is it
regulated by a single gene. They are a series of sequential events which require a sequential
expressions and gene products. The totality of this highly regulated gene expression results
in the transformation of vegetative buds into highly organized floral buds of a particular type.
As many plants belonging to different species produce flower where all are sepals, some
lacking petals, stamens or pistils or the combination of any of them. It is predictable that the
development of each of these structures is under the control of specific genes or gene
clusters.
It is to be remembered that the molecular biology of flower induction and development is in
progress and we have to wait the D-day for understanding the whole process in Toto.
Phytochrome Pfr activated gets autophosphorylated. It can act on membrane G protein
receptor that can lead to cGMP and calcium modulated Cam, both can enter into the nucleus.
What is their function is not clear. The activated PhyA-Pfr act as serine-threonine protein
kinase. Once it enters in to the nucleus it interacts and binds to PIF3 dimeric (phytochrome
interacting factor) proteins and also binds to HFR1-PIF3 dimers (HFR1 = long hypocotyl far-red
protein1 is a TF); then PhyA-p and receptor proteins bind to their respective gene regulatory
elements called G-box receptor and recruit TFs and RNAP II and activate genes like CCA (TF),
LHY (TF) and HY5 (TF). The fate of these gene products perhaps activate transcription of FT in
association with constans in leaf cells. Probably they activate FT gene in the vascular
bundles, especially companion cells, where the role of PhyB plays antagonistically in the
production of FT.
Vernalization pathway:
Many plants have to go through wintering to flower in the next season. The famous plant is
petkus rye, which is essential for bread makers in Russia. It is not the only plant that requires
cold treatment for the plant to flower. These plants, particularly seeds contain specific
proteins and its associated component which bind to loci that are involved flower induction
and silence the chromatin by heterochromatization. Heterochromatization is achieved by
histone methylation and histone deacetylation at specific loci. One such protein is known as
flowering locus C (FLC). FLC acts as a repressor. Cold treatment in fact induce few FLC
antogonizing genes and FLC dissociates from the loci and get degraded or remain free. There
are several genes that are involved are VRN1, VRN2, VRN3 and VIN3. Even Frigida (FRI)
proteins are involved. Frigida promotes FLC and FRI antagonize FLC. Once the chromatin is
free from FLA and its associated protein, they interact with floral integrators such as SOC, CO,
FT and LFY, which inturn activate genes for floral parts. In certain cases GA can overcome
vernalization.
Cold has positive effect on the activation of genes such as BMS genes
that leads to floral transition.
Major pathways: Pathways are shown in the following diagrams, copied from different
sources and acknowledged too- the pathways are Photoperiodic pathway, Autonomous
pathway, Vernalization pathway,
Vernalization pathway:
Many plants have to go through wintering to flower in the next season. The famous plant is
petkus rye, which is essential for bread makers in Russia. It is not the only plant that requires
cold treatment for the plant to flower. These plants, particularly seeds contain specific
proteins and its associated component which bind to loci that are involved flower induction
and silence the chromatin by heterochromatization. Heterochromatization is achieved by
histone methylation and histone deacetylation at specific loci. One such protein is known as
flowering locus C (FLC). FLC acts as a repressor. Cold treatment in fact induce few FLC
antogonizing genes and FLC dissociates from the loci and get degraded or remain free. There
are several genes that are involved are VRN1, VRN2, VRN3 and VIN3. Even Frigida (FRI)
proteins are involved. Frigida promotes FLC and FRI antagonize FLC. Once the chromatin is
free from FLA and its associated protein, they interact with floral integrators such as SOC, CO,
FT and LFY, which inturn activate genes for floral parts. In certain cases GA can overcome
vernalization.
Pathways in general- modalities of integration of signals generated by
each of the inducing stimuli:
light
Diiferent pathways integrating activate floral meristem and organ identity gene.
Genetics of Flowering:
Gregor John Mendel showed that the color of the flower id controlled by the action of a pair of
heritable factor, now called Genes. They are independent discrete units of heredity.
Inductive stimulus should transform the entire shoot apical meristem (SAM) into flower
inducing meristem; there again from the same meristem different organs like sepals, petals;
stamens and ovary have to develop. The flower is indeed is a modified shoot with leaves
representing different parts of the flower. Stamen and ovary differentiation is more complex
than sepals and petals for the simple reason they have highly specialized structures and they
are gamete producing super-specialized reproductive organs.
Whether it is light induced pathway or vernalization pathway or GA induced pathway or
autonomous pathway, signals are produced and translocated to phloem and the base of SAM.
The important component that induce FT is CO which activates the expression of FT which is
a 20Kda protein. The FT is association with FD (TF) activates AP1 and LFY; this leads to the
activation floral organ genes.
The above figure is self illustrative and shows how LD and SD effect the activation of gene,
and the products move into the sieve cells, from there they reach the SAM. Note Hd3a is a
homolog of FT in rice plants
Once FT and FD together act on AP1 and LFY floral Meristem evocates and the floral organs
start developing. Each of the structures is controlled by single or a combination of genes.
The famous called ABC model has been used to explain the genes involved. Mutants in said
gene exhibit their phenotypic characters. The gene-A produces sepals and combination of AB
generates petals and the combination of B and C generate stamens and C alone produces
Carpels. As stamens and carpels contain different set of structures, to explain them the ABC
model has be extended as ABCD and E model.
What is interesting thing about these genes is the said gene products are a group of MADS
genes and they are homeotic genes. Each of these have been identified and studied.
MADS box proteins in combination induce specific floral organs. Look at A,
AB, CD and D alone and the pervasive E overlaps ABCD. MADS are mostly
transcription factors and they act in combination with one another. They have
many domains from N to C end.
Ap3 expression with Gus as a reporter gene in apical meristems destined to
Become floral organs.
MADS=MCM, Agamous, Dificiens and SRF
Ap3 expression with Gus as a reporter gene in apical meristems destined to
Become floral organs.
Ap3 expression with Gus as a reporter gene in apical meristems destined to
Become floral organs.
Ap3 expression with Gus as a reporter gene in apical meristems destined to
Become floral organs.
This figure is an ultimate starting and finishing of flowering, starts at leaves and
ends in flowers.
I have taken few authors written articles and put them together so student can
understand experts views on flowering. I duly acknowdge the authors for their great
insight into the flowering process.
FT Protein, not mRNA, is the Phloem-Mobile Signal for Flowering
Jan A. D. Zeevaart, MSU-DOE Plant Research Laboratory, Michigan State University,
East Lansing, MI
September, 2007
Introduction
Florigen as a Physiological Concept:
Specific flower-inducing substances were first postulated by Julius Sachs (1865), but more
convincing evidence had to await the discovery of photoperiodism (Garner and Allard 1920).
The seminal finding was that in photo periodically sensitive plants the day length is perceived
by the leaves, whereas flower formation takes place in the shoot apical meristem (Knott
1934). This finding demonstrates that a long-distance signal, called the floral stimulus or
florigin (Chailkhyan 1936), moves from an induced leaf to the shoot apex. The floral stimulus
can be transmitted from a flowering partner (donor) via a graft union to a non-flowering
partner (receptor), as illustrated in Figure 25.29 of the textbook. Furthermore, it was shown
that florigen is exchangeable between related species and genera, as well as among different
photoperiodic response types (Lang 1965; Zeevaart 1976; Zeevaart 2006). These
observations led to the hypothesis that florigen is wide-spread, if not universal, in flowering
plants, and that only the conditions that regulate its production vary among the different
response types. Although certain physiological characteristics of florigen, such as its
movement in the phloem (e.g., King and Zeevaart 1973), could be investigated, its identity
remained unknown. Thus, florigen remained a physiological concept rather than a chemical
entity.
With the isolation of auxin as the first-identified plant growth hormone in the 1930s and the
discoveries of cytokinins and gibberellins in the 1950s, many attempts were made to extract
florigin. In the early approaches, it was assumed that like the classical plant hormones,
florigen would be a small organic molecule. Extracts prepared from flowering material were
tested for flower-promoting activity in vegetative plants. Positive results were reported
occasionally, but none of them was reproducible (reviewed in Zeevaart 1976). As a result,
skeptics challenged the adequacy of florigin as a single substance causing flowering. Instead,
it was proposed that florigen consists of multiple factors and that flowering is induced by a
specific ratio of known hormones and metabolites (Bernier 1988; Bernier et al. 1993).
Molecular-Genetic Research on Flowering
With the advent of Arabidopsis as a model plant for molecular-genetic studies, genetic and
molecular analyses became popular approaches in studies on flowering. Through
mutagenesis, many mutants were isolated in the quantitative long-day plant
(LDP) Arabidopsis thaliana. Of interest here are those mutants that exhibit changes in
flowering time in comparison with wild-type (WT) plants. Mutants flowering later than WT
plants represent a loss-of-function that must involve positive regulators of flowering.
Conversely, early-flowering mutants have lost an inhibitor of flowering. These molecular-
genetic studies have led to identification of four pathways that regulate flowering
in Arabidopsis: the photoperiod, vernalization, autonomous, and GA pathways (e.g., Komeda
2004; Corbesier and Coupland 2005, 2006; Imaizumi and Kay 2006). In the present essay, we
will deal mainly with the photoperiod pathway.
The genes called CONSTANS (CO) and FLOWERING LOCUS T (FT) are principlal to long day
induced flowering in Arabidopsis. CO encodes a nuclear zinc-finger protein, which in response
to LD induces transcription of FT in the phloem of leaves (?).Does it happens in phloem or in
leaf tissues, but where in the cells of leafs; is it mesophyll cells, bundle sheath cells or in cells
in contact with phloem progenitors. Visualize phloem food conducting vessels are like human
lymphocyte system. Neither CO nor FT is expressed in the shoot apex. Expression of CO from
a meristem-specific promoter does not enhance flowering, but early flowering is induced in
short days (SD) when FT is overexpressed in the shoot apex. Expression of CO from a phloem-
specific promoter is sufficient to generate a phloem-mobile stimulus that induces flowering, as
shown by grafting experiments between Arabidopsis donor plants over
expressing CO and co mutant shoots as receptor (An et al. 2004; Ayre and Turgeon 2004).
Because FT must act in the shoot apex in order to elicit flowering, this result gives a strong
indication that FT or its product is the signal that moves from an induced leaf to the shoot
apex and induces flowering.
FT acts in the shoot apex by forming a complex with the bZIP transcription factor FD. The
essential role of FD in flowering is demonstrated by the finding that fd mutants flower late and
that FT over expression is partially suppressed by fd (Abe et al. 2005; Wigge et al. 2005). The
FT/FD complex activates the downstream genesAPETALA1 (AP1) and SUPPRESSOR OF
OVEREXPRESSION OF CO1 (SOC1); the latter, in turn, activates LEAFY(LFY). So, although FT
and FD are produced at different sites, they act together in the shoot apex (Figure 1). This
finding suggests that the FT gene product has to move from the leaf to the shoot apex and
strongly implicates the FT gene product as part of florigin, if not florigin itself. The concept of
Ft as florigin, a protein of 20Kds how does it induce the SAM. Is it through the binding to FT
receptors found in the basement cell membranes of SAM or what?
Is the Phloem-Mobile Floral Stimulus FT mRNA or FT Protein?
A substance functioning as florigin must fulfill at least the following criteria:
1. It must be produced in the leaf, presumably only under inductive conditions for
flowering. At least, it would be expected to be more abundant in an induced than in a
non-induced leaf.
2. It must be phloem-mobile, moving from an induced leaf to the shoot apex.
3. It must be required for flowering in all plants.
From the hierarchy of regulatory proteins that controls flowering, it follows that FT is the
terminal gene expressed in the leaf, whereas its effect is in the shoot apex (see above;
Corbesier and Coupland 2006). Consequently, three possibilities can be envisaged:
1. FT mRNA is phloem-mobile and moves from an induced leaf to the shoot apex.
2. FT protein moves from an induced leaf to the shoot apex.
3. FT in the leaf controls the synthesis of a small compound in the leaf that then moves to
the shoot apex, where it induces FT expression to produce FT protein, which complexes
with FD. Results relevant to all three possibilities will be discussed below.
Huang et al. (2005) investigated the possibility that FT mRNA is the mobile stimulus. It was
shown in Arabidopsisthat FT under control of a heat shock promoter was transiently
expressed in a single heated leaf and FT mRNA was detected in the shoot apex by RT-PCR 6
hours later. In addition, the heat-treated leaf did induce flowering. Thus, in this work FT mRNA
would seem to fulfill some of the criteria of florigen. However, it was later reported that the
real-time RT-PCR data were analyzed incorrectly and that in new experiments the movement
of FT mRNA from leaf to shoot apex was not detected. Consequently, “…the conclusion
that FT mRNA is part of the floral inductive signal moving from leaf to shoot apex” was
retracted (Böhlenius et al. 2007). Other authors also failed to obtain evidence
favoring FT mRNA as a mobile floral stimulus. In the case of Arabidopsis and rice,FT mRNA as
measured by real-time RT-PCR was lower in shoot apices than in leaves (Corbesier et al. 2007;
Tamaki et al. 2007). Also, no FT mRNA could be detected in the shoot apex
of Arabidopsis by in situhybridization (Jaeger and Wigge 2007). In Cucurbita, FT mRNA was not
detected either in the phloem sap or in the shoot apex (Lin et al. 2007). Finally, in grafting
experiments with tomato SFT transcript of the donors did not move across the graft union to
the receptor shoots (Lifschitz et al. 2006; Lin et al. 2007).Thus, a rather extensive body of
evidence argues against FT mRNA functioning as florigen, although considering the presence
of mRNAs in phloem as components of a long-distance signaling network, the possibility
cannot be completely ruled out (Lough and Lucas 2006).
Simultaneously with the retraction of the publication on FT mRNA as a phloem-mobile signal,
two new publications reported results that FT protein rather than the mRNA is the mobile
signal in the phloem that induces floral initiation in Arabidopsis as well as in the SDP rice
(Figure 1). To determine the distribution and movement of FT protein, George Coup land’s
team at the Max Planck Institute for Plant Breeding Research, fused FT with the gene
encoding GREEN FLUORESCENT PROTEIN (GFP) and expressed this construct in ftmutant
plants. Expression from phloem-specific promoters demonstrated the presence of FT:GFP not
only in leaf phloem, but also in the shoot apex. FT:GFP transgenic plants also flowered earlier
than ft plants. Assuming that the promoters employed in these experiments are specific for
expression in phloem, these results demonstrate that the FT protein expressed in the leaf
phloem moves to the shoot apex to induce floral initiation (Corbesier et al.
2007). FT and FT:GFP were also expressed from the GALACTINOL SYNTHASE (GAS1) promoter,
which acts specifically in the phloem companion cells of the minor veins of leaves. In
transgenic plants expressing GAS1:FT:GFP, GFP fluorescence was observed only in the minor
veins of leaves. Transgenic GAS1:FTplants flowered early, but GAS1:FT:GFP plants flowered as
late as ft plants, although the transgene was active in the leaves, as shown by activation
of FRUITFULL (FUL). The authors speculated that the fusion protein was not mobile in the
minor veins and as a result did not move to the shoot apex and induce flowering. This result
confirms that FT protein is the floral stimulus in Arabidopsis and that no secondary product of
FT is involved (Corbesier et al. 2007).
In the SDP rice, Heading date 3a (Hd3a) is the ortholog of FT in Arabidopsis. Under SD
conditions, Hd3a shows highest expression in leaf blades of rice. Ko Shimamoto’s group at the
Nara Institute of Science and Technology, transformed rice with the Hd3a: GFP construct
under control of phloem-specific promoters. The transgenic plants flowered early and GFP
fluorescence was observed in the vascular tissues of the leaf blade, stem, and shoot apex.
Because the Hd3a:GFP construct was expressed only in the phloem of leaf blades, whereas
the protein was detected in the shoot apex, it must be concluded that Hd3a protein is moving
in the phloem and that Hd3a functions as florigin in rice (Tamaki et al. 2007).
Additional evidence obtained with Arabidopsis further supports the notion that FT protein
moves from an induced leaf to the shoot apex. When expressed from a phloem-specific
promoter, an epitope-tagged version of FT induced early flowering; the protein was detected
by immuno localization in the shoot apex. By contrast, when MycFT was targeted to the
nucleus (immobilized), it had no effect on flowering and remained localized in the phloem of
the leaf, whereas the constitutive 35S promoter did induce early flowering (Jaeger and Wigge
2007). Furthermore, FT expressed ectopically as a large fusion protein promoted flowering.
But in the case of a phloem-specific promoter, flowering was promoted only if the FT protein
was released from the complex by a specific protease (Mathieu et al. 2007). Thus, export of
FT from companion cells was required and correlated with flowering.
In the experimental approaches used in Arabidopsis to demonstrate the movement of FT
protein, FT was expressed as a fusion protein in transgenic plants for ready detection by
either confocal microscopy or immunolocalization. It should be realized that expression
of FT transgenes is usually much higher than that of the native FT gene. Although all the
results are consistent with movement of FT protein from induced leaves to the shoot apex,
the presence of native FT protein in the phloem translocation stream was not demonstrated in
these experiments. In a close relative of Arabidopsis, Brassica napus, FT protein has been
identified in phloem exudate of inflorescence stems (Giavalisco et al. 2006), so that it is
reasonable to assume that native FT protein is also present in the phloem sap of Arabidopsis.
Because of its small size, Arabidopsis is not a suitable plant for phloem translocation studies.
For this purpose,Cucurbita has been a favorite, especially because phloem exudate can be
readily collected from cut stems. However, cucurbits are day-neutral and had not been used
for flowering studies until Bill Lucas’s laboratory at the University of California, Davis,
surveyed 97 cucurbit accessions. They found one, C. moschata (Cmo), that flowered only
under SD conditions. The group tested this species, along with day-neutral C. maxima (Cm),
to determine whether long-distance movement of FT is required for flowering. They used the
Zucchini yellow mosaic virus (ZYMV) as vector for introducing the FT gene
of Arabidopsis (AtFT) into Cmo. Infection with this vector caused flowering of Cmo in LD. The
virus was present in developing leaves, but not in apical tissues (Lin et al. 2007). This result
demonstrates that the function of AtFT as an inducer of flowering is conserved when
expressed in Cmo, and further, that flowering was most likely induced by movement of FT
protein from virus-infected leaves to apical regions.
To investigate the role of FT-like (FTL) genes in Cucurbita, two orthologs of FT were isolated
from both Cm(Cm-FTL1 and Cm-FTL2) and Cmo (Cmo-FTL1 and Cmo-FTL2). Transcripts
of FTL genes were restricted to phloem of stems and leaves, but were not detected in phloem
sap. By contrast, the FTL proteins were detected in phloem sap by a combination of liquid
chromatography-tandem mass spectrometry. Cmo-FTL2 was approximately 10 times more
abundant in phloem exudate than was Cmo-FTL1. Both proteins were present only in phloem
sap obtained from SD-grown plants. Although transcript and protein levels of Cmo-FTL2 were
much up-regulated in the phloem of stems in SD, an important regulatory mechanism by the
photoperiod appeared to be entry of FTL proteins into the phloem translocation stream, in
addition to transcriptional control (Lin et al. 2007). Finally, Cmo(receptor) was grafted
onto Cm (donor) in long days. All receptor shoots were induced to flower and the CmFTL2
protein was identified in phloem sap collected from Cmo scions. These results show
convincingly that FTL proteins are present in the phloem translocation stream
of Cucurbita and that they can move across a graft union in a heterograft to induce flowering
in a vegetative receptor shoot (Lin et al. 2007). This experiment elegantly demonstrates that
transmission of florigen from a donor to a receptor plant is associated with transfer of FT
protein from donor to receptor.
FT Protein Is the Universal Signal for Flowering
The basic tenet of the florigin hypothesis is that florigin is common to all flowering plants.
Physiological evidence for the universality of florigin is based on results of grafting
experiments between closely related species in which one response type (e.g., a LDP) induces
flowering in a related species of another response type (e.g., a SDP). However, this approach
has been limited by graft-incompatibility between unrelated species. With the advent of
molecular genetics and plant transformation, this barrier can now be readily overcome.
Instead of grafting, a specific gene from one species can be “transplanted” into an unrelated
species and its role in flowering demonstrated. For example, the SFT gene of day-neutral
tomato can substitute for the LD requirement in Arabidopsis (Lifschitz et al. 2006).
Likewise, FT expressed in the SDP C. moschata induces flowering under LD conditions (Lin et
al. 2007).
One of the criticisms of the universality of florigin was that there were many examples of
grafting experiments in which receptor shoots failed to flower (reviewed in Zeevaart 1976).
Does this mean non-identity of florigin in the two grafting partners? Work with tomato
provides an answer to this question. Transgenic tomato plants over expressing SINGLE-
FLOWER TRUSS (SFT), an ortholog of FT, under control of the 35S promoter, were excellent
donors, but wild-type tomato could not complement sft mutant plants in grafting experiments
(Lifschitz et al. 2006). This result suggests that the failure to induce flowering in receptors is
not due to non-identity of florigin, but most likely due to a low level of florigin in the donor
and/or rapid decay of florigin in the receptor.
The functions of FT orthologs appear highly conserved in flowering plants regardless of
response type, and in monocots as well as in dicots. Although the control of expression
of FT varies across response types, the end product, FT protein, appears to be always the
same. The evidence obtained so far provides strong support for the universality of FT protein
as florigin not only in herbaceous plants, but also in trees (Böhlenius et al. 2006; Hsu et al.
2006). In Lolium temulentum, GAs, specifically GA5 and GA6, have been assigned a role as
florigen (Web Essay 25.1). But it is of interest that also in this species LtFT was strongly up-
regulated in the leaves after plants had been shifted from SD to LD (King et al. 2006).
The question is often asked: Why did it take so long to elucidate the molecular nature of
florigin? There are several aspects to a complete answer. For many years it was assumed that
florigin, like the classical plant hormones, would be a small molecule that could be extracted
and re-introduced into test plants. Some physiological evidence indicated that in some
species florigen has virus-like properties, but techniques to extract nucleic acid or proteins
and apply them to assay plants were not available. So, further progress had to await the
application of molecular-genetic techniques to studies on physiology of flowering. It then took
considerable time before the various flowering pathways had been worked out
in Arabidopsis and the pivotal role of FT in flowering became apparent (e.g., An et al. 2004;
Ayre and Turgeon 2004; Abe et al. 2005; Wigge et al. 2005; Imaizumi and Kay 2006). Rather
than applying extracts to test plants, transgenes could now be expressed and their products,
mRNA and protein, could be visualized by techniques of cell biology, or identified by mass
spectrometry. It took 70 years, but finally florigin has been identified as FT, a mobile protein
of approximately 20 Kda.
Perspective
With the identification of FT protein as florigin, questions regarding its production, transport,
and persistence can be studied at the molecular level. For example, is FT permanently
activated in plants with localized induction? Does FT induce its own production via a positive
feedback loop in species that exhibit the phenomenon of indirect induction of flowering? (see
textbook pp. 661–662). These intriguing phenomena, as well as other classical observations
on the physiology of flowering, can now be studied from a molecular-genetic perspective. It is
expected that results of further studies will provide a solid underpinning for the florigen
theory.
References
Abe, M., Kobayashi, Y., Yamamoto, S., Daimon, Y, Yamaguchi, A., Ikeda, Y., Ichinoki, H.,
Notaguchi, M., Goto, K., and Araki, T. (2005) FD, a bZIP protein mediating signals from the
floral pathway integrator FT at the shoot apex. Science 309: 1052–1056.
An, H.L., Roussot, C., Suárez-López, P., Corbesier, L., Vincent, C., Piñeiro, M., Hepworth, S.,
Mouradov, A., Justin, S., Turnbull, C., and Coupland, G. (2004) CONSTANS acts in the phloem
to regulate a systemic signal that induces photoperiodic flowering
of Arabidopsis. Development 131: 3615–3626
The Role of Gibberellins in Floral Evocation of the Grass Lolium temulentum
Rod W. King and Lloyd T. Evans, CSIRO, Plant Industry, GPO Box 1600 Canberra,
ACT 2601, Australia
September, 2006
Introduction
Almost 70 years ago it was recognized that the leaf is the site of the initial response in
daylength-regulated flowering. However, the nature of the factors transported from the leaf to
the shoot apex where the flowers form has remained elusive. Here we summarize the
evidence from studies with the grass Lolium temulentum, which shows that specific
gibberellins (GAs) act as its "floral stimulus." This evidence satisfies five requirements,
namely:
The production of florigenic GAs in the leaf when exposed to a photo inductive long
day
Their transport from the leaf to the shoot apex
Their sufficient increase in the shoot apex when floral evocation occurs
Morphological and molecular changes at the apex, especially involving floral organ
identity genes
Replacement of the long-day requirement by specific GAs
Our studies over the last 40 years, reviewed by King and Evans (2003), have established a
continuous trail of evidence for the gibberellin (GA) class of plant hormone as a floral stimulus
in the long-day regulated flowering of the grass, Lolium temulentum. Its long day (LD)
photoresponse in the leaf blade involves far-red rich phytochrome-active light, which
enhances the expression of a GA biosynthetic enzyme and alters GA precursor and product
levels. Most significantly, the florigenic GAs increase in the shoot apex not long after they rise
in the leaf, and at the time of the earliest molecular and morphological changes at the shoot
apex. Finding the final pieces of this scientific jigsaw has involved GA application studies. Not
only was it necessary to show that applied GAs could replace the need for a LD, but by
utilizing a range of structurally related synthetic and natural GAs as well as inhibitors of GA
synthesis, we have established that some GAs promote inflorescence initiation while others
are more active for stem elongation or may participate in the later stages of flowering of L.
temulentum.
Gibberellins for Flowering
In 1956, Anton Lang reported for the first time that applied gibberellic acid (GA3), like
vernalization or exposure to long days (LDs), could cause plants of Hyoscyamus niger first to
bolt, and subsequently, to flower. His finding was soon confirmed both for other dicot species,
for grasses and for GAs other than GA3 (Lang 1957).
Flowering of many temperate grasses is induced by LD or by treatment with GA3. For
example, the grass Lolium temulentum requires a single LD for inflorescence initiation, and
this requirement can be met in noninductive short days (SD) by a single application of some
GAs, either to the leaf blade or to the shoot apex (Evans 1964a, 1969; Evans et al. 1990).
However, there are differences between some GAs and LD in this response. Over at least the
first three weeks of inflorescence development, there is little or no stem elongation/bolting
associated with the LD-induced flowering of L. temulentum and perhaps other grasses—a
response duplicated by application of some GAs—but many GAs that induce flowering of
grasses also cause early and excessive stem elongation.
The responses to GAs may be similar across dicot and monocot species even though the
effects on stem elongation and flowering may occur in a different order, presumably reflecting
a different order of gene activation associated with different GAs. For example, GAs that
stimulate stem elongation may not be involved in the process of floral evocation in grasses,
although they could be involved in subsequent inflorescence development. Conversely, other
GAs may be florally effective because they cause inflorescence initiation with little or no effect
on stem elongation.
The Search for GAs that are Florigenic but Ineffective for Stem Elongation
Beginning in the mid 1980s, and in collaboration with Dick Pharis and Lew Mander, we
compared the effects of many different gibberellins on floral evocation and stem elongation
in L. temulentum. Some, like GA8 and GA9, influence neither process. 16,17-dihydro
GA5 promotes flowering while inhibiting stem growth (Evans et al. 1994b), while GA1 and
GA3 enhance both and are therefore unlikely to be the floral stimulus in L. temulentum(Evans
et al. 1990). However, exogenous GA5 and GA6 are both able to cause floral evocation and
induce the early stages of inflorescence development in SD at doses that have no effect on
stem elongation (King et al. 1993, 2003). Two questions arise from such findings. Firstly, are
either or both GA5 and GA6 agents of floral evocation by one LD in L. temulentum? Secondly,
are the more growth-effective GAs such as GA1 and GA4involved in the subsequent processes
of inflorescence development in this grass? To answer these questions, in the following
sections we consider evidence based on endogenous gibberellin contents of the leaf and apex
of L. temulentum along with information on metabolic pathways involved in GA synthesis and
degradation.
Critical Steps in GA Metabolism
The relevant steps in GA metabolism as summarized by Hedden and Phillips (2000) are shown
below:
Figure 1 (Click image to enlarge.)
The key to biological activity among many gibberellins is the closure of the lactone ring with
the conversion of GA19 to GA20 and GA24 to GA9. This step involves a 20-oxidase whose activity
increases in LD, as shown for spinach and Arabidopsis (Wu et al. 1996; Xu et al. 1997). A
multifunctional 3-oxidase completes the conversion of the biologically inactive GA20 or GA9 to
bioactive GAs—including GA5 and GA6—and to the 3-hydroxylated GA1, GA3, and GA4. A key
inactivation step involves the 2-oxidase responsible for adding a hydroxyl at C-2 to give GA8,
GA29, or GA34.
GAs for Floral Evocation: Production in the LD Leaf and Transport to the Shoot Apex
In L. temulentum, when the leaf is exposed to a single photo inductive LD, expression of the
messenger RNA for a 20-oxidase increases dramatically after 16 hours of light and peaks 4
hours later (Figure 2). Most significantly, the timing of the increase in the 20-oxidase in LD
matches the minimum duration of light required for floral induction of L. temulentum by one
LD. In SD, the expression level of mRNA for this key enzyme is much weaker and peaks 12
hours later during the next 8-hour-daylight period.
Figure 2 Expression of a L. temulentum 20-oxidase GA biosynthesis gene.
Compared with an 8h short day, LD exposure of the leaf (8h SD extended by
9.5h using incandescent lamps) led to a large increase in gene expression.
(Semi-quantitative RT PCR assays from Blundell, MacMillan, and King;
unpublished).
The consequence of increased 20-oxidase activity in L. temulentum leaves is a fall in
GA19 levels when the critical day length is reached around midnight, and an equivalent rise in
GA20 (GA19 falls from a peak of 35 ng g-1 DW to a minimum of 12 ng g-1 around midnight, while
GA20 rises from 3 ng g-1 to 23 ng g-1; King et al. in preparation). GA5, an immediate product of
GA20, also increases in LD but not in SD leaves so that the difference by midnight (i.e., after 16
hours of light) is fourfold. GA1 could also be expected to rise in the LD leaves, but this is not
apparent until the following day.
The next step, the export of GA5 from the leaf blades and down the leaf sheath has not been
directly documented. The difficulty is to isolate specific vascular tissue and to sense
potentially small changes in GAs. As an alternative, we have assessed movement from the
leaf to the apex of labeled GA5. In LD, 2H4-GA5 applied to the leaf blade was transported intact
and quantitatively to the shoot apex (King et al. 2001). Earlier experiments in which the blade
and sheath of the single LD leaf were cut off at various positions and times suggest that the
LD stimulus is translocated to the shoot apex at a speed of 1–2.4 cm h-1, compared with the
simultaneous transport of sugars at about 80–100 cm h-1 (Evans and Wardlaw 1966).
Furthermore, evocation can be maximal in shoot apices, which throughout the low intensity
daylength extension, show no increase in their sucrose content over the 2% found in the
vegetative plants exposed to SD (King and Evans 1991). The LD florigenic stimulus in L.
temulentum is clearly not sucrose, and an increase in the content of sucrose in the shoot apex
is neither necessary nor sufficient for its floral evocation.
Based on its apparent speed of translocation, the LD floral stimulus should begin to arrive at
the shoot apex and floral evocation begins on the morning after the LD. Such timing of floral
evocation was confirmed by excising shoot apices at various times after the LD, onto media
supporting apex development (McDaniel et al. 1991). King et al. (1993) confirmed these
results but also found with plants of various ages that shoot apices excised from vegetative
plants in SD were induced to flower by supplying GA3 in the medium and could reach a similar
stage of flowering to those from plants given one LD but with no GA3 in the medium.
Floral evocation in plants of L. temulentum is associated with an increase on the day after the
LD in 32P incorporation into RNA, and of 35S into protein in shoot apices (Rijven and Evans
1967; Evans and Rijven 1967). These increases occur in the dome of the apex and in the sites
of future spike lets, down both sides (Knox and Evans 1968). Thus, the timing of these
changes fits with the estimated time of arrival of the LD photoperiodic stimulus in the apex
(Evans and Wardlaw 1966; McDaniel et al. 1991; King et al. 1993). However, to complete the
evidence relating GAs to floral evocation in L. temulentum, it was essential to measure their
content in the shoot apex.
The minute size of the shoot apex (<3 µg dry weight) had made it difficult to measure its GA
content, but this became feasible through a recent collaboration with Thomas Moritz in
Sweden. Using highly sensitive GCMS techniques, he analyzed GAs at subpicogram levels in
the shoot apex of L. temulentum. As shown in Table 1, by the end of the daylight period
following the overnight LD, the content of the highly florigenic GA5 and GA6 at least doubles in
the shoot apex (King et al. 2001, 2003). Moreover, at this time the maximum
GA5 concentration reached in the shoot apices is 3 × 10-7M which is close to that needed in an
agar medium if shoot apices excised from plants in SD are to flower (about 5 × 10-7M GA5;
King et al. 1993). It is highly probable, therefore, that translocation of these two GAs from leaf
blades in LD causes floral evocation of L. temulentum.
Table 1
Changing the Guard: Inflorescence Development and Growth-Active GAs
A number of bioactive GAs and precursors including GA1, GA4, and GA9 are notably absent
from the shoot apex or minor in SD and on the day after the inductive LD, and remain so for
6–10 days. After exposure of the leaf to 2 or 3 LD, they all increase in content at the apex
(King et al. 2001), whereas the content of GA5, GA19, and GA24 falls more rapidly with
additional LD. Such additional LDs accelerate inflorescence development (Evans 1958, 1960;
Evans and Blundell 1996) and, as well, increase the content of GA1, GA4, and GA9 in the LD
leaf (Gocal et al. 1999). Thus, as for floral evocation, for inflorescence development there are
increases in GAs in LD but now for a new group of GAs that are highly active for stem
elongation.
During inflorescence development, the many fold increase in the shoot apex of the "new
guard" GAs—GA1 and GA4—along with evidence that their application can now promote
flowering (King et al. 2001), shows their importance for inflorescence development. That a GA
input is effective during floral development is also evident from studies with apices induced to
flower by one LD, and then excised. Progress to flowering is weak unless GAs are supplied by
about six days after the end of the LD (King et al. 1993). Which GAs are important at this time
is indicated by the response to application of two growth retardants, Trinexapac Ethyl and
LAB 198 999. These retardants block synthesis of GA1 and GA4 by 3-oxidases and inhibit
inflorescence development but show no inhibition when applied earlier during floral evocation
(Evans et al. 1994a). Thus, whereas GA5 and GA6 are the gibberellins most active in floral
evocation of L. temulentum, it is the elongation-active, C-3-hydroxylated GAs such as GA1 and
GA4 that play a role in subsequent inflorescence development.
Since GA1 and GA4 can be detected in leaves of L. temulentum—whether from vegetative or
floral plants—there must be some mechanism first to exclude them from the shoot apex and
then allow them to access the shoot apex late in inflorescence development. As discussed
below, this exclusion mechanism may involve: (i) degradation of specific GAs by a 2-oxidase;
(ii) localization of a 2-oxidase just below the apex of vegetative plants and; (iii) disappearance
of this 2-oxidase during inflorescence development. The implication that some GAs are more
readily degraded than others (e.g., GA1 or GA4 vs GA5 or GA6) also provides a focus in the
following section for understanding how GA structure affects response.
Structural Considerations: The Lord of the Rings
The functional groups contributing to GA activity are indicated in Figure 3, and from our
comparisons of many GAs (Evans et al. 1990, 1994 a,b; King et al. 2003; Mander et al.
1998 a,b), it is clear that the structural requirements for florigenicity are quite different from
those for stem elongation. As an example, for four GAs—2,2-dimethyl GA4, GA32, GA1, and GA4
—although not greatly different in their effectiveness in promoting stem elongation, there is
up to a ten thousandfold range in their florigenic activity.
Figure 3 Gibberellin structure and carbon numbering.
Differences in activity of an applied GA are likely to be determined not only by their ability to
resist degradation (see above) but also by structural features altering uptake and transport,
their inherent bioactivity, their potential as a biosynthetic substrate, and their capacity to
interfere with endogenous GA synthesis and degradation. GA uptake and transport is unlikely
to be critical to florigenicity, as GAs could variously promote either or both stem elongation
and flowering. Those structural elements favoring florigenicity in particular include:
(i) Formation of the lactone bridge between carbons 4 and 10, so adding a fifth ring to the 4-
ringed structure of the biologically inactive GAs. This requirement seems to apply to all the LD
grasses whose flowering is induced by gibberellins. For example, GA19 (a 20-carbon precursor
GA) is wholly inactive, whereas GA3, GA5, GA7, and several other 19-carbon GAs have reported
florigenic activity in other species besides L. temulentum. However, formation of the lactone
ring itself is insufficient, and a further step involving activity of a 3-oxidase enzyme is also
essential (see iii).
(ii) A free carbon-7 carboxy group is an absolute requirement for all plant GA responses and
may reflect ability to bind to a GA receptor.
(iii) Hydroxylation at carbons 3, 12, 13, and 15 and/or the presence of a C-1, 2 double (C-C)
bond (as in GA3), or a 2,3 double bond (as in GA5), or a 2,3 epoxide (as in GA6).
(iv) Absence of a C-2 hydroxyl.
While features listed under (iii) may enhance the stability of a GA against inactivation by 2β-
hydroxylation, structural elements at C-2 are the most significant in this regard. A good
comparison involves the responses to applications of three closely related GAs: the highly
florigenic and growth active 2,2-dimethyl GA4, the modestly florigenic but reasonably growth-
active 2α-methyl GA4, and the nonflorigenic, growth-active GA4 (Evans et al. 1990). Because it
lacks any C-2 structural elements, 2-hydroxylation of GA4 is highly likely and this apparently
rings the death knell for any florigenic activity. GA4 is taken up by—and transported in—the
plant as it is highly active for stem elongation but quite clearly it is not transported intact into
the shoot apex. The most likely explanation, for this observation would involve the presence
of a concentrated zone of 2-oxidase just below the vegetative apex, and while this has yet to
be demonstrated for L. temulentum, such a highly localized zone of 2-oxidase RNA expression
has been reported recently for rice (Sakamoto et al. 2001).
The concept for rice of the exclusion of 2-hydroxylation-sensitive GAs from the shoot apex fits
well with the structures of the GAs that are naturally found in the shoot apex of L.
temulentum. Neither of the readily 2-hydroxylated GAs we examined, GA1 and GA4, was
detectable in the shoot apex of vegetative plants or for about the first week after floral
evocation (King et al. 2001) although their content increased in LD leaves (Gocal et al. 1999).
By contrast, we detected highly florigenic but weakly growth-active GAs—such as GA5 and GA6
—these GAs probably being protected from C-2 hydroxylation by the C-2,3 double bond in
GA5 and the C-2,3 epoxide in GA6. High florigenicity with limited growth activity for GA32 also
fits with its having a C-1,2 double bond. Later, as floral development proceeds GA5 and
GA6 decrease in the apex while there is a dramatic increase in GA1 and GA4 (King et al. 2001)
and it is at this stage of flower development that the localized band of 2-oxidase disappears
from just below the rice inflorescence (Sakamoto et al. 2001).
Apparently, the localized band of 2-oxidase activity protects the shoot apex from "growth-
active" GAs, so guaranteeing the integrity of the apex during vegetative growth, floral
evocation, and early floral differentiation. Later, during inflorescence development,
disappearance of the 2-oxidase barrier allows the influx of highly growth-active GAs.
Explaining a Paradox: Flowering Promotion by GA Biosynthesis Inhibitors
Classic studies by Baldev and Lang (1965) with the rosette LDP Samolus parviflorus showed
that flowering in LD is inhibited when GA synthesis is blocked using either of the GA
biosynthesis inhibitors, Amo-1618 and Cycocel (CCC). Such results supported a role for GAs in
the LD response. Paradoxically, however, with L. temulentum, several "anti-gibberellins"—
which might be expected to inhibit floral induction—actually promote it, and act
synergistically with some GAs. For example, CCC alone did not change the flowering response
to one LD, but greatly enhanced the promotive effect of GA3, although other "anti-
gibberellins" such as B9 and Amo 1618 gave the expected inhibiting effect on flowering
(Evans 1969). With the acylcyclohexanedione inhibitors, LAB 198 999 and Trinexapac Ethyl,
promotion of flowering could reflect an interference with the 2-oxidase. Whether or not this
enzyme is localized below the vegetative shoot apex, endogenous or applied bioactive GAs
would be spared from inactivation. Another explanation involves either or both inhibition of
the 3-oxidase(s) responsible for conversion of GA20 to GA1 and inhibition of the 2-oxidase
involved in converting GA20 to inactive GA29. By inhibiting the conversion of GA20 to either
GA1 or GA29, LAB 198 999 and Trinexapac Ethyl treatments could promote GA20 conversion to
GA5, thereby accounting for their strongly promotive effects on floral evocation inL.
temulentum. Their subsequently inhibitory effects on inflorescence development (Evans et al
1994a) when synthesis of GA1 and GA4 becomes important, fits with their known action as 3-
oxidase competitors. The greater inhibition of the 2-oxidase by the derivative 16,17-dihydro
GA5 than by LAB 198 999 (Junttila et al. 1997), indicates further interesting effects of "ant
gibberellins," which preclude drawing too simple an interpretation of how they may alter GA
metabolism.
The Genes Involved at the Shoot Apex
Of the various genes known to be involved in floral determination and differentiation, the
earliest to be expressed in the shoot apex of L. temulentum is LtCDC2, which greatly
increases in activity in the future spikelet sites by the afternoon after the long day, just when
floral evocation is completed (Gocal 1997). By the next day, the API-like gene LtMADS2 and a
related gene LtMADS1 have also increased their expression at the spikelet sites and
subsequently at the floret sites (Gocal et al. 2001).
Whereas the LEAFY (LFY) gene is expressed early in the floral apices of Arabidopsis, Gocal et
al. (2001) found it not to be expressed until about 12 days after LD induction in L. temulentum
—possibly associated with the great difference between the two species in the timing of stem
elongation vis-à-vis floral evocation, and, as well, in the GAs involved.
Conclusion
For no other species is there such a complete and consistent trail of evidence on the identity
of the LD floral stimulus as in L. temulentum, from the daylength-sensitive, physiologically
mobile GA5, and probably GA6, in the leaf blades, translocated intact and at appropriate
velocity to reach the shoot apex in sufficient concentration to effect floral evocation at the
clearly identified time.
Moreover, several previously puzzling features of this investigation, especially those relating
to the great range in florigenicity among the gibberellins and to the synergistic effects of
several inhibitors of GA synthesis, can now be resolved. If, as in rice, the vegetative shoot
apex in L. temulentum is protected by a ring of 2-oxidase, a very coherent picture of the
control of flowering in L. temulentum by gibberellins emerges. In addition, such localized
action of the 2-oxidase explains much of the variation among bioactive GAs in their relative
promotion of floral evocation vis-à-vis stem elongation.
Our findings also highlight a feature of the growth habit of temperate grasses, which may
have been crucial to their evolutionary success when close-grazed by ungulates. Except for
the relatively brief stage when the inflorescences must grow upwards for wind pollination and
seed dispersal, the terminal meristems are kept close to the ground by the absence of stem
elongation until the later stages of inflorescence development. The initial involvement at floral
evocation of GAs such as GA5 and GA6, which do not cause stem elongation at concentrations
that are florigenic, and the initial exclusion from the apex of GAs such as GA1 and GA4, which
do cause stem elongation, presumably aids survival under both close-grazing and adverse
environmental conditions. Such an evolutionary explanation is required, because stem
elongation per se is not antagonistic to flowering. Applied 2,2-dimethyl GA4, for example, not
only induces flowering but also causes massive stem elongation.
For a number of reasons, our findings that specific GAs are florigens in grasses, cannot be
generalized to all species. Based on their evolutionary relatedness (see Kellogg 2001), we
believe our findings are applicable to other temperate grasses and cereals, which retain a LD
response—as most do (Evans 1964b; Heide 1994). However, the warm climate grasses and
cereals—including rice, corn, and sugarcane—often have no photoperiodic response and if
they are sensitive, they often respond to SD, which may lead to decreases, not increases, in
GA content. Where a species is insensitive to photoperiod, a role for GAs appears unlikely and
one of many other florigenic factors must be limiting. In temperate, LD-responsive dicots, GA
increases have often been documented but it is also clear that their primary action may be on
stem elongation rather than on flowering. This latter observation is especially interesting as
the GAs detected in dicots have always been the growth-active ones, and the florigenic but
weakly growth-active GAs have yet to be examined in such dicots. Nevertheless, there is no
reason at present to suggest that the growth-inactive GAs that are florigens in grasses play
any role in flowering of dicots.
References
Baldev, B., and Lang, A. (1965) Control of flower formation by growth retardants and
gibberellins in Samolus parviflora, a long-day plant. Amer. J. Bot. 52: 408–417.
Evans, L. T. (1958) Lolium temulentum L., a long-day plant requiring only one inductive photo
cycle. Nature 182: 197–198.
Evans, L. T. (1960) The influence of environmental conditions on inflorescence development in
some long-day grasses. New Phytol. 59: 163–174.
Evans, L. T. (1964a) Inflorescence initiation in Lolium temulentum L. V. The role of auxins and
gibberellins. Aust. J. Biol. Sci. l7: l0–23
Signals produced in leaves are transported to the shoot apex where they cause flowering.
Protein of the geneFLOWERING LOCUS T (FT) is probably a long day (LD) signal in Arabidopsis.
In the companion paper, rapid LD increases in FT expression associated with flowering driven
photosynthetically in red light were documented. In a far red (FR)-rich LD, along with FT there
was a potential role for gibberellin (GA). Here, with the GA biosynthesis dwarf mutant ga1-3,
GA4-treated plants flowered after 26 d in short days (SD) but untreated plants were still
vegetative after 6 months. Not only was FT expression low in SD but applied GA bypassed
some of the block to flowering in ft-1. On transfer to LD, ga1-3 only flowered when treated
simultaneously with GA, and FT expression increased rapidly (<19.5 h) and dramatically (15-
fold). In contrast, in the wild type in LD there was little requirement for GA for FT increase and
flowering so its endogenous GA content was near to saturating. Despite this permissive
role for endogenous GA in Columbia, RNA interference (RNAi) silencing of the GA biosynthesis
gene, GA 20-OXIDASE2, revealed an additional, direct role for GA in LD. Flowering took twice
as long after silencing the LD-regulated gene, GA 20-OXIDASE2. Such independent LD input
by FT and GA reflects their non-sympatric expression (FT in the leaf blade and GA 20-
OXIDASE2 in the petiole). Overall, FTacts as the main LD floral signal in Columbia and GA
acts on flowering both via and independently of FT
Fig. 7. Summary of findings here and in the companion paper of positive effects (arrows) on
flowering andCO/FT for two commonly used LD photoresponses. This schematic incorporates
effects on FT and flowering of: mutants; gene silencing; change in light intensity; and a block
to photosynthesis. Predominantly, in LD, photosynthetic sucrose amplifies CO/FT expression
(see companion paper) while phytochrome acts directly and also via GA, which plays a
permissive and, often, non-limiting role. There is also a direct but lesser LD-mediated increase
in GA supply via the petiole response to FR-rich light. A dashed arrow indicates a potential
step of regulation, and weaker responses are indicated by thinner arrows. The electronics
symbol for a speaker is used to show sucrose amplification of CO/FT expression
GA4 applied to ga1-3 shows an FT-independent effect on flowering in SD and a permissive
effect involving FTexpression in LD. A 10 μl drop of GA4 [1 mM in 20% ethanol: water (v:v)]
was applied to each of three leaves on consecutive days either in SD or at the start of a far-
red-rich LD (LD-FR). Plants of ga1-3 flowered, bolted, and leaves grew (A). Its FT expression
increased most after GA treatment in LD (B), and (C) shows the effect of GA4 on FT expression
in Columbia. Prior to treatment, the plants of ga1-3 had been grown in SD for 12 weeks and
those of Columbia for 5 weeks. The low intensity FR-rich LD exposure was for 2 d. GA4 was
applied 8 h after starting the day, and leaf blades were harvested 19.5 h later for assays
of FT expression (leaves harvested at 16 h showed similar increases; not shown). There was
no effect of solvent application on flowering or gene expression (not shown). All FT expression
was normalized to the value in SD without GA application. The means and SE were based on
three replicates for FT assays and 10 replicates for flowering time.
Intergrative pathway
In Arabidopsis, the four floral pathways converge through genes of the integrative
pathway [7]. The activation of floral integrators, such as AtFLOWERING LOCUS T (AtFT)
and AtSUPPRESSOR OF CONSTANS 1(AtSOC1), in turn, lead to the activation of floral meristem
identity genes such as LEAFY (LFY) and APETALA1(AP1). We did not observe differential
expression of TaFT (see above discussion of TaVRN3). On the other hand, transcripts for
wheat genes that share sequence similarity with AtSOC1 did show differential expression.
Zhaoet al. [38] identified seven MADS-box genes
(TaAGL1, TaAGL7, TaAGL18, TaAGL20, TaAGL21, TaAGL23 andTaAGL38) that, upon
phylogenetic analysis, were placed in the SOC1-like clade of MADS-box genes. Only three
probe-sets on the wheat array corresponded with these seven genes (see Additional file 5).
The gene TaAGL7corresponds with the probe set Ta.25343. The
genes TaAGL1, TaAGL18 and TaAGL23 (the most similar toAtSOC1) all correspond to one
probe-set (Ta.21250), and TaAGL20, TaAGL21 and TaAGL38 (the least similar toAtSOC1) to
another (TaAffx.19661) – given the high sequence similarity between the genes in the
respective groups, they are probably homoeologues and so we cannot report on their
individual behavior. However, the three probe-sets that correspond to the genes of the SOC1
clade of MADS-box genes all evidenced essentially the same profile of abundance (data not
shown). That is, in both leaf and crown tissue of all three varieties, transcript increased
slightly (Figure 5).
Photoperiod pathway
The principal components of the photoperiod pathway are conserved in the monocots and,
more pertinent to this discussion, in the cereals On the Wheat Genome Array, there are
probe-sets that correspond to many of the genes belonging to the photoperiod pathway.
In Arabidopsis, AtCONSTANS (AtCO) encodes a transcription factor that activates genes
required for floral initiation. It integrates circadian clock and day-length signals and, under
long-days, activates the floral promoters AtFT, AtSOC1 and AtLFY [8]. The two circadian clock
genesAtLHY and AtTOC1 influence the expression of AtCO. They form part of a feedback
mechanism, each directly affecting the expression of the other: AtLhy.p is a repressor
of AtTOC1, and AtToc1.p is required for the expression of AtLHY [8]. The cyclic expression of
these two genes, which occurs over a 24 hour period, entrains that of AtGIGANTEA (AtGI). This
latter activates AtCONSTANS.
In this study, the profiles of abundance for TaLHY and TaTOC1 were complementary to each
other, as one might expect from their relationship to each other in circadian cycling. In crown
tissue, transcript of TaLHYincreased in abundance and then declined; conversely, that
of TaTOC1 declined and later increased. The profile for TaGI was very similar to that
of TaTOC1 (see Additional file 4). Given that in both rice and Arabidopsis, GI is a promoter
of CO expression [8], one might have expected the transcript for HEADING DATE 1 (TaHD1),
the supposed wheat orthologue of AtCONSTANS, to follow the profile of TaGI. However, it did
not show differential expression in this study. Interestingly, other transcripts that appear to be
members of theCONSTANS-like family of genes exhibited profiles that did reflect those
of TaLHY, and TaTOC1, and TaGI. In particular, a sequence highly similar to barley CONSTANS-
like 9 (the most divergent of the barley CONSTANS-like genes which has no counterpart
in Arabidopsis [35]) had a profile of abundance very similar to that ofTaLHY (see Additional
file 4). A transcript with similarity to CONSTANS-like 1 in Lolium perenne, a gene which has
been reported to increase after extended periods of exposure to cold [36], had a profile of
transcript abundance that echoed that of TaTOC1 and TaGI. Ciannamea et al., using a similar
experimental approach to that used in this study, suggested that the profile of transcript
abundance for LpCOL1 was suggestive of the gene being involved in the vernalisation
response [36]. We observed a very similar profile of responses in both the cold treated plants
and the controls. This would suggest that this gene in wheat is responding to shortening day
length.
GA pathway genes
The Affymetrix array doesn't include probe-sets for AtGA1 (codes for ent-copalyl diphosphate
synthase) orAtGA INSENSITIVE (the wheat orthologue is REDUCED HEIGHT B1 [RHT B1]).
There is a probe-set forAtRGA1 (the wheat orthologue of RHT D1), but we did not observe
differential accumulation of this gene. However, there was a clear genotype-dependent, cold
response of some components of the gibberellin pathway: transcripts for ent-kaurene
synthase and ent-kaurene oxidase (correspond to AtGA2 and AtGA3, respectively), showed
leaf specific accumulation (> 20-fold increase after 12 weeks) in the two winter varieties and
no response at all in Paragon (Figure 4). This result was confirmed by qRT-PCR (Pearson
correlation = 0.99). Ent-kaurene synthase and ent-kaurene oxidase are two of the principal
enzymes of the gibberellin biosynthetic pathway [30,31]. Thus, given the profiles of
abundance that we observed, one might assume that, in the two winter varieties, there was
an increase in gibberellins. In Arabidopsis, gibberellic acid (GA) activates the expression
of AtSOC1(SUPPRESSOR OF OVER EXPRESSION OF CO 1) [32], an important integrator of
several flowering pathways (discussed below), which in turn promotes flowering through its
action on floral meristem identity genes or their products (Komeda 2004). What's more,
Moon et al. [32] report that the gibberellin pathway is the only pathway to promote flowering
under short days. Thus, it would appear that we have evidence to show that in wheat the
gibberellin pathway functions in a similar manner to that in Arabidopsis, and that as a
consequence of vernalization under short-days it tends to promote flowering. However, the
complete lack of response in the spring variety, Paragon, is intriguing: does the gibberellin
pathway not function to promote flowering in spring varieties of wheat.
Vernalization pathway:
Prolonged exposure to low temperatures (vernalization) accelerates the transition to
reproductive growth in many plant species, including the model plant Arabidopsis
thaliana and the economically important cereal crops, wheat and barley. Vernalization-
induced flowering is an epigenetic phenomenon. In Arabidopsis, stable down-regulation
of FLOWERING LOCUS C (FLC) by vernalization is associated with changes in histone
modifications atFLC chromatin. In cereals, the vernalization response is mediated by stable
induction of the floral promoterVERNALIZATION1 (VRN1), which initiates reproductive
development at the shoot apex. We show that in barley (Hordeum vulgare), repression
of HvVRN1 before vernalization is associated with high levels of histone 3 lysine 27
trimethylation (H3K27me3) at HvVRN1 chromatin. Vernalization caused increased levels of
histone 3 lysine 4 trimethylation (H3K4me3) and a loss of H3K27me3 at HvVRN1, suggesting
that vernalization promotes an active chromatin state at VRN1. Levels of these histone
modifications at 2 other flowering-time genes,VERNALIZATION2 and FLOWERING LOCUS T,
were not altered by vernalization. Our study suggests that maintenance of an active
chromatin state at VRN1 is likely to be the basis for epigenetic memory of vernalization in
cereals. Thus, regulation of chromatin state is a feature of epigenetic memory of vernalization
in Arabidopsis and the cereals; however, whereas vernalization-induced flowering in
Arabidopsis is mediated by epigenetic regulation of the floral repressor FLC, this phenomenon
in cereals is mediated by epigenetic regulation of the floral activator, VRN1.
Vernalization-induced flowering in cereals is associated with changes in histone
methylation at theVERNALIZATION1 gene.
1. Contributed by W. James Peacock, April 1, 2009 (received for review March 17, 2009)
epigenetic
MADS
intron
barley
chromatin
Plants respond to seasonal cues, such as temperature and day-length, to ensure that
flowering coincides with favorable conditions. Prolonged exposure to low winter temperatures
(vernalization) accelerates the progression from vegetative to reproductive growth in many
plant species, including the temperate cereals (such as wheat and barley) and dicot species
(such as Arabidopsis) (1–3). In both these lineages, plants retain a “memory” of the prolonged
cold of winter, which stimulates flowering when days lengthen during spring (1–3). The
memory of cold is then reset in the next sexual generation to ensure progeny are competent
to respond to vernalization (1–3).
In Arabidopsis, the vernalization response is mediated by epigenetic regulation of the floral
repressor,FLOWERING LOCUS C (FLC), which encodes a MADS-box transcription factor that
represses genes involved in floral initiation, including SUPPRESSOR OF CONSTANS
1 and FLOWERING LOCUS T (FT) (1, 4–6). FLC is expressed before vernalization and delays
flowering, but its expression is repressed by vernalization (1, 4). FLCremains repressed when
plants are subsequently exposed to warm temperatures, allowing activation of FT, which
promotes flowering (1, 4). The stable down-regulation of FLC by vernalization is associated
with an increase in the levels of repressive histone modifications at FLC chromatin, such as
histone H3 lysine 27 di- and trimethylation (H3K27me2, H3K27me3), histone H3 lysine 9
dimethylation, and histone H4 arginine 3 symmetrical dimethylation, as well as the loss of
histone modifications associated with active transcription, such as histone H3 acetylation and
histone H3 lysine 4 di- and trimethylation (H3K4me2, H3K4me3) (7–13). Repression of FLC by
vernalization involves the vernalization-dependent association of Polycomb-Group (PcG)
complexes to FLC chromatin, which are required for addition and maintenance of H3K27me3
at FLC (14, 15). Taken together, these studies indicate that vernalization induces an alteration
of FLC chromatin state from actively transcribed to stably repressed (7–15). The cellular
memory of transcriptional repression of FLC is maintained during successive cell divisions by
mitotic inheritance of repressive histone modifications at the gene (11), but
active FLC transcription is restored in progeny, ensuring that the next generation is
competent to respond to vernalization (1, 4, 16).
In temperate cereals, the vernalization response is mediated by the stable induction of a floral
promoter,VERNALIZATION1 (VRN1) (3, 17–19). VRN1 encodes a FRUITFULL-like MADS-box
transcription factor required for the initiation of reproductive development at the shoot apex
(20–22). In vernalization-requiring cereal plants, VRN1 is expressed at low levels and is
induced by vernalization, with the level of expression being dependent on the length of cold
exposure (17–19, 23–25). VRN1 expression remains high when plants are exposed to warm
temperatures following vernalization, and promotes the transition to reproductive
development (17–19,23–25). VRN1 down-regulates the floral
repressor VERNALIZATION2 (VRN2), and allows long-day induction of the floral activator FT to
accelerate subsequent stages of floral development (3, 24–26).
The vernalization response of VRN1 shows characteristics of epigenetic regulation, in
that VRN1 is induced by vernalization, expression is maintained following vernalization, and
the prevernalization level of VRN1 expression is reset in the next generation (17–19, 23–25).
In this article we analyze the effect of vernalization on the levels of histone modifications at
the barley (Hordeum vulgare) VRN1 gene (HvVRN1). Our study indicates that vernalization-
induced flowering in cereals is mediated by epigenetic regulation of VRN1 chromatin state.
Our results suggest that regulation of the histone methylation status of VRN1 chromatin is
important for repression of VRN1 before vernalization, for activation of VRN1 by vernalization,
and for maintaining a memory of vernalization following cold exposure
Flowering time genes in Arab
Co induced FT with FD and soc1 activate genes like LFY and AP1 in SAM; they take
their own course of development of structures.
The monocot Rice or dicot Arabidopsis have their homologs that operate in
inducing flowering.
Environmental signals that favor GA synthesis leads to the bonding of GA to its receptor
and interact with their binding proteins, where the regulator of GA called RGA gets degraded
through proteosomes. Now the GA signals SOC1/PFP/GAMYB like components that lead to the
production FT and FT with FD acts on apical meristem
This figure shows how the GA synthesis leads activation of different response factors that
leads to the synthesis of SOC1 and FT, where FT acts on AP1 (?) and SOC1 acts on LFY (?)
which ultimately act on flower identity genes.
Below some general and specific flowering pathways have been given and they are self
illustrative.
Integration of different pathways
The role of GA in stem elongation seed germination and flower development.
The effect of combinatorial expression of gene in the presence and
absence of VRN on phenotype is wells illustrated. The figure below shows even
Rice palnts produce a FT homolog called Hd3a and it has the same effect as FT of
dicots.
Ft homolog Hd3a operates in rice plants, they move from leaves to stem apex, Hd3a in combination with FD (?) induce flowering
Integration of different pathways on meristem identity genes
MADS box proteins action on floral development
MADS
MADS protein domains
Hour glass model showing the effects of PhyB and Phy A on CO synthesis and degradation. Co is transcription factor for the expression of FT.
PHYSIOLOGY OF PLANT MOVEMENTS
Higher plants, being fixed to soil cannot move from place to place. But within the plant body
various protoplasmic components are in constant motion, ex., movement of water, minerals,
food etc. But certain parts of the plant body in response to external stimuli, exhibit physical
displacement called movement. Lower unicellular plants also show movement from place to
place. Such movements may be autonomic or induced.
The movement of plant structures in response to stimuli is very interesting. The stimuli may
be in the form of light, touch, chemicals, temperature, gravity or water. Thus the agency or
factor that causes movement is called stimulus. Not all parts of the plant act as the site of
perception to stimuli. Only certain regions or structures are capable of receiving the stimuli
and such structures or organs are called perception site. The site of perception need not be
the structure that responds to movement. In fact, in many cases the site of response and the
site of perception are different. Nonetheless, the stimulus has to cross through the plasma
membrane of the cell or cells found in such structures that receives the stimulus. Plasma
membrane has all the inbuilt components and the potentiality to receive stimulus and
transmit it into intracellular mileu for proper response.
There is a time lag between the time at which the stimulus is applied and the time at which
the response begins. This time is called reaction time. It may vary and depends upon the
intensity of stimulus and the kind of response. If the stimulus is weak, there may not be any
response at all, but if the stimulus is adequate or in right quantity the response is positive.
The time required to cause the proper stimulus is called presentation time.
Once the plant body responds to the stimulus say sleeping movement (change in the position
direction), structures involves always come back to their original position. This process is
called recovery and the time required is referred to as relaxation time, which again varies
from species to species. If the stimulus is provided repeatedly the receiver structures do not
respond with the same intensity as the first instance, but it slows down. This effect is due to
fatigue. Such behavior is probably due to the loss of same material components required for
the response. If the stimulation is continued at increased frequency, the plant organs do not
respond and behave as if they are dead structures. Such a state is called Tetanus or extreme
fatigue. Now it is believed that stimulation (quantity) causes irritation at the site of
perception and the product of irritation are then transported to the site of response, where
the structures respond and perform movement. All these events initiate with signal, and the
receptor that receives the signal becomes active and induces signal transduction pathway in
the cell cells. There will be a cascade of events that finally leads to the response.
Plants endowed with different structural adaptation and different potentialities exhibit
different types of movement either voluntarily or involuntarily. Based on the behavior as
pattern, movement’s stimulus, the plant movements have been grouped into physical
movements and vital movements.
Physical Movements:
The structures involved in this type of movement are mostly dead. Xerochasy: Structures
like the wall of fruits, sporangia, capsules, have differential wall thickenings. During dry
weather conditions, they loose water to atmosphere. Because of this, the thick walls contract
as a result they break open along with the line of dehiscence, where the cell walls are thin
and susceptible. Hydrochasy:Certain structures, made up of hydrophilic substances, are
capable of imbibing water as well. Due to imbibition of water they swell. Due to imbibition of
water peristomial teeth in Moss capsules, elators in equisetum, etc. show movements. In fact
such movements help in the dispersal of spores.
Vital Movements:
Plant movements due to the activity of living structures are called vital movements. They are
further classified into different kinds. In some plants, particularly unicellular algae, the entire
cell moves from place to place or from one position to the other or the protoplasm by itself
shows continuous flux by physical displacement. In others, where the plant body is fixed in
the soil, certain structures show bending or curvature movements. Furthermore, some of the
movements are auto regulated and propelled by innate mechanisms but other movements
are induced by stimuli.
In this case the entire living cell is involved either in the movement of protoplasm or the
entire body of the plant cell from one place to another. These movements may be
autonomous and induced.
Autonomous: Protoplasmic Streaming:
Protoplasm is not a static fluid. With all its complicated structures the entire protoplasmic
fluid is in constant sweeping motions. These streaming movements can be observed under
high resolution light microscopes. For example, in the cells of staminal hairs of tradescantia,
the streaming is localized and in each of these areas the direction is clockwise or
anticlockwise. Tiny particulates are seen swept along with the stream in a particular direction
and particular directed path. Such compartmentalized movements are known as Cyclosis.
But in Elodea and other plants, the protoplasm shows uniform movement but in one
direction. One can observe chloroplasts movement along the cytoplasmic streaming. Such
movements are called Rotational Movements.
Protoplasmic streaming is due to the activity of contractile proteins found associated with
other microtubule network found within the cytoplasm. It is an active process microtubules
play an important role in such intra cellular movements. These structural elements contain
motor proteins and they perform the movements. The energy required for this process is
derived from ATP molecules. These movements help in the even distribution of chemical
components. And the protoplasmic movement across the plasmodesmata brings about the
transportation of materials from one cell to another. Auxin has been found to accelerate the
rate of protoplasmic movements. Addition of colchicine and cytochalasin B totally inhibits the
movement, thereby indicating the involvement of microtubules and microfilaments.
Respiratory poisons like DPN, KCN, also inhibit the movement, thus suggesting that these are
active movements. Even amoebae show such movements. For that matter all living cells
exhibit autonomous movements.
Paratonic movements:
These movements are stimulated by external agents like light, chemical heat, etc. Hence
they are called taxis or tactic
movements.
Phototactic Movement: Unicellular algae suspended in a test tube moves towards the light
source to obtain solar energy for photosynthesis. If the light is very intense, they move away
from light; this may be due to the raise in temperature. The movement in these cases is due
to locomotor structures like flagella or cilia. The beating of these structures propels the cells
towards light. The flagellar activity utilizes ATP. Hence these movements are active.
Chemotactic Movement; Certain flagellated or non-flagellated bacteria move towards the
source of food by lashing their flagella or by tumbling. In these cases, the stimulus is
chemicals. Similarly, the movement of spermatozoids in lower organisms like Bryophytes is
directional, because the chemicals released by mature archegonia provide the chemo
stimulus. Sensing the chemicals, the flagellated spermatozoids swim towards archegonia and
enter the neck canal and bring about fertilization.
Thermotactic: Again lower organisms sense the temperature and move towards the
compatible temperature or move away if the temperature is incompatible. Sensing the
change in the temperature by the plants is autonomic but the cells always show directional
movements.
All the above mentioned movements involve signal transduction pathway. Elucidation of
these pathways at molecular level is necessary and exciting.
Movements of Curvature:
Plants with their fixed plant body cannot move from place to place, but certain structures
show bending movements which may be directional or non-directional or they may be
autonomous or induced. However, some of the plant movements are due to the growth of the
cells or due to change in the turgidity of the cells. Based on the above features movement of
curvature has been further classified such as:
Autonomic Growth Movements:
Natatory; Plant structures like tendrils and stem tips exhibit differential growth alternately.
This results either in sideward or circular movements. Certain sub cylindrical stems (flat or
angular) due to differential growth at the sides show zig zag movement. On the other hand,
cylindrical tendrils in search of getting a hold on to a substratum, a waves of growth activity
takes place around the tendril. That is why it appears as if it is moving in circles. Such type
of movements is called circumnutatory movement. The growth activity of the above said
structures is in-built and they show a rhythmic pattern. Whether the hormonal fluxes are
responsible for this type of growth movement or any other innate mechanism involved is not
known. But the treatment of these stems with ABA inhibits the movements, indicating that
these are phytohormone mediated.
Ephemeral Movements:
During the development of leaves and floral organs, the growth pattern determines the
growth direction of the structures. For example, leaves expand laterally by continuous radial
divisions and expansions. Similarly sepals and petals because of continuous growth of cells at
the base on the inner surface make the flower open. The hypocotyl hook of the bean seed
straightens up because of one sided growth due to expansion cells on that side. Such growth
movements are called Ephemeral. Once the movement reaches certain stage, the bending
movement stops.
Autonomic Turgour Movement:
Desmodium gyrans (Indian telegraphic plant) and Eleiotis sorria have trifoliate compound
leaves. In the former case, central leaflet is larger and straight. But the lateral two leaflets
are smaller which show regular upward and downward movements. Such movements are
observed only during daytimes but not at night. Such rhythmic gyratory movements require
about 5-8 minutes for the completion of one cycle. This is due to autonomic turgour changes
in the cells found in the swollen pulvinus of the leaflets. How light brings about turgour
changes or is it due to phytochrome mediated responses or hormonal responses is not clear.
Paratonic Nastic Movements:
Paratonic movements are induced by external stimuli. Whatever may be the point or
direction of stimulus applied the movement of the plant structures is already predetermined
and they exhibit movement only in one direction. Most of the movements are turgour
movements, but growth movements are not uncommon. For example, the opening of
hypocotyl hook of germinating bean seedling, opening of circinately coiled leaves of ferns and
cycas are the examples of growth mediated nastic movements. Differential distribution of
growth hormone in the adaxial (dorsal) surfaces of the leaves; it is this that is mainly
responsible for such growth movements. These movements are permanent and they do not
show temporary day and night fluctuations.
Photonasty: Many leguminous plants, with their pulvinous bases, show characteristic
sleeping movements and they show a rhythmic pattern of opening of leaf-lets in the day and
closing in the evening with a precision of a clock. In fact, such movements are attributed to
circadian rhythm operated by an inbuilt biological clock.
The photonastic mechanism has been explained on the basis if hormonal distribution in the
cells of the pulvinus. During daytimes, auxin is found in greater amount at the upper region
of the pulvinus, because of this the cells become more turgid and leaf lets open. The same
process is reversed during night because of the redistribution of auxin to lower side which
causes folding by the way of turgour changes.
Investigations into such movements have shown that these are phytochrome mediated,
because red and far red lights are very effective in opening and folding of leaflets.
Phytochrome being an excitable molecule in response to light it is known to bring about
changes in the permeability of membranes. Thus the turgour movements bring about so
called sleeping movements. Added to this the change in the permeability also involves the
efflux and influx of K+ ions. The loading or unloading of potassium ions causes increased or
decreased DPD which acts as the driving force for the entry or the exit of water which results
is turgour movements.
Chemonasty: Drosera, Venus fly trap and such insectivorous plants have devised
mechanisms to trap insects to obtain nitrogenous compounds. These plants are found
growing in boggy areas and they are incapable of utilizing soil nitrogen.
The plant Drosera consists of leaves arranged in a rosette pattern. The leaf blades are flat
and racket shaped. The upper surface is made up of colorful glands which secrete sticky juice,
where you very high activity of golgi complex. There are a large number of sensitive
tentacles spread out at the margins. The tentacles have a sensitive broad base and a
terminal glandular globosely head which also secretes juice. In sunlight, these structures
glisten like dew drops; hence the name sundew plant.
Insects, mistaking the glistening juice for honey, settle down on these surfaces where their
legs get stuck because of the sticky juice and they cannot escape. As insects have
nitrogenous compounds in their body, they diffuse and sensitize the tentacles, because
nitrogen compounds act as signals, in response, they immediately fold upon the insect and
the same gets trapped. In this case nitrogenous compounds found in insects provide the
stimulus. On the other hand, if a metal piece is placed, tentacles do not fold, instead if a
piece of meat is dropped, the tentacles close immediately. However, after digestions,
tentacles open slowly and this process takes few hours. Such chemonastic movements are
found in utricularia, Venus fly trap, etc.
Thermonasty: Flowers in Tulips and Crocus are very sensitive to temperature variations.
Accordingly the accessory whorls close or open. These movements are known to be due to
turgour changes rather than growth movements.
Siesmonasty: Mimosa pudica (Touch me not plant), Biophytum, Neptunia oleracea,
Desmanthus are very sensitive to touch, rather shock generated by the touch. Mimosa pudica
has pinnately compound leafless with a swollen pulvinus at the base every leaflet. Touching
the leaves is believed to cause a seismic shock to the leaves and this stimulus is transmitted
all along the rachis downwards and reaches the basal pulvinus of the leaf. The irritability
caused, due to touch is actually transmitted through sieve tubes, because these are the only
structures which are capable of transmitting the stimulus as fast as 1.5-20 cm/sec. The
material basis for the transpiration stimulus has been found to be ABA and ABA mediated
ions. As the ABA ions diffuse along the rachis, it also trans-diffuses into pulvinus of the
leaflets.
The mechanism of upward movement of pinnules and downward bending of the entire leaf at
the base is now considered as due to the activity of the motor cells found in the pulvinus.
Anatomical studies indicate that at the lower region the pulvinus consist of thin walled
parenchymatous cells, which are loosely arranged with lot of intercellular spaces. Cells also
contain many contractile vacuoles. The cell membranes and vacuolar structures have
contractile proteins. These vacuoles are loaded with K+ ions when the leaflets are open.
These motor cells are highly sensitive and active. Such cellular organization is also found at
the upper region of the pulvinus found at the base of the leaflets.
When the stimulatory hormone ABA ,which is also called as stress hormone (released due to
irritability) reaches these specialized cells, they are stimulated to contract, collapse and
extrude water and K+ ions into intercellular spaces. This action brings about the collapsing of
cells on this side and the leaf downwards. Similarly mechanism is involved in the folding of
leaflets upwards. The recovery takes place within 5-10 minutes. The opening process takes
place by active pumping in of K+ ions back into cells. The K/ATPase pumps are supposed to
be found in the cell membranes. This active process requires energy. If the production of ATP
is inhibited by respiratory inhibitors like DNP or KCN recovery does not take place. If plants
are kept is continuous dark, they fail to produce any siesmonastic responses, which suggest
the siesmonastic movements are ATP dependent active processes.
Paratonic Tropic Movements:
These movements are due to growth activity. The curvature movement is always directional,
either towards the stimulus or away from the stimulus. Most of these are phytohormone
mediated movements.
Phototropism: Phototropic movement is mostly exhibited by stem tips; for they always bend
towards the light source. The action spectrum of the light has been found to be at blue light.
The blue light is now known to bring about unequal distribution of active auxins than the total
auxin content. The pigment for absorption of blue light has been found to be Cryptochrome.
The older view of destruction of auxin and the lateral movements of auxins in response to
light has been more or less ruled out. The difference in the quantity of active auxin brings
about differential growth and also curvature. Details of this process have been explained
elsewhere.
Photropic movement in in one of the fungi called Phycomycs blacesleeanus
The apoprotein cry in association with flavins bring about photoptropism, it gets activated by
absorbing blue light and autophosphorylation of ser/thr residues, that leads to transport of
ions to one side and brings about bending movement.
Geotropism: Growth of roots towards soil and the stem away from the soil in response to
gravitational force is an inbuilt mechanism endowed in stem tip and root tip of the plant.
Curvature movement of roots towards gravitational is termed as positive geotropism and the
movement of the stem away or against gravitational force is called negative geotropism.
However in some plants, rhizomes and runners grow parallel to the surface of the soil. Such a
response is called digeotropism. The obliquely growing stems show plagio geotropism (For
details refer the Chapter Auxin).
The differential response of stem tip and root tip to the same gravitational force is due to their
different innate structural and functional potentials. Though both structures are derived from
the same embryonic cell and possess the same genetic potential they behave differently
probably this is because of the programming of process driving the development of in such
structures. The mechanism of geotropism has been explained on the assumption that
differential responses are due to different concentrations of auxins. The concentration of
auxin that promotes the growth of the stem apex inhibits the growth of the root tip. On the
other hand, the concentration of auxin that is effective in the growth of the root tip is not
adequate for the growth of the stem tip. This concept suggests that stem apex requires
higher concentration of auxins for its growth and roots require low levels of auxins as optimal
concentration for the normal growth.
If a seedling is placed on the soil horizontally, due to mass action of gravitational force, auxins
move downwards in both stem apex and root apex. In the stem apex, as more and more of
auxin accumulates at the lower surfaces the cells found in this region grow faster than the
upper cells, thus the stem curls upwards. But in roots, as more of auxin accumulates at the
lower region, the higher concentration inhibits the growth of cells found at the lower surface,
but the cells at upper surface grow faster because of lower concentration of auxin.
Furthermore, the root cap being the preceptor of gravitaropism produces ABA, which on
translocation basipetally, reaches the lower cells by sheer gravity. With the accumulation of
more and more of ABA at lower cells, the growth of these cells is further inhibited, but the
cells at the upper region grow normally and thus bring about the downward growth curvature
for roots.
Chemotropism: Pollen tubes and certain fungal hyphae exhibit chemotropic movements,
because they grow towards organic nutrient rich media. In the case of pollen tubes, the
direction of growth is dictated by the chemical gradient generated by the embryo sac. This
chemical gradient greatly facilitates the growth movement of pollen tube towards the embryo
sac, irrespective of the position of ovules in the ovary.
Hydrotropism: Growth of roots towards water is called hydrotropism. Whatever may be the
positions of seedlings, as shown in the figure, the roots curl towards water. This positive
growth curvature might be due to greater water potential, probably acts as the motive force
for the growth of root tip. Stem and other structures are insensitive to water and they do not
show any hydrotropic curvature movements.
Thigmotropism: Plants with weaker stem spread around and require support for their
growth. Many of the climbers have developed hooks, pads and tendrils for getting a firm foot
hold onto the substrate; which may be a rock or a wooden stick or any such hard structure.
For example, in the case of tendrils of cucurbita and other species the terminal regions are
soft, tender. The terminal region of the tendrils consists of a number of fine pits, which are
highly sensitive to touch. When the tendril comes in contact with any supporting structures,
the pits get stimulated and the same is transmitted a few millimeters basally. This results in
the differential growth of the tendril on one side. This causes the curvature in the tendrils and
finally they coil around the stem the supporting structure. The time required for such coiling
after the stimulus is just 3 to 5 minutes. Once the initial reaction is set in at the basal region,
the tendrils coil round the support and thus draw the climber nearer to the support. It is
suspected that auxins and ABA are involved in thigmotropic curvatures. ABA inhibits the
growth of the cells in the region of the contact and auxins stimulate the growth of the cells on
the opposite side and bring about eh growth curvature called ‘coiling’.