Introduction_

In the simplest terms reproduction is defined as the creation of a new

individual or individuals from previously existing individuals. Reproduction is of great

importance for the continuation of any species. All living organisms multiply either by

means of asexual (vegetative) or by sexual reproduction.

Asexual (vegetative) reproduction

It is the simplest form of reproduction where the genetic information comes

from only one parent and the offspring produced are genetically identical to the

parent. All forms of asexual reproduction are basically variations of the cell division

process of mitosis and do not involve meiosis or syngamy. It is common among

prokaryotes and lower eukaryotes like algae, fungi, and lower invertebrates. Binary

fission, fragmentation, budding, and sporulation are the common ways of vegetative

reproduction in lower organisms.

In higher eukaryotes it is common in plants but less so in animals. Asexual

reproduction is generally favoured when a species lives in a more or less stable

environment. Many plants have developed specialized structures such as rhizome,

corms, tubers, bulbs, and stolons for vegetative/ asexual propagation. Several plants

bud off tissue that develops into a miniature version of the parent plant called

budding plantlets (e.g. bryophyllum). Besides these structural modifications, apomixis

(production of seeds without fertilization) and parthenogenesis are other methods

employed by higher plants to multiply asexually.

Asexual reproduction is an efficient way to reproduce as it allows the

beneficial combinations of alleles of an organism to continue unchanged but this has

serious disadvantages. The asexually reproducing population tends to become

genetically static and hence less adaptable to the changing environment. Asexual

reproduction is still used by some organisms but in general has failed to pass the test

of natural selection and has been replaced by the sexual mode of reproduction.

The Sexual mode of reproduction

Sexual reproduction is the favoured way of reproducing for many higher

organisms though it is an expensive alternate to simple asexual reproduction. The

two key features of sexual reproduction are meiosis (production of haploid gametes

2

Introduction

and genetic recombination during this process) and syngamy (fusion of two haploid

gametes to produce a diploid zygote). These features result in production of offspring

significantly varied from the parents. This variation enables a species to adapt easily

to environmental changes. Sexually reproducing groups can evolve more quickly as

compared to the asexual counterparts. Even in microorganisms that reproduce

asexually (e.g. bacteria), there are mechanisms allowing the exchange of hereditary

material.

Sexual reproduction in plants

Alternation of generations

The sexual life cycle in plants consists of an alternation between a haploid,

gametophyte generation, and a diploid, sporophyte generation. In the case of

unicellular algae, the haploid gametophyte represents the sexual generation and is

dominant and free-living. During the gametophytic phase, haploid gametes (egg and

sperm) are produced which fuse to form a diploid zygote. The zygote represents the

sporophytic phase, and is very short lived. The zygotes after a period of maturation

undergo meiosis and germinate to start the cycle afresh.

In lower plants such as the bryophytes (mosses and liverworts), the

gametophyte (haploid phase) is a highly developed, differentiated, and independent

plant. It bears multicellular reproductive organs, the antheridia and archegonia, which

produce the gametes. The gametes fuse to give rise to the diploid sporophyte. The

sporophyte is a simple structure, which depends on the gametophyte for nutrients

and water and bears the diploid spore mother cells that undergo meiosis to generate

spores.

During evolution of vascular land plants the gametophytic generation was

reduced while the sporophytic generation gained dominance. In pteridophytes the

sporophyte is independent, well developed and bears spores (1 n) on the abaxial

surface of the leaves. These spores germinate to give rise to a small and

inconspicuous structure the prothallus that represents the gametophyte and bears

the antheridia and archegonia. Though the gametophyte is small and short lived it is

still independent and free living. The evolutionary trend has been towards reduction

3

Introduction

of the gametophyte to being small in size and dependent on the sporophyte for

nutrition.

The flowering plants are the most evolved vascular land plants. In these

plants the sporophytic phase is dominant and gametophytic phase is highly reduced

and dependent on the sporophyte. The gametophyte generation consists of the

pollen grain (formed within the anther) and the egg cell (enclosed within a

multicellular ovule) contained within the flowers.

Sexual Reproduction in Lower Plants

In lower eukaryotes such as yeast (budding and fission), environmental

factors, such as nitrogen starvation, mating pheromones, and other stresses in

general induce the onset of the reproductive phase (Watanabe et a/., 1997).

Extensive studies in the unicellular alga Chlamydomonas reinhardtii have shown the

requirement of two extrinsic signals, nutritional starvation and blue light for the

induction of the sexual phase. In C. reindhardtii the formation of gametes can be

divided into two steps: the formation of pregametes followed by maturation and

formation of sexually mature gametes. Removal of the utilizable nitrogen source acts

as a signal for initiation of gametogenesis. The blue light signal is required for the

maturation of the pre-gametes and during zygote germination {Gloeckner and Beck,

1995). Blue light has been shown to be required in the last step of the sexual life

cycle i.e. the germination of the zygote.

In vitro experiments on ferns (pteridophytes) like Osmunda, Leptopteris, and

Toeda have shown that various environmental factors influence the time of

sporangia! production on the fronds. In Osmunda high levels of sucrose in the media

increases fertility. The maximum induction of fertile leaves is observed when the

leaves are grown in continuous darkness on media containing high concentrations of

sucrose. Reduction in the length of the light duration results in an increase in the

induction of fertile leaves. These results suggested a negative correlation between

day length and sporangia! initiation in ferns (Harvey and Caponetti, 1972). In addition

a high carbohydrate to inorganic nitrogen ratio is another important factor that brings

about fertility responses in ferns under in vitro conditions.

Sexual Reproduction in flowering plants

4

Introduction

In broad terms the life cycle of flowering plants can be divided into a

vegetative and a reproductive phase. The transition from the vegetative to the

reproductive phase is a key event in the life cycle of flowering plants and is called

"the floral transition". The correct timing of this transition is essential for the

reproductive success of flowering plants.

In the highly evolved flowering plants this developmental switch is under the

control of multiple endogenous and environmental factors. (Simpson et a/., 1999;

Reeves and Coupland, 2000). The endogenous cues include developmental age,

genetic background, and phytohormones. The environmental cues include light

quality and quantity, ambient temperature, and nutrient supply. The floral transition

signals from multiple regulatory pathways integrate and bring about changes in the

developmental fate of the Shoot Apical Meristem (SAM).

The Shoot Apical Meristem is a small population of highly organized,

morphologically undifferentiated dividing cells that remain uncommitted to any

specific program. During vegetative growth the SAM initiates leaf primordia on its

flanks in a spiral fashion (in Arabidopsis). When plant switches from the vegetative to

reproductive phase, the developmental fate of the SAM is changed and it starts

producing floral primordia on its flanks in place of leaf primordia. This switch from

vegetative to floral identity is brought about by expression of a number of floral

meristem identity genes such as LEAFY (LFY), APETALA t (APt), CAULIFLOWER

(CAL), and FRUITFUL (FUL) (Simpson eta/., 1999). Genetic analyses of flowering

time mutants have shown that floral inducers and repressors collectively act to

regulate the timing of floral transition (Pidkowich eta/., 1999).

Flora/repressors

Floral repressors play a crucial role in determining the length of the vegetative

growth phase. The molecular mechanisms that prevent plants from acquiring

reproductive fate early during embryo and seedling development are being

investigated. The repressors of the flowering programme were identified as mutants

that flower early indicating that the wild type gene product is involved in delaying the

onset of the reproductive phase. Floral repressors basically act by repressing the

expression of floral meristem identity genes such as LFY, APt, AGAMOUS (AG)

5

Introduction

(Simpson et a/., 1999; Sung et a/., 2003). Based on the mode of action the floral

repressors can be classified into two types: those that repress the floral signaling

pathway like FLC or TFL t and those that act as epigenetic repressors, regulating the

expression of floral meristem genes at the chromatin level.

FLOWERING LOCUS C (FLC) is a key repressor of flowering, which encodes

a MADS box transcription factor protein. It represses flowering by negatively

regulating the activity of FLOWERING LOCUS T (FT), a floral meristem identity gene

(Sheldon eta/., 1999; Michaels and Amasino, 1999). Moreover FLC also inhibits the

expression of SOCt, another positive regulator of flowering, which integrates signals

from various promotion pathways (Lee eta/., 2000).

TERMINAL FLOWER (TFL t) encodes a protein similar to a mammalian

membrane associated protein Phosphatidyl Ethanolamine Binding Protein (PEBP)

that can bind to hydrophibic ligands (Pidkowich et a/., 1999). It delays the floral

transition by negatively regulating the expression of the floral meristem identity genes

such as LFY and APt and conversely. Plants constitutively expressing LFY or APt

(under the 35SCaMV promoter) show reduced levels of TFL t expression (Liljegren et

a/., 1999 and Ratcliffe et al., 1999). TFL t and the floral meristem identity genes are

expressed in mutually exclusive domains. TFL t is expressed in the centre of the

shoot apex whereas LFY and APt are expressed at the flanks. TFL t represses the

activity of the floral meristem identity genes at the centre of the shoot apex. This

negative regulation is brought about in two ways firstly by delaying the up regulation,

and secondly, by preventing the meristem from responding to LFY or APt. On the

other hand expression of LFY, APt and CAL at the flanks of the SAM inhibits the

expression of TFL t in floral meristems thus promoting the formation of floral organs

(Liljegren eta/., 1999; Ratcliffe eta/., 1999). The loss of function mutation tflt-t

causes early flowering under both LD and SD conditions. Moreover, loss of TFL t

expression at the shoot apex promotes formation of a terminal floral meristem,

resulting in termination of the otherwise indeterminate inflorescence. The conversion

of the apical meristem into a flower in the tflt mutant is consistent with its role as a

suppressor of flowering (Shannon and Meeks-Wagner, 1991; Pidkowich eta/., 1999).

EMBRYONIC FLOWER t&2 (EMFt, 2) play a major role in floral repression

by repressing the expression of meristem identity genes. EMFt encodes a putative

transcriptional regulator, and EMF2 encodes a Polycomb group (PeG) protein

homologue (Aubert et a/., 2001; Yoshida et a/., 2001 ). Mutations in the EMF genes

6

Lnrroaucnon

result in early flowering with a complete elimination of the vegetative phase.

Moreover, a comparison of gene expression profiles of emf mutant seedlings and the

wild type floral buds shows a significant amount of overlap in gene expression. Loss

of emf function causes ectopic expression of the floral organ genes APt, AP3, PI,

and AG indicating that the germinating emf seedlings are committed to the

reproductive fate at a very early stage of plant development. These results suggest

that reproductive development is a default pathway and the vegetative growth is a

result of repression of the reproductive programme by EMF (Chen eta/., 1997; Moon

et a/., 2003). As EMF2 encodes a PeG protein homologue, it may repress the

expression of meristem identity genes at the chromatin level.

Floral repressors like, CURLY LEAF (CLF}, and FERTILIZATION

INDEPENDENT ENDOSPOERM (FIE) also encode proteins that are homologous to

the Drosophila PeG protein. These genes act as epigenetic repressors and regulate

the expression of floral meristem genes at the chromatin level (Yoshida eta/., 2001,

Kinoshita et a/., 2001 ). The elf mutants show a pleiotropic effect including early

flowering, indicating that the wild type protein is required for repressing the onset of

the reproductive program. CLF acts by repressing the floral homeotic gene

(AGAMOUS) in vegetative tissues (Goodrich et a/., 1997). In fie mutants, the

embryos germinate to give rise to emf like plants and show ectopic expression of

LFY and other floral identity genes. The floral repressors belonging to the polycomb

group of genes directly or indirectly tend to repress the floral homeotic gene

expression (Chen eta/., 1997).

TFL2 also called the Like Heterochromatin Protein1 (LHP1) resembles the

Drosophila Heterochromatin associated protein (HP1). It is proposed to regulate

various plant development pathways, including the vegetative to reproductive

transition by forming heterochromatin like repressive complexes (Gaudin et a/.,

2001 ). Wagner and Meyerowitz, 2002, proposed the role of SPLA YEO (SYD) a

SWI/SNF ATPase homolog in flowering time. SYD is thought to regulate flowering

time by modulating the activity of LFY at the chromatin level.

Floral inducers

The multifactorial model for floral promotion (reviewed in Bernier, 1988; Levy

and Dean, 1998) proposes that the time of floral transition is regulated by a number

7

Introduction

of environmental and endogenous factors. Environmental signals such as

photoperiod and temperature provide plants with seasonal information allowing them

to initiate flowering when the conditions are favourable. Analysis of a large collection

of flowering time mutants has allowed investigators to genetically define various

pathways regulating the time of flowering. These are: the photoperiod pathway,

autonomous, vernalization, and GA pathways.

The Photoperiod Pathway

Light is one of the major environmental factors controlling plant growth and

development. In most flowering plants the onset of sexual reproduction, flowering, is

triggered by a change in the photoperiod. The role of light in flowering was initially

demonstrated by Garner and Allard in the 1920's when they recognized that day

length controlled the initiation of flowering in many plant species. The Short Day

plants maintain the vegetative phase of development until they receive day length

that is shorter than a particular threshold, called the critical length. In contrast the

Long Day plants flower only when the day length exceeds the critical limit. Grafting

experiments suggest that the leaves perceive the light signals and transmit a

diffusible signal (Fiorigen) to the shoot apical meristem that triggers the flowering

programme (Bernier eta/., 1993}.

A mechanism by which plants can measure the rhythms of light and dark

phase is essential for the photoperiod control of flowering time. The circadian clock

regulates the daily rhythms in gene expression and performs the role of a timekeeper

in the photoperiod response. The circadian clock comprises of three parts: the input

system (the photoreceptors), the oscillator, and the output system (the clock

regulated genes). The photoreceptors present in the leaves receive the light signals

and signals initiating from these photoreceptors in turn entrain the components of the

circadian clock. The central oscillator is involved in regulating the expression of the

down stream effector genes (Samach and Coupland, 2000; Mouradov eta/., 2002).

Recent genetic studies have begun to identify the molecular components of

the photoperiod promotion pathway. Two photoreceptors CRYPTOCHROME 2

(CRY2) and PHYTOCHROME A (PHY A) encode proteins that are light labile and

are the major day length sensors. CRY2 and PHY A show oscillations in protein levels

in a photoperiod dependent manner only in plants grown under SD conditions but not

8

Introduction

when plants are grown under LD conditions. The abundance of these proteins

reduces during daytime (light labile) and increases during the night. The phyA mutant

of Arabidopsis shows delayed flowering when grown under LD (white light) or under

continuous Red + Far-Red light. Failure of phyA mutants to flower when grown under

continuous Far-Red light indicates that PHYA is involved in mediating the Far-Red

light promotion of flowering (Mockler eta/., 2003). CRY2 is a blue light receptor that

regulates the expression of the flowering time gene CONSTANS (CO). CO is a key

component of the photoperiod pathway, which integrates the signals from the

circadian clock as well as the photoreceptors. Mutation of CRY2 delays flowering

under long days but the time of flowering under SD conditions is not significantly

different from the wild type plants grown under similar conditions. In the cry2 mutants

the levels of CO mRNA are significantly reduced under LD but only slightly reduced

under SD conditions. Over expression of CRY2 results in early flowering and a

corresponding increase in the levels of CO mRNA is also observed (Guo eta/.,

1998). Over expression of CO in the cry2 mutant can suppress the late flowering

phenotype suggesting that CO acts downstream to CRY2.

CONST ANS (CO) is a putative transcription factor of the Zn finger family and

is shown to accelerate flowering in response to long days (Putterill et al., 1995). A

loss of function mutation of CO results in a complete lack of response to florally

inductive long days. Plants over expressing CO flower early under both long day and

short day length and are not responsive to the photoperiod, suggesting that CO

promotes flowering in response to photoperiod (Onouchi eta/., 2000; Suarez-Lopez

et a/., 2001 ). CO promotes flowering by up-regulating the expression of flowering

time genes FT and SOC1/ AGL20 (Onouchi eta/., 2000).

The expression of CO mRNA oscillates in a diurnal pattern under long day

condition. The CO mRNA expression increases between 12-24 hrs after dawn with

the maximum level at 16 hrs after dawn. Under long days the expression of CO

mRNA is moderately high at dusk (16hrs) and at dawn (end of 24 hrs cycle). Under

long day conditions expression of FT is also high at dusk, which coincides with the

high levels of CO and the light phase of the cycle. All these events together promote

floral transition. Under short day conditions the CO expression increases between

12-20 hrs after dawn (dark phase) therefore the high levels of CO do not coincide

with the light phase (Suarez-Lopez eta/., 2001, Roden eta/., 2002). CO seems to act

as a connector between the circadian clock and the flowering time gene FT and

SOC1 (Suarez-Lopez eta/., 2001; Lee eta/., 2000).

9

Introduction

Besides the amount of light received, the quality of light received also affects

the timing of flowering. It is observed that plants flower early when grown in shade.

PHYTOCHROME B (PHY B) plays a significant role in shade-avoidance responses

and is a negative regulator of flowering. Mutation of PHYB results in early flowering

under LD and SD conditions. PHYTOCHROME AND FLOWERING TIME 1 (PFT1)

encodes a nuclear protein, which contains a N-terminal von Wilder factor type A

domain and a C-terminal glutamate rich domain. The C-terminal glutamate rich

domain is shown to act as a transcriptional activator in yeast. The pft mutant shows

delayed flowering and is efficient in suppressing the early flowering phenotype of the

phyB mutant indicating that it acts downstream to PHYB. Under conditions that mimic

shade, pft1 mutant fails to promote flowering indicating that it is involved in promoting

flowering under low light conditions. In the pft and phyb pft mutants the levels of FT

are reduced under both LD and SD conditions indicating that PFT might regulate

flowering by regulating the expression of FT (Cerdan and Chery, 2003}.

CIRCADIAN CLOCK ASSOCIATED1 (CCA1), LONG ELONGATED

HYPOCOTYL 1 (LHY1) and TIMING OF CAB (TOC1) components of the oscillator of

the circadian clock are involved in the photoperiodic sensitivity. LHY1 and CCA 1

encode MYB related transcription factors. The expression of LHY and CCA 1 show

rhythmic oscillations, which reaches the maximum levels just after dawn. A

constitutive expression of these genes results in disruption of multiple circadian

responses resulting in a delay in flowering (Levy and Dean, 1998; Lin, 2000}

TOC1, a component of the oscillator codes for an atypical response regulator,

which has aN-terminal motif similar to the receiver domain of the response regulator,

and a distinct motif in the C-Terminal domain similar to that identified in the CO family

of transcription factors. Disruption of the TOC1 gene expression results in reduction

in the length of the free running cycle where as an increase in the TOC1 expression

has a reverse effect (Mas et a/., 2003}. In semi dominant toc1 mutant plants the

length of the circadian period is reduced by -3 hours therefore toc1 plants grown

under a 24 hr SD condition show early flowering. In these mutants grown the phase

of CO expression is significantly advanced such that high levels of CO coincidence

with the light phase at dusk, thus explaining the reason for early flowering. However,

when the toc1 mutant is grown under circadian cycle of 21 hrs the early flowering

phenotype under SD conditions is not observed because the light cycles match the

10

lntroauctwn

endogenous clock. In the toc1 mutant the circadian expression of other genes like

LHY and Gl is not affected under the 24 hr photoperiod (Yanovsky and Kay, 2002}.

GIGANTEA (G~ is another gene involved in photoperiod control of flowering.

It encodes a protein, with six putative transmembrane domains, is nucleoplasmically

localized, and is proposed to have a role in PHY B signaling (Huq eta/., 2000}.

Mutation of Gl causes photoperiod-insensitive flowering and alteration of circadian

rhythm (Park et a/., 1999}. The expression of G/ is under the regulation of the

circadian clock, with maximum expression detected at 8-1 0 h after dawn (Fowler et

a/., 1999).

The Autonomous Pathway

The German plant physiologist Klebs postulated that in each plant species, a

certain stage of development must be reached before it could start flowering. Only

very few plants respond to floral stimulus at the seedling stage. The Japanese

morning glory responds to SDs at the cotyledonary stage. Most species for example

Cocklebur, are not responsive to the floral signals at the cotyledonary stage and the

plants need to attain a minimum size to respond to the floral stimulus. Certain

monocarpic bamboo species and several other polycarpic plants (fruit trees) do not

flower until they reach a particular age. This phenomenon is called "ripeness to

flower''.

In Arabidopsis, the post germination vegetative phase can be divided into

juvenile and adult vegetative phases (Lawson and Poethig, 1995). A plant in the

juvenile vegetative phase fails to produce flowers even when grown under conditions

that are favourable for flowering. As the plant progresses from juvenile to the adult

vegetative phase the SAM acquires the competence to respond to the floral stimulus.

The autonomous pathway monitors the status of the endogenous developmental

clock of the plant. Mutation of HASTY (HS1) reduces the juvenile phase of shoot

development but has no effect on the adult phase. In the hst mutant the time of

flowering is accelerated due to an early transition of the SAM to the reproductively

competent developmental stage. HST appears to be a part of the developmental

clock that regulates the timing of shoot maturation. It promotes juvenile vegetative

growth and inhibits flowering by reducing the competence of the shoot apical

meristem to respond to LFYand AP1 (Telfer and Poethig, 1998}.

11

lfUTUUUCilUn

Mutants (in the early flowering background) that flower late under both LD

and SO conditions and are highly sensitive to vernalization classify the Autonomous

pathway. These mutants are different from the photoperiod pathway mutants, which

flower late under LD conditions but are insensitive to vernalizaton. Thus, the wild type

products of the genes of the autonomous pathway promote flowering independently

of the photoperiod.

The autonomous pathway promotes flowering by down regulating the

expression of FLC, a strong floral repressor. FCA, FPA, FY, LD, FVE are genes that

act in the autonomous pathway. Mutation of any of these genes results in increased

levels of FLC expression and a corresponding delay in flowering time. The late

flowering phenotype of the autonomous pathway mutations can be suppressed by

reducing the levels of FLC transcripts. However, the down regulation of FLC

expression does not affect the vernalization response of the autonomous pathway

mutants. Thus, suggesting that the autonomous pathway acts mainly by regulating

FLC expression, whereas vernalization responses can promote flowering by FLC­

independent mechanisms (Michaels and Amasino, 2001 ).

FCA is a nuclear RNA binding protein, which promotes reproductive

development in Arabidopsis by regulating the expression of FLC mRNA. Constitutive

expression of FCA results in reduced expression of FLC and hence an early

transition to the flowering phase. Conversely the loss of function mutant of FCA

shows delayed flowering. FCA also contains a WW protein interaction domain that is

essential for FCA function (Macknight eta/., 1997). The FCA transcript is alternatively

processed to yield four transcripts (a-8), the most abundant of which (~) is

polyadenylated within intron 3. FCA in combination with another protein (FY)

negatively regulates its own expression by promoting cleavage and polyadenylation

within intron 3. This results in production of a truncated, inactive transcript thus

limiting the expression of active FCA protein. For the efficient selection of the

promoter-proximal polyadenylation site the FCA/FY interaction is essential. FY shows

homology to S. cerevisiae Pfs2p a 3' RNA end processing factor (Quesada et a/.,

2003; Simpson eta/., 2003).

FPA is another RNA binding protein involved in the autonomous pathway.

Mutation of FPA result in extremely delayed flowering whereas over expression

causes early flowering in non-inductive short days. FPA is expressed most strongly in

12

Introduction

developing tissues, this pattern of expression is similar to the expression of other

autonomous pathway genes FCA and LD (Schomburg eta/., 2001)

Another component of the autonomous pathway is LUMINIDEPENDENS

(LD), which encodes a putative transcription factor (Lee eta/., 1994; Aukerman eta/.,

1999}. Like FCA, LD promotes flowering by negatively regulating the expression of

FLC (Sheldon eta/., 1999 Michaels and Amasino, 1999). It has been reported that

some of the homeodomain proteins can bind RNA directly through their

homoedomain-encoding region and participate in RNA processing (Dubnau and

Struhl, 1996; Zamore and Lehmann, 1996}.

As most of the genes in this pathway (FCA, FPA, FY, and probably LD) have

RNA binding ability, it is speculated that the autonomous pathway may work by post­

transcriptionally regulating the expression of FLC (Simpson and Dean, 2002;

Simpson eta/., 2003).

The Vernalization Pathway

Exposure of germinating seeds or juvenile plants to low temperatures for

several weeks often accelerates flowering in the adult plant and this phenomenon is

known as vernalization. In nature the winter annuals germinate in summer, grow

vegetatively through winter and flower only in the following spring. These varieties do

not flower until they have been exposed to winter conditions. The duration of the cold

period and the range of effective temperature vary from species to species.

In 1960's Lang had shown that the vernalization signal is perceived at the

shoot apical meristem. He has shown that exposure of the intact shoot apex to low

temperatures is required for vernalization to occur (reviewed in Michaels and

Amasino, 2000}. These observations indicate that the exposure to low temperatures

imparts competence to the SAM to respond to flowering signals.

The two dominant genes FR/GIDA (FR~ and FLC play a central role in

vernalization response and are responsible for delayed flowering in the naturally

occurring late flowering ecotypes of Arabidopsis (Michaels and Amasino, 1999;

2001 ). FRI delays the floral transition by increasing the levels of FLC transcript. FLC,

a MADS box transcription factor is a strong repressor of the floral initiation pathway.

13

Introduction

Late flowering ecotypes when exposed to low temperature show reduced levels of

FLC transcripts, which correlates with the time of flowering in these plants (Sheldon

eta/., 1999; Rouse eta/., 2002). Transgenic plants that over express FLC show

delayed flowering and do not respond to vernalization treatments indicating that

reduction in the FLC levels is required for vernalization to be effective. However, FLC

is probably not the only target of this pathway. Mutation of FLC can suppress the late

flowering effect of FRI but the ftc mutant retains the ability to respond to vernalization.

These observations suggest that besides repressing FLC expression, vernalization

might promote flowering by activating some flowering time genes (Michaels and

Amasino, 2001).

During vernalization germinating seeds and young plants are exposed to low

temperatures but the effects of this cold treatment is observed in the adult plants. A

long time span between the cold treatment and its effects on flowering time indicates

that the vernalization response is transmitted mitotically during the development of

the vernalized plant. On the other hand the vernalized state is not a heritable trait

suggesting that the vernalization signal is meiotically unstable. The ability of the

signal to be transmitted mitotically but not meiotically suggests that these signal act

by bringing about epigenetic modification of DNA such as methylation (Burn et a/.,

1993). This hypothesis is supported by observations that treatment of late flowering

mutants with demethylating agents causes early flowering. Moreover, the vernalized

plants show a transient reduction in the levels of methylation (Burn et a/., 1993;

Finnegan eta/., 1998).

A genetic approach to identify the genes required for vernalization response

revealed the role of VRN1&2 and HOS1 genes in this pathway. VRN1 is a nuclear

protein that has two B3 DNA binding domains and it binds DNA in vitro in a non­

sequence specific manner. FLC levels are reduced in the vernalized plants and

VRN1 is required for the maintenance of the repressed state of FLC when the plants

are transferred back to the ambient temperature (Levy eta/., 2002). Over expression

(35SCaMV::VRN1) in a vrn1 tea mutant background results in early flowering of

these transgenic plants without vernalization treatment. Exposure of these plants to

cold further accelerates flowering suggesting that VRN1 has a vernalization

independent function also. (Levy eta/., 2002).

Like VRN1, VRN2 is also required for the maintenance of the vernalized

state and not for the initial response to cold (Gendall et a/., 2001 ). The vrn2 mutant

14

Introduction

when exposed to cold shows reduction in the level of FLC transcript but when these

plants are returned to normal temperatures the level of FLC increases again. These

observations indicate that there are two distinct components of the vernalization

response machinery, one for the initial response to cold and second to maintain the

response. VRN2 encodes a zinc finger protein with similarities to Polycomb group

(PeG) proteins. It may be involved in repressing the FLC function through chromatin

remodelling, supporting the hypothesis that vernalization signals are transmitted

epigenetically (Gendall eta/., 2001 ).

HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENEt (HOSt)

appears to regulate the response to cold. It represses the transcription of genes that

are induced in response to cold treatment. Plants carrying mutations in the HOSt

gene flower early and have low levels of FLC expression. It encodes a RING finger

protein that may serve as an ubiquitin ligase (Lee eta/., 2001; lshitani eta/., 1998).

GA Pathway

The phytohormone GA promotes flowering in many plant species including

Arabidopsis (Bernier 1988) and this promotive effect is independent of the

photoperiod effect (Wilson et a/., 1992; Blazquez et a/., 1998). Application of GA

accelerates flowering time of wild type plants under short days. Mutations in the GA

biosynthesis pathway (gat, ga4 and gaS) delay the vegetative to floral transition. One

such mutant is gat-3, which has a deletion in the gene coding for ent kaurene

synthetaseA, the first enzyme in the synthesis of gibberellic acid (Sun and Kamiya

1994). The gat-3 mutant requires exogenous GA for germination, fails to flower

under SD and shows delayed flowering under long days (Wilson et a/., 1992,

Michaels and Amasino, 1999). Other mutants in the biosynthesis pathway, ga4 and

gaS have less severe defects.

Mutations in the GA signalling pathway also cause alteration in the flowering

time. These mutants are either GA insensitive or show a constitutive response. The

GA insensitive mutants resemble the GA-deficient mutants but cannot be rescued by

GA treatment. Constitutive response mutants appear as though they are exposed to

high concentrations of GA.

15

Introduction

SPINDLY (SPY) is involved in gibberellin signalling in Arabidopsis. SPY

encodes a ser(thr)-0-linked GlcNAc transferase. The spy mutant exhibits constitutive

GA mediated signal transduction and flowers early, suggesting that the wild type

protein negatively regulates the gibberellin signalling (Jacobsen eta/., 1996).

Other genes implicated in GA signalling and flowering time regulation are GA

INSENSITIVE (GAl), REPRESSOR of GA 1-3 (RGA) and REPRESSOR of GA LIKE

(RGL-1). These genes belong to the GRAS family of regulatory proteins (Peng eta/.,

1997; Silverstone et a/., 1998). RGA and GAl are thought to be nuclear-localized

transcriptional regulators. These genes act as repressors of GA signal transduction.

Treatment with GA or increase in the levels of endogenous GA promotes rapid

degradation of RGA (Silverstone eta/., 1998; 2001). The RGA, GAl, RGL-1 proteins

contain a conserved DELLA motif which is required for the mechanisms by which GA

promotes the degradation of these genes. Deletion of the DELLA domain causes GAl

and RGA to become insensitive to GA (Peng eta/., 1997). RGA proteins lacking the

DELLA domain cannot be degraded by GA mediated mechanisms suggesting that

the RGA regulation by GA requires the DELLA domain (Dill eta/., 2001). Moreover,

plants over expressing a RGL 1 protein lacking the DELLA domain show repression

of GA response and were phenotypically similar to GA-deficient plants 0fl/en and

Chang, 2002).

Genetic interaction studies between GA and the photoperiod pathway

suggest that the GA pathway acts independently of the photoperiod pathway. The

ga1-3 mutant is sensitive to photoperiod and shows a slight delay in flowering under

long day but the ga 1-3 co double mutant fails to flower under long days suggesting

that CO and GA 1 are in separate pathways (Putterill et a/., 1995; Reeves and

Coupland, 2001 ). GA promotes flowering by increasing the transcriptional activity of

the floral meristem identity gene LFY. The expression of LFY::GUS in mutants

defective in GA biosynthesis (gat-3) is reduced but increases in mutants with

constitutive GA signalling, such as spy (Biazquez eta/., 1998). Blazquez and Weigel

(2000) have shown that Gibberellins activate the LFY promoter through cis elements

that are different from those that are required for the day length response further

supporting that the GA pathway acts independently of the photoperiod pathway.

Besides this other known phytohormones (auxins, cytokinins, and

brassinosteroids) have been shown to be associated with flowering time but most of

the genetic studies have focused on the role of GAs (Bernier 1988).

16

l

Interaction of the floral pathways

In flowering plants there are separate genetic pathways regulating the

flowering time program in response to different environmental and endogenous cues.

Inputs from multiple pathways eventually converge to regulate the expression of the

common downstream genes. Identification of genes whose expression is regulated

by more than one floral transition pathway shows that there is an intricate network of

cross talk between signals from different pathways. These genes are termed as

''floral pathway integrators". Three such genes that integrate these signals are: LFY,

FT, and AGL20/SOC1 (Samach et a/., 2000; Weigel, 1992; Lee et a/., 2000). The

floral integrators serve a similar function but redundancy of these genes in different

pathways is partial. One reason for this is that different pathways regulate the

expression of these genes to different extents.

The autonomous and the photoperiod pathway are separate until FLC and

CO but they converge at the same downstream gene SOC1/ AGL20. SOC1, a direct

target of CO is positively regulated by the photoperiod pathway (Onouchi et a/.,

2000). On the other hand SOC1 expression is negatively regulated by FLC (Lee et

a/., 2000). SOC1 is also shown to integrate signals from the GA pathway and this

integration is necessary for flowering under short days (Moon eta/., 2003).

In addition to SOC1, FT and LFY also act as flowering pathway integrators.

FT is another immediate target of CO, and is positively regulated by the photoperiod

pathway but negatively regulated by FLC, suggesting that FT integrates the

photoperiod and the autonomous/ vernalization pathway (Samach et a/., 2000). It

encodes a protein homologous to the floral repressor TFL 1 and acts to promote

flowering in a parallel pathway as LFY (Simpson eta/., 1999). FT is proposed to act

via up-regulating the expression of floral meristem identity genes other than LFY

such as AP1 (Ruiz-Garcia eta/., 1997).

LFY, a floral meristem identity gene encodes a protein that shows no

homology to any protein of known function (Biazquez and Weigel 2000; Blazquez et

a/., 1998). LFY localizes to the nucleus and binds DNA in a sequence dependent

manner (Parcy eta/., 1998) suggesting that it may act as a transcriptional activator.

Over expression of LFY (CaMV35S::LFY) causes early flowering and promotes

formation of determinate floral meristems (Weigel and Nilsson, 1995 ). A loss of

function mutation results in conversion of the first few flowers into shoot like

17

Introduction

structures. Signals from the photoperiod, autonomous, and GA pathways regulate

flowering by up regulating the expression of LFY (Nilsson eta/., 1998; Blazquez and

Weigel, 2000) suggesting integration of signals from multiple pathways at the LFY

promoter (Fig. 1.1 ).

In higher plants the entry into the reproductive phase is strictly regulated by

inputs from multiple pathways. This stringent control on the timing of transition is a

mechanism to ensure the reproductive success of the plant and results in the

formation of flowers under conditions that are favourable for fertilization and seed

development. The events of meiosis and gametogenesis take place within the

reproductive organs of the flower and are under developmental control.

MEIOSIS in sexual plant reproduction:

Meiosis and syngamy play a pivotal role in bringing about alternation of

generation in flowering plants. During meiosis, a single round of DNA replication (pre­

meiotic S phase) is followed by two successive rounds of chromosome segregation

(meiosis I and II). The first meiotic division is a reductional division and is

accompanied by meiotic recombination whereas the second division is a simple

equational division. Recombination is the key factor that brings in genetic diversity.

Though there are striking differences in meiosis between different organisms, the

basic features of meiosis are remarkably conserved throughout evolution. Based on

the chromosome behaviour as observed in cytological studies in various organisms,

meiosis can be divided into different stages. The meiotic cycle begins with pre­

meiotic DNA synthesis. Following pre-meiotic S phase the chromosomes condense

and pairing between the homologous chromosomes is initiated. The synaptonemal

complex is formed along the entire length of the bivalents and holds the homologous

chromosomes together for recombination. The synaptonemal complex is

progressively shed at a later stage (post recombination) and the homologues

separate out except at chiasmata, the sites of recombination. The bivalents continue

to condense and compact further throughout the remainder of prophase till they enter

metaphase1. At anaphase I the homologues, with sister chromatids still held

together at the centromere, are pulled towards opposite poles thus segregating the

homologous chromosomes to different poles.

The biological simplicity and the availability of genetic methodology make

yeast a favourable model system to study the molecular basis of meiosis. In yeast

18

Circadian clock Autonr mous V) ernalization

Photop~ ~ GA Pathway

co~11 l A~l24

APloc: •LFY

Floral Transition

Phase Phase

Figure 1.1 Overview of the relationship between different pathways controlling the flowering time in Arabidopsis

Flowering integrators SOC1, LFY, and FT , integrate the floral signals frorr the photoperiod, autonomous , vernalization and the GA pathways anc activate a common set of flower meristem genes.

Introduction

( S. pombe and S. cerevisiae) the meiotic pathway is well dissected at the genetic as

well as the molecular level. Genes regulating different steps of the meiotic cycle have

been identified and their function at the molecular level is known. Most of our present

day understanding about the regulation of meiosis at the molecular level comes from

studies carried out in yeast.

As the process of meiosis is conserved throughout evolution one can

hypothesize that some of the basic mechanisms of regulation might also be common.

Therefore the information available from lower eukaryotes can be applied to higher

eukaryotes in understanding the similar processes. Many of the genes like RecB,

Hop1, Rad50, Spo11, Dmc1 known to be involved in chromosome organization and

recombination in yeast meiosis are conserved through evolution and are involved in

meiosis in other organisms also including plants (Caryl eta/., 2000; Bhatt eta/., 1999;

Bai eta/., 1999; Grelon eta/., 2001; Couteau eta/., 1999) Table 1.1 (list of known

plant meiotic genes and their homologues in yeast or other organisms).

Meiosis in plants

Meiosis in plants has been a subject of interest for many years and a large

number of meiotic mutants have been identified and cytogenetically characterized in

various plant species especially maize (Golubovskaya, 1989; Staiger et a/., 1993;

Dawe, 1998). Presence of a large number of male meiocytes in the anther and their

relatively prolonged prophase has enabled cytologists to carry out detailed studies to

define various events occurring during meiosis. In recent years, beside the detailed

cytological studies, attention has been given towards characterization of meiotic

mutants at the molecular level. Arabidopsis thaliana because of its small genome,

complete genome sequence information and powerful genetic and molecular tools is

the favoured model organism used for isolating and characterizing meiotic mutants.

Moreover developments in cytogenetic techniques have helped to examine in detail

the chromosome behaviour during meiosis (Ross eta/., 1996; 1997; Armstrong and

Jones, 2003). Two different approaches have been used: 1) Forward genetics,

which is based on screening mutants (EMS, fast neutrons, T-DNA insertion,

Transposon insertions) for meiotic defects and then identifying and characterizing

those genes (Sanders et a/., 1999). 2) Reverse genetics, an alternate approach

where one analyses mutations in genes that either show homology to meiotic genes

from other species or have an expression pattern suggestive of the meiotic role.

20

S.No. Gene name Gene Product Sterility r;;_1r3 Function

Meiotic recombination, SC 1. AtSP011 Topoisomerase r;;.&r3 formation, Cohesion

Meiotic recombination and 2. AtDMC1 RecA homologue r;;.&r3 strand exchange

3. ASK1 SKP1 r3 Homologue segregation

4. AtRADSO SMC N-terminal ATP

r;;.&r3 Double-strand break repair and

binding protein Telomere maintenance

5. ASYNAPTIC1

DNA binding protein r;;.&r3 Homologous chromosome

(ASY1) synapsis, SC morphogenesis

DIF1/ Cohesin Chromosome condensation and

6. SYNAPSIS1 r;;.&r3 (SYN1)

(RAD21 like) Pairing

7 SOLODANCER

Cyclin like protein r3 Homologue synapsis and

(SDS) bivalent formation

8 AHP2 Histidine containing

r;;.&r3 Pairing and segregation of

phosphotransferase homologous chromosomes

AtATM Protein with

9 phosphatidylinositol r;;.&r3 DNA repair 3-kinase domain

Table 1.1 List of Arabidopsis meiotic genes that have orthologs in other organisms Sc: Saccharomyces cerevisiae; Sp: Schizosaccharomyces pombe

Homologues in yeast or References

other organisms Grelon eta/., 2001

Spo11 (Sc) Hartung and Puchta, 2000

Klimyuk and Jones Dmc1 (Sc) 1997, Couteau et

a/., 1999 Skp1 (Sc) Yang eta/., 1999

RadSO(Sc) Gallego eta/., 2001

Ross eta/., 1997,

Hop1 (Sc) Caryl eta/., 2000, Armstrong eta/.,

2002

Bhatt eta/., 1999 RecB(Sc)

Bai eta/., 1999

Yeast Cyclins Azumi et at., 2002

Meu13 (Sp) Schommer eta/.,

2003

Mammalian ATM Garcia eta/., 2003

Introduction

Using different approaches a number of meiotic mutants have been identified and

characterized at the molecular level {Table 1.2).

Considerable information is available regarding the molecular mechanisms

involved in later stages of sporogenesis but limited information is available on the

mechanism of germline specification and initiation of meiosis. In animals the germline

specification occurs early during embryo development when the primordial germ cells

are defined (Wylie, 1999). In plants the cell fate is determined late in development as

a result of positional information (Dawe and Freeling, 1992; Huala and Sussex,

1993). In plants the germ line arises late in the development from the L2 layer of the

anther and ovules. An early event in anther and ovule development is the switch in

the developmental fate from the mitotic to the meiotic cycle.

During sporogenesis a small number of hypodermal cells in the anther and

the ovule differentiate into the archesporia! cells, the progenitors of the meiocytes. In

the ovule the archesporia! cell directly differentiates into the megaspore mother cell

(MMC) in most plants. In the anther the archesporia! cell undergoes mitotic divisions

before it gives rise to the pollen mother cell (pmc). During the differentiation from the

archesporia! cell to mmc or pmc, the cells switch from the mitotic cell cycle to the

meiotic cell cycle. The molecular identities of the signals that recruit certain mitotic

cells into the meiotic pathway are not known in plants. A few genes involved in

specification of the reproductive cell types and the meiotic to mitotic switch have

been identified in maize and Arabidopsis.

SPOROCYTELESS (SPL) I NOZZLE (NZZ) encodes a nuclear protein that is

similar to transcription factors, and is involved in early germ line specification (both

male and female) (Yang eta/., 1999, Schiefthaler eta/., 1999). In the sp/ /nzz ovule

the archesporia! cell fails to differentiate into a megasporocyte and the nucellar

development is defective. In the spl anther the archesporia! cell is formed and

undergoes mitotic division to form normal primary parietal and primary sporogenous

cells. The further differentiation of primary sporogenous cells into microsporocytes is

blocked.

During ovule development a single hypodermal cell develops into a

archesporia! cell. The surrounding cells are inhibited from acquiring the archesporia!

cell fate. In maize, the MULTIPLE ARCHESPOR/AL CELLS (MAC1) gene plays an

important role in switching the hypodermal cell from the vegetative pathway to the

22

S. No. Gene name Species Stage Mutant Phenotype Sterility

Gene Product References Cj_f(!;

1 MAC1 Maize Sporocyte Multiple archesporia! cells,

Cjl&c)' Not Cloned Sheridan eta/., 1996,

formation defective male meiosis 1999

2 SPUNZZ Arabidopsis Sporocyte No sporocytes formed

Cjl&c)' Nuclear protein similar to Schiefthaler eta/., 1999

formation MADS box transcrlJ:>tion factors Yang eta/., 1999

3 AM Maize Sporogenesis Sporocytes undergo mitosis

Cjl&c)' Not Cloned Golubovskaya, 1993

instead of meiosis

4 AFD " Sporogenesis Premature sister kinetochore

Cjl&c)' Not Cloned Yu and Dawe, 2000

separation

4 SAP1 Arabidopsis Sporogenesis Arrest after first meiotic division

Cjl&c)' Novel protein with hallmarks of Byzova eta/., 1999

a Transcriptional regulator Sporocyte Supernumerary microsporocytes, Canales eta/., 2002

5 EXSIEMS Arabidopsis formation & defective male meiosis I 0' LRR receptor like Kinase Zhao eta/., 2002 sporOQenesis

Sporocyte Supernumerary megasporocytes Nonomura eta/., 2003 6 MSP1 Rice formation & and microsporocytes, defective Cjl&c)' LRR receptor like Kinase

sporOQenesis male meiosis

7 DYAD/SW/1 Arabidopsis Sporogenesis Defective meiosis, progression

Cjl&c)' Cohesin /Structural Agashe et a/., 2002

defects maintenance protein Mercier eta/., 2001

8 DUETIMMD1 Arabidopsis " Defective male meiosis, 0' PHD finger protein

Reddy eta/., 2003 progression defects Yang eta/., 2003

9 ATK1 Arabidopsis " Polyads formed 0' Kines in Yang eta/., 2003 Chen eta/., 2002

10 ME/1 Arabidopsis " Meiotic fragmentation

Cjl&O' Protein containing BACT Mathilde eta/., 2003

domains

11 TESISTUD Arabidopsis " Defective male meiotic 0' Kines in

Yang eta/., 2003 cvtokinesis Spielman eta/., 1997

12 PAM1 Maize " Impaired homologous pairing and

Cjl&c)' Not cloned Golubovskaya eta/.,

telomere clusterina 2002

13 MSS Arabidopsis Sporogenesis Polyads formed 0'

Homology to Synaptonemal Glover eta/., 1998 Complex Proteins

Table 1.2 List of meiotic mutants in plants

Jntroauctwn

meiotic cell cycle. In mac1 mutant ovules several hypodermal cells develop into

archesporia! cells, and the resulting megasporocytes undergo a normal meiosis

forming multiple embryo sacs {Sheridan eta/., 1996). In mac1 anthers, the three wall

layers do not form, and the archesporia! cells divide to produce abnormal

microsporocytes that fail to proceed through meiosis {Sheridan eta/., 1996; 1999).

During anther development the archesporia! cell undergoes a series of mitotic

divisions to give rise to a fixed number of PMCs. Mutation of EXTRA

SPOROGENOUS CELLS (EXS)/ EXCESS MICROSPOROCYTES (EMS) in

Arabidopsis and MULTIPLE SPOROCYTES (MSP1) in rice results in the formation of

supernumerary sporogenous cells (Canales et a/., 2002; Zhao et a/., 2002;

Nonomura eta/., 2003). Both the genes encode a putative LRR receptor kinase. A

mutation in EXS/EMS results in increased cell division in the L2 layer of the

developing anther. In the msp1 mutant the cells originally programmed to be the

wall layers are converted into sporogenous cells resulting in an increase in the

number of pmcs. In the msp1 ovules multiple precursor cells develop into the mmcs.

The mmcs in the msp1 mutant do not show any defect in meiotic progression

whereas meiotic progression in the PMC is arrested at various stages between

leptotene and diakinesis. The rice MSP1 appears to be an ortholog of the EXSIEMS

gene of Arabidopsis. EXS/ESM and MSP1 genes participate in a signaling pathway

to limit the number of male and female hypodermal cells entering into sporogenesis

(Nonomura eta/., 2003). In the ems/exs and the msp1 mutants the archesporia! cell

formation is normal indicating that these genes are downstream to SPUNZZ.

Initiation of Meiosis in S. pombe

In S. pombe the mechanism of the switch from the mitotic to meiotic cycle is

well dissected at the genetic and molecular level. S. pombe is predominantly a

haploid organism, which exists in two mating types h+ and h .. The switch from mitosis

to meiosis is in response to nutritional starvation and mating pheromone signals

(Sugimoto eta/., 1991 ).

Nutritional starvation results in reduction in the levels of cyclic AMP (cAMP) in

the cell. The cAMP cascade induces the expression of Ste11 p, a High Mobility Group

(HMG) family transcription factor (Sugimoto eta/., 1991 ). Ste11 p in turn induces the

24

Introduction

expression of genes like matt-Pc, matt-Me, and Mei2 that are involved in

conjugation and meiosis.

The two key players that bring about the mitotic to meiotic switch are Pat1

kinase and Mei2 (an RNA binding protein). Pat1 kinase is required for inhibiting the

initiation of meiosis during the mitotic cycle. In the absence of Patt kinase (Lpatt)

the haploid heterothallic cells growing on a nutrient rich, differentiation-inhibiting

medium can enter meiosis, resulting in production of inviable spores containing less

than 1 c DNA content (lino and Yamamoto, 1985; Nurse, 1985). Pat1 kinase inhibits

meiosis during the haploid phase by phoshorylating a meiotic inducer Mei2p

(Watanabe eta/., 1997; Li and Mcleod, 1996). Presence of unphosphorylated Mei2p

is sufficient to initiate meiosis under conditions considered unfavourable for meiosis.

The phosporylated Mei2p is susceptible to ubiquitin mediated protein degradation

(Kitamura et a/., 2001) and is preferentially bound to Rad24p. Binding to Rad24p

decreases the affinity of Mei2p for RNA (Sato eta/., 2002}.

Entry into meiosis requires inactivation of Pat1 p, which is brought about by its

binding to Mei3p, a pseudo substrate for Pat1 p. Expression of Mei3 depends upon

heterozygosity at the mating loci and the co-expression of matt-Pi and matt-Mi

(Mcleod et a/., 1987; Li and Mcleod, 1996}. This stringent requirement for the

expression of Mei3 prevents untimely inactivation of Pat1 p kinase and entry of

haploid cells into meiosis.

The inactivation of Pat1 p kinase in diploid cells leads to accumulation of the

active form of Mei2p (unphosphorylated) required for the initiation of meiotic

processes. Mei2p is an RNA binding protein, which is required for premeiotic DNA

replication, and entry into meiosis1 (Watanabe and Yamamoto, 1994). Mei2p shuttles

between the cytoplasm and the nucleus (Sato eta/., 2002}. Its RNA binding ability is

essential for its activity to stimulate meiosis and its translocation into the nucleus

(Watanabe and Yamamoto, 1994}. Mei2p is cytoplasmic during interphase and this

cytoplasmic form is essential for the premeiotic DNA replication function of Mei2p

(Yamashita et a/., 1998}. Mei2p forms a single dot at an apparently fixed position in

the horsetail nucleus during the meiotic prophase (Yamashita eta/., 1998; Watanabe

eta/., 1997). Mei RNA, a meiosis specific non-coding RNA species play a major role

in the transport of Mei2p into the nucleus.

25

Introduction

Formation of the Mei2p dot has been shown to be associated with the sme2

locus, which codes for the Mei RNA (Shimada eta/., 2003}. Transcriptional activity of

the sme2 locus is required for the formation of the Mei2p dot and Mei RNA plays a

major role in the positioning of the dot. Mei2 has been known to be a key positive

regulator of meiosis but the mechanism by which it regulates is still a mystery.

Hirayama et al., 1997 employed a trans-complementation screen in a meiosis

defective yeast mutant (mam2/mam2) to identify cDNAs from Arabidopsis that

encode signal transducers and components of meiosis. Expression of the

ARABIDOPSIS ME/2 LIKE1 (AML 1) gene was able to rescue the meiotic defects and

induce sporulation by bypassing the pheromone signalling. However, the efficiency of

sporulation was less than the mamz+"lmamz+- strain. The over expression of the full

length AML 1 failed to complement the ii.mei2 (null) or mei2-33 a temperature

sensitive allele at semi-restrictive temperature where the pre-meiotic DNA replication

function was normal but the entry into meiosis1 was defective. This suggested that

AML 1 might be capable of the initiation of pre-meiotic DNA replication function of

Mei2p. There are five genes in the Arabidopsis genome that show homology to Mei2

protein. The AML genes thus are potential candidates for a role in reproductive

development in Arabidopsis.

Objective of this study.

Many of the yeast meiotic genes are known to have orthologs in plants,

C.e/egans, and Drosophila (Table 1.1 ). In the course of evolution some of these

genes have also acquired additional functions besides their meiotic roles. I have

investigated the role of the AML gene family in plant reproductive development,

including meiosis. As discussed earlier, the floral transition marks the onset of the

reproductive phase in plants. To understand the role of AML genes in reproduction, I

have examined the expression profile of the AML genes in Chapter 2 and show that

AML genes are targets of the flowering time program. In Chapter 3, I have

characterized expression of two representative members (AML2 and AML3} at the

tissue and cellular level. In order to address the function of AML genes I have used

T -DNA insertion mutants as well as anti-sense and RNAi approaches to study the

effect of down regulation of these genes on vegetative and reproductive development

in Arabidopsis. These experiments are presented in Chapter 4. The results indicate

26

that the AML genes function in multiple aspects of development including leaf

development and gametogenesis.

27