chapter 2 review on literature -...

Chapter 2 Review on literature 2015

66

Chapter-2

REVIEW ON LITERATURE


67

6. Influence of altitudinal variation on secondary metabolites contents

6.1 Introduction

Medicinal plants are natural resource which constitutes one of the sources of new compound

or bioactive compounds for drug development1. About 60% of the world population and

80% of the population of developing countries rely on tradition medicine, mostly plant

drugs, for their primary health care needs2. In India, about 7500 species of higher plants out

of 1700 species, which comprises a considerable proportion of total flowering plants3are

known for medicinal uses. Plants are used for medicinal purpose in India has long history,

and the proportion of medicinal plants is the biggest proportion of plants known for their

medicinal purpose in any country of the world for the existing flora of the respective

country4.

Damaging and rapidly changing external environmental factors interrelate with Plants. To

avoid damage against external environment plants have evolved alternative defence

strategies, which involve production of several different varieties of chemical known as

secondary metabolites act as defense system to defeat damages caused by stress conditions.

Secondary metabolites also help plants in adaptation to the changing environment. It is

found that plants have ability to produce limitless secondary metabolites in response to

external attack. Mostly stresses are considered as biotic and abiotic stresses which include

microbial and herbivore attack, temperature and light intensity etc. Theses defense

mechanisms are generated by plants via triggering several simple to complex biochemical

processes5. Imapct of stress conditions have been documented at genetic and protein level


68

and its reflection is reported in alteration of the secondary metabolite pool of the affected

plants6,7,8

.

Yet, the biosynthesis of secondary metabolites is commonly either restricted to particular

plant tissues or developmental stage, is firmly regulated, or produced in response to external

stimulation9,10

. Different classes of secondary metabolites are produced for survival under

adverse conditions and causes diversity at organism level but restricted to specific plants

groups. In response to external factors plants produce variety of chemical compounds by in

vivo chemistry by matching, mixing and developing the intermediates needed for

biosynthesis of secondary metabolites. Humans exploit these secondary metabolites to their

benefits in different fields include medicinal properties.

Recent findings in research on secondary metabolites have shown overlapping of boundaries

of different research fields like ecology, biochemistry, molecular biology and plant

physiology and recognized the variation in metabolites compositions basis of the diversity

and production of secondary metabolites in plants. This review literature is on impact of

altitudinal variation on secondary metabolites contents of medicinal benefits and this study

can be incorporated in medicinal quality assurance of phytomedicines.

6.2 Primary and Secondary metabolism

In living organism several enzymatic regulted chemical reactions which term as metabolism

take place to maintain life. These chemical reactions produce compounds refer as primaty

and secondary metabolites necessary for growth and development of cell or enable organism

to survive in adverse environmental conditions. However, diversity in primary metabolites

compare to secondary metabolites is rare between species. Primary metabolites are


69

synthesizing via primary metabolic pathways and include molecules like sugars, amino acids

and citric acid.

On the other hand molecules like nucleic acids, proteins, peptides and polysaccharides that

are reported as universal building blocks of life shows structural diversity from one

organism to another but their functions are common11

.

Secondary metabolites possess multiple functionality and bioactivity because of presence of

more than one functional group12

. Secondary metabolites are biosynthesized by using

intermediates of primary metabolism13

.

Current report on function of secondary metabolites in organism is limited14

. Some of them

reported as responsible for pollination and seed dispersion and natural pesticide, while

function of many other secondary metabolites is not yet clear14,15

. Biosynthesis of secondary

metabolites is not random process but occur higly specific cells, organs and organelles9. In

biosynthesis of secondary metabolites several enzymes are involved in chemical reactions

and their metabolism is incorporated into morphological and biochemical pattern. There is

several reports are available reavealing the highly managed interaction between plants and

stresses as being involve in major role as driving force behind the biosynthesis of secondary

metabolites16

.

On the basis of experimental findings of plants and envirrnmenal interactions caused

diversity in metabolic profile and associated and adjacent genetic mechanism on regulation

of metabolite chemical profile, the question develop; what enviorment of plants can be

adjusted in favor of production of required secondary metabolites? To understand the

accurate reason and answer of develop question study of impact of environmental factors on

secondary metabolites profile is need to explore.


70

6.3 Role of environmental factors in secondary metabolite biosynthesis

The main crucial question in survival of plants in adverse environment is the recognistion of

the driving force of diversity within the plant’s instrinsic mechanism to find these and

populations in which diversity is propogated. Harborne17

, 1982 have reported several

environmental factors like season, grazing, age of the plant, nutritional status and microbial

attack have impact on secondary metabolites profile. There is experimental evidence

available on effect of external factors on contents of secondary metabolites through their

impact on plant’s growth rates, and development. Effect of these factors is also reported on

qualitative variation in secondary metabolites18,19,20

.

In response to different environmental conditions, plant’s defence mechanisms respond

differently and are summarized in (Table-1). Plants are exposed to different biotic and

abiotic factors and in response activate their defense systems throughout their life span.


71

Plant Species Secondary metabolites Environmental factor References

Table-1 Effect of environmental factors on secondary metabolites biosynthesis in some

medicinal plants

UV Light/

Solar radiation

Hypericum

perforatum

Achnatherum

inebrians,

Arnica montana,

Quercus spp.,

Pachypodium

saundersii,

Petunia×hybrida

Terpenoids,

anthocyanins,

Phenolic

compounds

alkaloids,

flavonoids,

tannins;

Christie et al. (1994),

Pennycooke et al. (2005),

Albert et al. (2009), Sharkey

and Loreto (1993), Singsaas

and Sharkey (2000), Solecka

and Kacperska (1995),

Zhang et al. (2011) and

Zobayed et al. (2005)

Temperature

Betula spp., Salix

myrsinifolia,

Secale cereal, Artemisia

annua, Sambucus nigra,

Prunus serotina,

Frangula alnus, Corylus

avellana, Pteridium

arachnoideum, Solanum

tuberosum, Diplacus

spp., Larrea,

Marchantia

polymorpha, Pinus

taeda, Arabidopsis

thaliana, Cornus

sanguinea,

Terpenoids,

flavonoids,

hydroxycinnamic

acids, tannins,

artemisinin,

phytosterols,

glycoalkaloids,

luteolin, apigenin,

alkaloids, flavonol

glycosides,

phenolic acids;

Alonso-Amelot et al. (2007),

Burchard et al. (2000),

Kliebenstein (2004), Laakso et

al. (2000), Lavola et al.

(1997), Markham et al.

(1998), Rhoades (1977),

Tegelberg and Julkunen-Tiitto

(2001) and Winkel-Shirley

(2002), Arnqvist et al. (2003) ,

Karolewski et al., (2010),

Lommen et al. (2008)

Ceratonia siliqua,

Lithopermum

erythrorhizon, Betula

spp., Eucalyptus

cladocalyx, Rhodiola

sachalinensis ,

Aradopsis thaliana,

Phenolic

compounds,

gallotannins,

shikonin,

cyanogenic,

glycosides,

Salidroside,

condensed tannins

Soil nutrients Gleadow et al. (1998),

Kim and Chang (1990),

Kouki and Manetas (2002),

Riipi et al. (2002),

Simon et al. (2010) and Yan

et al. (2004), Dixon and

Paiva (1995), Kliebenstein

(2004)

Artemisia annua,

Pachypodium

saundersii,

Achnatherum

Inebrians, Pteridium

arachnoideum,

Lipophilic resins,

artemisinin, tannins,

isoprene,

anthocyanins,

alkaloids, Phenolic

compounds

Soil water

availability

Horner (1990),

Lommen et al. (2008),

Sharkey and

Loreto (1993) and

Zhang et al. (2011),

Alonso-Amelot et al.

(2007)


72

6.3.1 Effect of altitudinal variation on secondary metabolites contents

Effect of altitudinal variation on contents of secondary metabolites of higher plants is not

well reported so far. In experiments of effect of altitudinal variation study in genetically

uniform plants, significant variation in phenolic contents was found. Furthermore the results

were confirmed by antioxidant activity which confirmed that higher antioxidant activity is

found in extract of plants at higher altitude compare to plants at lower altitude21,22,23

. On

varying altitudinal zone many environmental factors such as mean temperature, high and

low temperature extremes, precipitation, soil fecundity etc changes of growing site24

.

Blumthaler et al., 1997 have reported an increase of UV-B (280-315 nm) irradiance in

European Alps, an increase of total 8% for irradiance, an increase of 9% for UV-A radiation,

and an increase of 18% for erythermal effective radiation per 1000 m of altitude. In high

altitudes effect of enhanced UV-B radiation on contents of secondary metabolites has been

discussed by Korner24

1999 and Bilger25

et al., 2007 linked low temperature with increase of

antioxidant secondary metabolites. Enhanced productivity of UV-B absorbing and

antioxidant phenolic compounds is reported in response to enhanced UV-B radiation and

interpreted the result of enhanced production is as a protective response against damage

from excessive UV-B radiation due to their UV-shielding properties26,24

. Wellmann27

, 1975;

Jaakola and Maatta-Riihinen28

, 2004 have reported the induction of enzymes catalyzes the

biosynthesis of UV-B absorbing compounds under enhance UV radiation. Phenolic

compounds possess antioxidant acitivity and contribute to their protective properties against

damage by enhance UV-B radiation26,22

.

6.3.2 Report on altitudinal variation in general

http://link.springer.com/article/10.1007/s11101-009-9143-7/fulltext.html#CR5


73

Effect of altitudinal variation on secondary metabolites contents is subject with more

approximate theories than real scientific data. There are two contradictory theories are

reported. Out of both theories one theory rely on climatic extremes than selective pressure

induced by herbivores. According to this theory contents of secondary metabolites decreases

from the equator to the poles29,30

. The effect of temperature is understood as direct and

indirect effect. Low terpertaure causes indirect effect while enhanced UV-B radiation has

direct effect on DNA. Bilger25

et al., 2007 documented that enzymatic repair process at low

temperature is more inhibited as compare to photochemical damage at high temperature.

In support of both theories few experimental supports are given. They reported lower

amounts of toxic quinolizidine alkaloids in populations of Lupinus argenteus Purse grown at

high altitude. As a result of this theory the obtained difference was found least inheritable

and genetically encoded. Second theory was validated by Alonso-Amelot31,32

et al., 2004, 2007. They reported higher amount of antioxidant phenolic compounds in ferns

Pteridium arachoideum (Kaulf.) Maxon and Pteridium caudatum (L.) Maxon are grown at

higher altitudes compare to grown at lower altitude. According to both theories and

experimental data altitudinal trenda for different classes of secondary metabolites are shown

in Figure-14.





74

6.3.3 Temperature

Among several environmental factor termerature stress is one of important factot known to

stimulate the production free radical scavenging enzymes such as superoxide dismutase,

catalase, and peroxidase along with various antioxidants. Impact of temperature stress on

several biochemicals, physiochemical and molecular level in plant metabolism like

perturbation of membrane integrity or protein denaturation is reported. As a result of these

changes secondary metabolites contents are vary in the plants tissue33

. There are several

reports are available on effect of temperature on composition or contents of phenolic or

phenolic derivatives. High and low both temperatures have different effect of plants

Figure-14 Altitudinal variation effect on various classes of plant secondary metabolites with

varying ecological functions

High altitudinal zone

Low altitudinal zone

Anti-herbivory

substance

Allelopathic

compounds e.g.

Iridoids,

Alkaloids,

Polyacetylenes,

Sesquiterpenes

lactones

Free radical

scavenging

compounds

UV-B

protective

compounds

e.g.

Anthocyani

n

Ascorbic

acid

Carotenes

Flavonoids

Glutathione

Phenolic

acid


75

secondary metabolites. Chalker-Scott34

1999; Choi35

et al., 2009 have reported low

temperature induced anthocyanin biosynthesis in several species.

Parker36

, 1977; Sharkey & Yeh37

, 2001 have described effect of elevated temperature on

contents of volatile compounds which were found to be increased. Increased biosynthesis of

anthrocyanin is considered to protect plants from cold stress38

. Results of Pennycooke38

et

al. studies have been supported by study have done by Ncube39

et al. (2011a). Ncube et al.

2011a stated higher production of phenolic compounds in winter season which supports the

previous findings given by Pennycooke38

et al. Decrease in anthocyanin contents under high

temperature is caused by low gene expression and decline enzymatic activity necessary for

biosynthesis of anthocyanin. This effect has been reported by Dela40

et al., 2003 in rose and

in petunia by Shvarts, Borochov & Weiss41

, 1997. There is several reports are available on

effect of temperature of contents of antioxidant compounds.

Quanlitative variation in addition to quantive variation, in secondary metabolites was also

found by variation in temperature. Albert42

et al., 2009 reported increase in ratio of quercetin

: kaempferol in flowering heads of Arnica Montana by decreasing temperature with 5°C in

alpine climate system. In very recent study is done by Schmidt43

et al., 2010 who have study

the together influence of climatic and genotypic on composition and contents of flavonoid

compounds in Brassica oleracea growing in cool (0.3–9.6 °C) temperatures. In his study he

collected samples during month period at four week intervals and found flavonol kaempferol

as main flavonoid aglycon followed by quercetin and isorhamnetin.

While, no significant variation is observed in total flavonoid contents but ratio of quercetin:

kaempferol is found increased with decline in temperature. On the basis of all reported

studies it was concluded that plants are sensitive to temperature variation and it can be


76

detected by quantification of free radical scavenging compounds in species-specific way.

Too high temperature has negative impact of flavonoid biosynthesis while the low

temperature can enhance the production of flavonoid although its accumulation is light

dependent in low temperature.

6.3.4 UV Light/Solar radiation

Amonng environmental factors solar radition is one of the most important factors for the

plant growth. Quality and period of radition are different in all area of the universe. Even in

the same area quality of light is not stable. Solar angle which depend on latitude and day

time have effect on light quality. In northern side above 69°N latitude, in summer sun

remains above the horizon, and evening shows comparatively long ‘end-of-day’ period,

which ends for equal to 20% of per day44

. Varition of R: FR ratio has effect on plants

growth. During this period, ration of R: FR is reduced and impact on plant development. On

increasing solar angle from maximum zenith the ration of Red: Far Red (R: FR) decreases

under blue skies45

.

Plants growth is dependent on quality of light, duration of solar radition. Alokam,

Chinnappa & Reid, 2002 have studies the effect of different R: FR ratios on accumulation of

anthocyanin in plants of Stellaria longipes under same environmental conditions. Under

high R: FR ratio high level of accumulation of anthocyanins is found in prairie plants.

However, both alpine and prairie plants are found to contain same levels of anthocyanins

under low R: FR ratio. Kazan & Manners46

, 2011 have described the ability of plants to

sense the variable light spectra and UV radition and plants accordingly shows adaptation in

response to surrounding changes.


77

In response to use maximum benefits from incidence light, several biochemical mechanism

operates in plants against increase UV radition and high light intensities. According

mechanisms do adjustment in plants to optimize absorption of needful light spectra. Plants

have intricate signaling network system in which light signals are incorporated in plants

development and resulting into light output from inputs. Among light raditions UV-B

radition is more damaging to plants, impairing gene transcription and translation and

photosynthesis.

Kazan & Manners46

, 2011; Rozema47

et al., 1997 have reported significant variation in

contents of seconday metabolites including terpenoids, phenolic compounds and alkaloids.

The ability of each species to resist to the variation in environmental factors and to produce

phenolic compounds can be explained as changes occur due to altitudinal segragartion.

Phenylpropanoid derivatives particularly absorb in UV-B spectral region exclusive of

minimizing penetration of radition needed for photosynthesis in leaves. On the basis of

results it is concluded that these compounds act as sunscreens to take up UV-B light. In

contrast of these findings some reports reveal decrease in contents of these metabolites in

some species while some studies using phenylpropanoid mutants and different UV-B

radiation levels have produced same results consistent with previous opinion48

. Results of

these studies brought forward dissimilar conclusion on group of compounds as flavonoids,

hydroxycinnamic acids, phenolics, complex polymeric lignin or tannin and their derivatives

as esters fuction as predominant UV-B protectants in several plant species49

.

Overall experimental results are concluded as solar radition have profound contribution in

production of phenolic compounds and their derivative as flavonoids in plants. Being

pharmacological active in nature phenolic, flavonoids and its derivatives have attracted


78

research interest in pharmaceutical industries and flavonoids have been proven to possess

various pharmacological activities like anti-inflammatory, antioxidant, antifungal,

antibacterial, anti-hepatotoxic, vasodilator, antiallergic agents and

immunostimulant50,51,52,53,54

. Phenolic compounds have been reported for numerous

medicinal activities like antidiarrhoeal, free radical scavenging, immunomodulatory,

antiviral, antibacterial, anti-inflammatory and antifungal52,55

. Haslam56

, 1989 have reported

phenolic compounds are active as antibiotic and antifeedant on multiple range of organism

that consume them. Being major contribution of these compounds in human health systems

it is important to consider their quality in plants and quality of these compounds are largely

depends on the external environmental factors. Effect of solar radition in not only limited to

phenolic compounds but also cover broad spectrum of other secondary metabolites, some of

them retain pharmacological activities while some of them are toxic to human beings. For

example α-chaconine and α-solanine are produced more in potato tubers due to mechanical

stress and cause gastro-intestinal or neurological disorders in human beings. While

deposition of phytosterols led more deposition of these compounds and has positive impact

of human beings. Phytosterol bound absorption of cholesterol from fat matrices which

results in lower level of cholesterol into the intestinal tract in human consumers and prevents

from cardiovascular diseases. Results show that there is direct link between light stimulated

secondary metabolites and therapeutic benefits. Stadarization, safety and quality assessment

and to improve phytopharmaceuticals and human healths, there is need to understand and

study of impact of factors controlling these metabolites would therefore become a basic

prerequisite. Several experimental data have proven that altitude has an effect on the

contents of secondary metabolites in higher plants. Many climatic differences and quality of


79

radition vary on varying altitudinal zone. Barnes, Flint & Caldwell57

1987 have documented

higher UV-B radiation in alpine sites compared with lower habitats. Impact of solar radition

on secondary metabolites contents in plants are grown at different altitudes has also been

reported. In response to high UV radiation at high altitude, an increase in phenolic contents

has been reported.

6.3.5 Soil nutrients

Soil is the most important factors for the plants growth, mainly governed by weathering and

climatic conditions. Soil is formed from weathering of rocks. The rate of plants growth and

type of species depend upon the mineral composition of soil and parent rock. Plant nutrients

are mainly derived from weathering of minerals. The mineralogical composition studies

have importance in forestry where the plants growth lasts over a long period and depends to

a large extent on the mineral as a source of plant nutrients in the soil.

Coley58

et al., 1985 has reported variation in secondary metabolites contents occur with

resource availability.

By supply of varying nutrients in experiments, the effect of nutrients availiabilty on plants

development, physiology and tissue chemistry or stress tolerance have been investigated.

Induced biosynthesis of phenolic compounds is reported in soil contains low level of iron59

.

On the other side, increased proanthocyanidins contents were reported in soil contains

limited phosphate60

.

Soil nutrients are directly linked with biosynthesis of secondary metabolites as well as their

abudance or limited availability has effect on contents of secondary metabolites in plants61

.

There are several hypothesis deals with soil nutrients. Bryant et al., (1983) proposed the


80

carbon/nitrogen balance (CNB) hypothesis and postulated that fertilization in presence of

limited available nutrients will cause low production of carbon-based secondary metabolites.

According to CNB hypothesis contents of secondary metabolites deposition in plant tissues

depends on relative excess of plant resources, especially nitrogen. Generally, the primary

metabolism is necessary for plants growth and development and in priority for plants over

secondary metabolism, after fullfiment of requirement of carbon and nitrogen in primary

metabolism plants growth, they are allocated for secondary metabolites production.

However intermediates of primary metabolism are precursor of secondary metabolism and

precursor contents determine the production rate of secondary metabolites62

. In deficiency of

nitrogen in plants, CNB predicts that carbohydrates will accumulate in plant tissues and

stimulate the production carbon-based secondary compounds. From the result obtained it is

interpreted that the accumulation of carbon-based/N-based compounds depends on the

availability of both elements. In deficiency of nitrogen elements, biosynthesis of C-based

compounds increases and vice versa. Nitrogen containing compounds like cyanogenic

glycosides and alkaloids contents was reported low in soil of nitrogen deficiencies and

results of more experiments has found consistent with these predictions59,60,63

, yet CNB also

unsuccessful repeatedly in other similar studies64,65

. Jones & Hartley66

1999 have given

protein competition model (PCM) of phenolic distribution in plants, in line with CNB

theory. According to protein competition model (PCM) production of phenolic compounds

depend on constituetnt derived from metabolic pathways or pathways precursors and are

regulated by biochemical mechanism. Diallinas & Kanellis, 1994 have reported that

biosynthesis of alkaloid and phenolic compounds are catalysed by Phenylalanine ammonia-

lyase (PAL) and utilize the amino acid phenylalanine as a precursor. Findings show that


81

there is competition between protein and metabolite synthesis for the limiting phenylalanine

and leading to prcess level exchange among rate of protein vs metabolite synthesis. Jones &

Hartley66

, 1999 have concluded in terms of synthesis rate, if rate of protein synthesis are

high the rate of phenolic synthesis should be low and vice versa. Finding of these two

theories clearly revealed that rate of secondary metabolite biosynthesis is consequence of the

interaction between the plant’s intrinsic (genetic) and extrinsic (environmental) factors.

6.3.6 Soil water availability

In continuation of effect of soil nutrients, soil water availability is also one of the important

factors in plants growth and development. Magnitude of drought and period are usually

documented in many environments and can severely impact on plant’s stress tolerance and

survival. Photosynthetic rate are reduced under less availability of soil water or drought

condition. As of result of reduction of photosynthetic rate, the rate of plants growth is also

reduced. Alonso-Amelot32

et al., 2007; Glynn67

et al., 2004 have reported induce phenolic

biosynthesis in high temperature and reduced water availability. Biosynthesis of secondary

metabolites is dependent on primary metabolism and primary metabolism are effected by

photosynthetic rate.During reduced water availability, plants close their stomata and restrict

photosynthesis and therefore one might expect negative relationship between biosynthesis of

secondary metabolites and water shortage. That why phenolic and saponin contents and their

pharmacological activity were reported to vary seasonally in medicinal bulbs39

. Horner68

,

1990 have suggested that there is relation between tannin synthesis and xylem pressure,

however it can be positive or negative depends on soil water availability to the plants.

6.3.7 Stress factors and their effect on quality


82

Plants are faced to several fluctuations in environmental conditions during the day and night,

between the days and even during growng season. Not only abiotic factors, but also biotic

factor exerts pressure on plants. As a result of these pressures several changes in

physiological and biochemical processes in plants have been reported. According to

Niinemets69

, 2010 the ability of plants to sense stress rely on its timing, severity

physiological status of the plant. Biosynthesis of secondary metabolites in plants is

dependent of photosynthetic rate which affected by duration and intensities of stress factors

and limites carbon and energy distribution for the biosynthesis of secondary metabolites and

therefore quantitatively affect their levels in plants. However effect of different stress factors

is different on quality and quantity of the secondary metabolites pool plants.

For pharmaceutical purpose or on industrial level it is important to account effect of each

stress factors on production of secondary metabolites for their commercialization. Studying

and monitoring of these stress factors duration and frequency that favour optimum

deposition of desired active metabolites of interest open an opportunity for people to exploit

plants as factories of natural chemical for producing pharmaceuticals.

6.3.7. a Biotic stress factor

In addition to climatic factors, biotic stress factor is one of the important factors in which

plants are exposed in environment of multiple herbivore and pathogenic attacks59,5

.

Increased release of inducible secondary metabolites is reported in plants parts exposed to

herbivores70,71

. Numerous agents counting induced hormones participated in perception,

transduction and proliferation of stress signal and foremost to a huge number of secondary

metabolites being free as a result of activation of biosynthesis pathways in addition to the


83

omnipresent stress compounds. In case of biotic stress, the type of compound relies on the

attacking organism69

.

6.3.7. b Abiotic stress factor

Abiotic stress represents non-living environment factors and also considere as important

factor which have impact of biosynthesis of secondary metabolites. Holopainen and

Gershenzon5 2010 have reported that on Mechanical damage to plant foliage stimulated

biosynthesis of second metbaolites while artificial damage and compositional diversity, as

biotic stress do not produce the same result.

Effect of ozone on secondary metabolites biosynthesis in plants is reported by Eckey-

Kaltenbach72

et al., 1994; Jordan73

et al., 1991. In experiment under greenhouse condition,

elevated ozone levels increased the contents of terpenes, but decreased the phenolic contents

in Ginkgo biloba leaves74

. Effect of inorganic salt stress factor has also been assessed in

plant secondary metabolism. Elevated levels of alkaloids are reported in Achnatherum

inebrians plants cultivated under salt stress.

6.3.8 Exposure to various stress factors

Plants are exposed to multiple stresses instead of single abiotic factors one by one, under

natural conditions. Holopainen & Gershenzon5, 2010 have reported unpredictable and

complex reponse of plants in case of seasonal climatic changes which include variety of

different stress combination factors. Gouinguene & Turlings75

2002 have documented effect

of two or more factors when co-occur are sometimes complementary, however in other

cases the influence of one is dominating over other. They have studied the combinational

effect of high temperature and simulated lepidopteran herbivory on plants secondary


84

metabolites contents, resulted in greater metabolite accumulation than when either stress

was applied alone by. On the basis of results obtained from experiments it can be concluded

that multiple stress factors are more diverse than single stress factor. Researchers are aimed

to understand this diversity and its importance in that the plants response to several stress

combinations and which cannot always be concluded from responses to individual stress

factors. Report on influence of multiple stresses on deposition of secondary metabolite in

plants will provide more information to assess the biological activities of these metabolites

in explanatory stress and give criteria for describing their optimum contents and quality and

therefore provide a means of ensuring quality in phytomedicine.

6.4 Solution to quality problem

Cultivation of medicinal plants would appear to be an important strategy to achieve the

continuous demand of medicinal plants with uniform quality76

. Yet, the total number of

cultivated species of medicinal plants on any scale is very small77

.

Schippmann78

et al., (2002) have reported that China is the country with the largest land of

medicinal plants under cultivation, but, nonetheless, only 100–250 species are cultivated.

It is assumed that the cultivated medicinal plants are qualitatively substandard when

compared with wild specimens78

. In several case studies, the boundaries of cultivation as an

alternative to wild harvest have been examined79,77,80

. Cultivation strategy is used to enhance

the production of most medicinal plant species will prolong to be harvested from the wild to

some level. Regulation on different factors that affect specific secondary metabolites of

interest can be achieved by cultivation of medicinal plants under controlled environments

like greenhouses. By altering various stress factors like mineral nutrition, temperature,

water, light and salt stress etc, in cultivation setups have previously been investigated, with


85

different level of success67,75,74,63,81

. In in vitro manipulation and preservation of external

stress factors in plant cultures and cell under controlled environments could be an additional

potential basis of ensuring uniform quality provide of phytotherapies82,83,84,85

.

Cultivation of medicinal plants under controlled environment, where stress factors and

disorder could be looked at as uniform and quality of metabolites could at least be

ascertained with some level of inevitability.

6.5 Conclusion

Study of influence altitudinal variation on secondary metabolites contents is not easy, due to

a huge diversity of possible added factors like herbivory and season, which might also basis

of variation in secondary metabolite profiles. There are different theories which explain

conflicting trends for different classes of secondary metabolites. Therefore, it is important to

study the effect of altitudinal variations on secondary metabolite contents systematically.

The effect of altitudinal variation on secondary metabolites contents in higher plants has

been assessed but there is no literature available on effect of altitudinal variation in

secondary metabolites contents of Bergenia ciliata. The studies on altitudinal variation

effect on secondary metabolite contents in higher plants depart few interesting questions

unanswered. Therefore, there is need to investigate more number of species to find out

whether the observed trends are taking place in the majority of plants or are restricted to the

investigated model species. In addition, it is important to investigate genetic adaption to

higher altitudes by growing wild plants resulting from changeable altitudes under uniform

conditions. Will these plants have variable secondary metabolite contents, too? An

additional question is the interaction of different abiotic ecologic factors, particularly

temperature and radiation. Are there any other effects in the impact of higher radiation and


86

low temperature in phenolic biosynthesis and storage in plants at high altitude? If extrinsic

factor (biotic and abiotic stresses) induces biosynthesis of secondary metabolites, then a

potential exists to use such stresses as a tool to improve the quality as well as

pharmacological properties of plant material. For quantitative assessment of secondary

metabolites it is important to understand the elicitation of induced metabolites and their

stability.

Understanding difficult interactions between external factors and the plant's metabolic

system is possibly the ultimate possible strategy of ensuring quality in phytochemicals.

However, it must be point out those plants responses to stress factors is species specific. A

combined approach includes ecology, molecular physiology and biochemistry would have

immense potential to precede this field and unravel the degree to which plant–environment

interactions contribute to phytochemicals. In conclusion, study of effect of altitudinal

variation in plant secondary metabolites contents offers a wide range for future studies,

applicable for both ecology and medicinal plant research.


87

REFERENCES

1. Gangwar, K.K., Deepali, Gangwar, R.S. (2010). Ethnomedicinal plant diversity in

Kumaun Himalaya of Uttarakhand, India. Nat. Sci., 8(5), 66-78.

2. Shrestha, P.M., Dhillion, S.S. (2003). Medicinal plant diversity and use in the

highlands of Dolakha district. Nepal. J. Ethnopharmacol., 86, 81-96.

3. Shiva, M.P. (1996). Inventory of Forestry Resources for Sustainable Management

and Biodiversity Conservation. New Delhi: Indus Publishing Company.

4. Kala, C.P., Dhyani, P.P., Sajwan, B.S. (2006). Developing the medicinal plants sector

in northern India: challenges and opportunities. Journal of Ethnobiology and

Ethnomedicine. (2): http.//www. ethnobiomed.com/content/ 2/1/32.

5. Holopainen, J.K., Gershenzon, J. (2010). Multiple stress factors and the emission of

plant VOCs. Trends in Plant Science, 15, 176–184.

6. Dangle, J.L., Jones, J.D.G. (2001). Plant pathogens and integrated defence responses

to infection. Nature, 411, 826–833.

7. Loreto, F., Schnitzler, J.-P. (2010). Abiotic stresses and induced biogenic volatile

organic compounds. Trends in Plant Science, 15, 154–166.

8. Szathmary, E., Jordán, F., Pal, C. (2001). Can genes explain biological complexity?

Science, 292, 1315–1316.

9. Osbourn, A.E., Qi, X., Townsend, B., Qin, B. (2003). Dissecting plant secondary

metabolism - constitutive chemical defenses in cereals. New Phytologist, 159, 101–108.

10. Wink, M. (2003). Evolution of secondary metabolites from an ecological and

molecular phylogenetic perspective. Phytochemistry, 64, 3–19.


88

11. Dewick, P.M. (2002). Medicinal Natural Products: A Biosynthetic Approach.

Chichester, West Sussex, England, Wiley.

12 .Dey, P.M., Harborne, J.B. (1989). Methods in Plant Biochemistry. Plant Phenolics,

Academic Press, New York, 1.

13. Crozier, A., Jaganath, I.B., Clifford, M.N. (2006). Phenols, polyphenols and tannins:

an overview. In: Crozier, A., Clifford, M.N., Ashihara, H. (Eds.), Plant Secondary

Metabolites - Occurrence, Structure and Role in the Diet. Blackwell Publishing, Oxford.

14. Oksman-Caldentey, K. & Inze, D. (2004). Plant cell factories in the post-genomic era:

New ways to produce designer secondary metabolites. Trends Plant Sci., 9(9), 433–440.

15. Taiz, L. & Ziegler, E. (2006). Plant Physiology. Sunderland, M.A, U.S.A., Sinauer

Associates.

16. Facchini, P. (1999). Plant secondary metabolism: out of the evolutionary abyss.

Trends in Plant Science, 4, 382–384.

17. Harborne, G. (1982). Introduction to Ecological Biochemistry. Academic Press,

London.

18. Laughlin, J.C. (1993). Effect of agronomic practices on plant yield and antimalarial

constituents of Artemisia annua L. Acta Horticulturae, 331, 53–61.

19. Lommen, W.J.M., Bouwmeester, H.J., Schenk, E., Verstappen, F.W.A., Elzinga, S.,

Struik, P.C. (2008). Modelling processes determining and limiting the production of

secondary metabolites during crop growth: the example of antimalarial artemisinin

produced in Artemisia annua. Acta Horticulturae, 765, 87–94.


89

20. Perez-Estrada, L.B., Cano-Santana, Z., Oyama, K. (2000). Variation in leaf trichomes

of Wigandia urens: environmental factors and physiological consequences. Tree

Physiology, 20, 629–632.

21. Spitaler, R., Schlorhaufer, P.D., Ellmerer, E.P., Merfort, I., Bortenschlager, S., Stuppner,

H., Zidorn, C. (2006). Altitudinal variation of secondary metabolite profiles in flowering

heads of Arnica montana cv. ARBO. Phytochemistry, 67, 409–417.

22. Spitaler, R., Winkler, A., Lins, I., Yanar, S., Stuppner, H., Zidorn, C. (2008). Altitudinal

variation of phenolic contents in flowering heads of Arnica montanacv. ARBO: a 3-year

comparison. J. Chem. Ecol., 34,369–375.

23 .Ganzera, M., Guggenberger, M., Stuppner, H., Zidorn, C. (2008). Altitudinal variation

of secondary metabolite profiles in flowering heads of Matricaria chamomilla cv.

BONA. Planta Med., 74, 453–457.

24. Korner, C. (1999). Alpine plant life. Functional plant ecology of high mountain

ecosystems. Springer, Berlin

25. Bilger, W., Rolland, M., Nybakken, L. (2007). UV Screening in higher plants induced

by low temperature in the absence of UV-B radiation. Photochem. Photobiol. Sci., 6,

190–195.

26. Markham, K.R., Ryan, K.G., Bloor, S.J., Mitchell, K.A. (1998a). An increase in the

luteolin : apigenin ratio in Marchantia polymorpha on UV-B enhancement.

Phytochemistry, 48, 791–794.

27. Wellmann, E. (1975). UV dose-dependent induction of enzymes related to flavonoid

biosynthesis in cell suspension cultures of parsley. FEBS Lett., 51, 105–107.


90

28. Jaakola, L., Maatta-Riihinen, K. (2004). Activation of flavonoid biosynthesis by solar

radiation in bilberry (Vaccinium myrtillus L.) leaves. Planta, 218, 721–728.

29. Bakus, G.J., Green, G. (1974). Toxicity in sponges and holothurians: a geographic

pattern. Science. 185, 951–953.

30. Siska, E.L., Pennings, S.C., Buck, T.L., Hanisak, D.M. (2002). Latitudinal variation in

palatability of salt-marsh plants: which traits are responsible? Ecology, 83, 3369–3381.

31. Alonso-Amelot, M.E., Oliveros-Bastidas, A., Calcagno-Pisarelli, M.P. (2004). Phenolics

and condensed tannins in relation to altitude in neotropicalPteridium spp. A field study

in the Venezuelan Andes. Biochem. Syst. Ecol. 32,969–981.

32. Alonso-Amelot, M.E., Oliveros-Bastidas, A., Calcagno-Pisarelli, M.P. (2007). Phenolics

and condensed tannins of high altitude Pteridium arachnoideum in relation to sunlight

exposure, elevation, and rain regime. Biochem. Syst. Ecol. 35, 1–10.

33. Zobayed, S.M.A., Afreen, F., Kozai, T. (2005). Temperature stress can alter the

photosynthetic efficiency and secondary metabolite concentrations in St. John's Wort.

Plant Physiology and Biochemistry, 43, 977–984.

34. Chalker-Scott, L. (1999). Environmental significance of anthocyanins in plant stress

response. Photochemistry and Photobiology, 70, 1–9.

35. Choi, S..,Kwon, Y.R., Hossain M.A.,Hong S.,Lee B.&Lee H. (2009). A mutation in

ELA1, an age dependent negative regulator of PAP1/MYB75, causes UV-and cold

stress tolerance in Arabidopsis thaliana seedlings. Plant Science, 176, 678–686.

36. Parker, J. (1977). Phenolics in black oak bark and leaves. Journal of Chemical

Ecology, 3, 489–496.


91

37. Sharkey, T.D., Yeh, S.S. (2001). Isoprene emission from plants. Annual review in

plant physiology. Plant Molecular Biology, 52, 407–436.

38. Pennycooke, J.C., Cox, S., Stushnoff, C. (2005). Relationship of cold acclimation,

total phenolic content and antioxidant capacity with chilling tolerance in petunia

(Petunia×hybrida). Environmental and Experimental Botany, 53, 225–232.

39. Ncube, B., Finnie, J.F., Van Staden, J. (2011a). Seasonal variation in antimicrobial

and phytochemical properties of frequently used medicinal bulbous plants from South

Africa. South African Journal of Botany, 77, 387–396.

40. Dela, G., Or, E., Ovadia, R., Nissim-Levi, A., Weiss, D. & Oren- Shamir, M. (2003).

Changes in anthocyanin concentration and composition in ‘Jaguar’ rose flowers due to

transient hightemperature conditions. Plant Science, 164, 333–340.

41. Shvarts, M., Borochov, A. & Weiss, D. (1997). Low temperature enhances petunia

flower pigmentation and induces chalcone synthase gene expression. Physiologia

Plantarum, 99, 67– 72.

42. Albert, A., Sareedenchai, V., Heller, W., Seidlitz, H.K. & Zidorn, C. (2009).

Temperature is the key to altitudinal variation of phenolics in Arnica montana L. c.v.

ARBO. Oecologia, 160, 1–8.

43. Schmidt, S., Zietz, M., Schreiner, M., Rohn, S., Kroh, L.W. & Krumbein, A. (2010).

Genotypic and climatic influences on the concentration and composition of flavonoids

in kale (Brassica oleracea var. sabellica). Food Chemistry, 119, 1293–1299.

44. Sarala, M.,Taulavuori, K.,Taulavuori, E., Karhu, J. & Laine, K. (2007). Elongation of

Scots pine seedlings under blue light depletion is independent of etiolation.

Environmental and Experimental Botany, 60, 340–343.


92

45. Pecot, S.D., Horsley, S.B., Battaglia, M.A. &Mitchell, R.J. (2005). The influence of

canopy, sky condition, and solar angle on light quality in a longleaf pine woodland.

Canadian Journal of Forest Research, 35, 1356–1366.

46. Kazan, K., Manners, J.M. (2011). The interplay between light and jasmonate

signalling during defence and development. Journal of Experimental Botany, 62, 4087–

4100.

47. Rozema, J., Van de Staaij, J., Bjorn, L.O., Calwell, M. (1997). UV-B as an

environmental factor in plant life: stress and regulation. Trends in Ecology &

Evolution, 12, 22–28.

48. Winkel-Shirley, B. (2002). Biosynthesis of flavonoids and effects of stress. Current

Opinion in Plant Biology, 5, 218–223.

49. Kliebenstein, D.J. (2004). Secondary metabolites and plant/environment

interactions: a view through Arabidopsis thaliana tinged glasses. Plant, Cell &

Environment, 27, 675–684.

50. Alias, Y., Awang, K., Hadi, H.A., Thoison, O., Seavenet, T., Paoas, M. (1995). An

antimitotic and cytotoxic chalcone from Fissistigma lanuginosum. Journal of Natural

Products, 58, 1160–1166.

51. Burda, S., Oleszek, W. (2001). Antioxidant and antiradical activities of flavonoids.

Journal of Agriculture and Food Chemistry, 49, 2774–2779.

52. Gurib-Fakim, A. (2006). Medicinal plants: traditions of yesterday and drugs of

tomorrow. Molecular Aspects of Medicine, 27, 1–93.


93

53. Iinuma, M., Tsuchiya, H., Sato, M., Yokoyama, J., Ohyama, M., Ohkawa, Y., Tanaka,

T., Fujiwara, S., Fujii, T. (1994). Flavanones with antibacterial 18 B activity against

Staphylococcus aureus. Journal of Pharmacy and Pharmacology, 46, 892–895.

54. Williams, C.A., Harborne, J.B., Geiger, H., Hoult, J.R.S. (1999). The flavonoids of

Tanacetum parthenium and T. vulgare and their anti-inflammatory properties.

Phytochemistry, 51, 417–423.

55. Okuda, T., Yoshida, T., Hatano, T. (1992). Pharmacologically active tannins isolated

from medicinal plants. In: Hemingway, R.W., Laks, P.E. (Eds.), Plant Polyphenols:

Synthesis, Properties, Significance. Plenum Press, New York.

56. Haslam, E. (1989). Plant Polyphenols: Vegetable Tannins Revisited. Cambridge

University Press, Melbourne.

57. Barnes, P.W., Flint, S.D. & Caldwell, M.M. (1987). Photosynthesis damage and

protective pigments in plants from a latitudinal arctic/alpine gradient exposed to

supplemental UV-B radiation in the field. Arctic and Alpine Research, 19, 21–27.

58. Coley, P.D., Bryant, J.P., Chapin, S.F. (1985). Resource availability and plant

antiherbivore defence. Science, 230, 895–899.

59. Dixon, R.A., Paiva, N.L. (1995). Stress-induced phenylpropanoid metabolism. The

Plant Cell, 7, 1085–1097.

60. Kouki, M., Manetas, Y. (2002). Resource availability affects differentially the levels

of gallotannins and condensed tannins in Ceratonia siliqua. Biochemical Systematics

and Ecology ,30, 631–639.

61. Herms, D.A., Mattson, W.J. (1992). The dilemma of plants: to grow or defend. The

Quarterly Review of Biology, 67, 282–335.


94

62. Reichardt, P.B., Chapin III, F.S., Bryant, J.P., Mattes, B.R., Clausen, T.P. (1991).

Carbon/nitrogen balance as a predictor to of plant defence in Alaskan balsam poplar:

potential importance of metabolite turnover. Oecologia, 88,401–406.

63. Simon, J., Gleadow, R.M., Woodrow, I.E. (2010). Allocation of nitrogen to chemical

defence and plant functional traits is constrained by soil. N. Tree Physiology, 30, 1111–

1117.

64. Koricheva, J., Larsson, S., Haukioja, E., Keinanem, M. (1998). Regulation of woody

plant secondary metabolism by resource availability: hypothesis testing by means of

meta-analysis. Oikos, 83, 212–226.

65. Riipi, M., Ossipov, V., Lempa, K., Haukioja, E., Koricheva, J., Ossipova, S., Pihlaja, K.

(2002). Seasonal changes in birch leaf chemistry: are there trade-offs between leaf

growth and accumulation of phenolics? Oecologia, 130, 380–390.

66. Jones, C.G., Hartley, S.E. (1999). A protein competition model of phenolic allocation.

Oikos, 86, 27–44.

67. Glynn, C., Ronnberg-Wastljung, A.C., Julkunen-Tiitto, R., Weih, M. (2004). Willow

genotype, but not drought treatment, affects foliar phenolic concentrations and leaf-

beetle resistance. Entomologia Experimentalis et Applicata, 113, 1–14.

68. Horner, J.D. (1990). Nonlinear effects of water deficits on foliar tannin

concentration. Biochemical Systematics and Ecology, 18, 211–213.

69. Niinemets, U. (2010). Mild versus severe stress and BVOCs: thresholds, priming

and consequences. Trends in Plant Science, 15, 145–153.

70. Bernays, E.A., Chapman, R.F. (2000). Plant secondary compounds and grasshoppers:

beyond plant defences. Journal of Chemical Ecology, 26, 1774–1794.


95

71. Hagerman, A.E., Butler, L.G. (1991). Tannins and lignins. In: Rosenthal, G.A.,

Berenbaum, M.R. (Eds.), Herbivores. Their Interactions with Secondary Plant

Metabolites, Academic Press, London.

72. Eckey-Kaltenbach, H., Ernst, D., Heller, W., Sandermann, H. (1994). Biochemical

plants responses to ozone. IV. Cross-induction of defensive pathways in parsley

(Petroselinum crispum L.) plants. Plant Physiology, 104, 67–74.

73. Jordan, D.N., Green, T.H., Chappelka, A.H., Lockaby, B.G., Meldahl, R.S., Gjerstad,

D.H. (1991). Response of total tannins and phenolics in loblolly pine foliage exposed to

ozone and acid rain. Journal of Chemical Ecology, 17, 505–513.

74. He, X., Huang, W., Chen, W., Dong, T., Liu, C., Chen, Z., Xu, S., Ruan, Y. (2009).

Changes of main secondary metabolites in leaves of Ginkgo biloba in response to ozone

fumigation. Journal of Environmental Sciences, 21, 199–203.

75. Gouinguene, S.P., Turlings, T.C.J. (2002). The effects of abiotic factors on induced

volatile emissions in corn plants. Plant Physiology, 129, 1296–1307.

76. Uniyal, R.C., Uniyal, M.R., Jain, P. (2000). Cultivation of Medicinal Plants in India.

TRAFFIC India and WWF India, New Delhi.

77. Hamilton, A.C. (2004). Medicinal plants, conservation and livelihoods. Biodiversity

and Conservation, 13, 1477–1517.

78. Schippmann, U., Leaman, D.J., Cunningham, A.B. (2002). Impact of Cultivation and

Gathering of Medicinal Plants on Biodiversity: Global Trends and Issues. FAO, Rome.

79. CBD. (1992). Convention on biological diversity. http://www.cbd.int/doc/legal/ cbd-

en.pdf (accessed on 01/08/2010).

http://www.cbd.int/doc/legal/


96

80. Sheldon, J.W., Balick, M.J., Laird, S.A. (1997). Medicinal plants: can utilization and

conservation coexist? Advances in Economic Botany, 12, 1–104.

81. Zhang, X.-X., Li, C.-J., Nan, Z.-B. (2011). Effects of salt and drought stress on

alkaloid production in endophyte-infected drunken horse grass (Achnatherum

inebrians). Biochemical Systematics and Ecology, 39, 471–476.

82. Aoyagi, H. (2011). Application of plant protoplasts for the production of useful

metabolites. Biochemical Engineering Journal, 56, 1–8.

83. Bruce, R.J., West, C.A. (1989). Elicitation of lignin biosynthesis and isoperoxidase

activity by pectic fragments in suspension cultures of castor bean. Plant Physiology, 91,

889–897.

84. Kim, D.J., Chang, H.N. (1990). Enhanced shikonin production fromLithospermum

erythrorhizon by in situ extraction and calcium alginate immobilization. Biotechnology

and Bioengineering, 36, 460–466.

85. Ncube, B., Ngunge, V.N.P., Finnie, J.F., Van Staden, J. (2011b). A comparative study

of the antimicrobial and phytochemical properties between outdoor grown and

micropropagated Tulbaghia violacea Harv. plants. Journal of Ethnopharmacology, 134,

775–780.


97

7. Review on biosynthesis of terpenoid compounds

7.1 Introduction

Plants are known as natural chemist and produce large number of low molecular weight

organic compounds known as secondary metabolites1. More of secondary metabolites are

molecules of lipophilic in nature with low boiling points and high vapor pressures at

ambient temperature. These secondary metabolites are commonly classified as isopernoids,

phenylpropanoids/benzenoids, amino acid derivatives and fatty acid derivatives. Out of them

isoprenoid compounds are released from flowers, fruits and leaves into surroundings.

Among all groups of secondary metabolites isoprenoid compounds are reported as one of

largest group of compounds; more than, approximately or more than 40,000 terpenoid

compounds have been documented, and increasing constantly. Figure-15 represents

chemical structure of vitamin A which is reported for photoreceptor activity in animals and

as the chromophoric element of a light-driven proton pump in certain bacteria. Figure-16

represents chemical structure of P-carotene which act as lightprotecting pigments in plants

and also known for having anticarcinogenic activity. Figure-17 represents chemical structure

of steroids, such as cholesterol which is understood as essential components of eucaryotic

cell membranes. The phytol (Figure-18) of chlorophyll, one of the most plentiful organic

molecules, serves to fix the photoreceptor molecule in lipophilic environments.


98

OH

RO

HO

Figure-15

Figure-16

Figure-17

Figure-18

Figure-15 Chemical structure of vitamin A

Figure-16 Chemical structure of P-carotene

Figure-17 Chemical structure of cholesterol

Figure-18 Chemical structure of chlorophyll

Other principal functions of volatiles compounds are to defend plants against pathogens and

herbivores, seed dispersers, to attract pollinators and others animals and microorganisms,


99

and to give out as signals in plant–plant interaction. Isoprenoid compounds are not only

contribute to plant survival but also responsible for in general reproductive success in

natural ecosystems. The impact of these compounds on agronomic and other commercial

traits, adding their yield and food quality, suggests that amendment of volatile production

via genetic engineering has the potential to improve cultivated plant species. These

compounds are classified as Monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids

(C20) and triterpenoids (C30) are their multiples and considered to be secondary metabolites

(Figure-19).


100

OPP OPP

IPP DMAPP

OPP

GDP

GDP Synthase

OHOH

Linalool Pinene Perilla alcohol

M

O

N

O

T

E

P

EN

O

I

DS

PPO

O

O

O

H

O

O

Artemisinin (E,E)-Farnesene

SE

S

Q

UI

T

E

RP

E

N

O

I

D

S

Sesquiterpene synthase+/- modifying enzymes

Monoterpenes synthase

+/- modifying enzymes

FDP synthase

Squalene synthase

Squalene epoxidase

OPP

GGDP

GGDP synthase

CHO

CHO

CHO

CHO

O

NHO

OAc O OH

OH OBz

HO

AcO

Diterpenes synthase


Polygodial

Paclitaxel

D

I

TE

R

P

E

N

O

I

D

S

OSqualene-2,3-epoxide

Triterpene synthase


OAc

OH

O

OH

OH

HO

H

O

H

Cucurbitacin C

O

O O

O

H

O

H

COOH

OH

HOHO

HO

HOOC

Glycyrrhizin

T

R

I

T

E

R

P

E

N

O

I

D

S

StrawberryAnti-malarial drugs

Myzus persicae Cucumber leaf

Figure-19 Schematic representation of biosynthesis of monoterpenoids, sesquiterpenoids,

diterpenoids and triterpenoids from IPP or DMAPP. Abbreviation; IPP, Isopentenyl

pyrophosphate and DMAPP; Dimethyl allyl pyrophosphate


101

Being aromatic in nature these terpenoid compounds are commercially interesting in

agriculture products, like the fragrance of flowers (e.g. linalool) and flavor of fruits (Figure-

19). Aharoni2 et al., 2004; Pichersky

3 et al., 1994 have reported the use of these compounds

as flavors and fragrances in foods and cosmetics (e.g. menthol, nootkatone and sclareol).

The ecological and commercial importance of these compounds makes their metabolic

engineering an attractive subject for investigation. Bascially, metabolic engineering needs

understanding of the steps in biochemical pathways and isolation and identification of genes

and enzymes involved in the biosynthesis of terpenoid compounds. While, via genetic

engineering many input and output traits in plants can be achieved. Alteration via genetic

engineering includes enhanced fragrance of ornamentals and pollination of seed crops,

prevent from disease and pest resistance, production of allelopathic compounds in weed

control and synthesis of pharmaceuticals in plants. Transgenic plants with improved

terpenoid compounds production could build an important contribution to basic studies of

the biosynthesis and their importance in ecological relationships.

Lucker4 et al., 2006; Dudareva & Pichersky

5, 2006; Degenhardt

6 et al., 2003; Aharoni

7,

2005 have reported importance of metabolic engineering in enhancement of aroma quality

of flowers and fruits. However, some project have been successful in achieving the desired

results, many of them have given insufficient increment of terpenoid contents or or in other

unexpected metabolic consequences like additional metabolism of the future end products or

deadly effects on plant growth and development. Lichtenthaler8, et al., 1997 have reported

two distinct pathways to branched five-carbon unit isopentenyl diphosphate (IPP) and its

interconvertible isomer dimethylallyl diphosphate (DMAPP) which lead biosynthesis of

terpenoid compounds. Out of both biosynthetic pathways eukaryotic mevalonic acid (MVA)


102

pathway localized in the cytoplasm, and 2-C-methyl-d-erythritol (ME) 4-phosphate (MEP)

pathway (Figure-20) that occurs in the plastids9,10

.

Some isoprenoids (i.e. sterols and the side chain of mitochondrial ubiquinone) are

biosynthesized from MVA-derived IPP, whereas others [i.e. isoprene (C5), monoterpenes

(C10), diterpenes (C20, including gibberellins and the phytyl tail of tocopherols and

chlorophylls) and carotenoids (C40)] originate from IPP and are biosynthesized via the

MEP/DXP pathway in plastids. Most sesquiterpenes (C15) are synthesized from the MVA

pathway in the cytosol, although others such as the phytohormone abscisic acid are

synthesized from the MEP/DXP pathway by the specific cleavage of carotenoids11

.

Folkers, Tavormina and coworkers11,12

indicated that the IPP and DMAPP are

biosynthetically derived from mevalonate. Details of steps of MVA pathways in yeast and

animal cells are explained by work Bloch, Cornforth, Lynen, Popjak and coworkers13,14,15,16

.

Since the breakthrough of the mevalonate pathway, many studies on the biosynthesis of

isoprenoids in a wide range of species have been published. Furthermore, there is facts that a

certain degree of crosstalk between the MVA and MEP/DXP pathways can occur, implying

that these pathways are not completely self-governing17

. In this review we highlight the

latest stimulating reported developments in the engineering of terpenoids compounds in

plants and discuss cited evidence regarding crosstalk between MVA and DXP pathways.

7.2 Screening of MVA pathways by feeding experiment

It was only much later (ca. 1955) shown that the biosynthesis of isoprenoid compounds does

indeed occur starting from isoprene-like C5 building blocks. Labeling experiments, using

14C-labelled acetic acid, showed early on that the steroid skeleton is constructed from this

C5 building block, but not simply through regular head-to-tail coupling reactions: First


103

incorporations are done in the biosynthesis of cholesterol in liver tissues and ergosterol in

yeast. Results of incorporation exposed that a biosynthetic pathway (Scheme-1), initial from

acetate 1, activated as acetyl CoA A 1, and yielding isopentenyl diphosphate 2 which is the

biological alike of isoprene unit and presents the fundamental branched C5 skeleton of the

isoprene unit18,19

. Further, prenyltransferase catalyze the condensation of IPP 5 into

monterpenes precursor gerenyl diphosphate (GPP), the sesquiterpene precursor farnesyl

diphosphate (FPP) and the diterpene and C40 carotenoid precursor (GGPP) (Scheme-1).

Squalene is produced via condensation of two units of (FPP) and FPP is precursor for

tritepenes and sterols.

SCoA

O

+

SCoA

O

SCoA

O O

O-

HO

SCoA

O

OO-

HO

OH

O

OPPOPP

1

2

3

4

5

SCoA

O

2 NADPH

1 2

34

3 ATP

5 6

Scheme-1 Mevalonic acid pathway for biosynthesis of isoprenoid compounds


104

In many cases, yet the results obtained on biosynthesis of isoprenoid compounds could not

be explained by MVA pathway. To elucidate the differing data some another hypothesis

were made, like connecting the biosynthesis of branched chain amino acids. Theses

hypothesize were reviewed repeatedly and lead into existence of another biosynthetic route

which is independent to MVA pathway16

. However, there is rising experimental facts that

there is exchange of intermediates between these building blocks units (IPP and

DMAPP)16,20,21,22,23,17,24

. The MVA pathway is usually considered to provide the precursor

for the invention of sesquiterpenes and their multiples. Similarly, in the plastids, the DXP

pathway supplies the precursor for biosynthesis of isoprene, monoterpenes, diterpenes and

their multiples.


105

O

CoA

HMG-CoA MVA IPP

DMAPP

Cytokinins

FDP

Squlaene

Plastid

GlyAld-3P Pyruvate

DXP

MEP

DMAPP IPP

GDP

Monoterpenes

DMAPP

Carotenoids

Diterpenes

GibberellinsPlastoquinonePhylloquinone

DXS

DXR

HDR+

ABA

Mitochondrion

IPP

DMAPP FDP

Ubiquinone

Terpenes Oxidised terpens

Terpenes Reduced terpenes

Reductase

Sterols

BRs

Triterpenes

Sesquiterpens

PolyprenolsPrenylation

Cystol

MVA biosynthesis (Scheme-1) was universally accepted for the biosynthesis of all

terpenoids in all livings notwithstanding some conflicting results obtained in the field of

isoprenoid biosynthesis in plants.

7.3 Contradictory results obtained in isoprenoid biosynthesis lead into discovery of

MVA independent pathway

Figure-20 Biosynthesis pathway of terpenoids in plants, Solid arrow, broken arrows with

short dashes and broken arrow with long dashes symbolize single and multiple enzymatic

steps and transport respectively.


106

Biosynthesis of terpenoid compounds via MVA pathways is universally acceptable until

contradictions with the MVA like isoprenoid precursor were made apparent in early 1950

and became more obvious. Result of incorporation of labeled MVA and acetate were very

poor into biosynthesis of carotenoids, monterpenes and diterpenes in plant

systems25,26,27,8,28,14

. In contrast to this well incorporation of labeled MVA and acetate were

found in biosynthesis of sterols, the triterpenoids and quite often also into the

sesquiterpenoids. The enzyme catalyzing the committed step of the MVA pathway,

mevinolin, an effective inhibitor of HMGCoA reductase, strongly inhibited biosynthesis of

sterol in plants, but did not influence the formation of the chloroplast pigments like

carotenoids and the chlorophylls containing the diterpenic phytyl side-chain29,30

. Such

contradictory findings were interpreted in stipulations of lack of permeability of the

chloroplast membrane towards the inhibitor or precursor. Lutke-Brinkhaus31

, 1987 reported

IPP biosynthesis via the MVA pathway in the chloroplasts, even though the probable

presence of second route was not excluded. From facts; the second MVA independent

metabolic route towards the terpenoids biosynthesis was first detected in bacteria and

afterward found to be extensive amongst phototrophic eukaryotes.

7.4 Discovery of DXP pathway

7.4.1 Biosynthesis of bacteria haponoids

Conflicting results lead into reconsideration of isoprenoid biosynthesis in plants and

microorganisms, starting in 1980s via independent studies by Rohmer, Sahm, Arigoni and

their coworkers. Flesch and Rohmer have used 13

C-lebelled acetate precursor in experiment

(Figure-21) exposed unpredicted labeling pattern in the isoprenoid moiety was found.


107

NH2OH

OHOH

In addition to existence of acetyl CoA pool which explains the labeling patterns could arise

via MVA pathway, other possibilities were also considered. Further with the help of findings

data a novel pathway the deoxyxylulose pathway was given (Scheme-2).

Figure-21 Labeling pattern of aminobacteriohopanetriol from Rhodopseudomonas palustris

supplied with [1-13

C] acetate (Flesch and Rohmer, 1988). Solid square shows the 13

C-

enrichment from MVA pathway and solid spherical dots shows the enriched 13

C from

experimental findings


108

2

H

OHH

CH2OP3

O1

C

C

O

O-

O

CH3

1

3

2

+

Glyceraldehyde-3-phosphate Pyruvate

5

432

OP

O

OH

OH

1

5

432*OP

O

O

OH

1

H

Glyceraldehydemoeity

Pyruvate moeity

5

4

32*

OP

HO

HO

1

(a)

(a) DXP Synthase(b) Thiamine diposphate(c) DXP reductoisomerase(d) MEP-Cytidylyltransferase(e) CDP-Me kinase(f) MECDP Synthase(g) HMBDP Synthase

(b)

O

5

42

*

OP

OH OH

1 OH

2-C-Methyl-D-erythritol 4-phosphate (MEP)

(c)3

5

42

*

OPOPO

OH OH

1 OH

3

N

NH2

ON

O

OHOH

HH

HH

4-(Cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-Me)

(d)

5

42

*

OPOPO

OH OH

1 OP

3

N

NH2

ON

O

OHOH

HH

HH

2-Phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol (CDP-Me2P)

(e)

5

42

*

OH OH

1 P

3O

P

O

OHO

OH

O

O (f)

2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP)

5

42

*

OPP

OH

1

3

1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBDP)

(g)

*C is derived from Glyceraldehyde-3-phosphate is derived from Pyruvate

*

*

Lost as CO2

OPPIPPDMAPP

Scheme 2 - DXP Pathway or Non-Mevalonate Pathway believed to be existing in Plastids and followed in the biosynthesis of mono and diterpenes

1-deoxy-D-xylulose 5-phosphate

**1

2

34 5

1

2

3 45

*

*

*

*

*

*

*

*

*

*

*


109

The first feeding of labeling precursor were formed using [1-13

C]- and [2-13

C]acetate by

means of two heterotrophically grown Rhodopseudomonas species (R. palustris and R.

acidophila) and by Methylobacterium organophilum32

. This experiment enable us to

determine the beginning of bacteriohopane side-chain from a D-pentose via non-oxidative

pentose phosphate metabolism and which is coupled to C-5 carbon atom to isoprenyl group

of the triterpenic moiety. However, result have showed different labeling pattern in the

isoprenic units compare to direct incorporation of acetate through the classical MVA

pathway. In Scheme-3 labeling pattern obtained from [1-13

C] were found clear. Bacteria

utilized [1-13

C] acetate as the single carbon supply following incorporation into the

glyoxylate and tricarboxylic acid cycles and were thus incorporated into IPP through further

metabolic routes that had to be recognized (Scheme-3). At first instant, these results were

taken as derive from route which had no basis to be discarded at that time, pretentious the

compartmentation of acetate metabolism and the presence of two different and non-

interconvertible activated acetate pools, even though a entirely different route could not be

barred32

. To get a conclusion about existence of other pathway, further study was performed

with Escherichia coli, [13

C] labeled acetate was used in feeding experiment in bacterium

making no hopanoids: once more the isoprenic units of the ubiquinone prenyl chain showed

the same labeling pattern from acetate as those beforehand found in the hopanoids from the

previous bacteria, representing a likely common distribution of this metabolic pathway.


110

COOH

CH3

O

CH3COOH

HOOC COOH

O

HOOCOP

CH2OP

O

OH

OH

OPP

CHO

OH

CH2OP

BA

CHO

CH

CH

CH

HC

OH

HO

OH

OH

CH2OH

COOH

C O

CH2

HC

HC

CH2OH

OH

OH

COOH

C O

CH3

CHO

HC OH

CH2OP

CH3

C O

CHHO

HC

CH2OP

OH

OPP

CHO

HC OH

CHHO

HC

HC

CH2OP

OH

OH

CH2OP

C

CH

HC

HC

CH2OP

O

HO

OH

OH

CH2OH

C O

CH2OH

CHO

CH OH

CH2OP

COOH

C O

CH3

CH3

C O

CHHO

HC OH

CH2OP

CH3

SCoA

O

OPP OPP

C

B

B

1- Glyoxylate and tricarboxylic acid cycles

2- Entner-Doudoroff pathway

3- Glycolysis

Scheme-3 Incorporation of glucose and acetate into isoprenoids; (A) Glyoxylate and tricarboxylic

acid cycles (B) GAP/Pyruvate pathway (C) MVA pathway. (B) via the non-MVA pathway (e.g. in

the isoprenoids of E. coli or of chloroplasts) or (C) via the acetyl-CoA/MVA route (e.g. in plant

sterols). (Flesch and Rohmer, 1988).


111

7.4.2 Biosynthesis of ginkgolides

Broers33

, 1994 and Schwarz34

, 1994 have reported Arigoni and his research group studies of

incorporation of dissimilar 13

C-labelled glucose samples into the sidechain of ubiquinone

isoprenoid via the bacterium E.coli and into ginkgolides in seedlings of the tree Ginkgo

bilolia. Figure-22 represents labeling pattern in ginkgolide A (15). Two 13

C atoms in five

building blocks and three 13

C atoms in one block (represented by red color) were revealed to

be transferred mutually from the 13

C-labelled carbohydrate to the ginkgolide. Schwarz34

,

1994; Nakanishi35

, & Habaguchi, 1971 have reported manifestation of the hexacyclic

diterpene results from reshuffling of the original C5-unit (linear precursors). Through

reorganization processes, C-4 of IPP (5), indicated by red color in (Figure-22), becomes

directly joined to C-Z. From the result it became clear that the three contiguous 13

C atoms

indicated that carbon atoms 1, 2 and 4 of IPP stem from a single, generally 13

C-labelled

glucose molecule. Throughout synthesis of IPP unit, one of linkages between carbon atoms

is disrupted and then rearranged (Figure-22).


112

OPP

OPP

O OO

HO

O

O

O

O

HO

Through various experimenta results analysis it was concluded that the formation of

isoprenoid precursors involved step of rearrangement and resulting in fragmentation of

three-carbon unit of glucose precursor into a two-carbon unit and an isolated carbon atom

forming part of the same precursor moiety. On the basis of above findings it is given that the

formation of these building blocks (IPP & DMAPP) proceeds via a three-carbon and a two-

carbon fragment from glucose. The obtained results could not be explained via the MVA

pathway. As mentioned above, evaluation of the biosynthetic association between glucose

and isoprenoid carbon atom is shown in Figure-23 from the studies of biosynthesis of

hopanoids and ubiquinones in bacteria Zymomonas mobilis, Methylobacterium fujisawaenes,

Figure-22 Incorporation of D-[U-13

C6] glucose into ginkgolide A (13) in seedlings

of G. biloba [10, 56]. Building blocks of carbon atoms that were jointly diverted to

the ginkgolide from individual, universally 13C-labeled glucose molecules are

connected by red bold line.


113

E.coli and Alicyclobacillus acidoterrestris by Rohmer, Sahm and their co-workers36

degradation of glucose (14) yields equimolar amounts of the triose phosphates

glyceraldehydes phosphate (15) and dihydroxyacetone phosphate (16). Both are

interconverted by triose phosphate isomerase, each triose carbon can acquire label from the

bottom as well as top part of the glucose molecule (Figure-23a). Labeled [1-13

C] or [6-13

C]

glucose incorporated into ubiquinone by E.coli33,36

and into hopanoids by A.acidoterrestris36

resulted in labeling at C-1 and C-5 atom of IPP unit (5) via the glycolytic degradation. If the

IPP was produced from MVA pathway, incorporation of [1-13

C] or [6-13

C] glucose should

have resulted in labeling of C-2, C-4 and C-5 atom of IPP (Figure-23a).


114

O

OH

OH

OH OH

HO

1

23

4

5

6

O- O

OH

O- OH

O

O

O

OHO

SCoA

PPO PPO

O

OH

HO

OH

OH

OH

O- O

OH

O

O

OH

O

OH

O

SCoA

O

OPPOPP

(a)

E.coli

Proferred glucose Degradation pathway Prediction via mevalonate Observed

(b)

Z.mobilis

+

14

15

16

17

5

5

14

15

16

17

1

1

7.5 Mechanism of DXP pathway; gene, enzymes and intermediates

Enzymes for MVA pathways are located in cytoplasm, while for DXP in plastids. The DXP

pathway is not known to function in mammals unlike MVA pathway. A variety of genes,

enzymes and intermediates involved were first revealed in E. coli and broadly studied

(Scheme-2; Eisenreich37

et al., 2001; Rohdich38

et al., 2001), but a few orthologous genes

encoding different enzymes of DXP pathway have also been confirmed in plant species.

Figure-23 a & b Metabolism of 13

C-labelled glucose in (a) E.coli via the Embden-Meyerhof

pathway and (b) Zymomonas mobilis via the Entner-Doudoroff pathway. The hypothetical

labeling pattern of IPP/DMAPP that could have been obtained via MVA pathway differs

substantially from the observed labeling patterns in both microorganisms. Coloured dots

indicate the metabolic fate of carbon atoms (Broers, 1994; Rohmer, et al., 1993).


115

The first step of the pathway is a transketolase-like condensation among pyruvate and D-

glyceraldehyde 3-phosphate to structure 1-deoxy-D-xylulose 5-phosphate (DOXP) (Scheme-

2).

Pyruvate formed thiamine diphosphate which undergoes condensation with the aldehyde

group of glyceraldehydes 3-phosphate. Lois39

, et al., 1998 have reported a genes dxs from

E.coli for encoding DXP synthase enzyme. Characterized gene was part of an operon which

also contains gene ispA which encode FPP synthase enzyme. Mechanism for proton transfer

transketolase reactions from enzyme binding site for thiamine diphosphate and a histidine

residue has been projected. Further studies on peppermint (Mentha X piperita) gene cloning

have also been reported, together with expression of the functional protein in E. coli40

.

This enzyme was found to contain a proposed plastid-targeting sequence. Extensive

sequence similarities between novel classes of transketolases dissimilar from the well-

characterized transketolases occupied in the pentose phosphate pathway, suggest that they

all are DXP synthases. A dxs gene in the cyanobacterium Synechococcus leopoliensis

(Anacystis nidulans) has been recognized and expressed successfully in E. coli, resulting in

increased synthesis of DMAPP41

. Gene dxs from Streptomyces sp. strain CL190 has been

clones and over expressed in E.coli to yield recombinant enzyme42

. This enzyme was found

similar in its activity as those of recombinant E. coli DXP synthase expect for pH optimum,

also over expressed and purified. Similarly, a gene CLA1, isolated from Arabidopsis

thaliana, has currently been exposed to encode DXP synthase43

.

In the second step of reaction DOXP is changed into 2-C-methyl- D-erythritol-4-phosphate

(MEP). Enzyme DOXP reductoisomerase (DXR) catalyze the intramolecular rearrangement

and reduction step involve in the production of MEP from DOXP in the presence of


116

NADPH. Scheme-2 represents intramolecular rearrangement is pinacol-like in which

aldehyde (2-C-methylerythrose 4-phosphate) intermediate is not unconfined from the

enzyme, but is concurrently reduced by the cofactor NADPH. Scheme-4 represents

rearrangement–reduction in the conversion of 2-acetolactate into 2,3-dihydroxyisovalerate

via ketol acid reductoisomerase in the biosynthesis of valine, isoleucine, and leucine.

CH3

C

C

COOH

O

H3C OH

CH3

C

C

COOH

HO CH3

O

CH3

C

CH

COOH

CH3HO

HO

2-acetoacetate 2-oxo-3-hydroxy-isovalerate

NADPH

2,3-dihydroxy-isovalerate

x

The third step of reaction is the formation of MEP from 4-(diphosphocytidyl)-2-C-methyl-

D-erythritol (CDP-ME) in a cytidine triphosphate (CTP)-dependent reaction. Rohdich, et al.,

2000a have cloned gene encoding CDP-ME synthase from plant A.thaliana, and use

recombinant E.coli for its expression.

The next step of the reaction is the Phosphorylation of the 2-hydroxy group of CDP-ME

gives 4- diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDPME2P) in the

presence of CDP-ME kinase (Scheme-2). Kuzuyama42

, et al., 2000 have repoted this

achievement by incubation of the gene encoded by ychB from E. coli with CDP-ME in the

Scheme-4 Enzyme x; Ketol acid reductase


117

presence of ATP. However, except few radiotracer studies in plants, which confirmed the

probable action of CDP-ME kinase in the formation of CDP-ME2P, the complete molecular

and enzymology from plant system is unavailable.

The last step in the reaction of DXP pathway is the transformation of 4-diphosphocytidyl-2-

C-methyl-D-erythritol 2-phosphate into 2-C-methyl-D-erythritol 2,4-cyclodiphosphate

(Scheme -2). This step is found to be catalyzed by the enzyme encoded by the ygbB gene,

found to be closely linked to ygbP which encodes CDP-ME synthase. Gene of E. coli was

expressed in a recombinant E. coli strain to give a soluble enzyme which converted into the

cyclodiphosphate and CMP. For activity enzyme require divalent cation Mn2+

or Mg2+

but

no other cofactors. The enzyme was also found to give cyclophosphate from CDP-ME

(Scheme-5), but the product was not incorporated into carotenoids, and is assumed not to be

a metabolic intermediate.

H

CH2

HO OH

OOP

O

O

OH

P

O

O

OHOHOH

OH

N

N

O

NH2

OH OP

O

OH

O

O-

15

4-(CDP)-2-C-methyl-D-erythritol

Scheme-5


118

The cyclic diphosphate had been extracted earlier from cultures of bacteria exposed to

oxidative stress, but had been interpreted as a dead-end product derived from the DXP

pathway. Formation of from is formulated as nucleophilic attack of the 2-phosphate to form

the phosphoanhydride, displacing CMP as leaving group (Scheme-2). The remaining step in

the pathway to IPP is suggested to be an intramolecular elimination followed by reductions

and dehydrations (Scheme -2)44

. In the MVA pathway IPP is converted into DMAPP by the

action of IPP isomerase enzyme, but there is evidence that this isomerism may not occur.

7.6 Crosstalk between MVA and DXP pathway

There is experimental evidence reported in literature that, in higher plants, MVA pathway

operates in the cytoplasm and mainly synthesize sterols, however, in the plastids isoprene

compounds are formed exclusively via DXP or MEP pathway45,46,37

. From studied both the

pathway using labeled tracers and reported that separation of the two IPP biosynthetic

pathways is not always complete, as in some cases at least one metabolite can be exchanged

between pathways in two separate compartments47,48

. In higher plants, both isoprene

pathways function simultaneously. From the result of studies on Matricaria recutita20

and

G. biloba34

it was found that both pathways MVA and DXP of isoprenoid compound

synthesis can work together to provide isoprene units for sesquiterpene and diterpene

synthesis, respectively. In present study of biosynthesis is based on 13

C labeled tracers

The [13

C]-labelling patterns of ginkgolides in G. biloba showed that three isoprene building

block units of the ginkgolide carried the [13

C]-labelling characteristics of cytoplasmic MVA

pathway, whereas the fourth isoprene unit was labelled via the DXP route34

. A mixed

labelling of isoprene building block units of phytol and other diterpenes from both MVA


119

and DXP pathways was also observed in liverwort (Heteroscyphus planus)49

and hornwort

(Anthoceros punctatus)50

.

Labeling patterns in the respective building block C-5 unit was quantitatively analyzed using

[13

C]-NMR spectroscopy of the sesquiterpenes bisabololoxide-A and chamazulene, isolated

from the hydrodistillate of the labelled chamomile (M. recutita) flowers showed that the two

of the isoprene building blocks were predominantly formed via the non-MVA pathway,

whereas the third unit was of mixed origin20

. Thus, the sub-cellular compartmentation of

two independent MVA and DXP pathways could play a crucial role in regulating the

biosynthesis of various classes of plant-derived isoprenoid compounds45,46,37

(Lichtenthaler,

1999; Rohmer, 1999; Eisenreich et al., 2001).


120

REFERENCES

1. Pichersky, E., Gang, D.R. (2000). Genetics and biochemistry of secondary metabolites

in plants: an evolutionary perspective. Trends Plant Sci., 5, 439-445.

2. Aharoni, A. et al. (2004). Gain and loss of fruit flavor compounds produced by wild

and cultivated strawberry species. Plant Cell, 16, 3110–3131.

3. Pichersky, E. et al. (1994). Floral scent production in Clarkia (Onagraceae): I.

Localization and developmental modulation of monoterpene emission and linalool

synthase activity. Plant Physiol., 106, 1533–1540.

4. Lucker, J., Verhoeven, H.A., Vander Plas, L.H.W., Bouwmeester, H.J. (2006). Molecular

engineering of floral scent. In Biology of Floral Scent. Edited by Dudareva N, Pichersky

E. CRC Press, 321-337.

5. Dudareva, N., Pichersky, E. (2006). Metabolic engineering of floral scent of

ornamentals. J Crop Improvement, 18, 325-346.

6. Degenhardt, J., Gershenzon, J., Baldwin, I.T., Kessler, A. (2003). Attracting friends to

feast on foes: engineering terpene emission to make crop plants more attractive to

herbivore enemies. Curr Opin Biotechnol.,14,169-176.

7. Aharoni, A., Jongsma, M.A., Bouwmeester, H.J. (2005). Volatile science? Metabolic

engineering of terpenoids in plants. Trends Plant Sci.,10, 594-602.Annu. Rev. Plant

Physiol., 46, 521.

8. Lichtenthaler, H. K., Rohmer, M. and Schwender, J. (1997).Two independent

biochemical pathways for isopentenyl diphosphate (IPP) and isoprenoid biosynthesis in

higher plants. Plant Physiol., 101, 643.


121

9. Mahmoud, S.S. and Croteau, R.B. (2002). Strategies for transgenic manipulation of

monoterpene biosynthesis in plants. Trends Plant Sci., 7, 366–373.

10. Rodriguez-Concepcion, M. and Boronat, A. (2002). Elucidation of the

methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and

plastids. A metabolic milestone achieved through genomics. Plant Physiol., 130, 1079–

1089.

11. Tan, B.C. et al. (1997). Genetic control of abscisic acid biosynthesis in maize. Proc.

Natl,. Acad. Sci. U. S. A. 94, 12235–12240.

12. Tavormina, P.A., Gibbs, M.H. & Huff, J.W. (1956). The utilization of β-hydroxy-β-

methyl-valerolactone in cholesterol biosynthesis. J. Am. Chem. Soc., 78, 4498-4499.

13. Queshi, N. & Porter, J.W. (1981). Conversion of acetyl-coenzyme A to isopentenyl

pyrophosphate. In Biosynthesis of Isoprenoid. Porter, J.W. & Spurgeon, S.L., eds. John

Wiley, New York, 1, 47-94.

14. Banthorpe, D.V., Charlwood, B.V. & Francis, M.J.O. (1972). The biosynthesis of

monoterpenes. Chem. Rev. 72, 115-155.

15. Bloch, K. (1992). Sterol molecule: structure, biosynthesis and function. Steroids, 57,

378-382.

16. McCaskill, D and Croteau, R. (1998). Some caveats for bioengineering terpenoid

metabolism in plants. Trends Biotechnol., 16, 349-355.

17. Laule, O. et al. (2003). Crosstalk between cytosolic and plastidial pathways of

isoprenoid biosynthesis in Arabidopsis thaliana. Proc. Natl., Acad. Sci. U. S. A. 100,

6866–6871.


122

18. Spurgeon. S.L. and Porter, J.W. (1981). In Biosynthesis of Isoprenoid compounds. ed.

J.W. Porter and S.L. Spurgeon., John Wiley and Sons, New York, 1, 1 and references cited

therein.

19. Quershi, N.and Porter, J.W. (1981). In Biosynthesis of Isoprenoid compounds. ed.

J.W. Porter and S.L. Spurgeon., John Wiley and Sons, New York, 1, 47 and references

cited therein.

20. Adam, K. P. and Zapp, J. (1998). Biosynthesis of the isoprene units of chamomile

sesquiterpenes, Phytochemistry, 48, 953–959.

21. Bick, J.A. and Lange, B.M. (2003). Metabolic cross talk between cystolic and

plastidial pathways of terpenoid biosynthesis: unidirectional transport of intermediates

across the chloroplast envelope membrane. Arch. Biochem. Biophys., 415, 146-154.

22. Dudareva, N. et al. (2005). The nonmevalonate pathway supports both monoterpene

and sesquiterpene formation in snapdragon flowers. Proc. Natl., Acad. Sci. U.S.A., 102,

933-938.

23. Hemmerlin, A. et al. (2003). Crosstalk between cystolic mevalonate and the plastidial

methylerythritol phosphate pathways in Tobacco Bright Yellow-2 cells. J. Biol. Chem.,

278, 26666-26676.

24. Schuhr, C.A. et al. (2003). Quantitative assessment of crosstalk between the

isoprenoid biosynthesis pathways in plants by NMR spectroscopy. Phytochem. Rev., 48,

953-959.

25. Kleinig, H. (1989). The Role of Plastids in Isoprenoid Biosynthesis. Annu. Rev.

Plant Physiol., 40, 39.


123

26. Chappel, J. (1995). The biochemistry and molecular biology of isoprenoid

metabolism.

27. Bach, T. J. (1995). Some new aspects of isoprenoid biosynthesis in plants - a

review. Lipids, 30, 191.

28. Treharne, K.J., Mercer, E.I. and Goodwin, T.W. (1966). Incorporation of [14

C]Carbon

Dioxide and [2-14

C]Mevalonic Acid into Terpenoids of Higher Plants during

Chloroplast Development. Biochem. J., 99, 239.

29. Bach, T. J. and Lichtenthaler, H. K. (1987). In Ecology and Metabolism of Plant

Lipids. ed. G. Fuller and W. D. Nes, Am. Chem. Soc. Symp. Series, American Chemical

Society, Washington DC, 325,109.

30. Bach, T.J., Motel, A. and Weber, T. (1990). Some properties of enzymes involved in

the biosynthesis and metabolism of 3-hydroxy-3-methylglutaryl-CoA in plants. Rec.

Adv. Phytochem., 24, 1.

31. Lutke-Brinkhaus, F. and Kleinig, H. (1987). Formation of isopentenyl diphosphate via

mevalonate does not occur within etioplasts and etiochloroplasts of mustard (Sinapis

alba L.) seedlings. Planta, 171, 406.

32. Flesch, G.and Rohmer, M. (1988). Prokaryontic hopanoids; the biosynthesis of the

bacteriohopan skeleton. Eur. J. Biochem., 175, 405.

33. Broers, S.T.J. (1994). Ober die fruhen Stufen der Biosyntheses von Isoprenoiden in

Escherichia coli. [On the early stage of isoprenoid biosynthesis in E.coli]. Thesis Nr.

10978, ETH Zurich, Schweiz.

34. Schwarz, K. M. (1994). Terpen-biosynthese in Ginkgo biloba: Eine Uberraschende

geschichte. Ph.D. thesis, Eidgenossischen Technischen Hochschule, Zurich, Switzerland.


124

35. Nakanishi, N. & Habaguchi, K. (1971). Biosynthesis of ginkgolide B, its diterpenoid

nature, and origin of tert-butyl group. J. Am. Chem. Soc., 93, 3546-3547.

36. Rohmer, M., Knani, M., Simonin, P., Sutter, B & Sahm, H. (1993). Isoprenoid

biosynthesis in bacteria; a novel pathway for the early steps leading to isopentenyl

diphosphate. Biochem. J., 295, 517-524.

37. Eisenreich, W., Rohdich, F. and Bacher, A. (2001). Deoxyxylulose phosphate pathway

to terpenoids. Trends Plant Sci., 6, 78–84.

38. Rohdich, F., Kis, K., Bacher, A. and Eisenreich, W. (2001). The nonmevalonate

pathway of isoprenoids: genes, enzymes and intermediates. Curr. Opin. Chem. Biol. 5,

535–540.

39. Lois, L. M., Campos, N., Putra, S. R., Danielsen, K., Rohmer, M. and Boronat, A.

(1998). Cloning and characterization of a gene from Escherichia coli encoding a

transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-

phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis.

Proc. Natl., Acad. Sci. U.S.A., 95, 2105.

40. Lange, B. M., Wilding, M. R., MaCaskill, D.and Croyeau, R. (1998). A family of

transketolases that directs isoprenoid biosynthesis via a mevalonate-

independent pathway. Proc. Natl., Acad. Sci. U.S.A., 95, 2100.

41. Miller, B., Heuser, T.and Zimmer, W. (1999). A Synechococcus leopoliensis SAUG

1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two

additional open reading frames is functionally involved in the dimethylallyl

diphosphate synthesis. FEBS Lett., 460, 485.


125

42. Kuzuyama, T., Takagi, M., Takahashi, S. and Seto, H. (2000). Cloning and

characterization of 1-deoxy-D-xylulose 5-phosphate synthase from Streptomyces sp.

Strain CL190, which uses both the mevalonate and nonmevalonate pathways for

isopentenyl diphosphate biosynthesis. J. Bacteriol., 182, 891.

43. Estevez, M., Cantero, A., Romero, C., Kawaide, H., Jimenez, L. F., Kuzuyama, T., Seto,

H., Kamiya, Y. and Leon, P. (2000). Analysis of the expression of CLA1, a gene that

encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-

phosphate pathway in Arabidopsis. Plant Physiol., 124, 95. FEBS Lett., 448, 115.

44. Lichtenthaler, H. K., Zeidler, J., Schwender, J. and Müller, C. (2000). The non-

mevalonate isoprenoid biosynthesis of plants as a test system for new herbicides and

drugs against pathogenic bacteria and the malaria parasite. Z. Naturforsch., Teil C, 55,

305.

45. Lichtenthaler, H. K. (1999). The 1-deoxy-D-xylulose-5-phosphate pathway of

isoprenoid biosynthesis in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol., 50 47–65.

46. Rohmer, M.1(999). The discovery of a mevalonate independent pathway for

isoprenoid biosynthesis in bacteria, algae and higher plants. Nat. Prod. Rep., 16, 565–

574.

47. Arigoni, D., Eisenreich, W., Latzel, C., Sagner, S., Radykewicz, T., Zenk, M. H. and

Bacher, A. (1999). Dimethylallyl pyrophosphate is not the committed precursor of

isopentenyl pyrophosphate during terpenoid biosynthesis from 1-deoxyxylulose in

higher plants. Proc. Natl., Acad. Sci. U.S.A., 96, 1309–1314.

48. Thiel, R. and Adam, K. P. (2002). Incorporation of [1-13

C]1-deoxyd-xylulose into

isoprenoids of the liverwort Conocephalum conicum, Phytochemistry, 59, 269–274.

http://www.ncbi.nlm.nih.gov/pubmed/10928537




126

49. Nabeta, K., Kawae, T., Kikuchi, T., Saitoh, T. and Okuyama, H. (1995). Biosynthesis of

chlorophyll a from 13

C-labelled mevalonate and glycine in liverwort: nonequivalent

labeling of phytyl side chain. J. Chem. Soc. Chem. Commun., 2529–2530.

50. Itoh, D., Karunagoda, R. P., Fushie, T., Katoh, K. and Nabeta, K. (2000).

Nonequivalent labeling of the phytyl side chain of chlorophyll a in callus of the

hornwort Anthoceros punctatus. J.Nat. Prod., 63, 1090–1093.

chapter 2 review on literature -...

Documents