Polyamines: An Overview and Prospects in Crop Improvement

21Polyamines: An Overview and Prospects in Crop Improvement

Neelam Setia and R.C. SetiaDepartment of Botany

Punjab Agricultural University, Ludhiana-141004, Indiaemail: [email protected]

ABSTRACT

Polyamines are low molecular weight polycationic compounds that are ubiquitously distributedin all living organisms. The basic mechanisms of polyamine biosynthesis and metabolismcoupled with identification of mutants with altered metabolism have greatly helped inunderstanding role of these compounds in a wide range of cellular, growth and developmentalprocesses in plants, such as cell division, embryogenesis, flower and fruit development,post-harvest fruit management and senescence. Different polyamines have been implicatedin imparting resistance/protection against various abiotic and biotic stresses. At physiologicalpH, polyamines being fully protonated perform various physiological functions by binding tothe negative charges of nucleic acids and phospholipids, and thereby stabilize the functionof nucleus and cell membranes. They are suggested as good candidates in protecting theplants against oxidative damage being induced by a wide variety of stress conditions. In thisreview, recent advances relevant to the molecular mechanism of polyamine action and theirinvolvement in signal transduction are also discussed keeping in view the future prospectivesof polyamine research in crop improvement.

Keywords: Polyamines, biosynthesis, metabolism, growth, embryogenesis, senescence, plantstresses, signal transduction

INTRODUCTIONPolyamines are low molecular weight nitrogen-containing aliphatic compounds found in all livingorganisms. The major polyamines found in plant cells are putrescine, spermine and spermidine. Inaddition, cadaverine, a diaminopentane, is a common constituent of legumes and is present in muchless concentration than putrescine. Less common polyamines like 1,3-diamine propane andhomospermidine, which differ from the common polyamines in the number of methylenic moietiesbetween amino groups, have been detected in a broad spectrum of biological systems includingplants, algae, bacteria and animals (Oshima, 1983; Cohen, 1998). Also nor-spermidine and nor-spermine types are reported in Medicago sativa (Rodriguez-Garay et al., 1989). Many lines ofresearch evidences, such as treatments with exogenous polyamines, quantification of their endogenouslevels, use of polyamine biosynthesis inhibitors and studies with mutants and transgenic plants have

Crop Improvement: Strategies and Applications Editors: R.C. Setia, Harsh Nayyar and Neelam Setia

© 2008 I.K. International Publishing House Pvt. Ltd., New Delhi, pp 376-393

Polyamines: An Overview and Prospects in Crop Improvement 377

shown their role in wide range of cellular, growth and developmental processes like DNA replication,transcription and translation, cell division, organogenesis, senescence, environmental stresses andinfection by fungi and viruses (Tabor and Tabor, 1984; Galston et al., 1997; Kaur-Sawhney et al.,2003). The uncommon polyamines have been postulated to serve specific protective roles underextreme environmental conditions both in bacteria and higher plants. The ability of polyamines toenable biological systems to grow or function under extreme conditions has provided opportunitiesfor new investigations into their potential functions.

Many plant growth and developmental processes known to be regulated by plant hormones,such as auxins, gibberellins, cytokinins and ethylene have also been correlated with changes inpolyamine metabolism (Galston,1983). Recent studies of Tun et al. (2006) using a fluorimetricmethod employing the cell-impermeable nitric oxide (NO) binding dye revealed that polyamines,spermidine and spermine induced NO biosynthesis in Arabidopsis seedlings. They observed thatinduction of NO synthesis was tissue specific being maximum in the elongation zone of Arabidopsisroot tip and primary leaves, especially in veins and trichomes. However, a little or no effect ofpolyamines on NO production was observed in cotyledons in presence of polyamines. Theinvolvement of polyamines in regulation of various plant responses in interaction with hormonessuggests their role as plant growth regulator. But unlike plant growth regulators, polyamines inplants are required in millimolar concentrations, rather than in micromolar levels typical of thetraditionally accepted plant hormones and play dual function, i.e., both structural and regulatory.Due to their versatile involvement in controlling various physiological, biochemical and developmentalprocesses in plants, polyamines are now recognized as a new class of plant growth regulators (Galstonand Kaur-Sawhney, 1995). This article, besides general introduction to polyamines, also presents anoverview of their involvement in various growth and developmental processes with focus on recentadvances in polyamine research in relation to crop improvement.

STRUCTURE, OCCURRENCE AND LOCALIZATION OF POLYAMINESPolyamines, the low molecular weight nitrogen containing aliphatic compounds, are positively chargedat physiological pH due to presence of amino groups and act as polycations, with charges distributedalong a flexible carbon chain. They occur in free or conjugated forms. Polyamine conjugates withphenolic acids are widespread in higher plants (Martin-Tanguy, 1985). Polyamines are also boundto some macromolecules like proteins and nucleic acids (Smith, 1985). The conjugates may act asstorage forms of polyamines from which free bases may be released and transported as and whenrequired. The level of polyamines in plant cells depends on their biosynthesis, degradation,conjugation, transport and conversion to other metabolites.

Structure of polyaminesH2N-(CH2)4 - NH2 Putrescine (Diamine)H2N-(CH2)3 - NH - (CH2)4- NH2 Spermidine (Triamine)NH2-(CH2)3 - NH - (CH2)4 - NH - (CH2)3 - NH2 Spermine (Tetramine)H2N-(CH2)5 - NH2 Cadaverine (Diaminopentane)

Some common polyamine-conjugatesCoumaroyl putrescine Alkyl cinnamoyl putrescineCaffeoyl putrescine Caffeoyl spermidine

378 Crop Improvement: Strategies and Applications

Feruloyl putrescine Alkyl cinnamoyl spermineHydroxycinnamoyl putrescine Coumaroyl agmatine

Polyamines being small, soluble, diffusible molecules at cellular pH, their immobilization in thecell for localization is difficult to achieve. Several approaches used to study the localization ofpolyamines include subcellular fractionation, cytochemical and immunocytochemical staining methods,and autoradiographic localization of labeled polyamines and the enzymes involved in their biosynthesis(Cohen, 1998). Spermine and spermidine are found mainly in the cell wall while putrescine is foundin cytoplasm fraction. Polyamines present in the cell wall are mainly associated with pecticpolysaccharides and are believed to control cell wall pH and lignification (Angelini et al., 1993).Studies on uptake of radiolabelled polyamines by carrot cells revealed that approximately two-thirdof the total polyamines are bound to the cell wall possibly due to adsorption of protonated polyaminesdue to net negative charge. Vacuole is suggested as the temporary reservoir of polyamines, as it canbe readily transported across the tonoplast and plasma membrane. Polyamines are also foundassociated with thylakoid membranes of chloroplasts and other subcellular organelles like mitochondria(Cowley and Walters, 2002).

POLYAMINE BIOSYNTHESIS AND METABOLISMA common biosynthetic pathway for polyamines occurs in plants, microorganisms and mammals.They are synthesized from L-arginine and L-ornithine by arginine decarboxylase (ADC) and ornithinedecarboxylase (ODC) enzymes which convert these amino acids into agmatine and putrescine,respectively (Fig. 1). Arginine is usually preferred for putrescine synthesis in higher plants due toits relative abundance in storage proteins, involvement in long distance transport, and position inplant economy with nitrogen to carbon ratio. Spermidine and spermine are synthesized by the additionof an aminopropyl group to one or both primary amino groups of putrescine by spermidine andspermine synthases, respectively. The aminopropyl moiety is derived from decarboxylated S-adenosylmethionine (SAM) which in turn is derived from methionine. SAM is also involved in ethylenebiosynthesis (Evans and Malmberg, 1989; Cohen, 1998). The genes for both ADC and ODC havebeen cloned from a variety of plants. Based on the expression pattern of the corresponding mRNAs,it is inferred that ODC is mainly expressed in dividing tissues, whereas ADC is expressed duringcell elongation and under the conditions of stress. The activity of ADC in most of the tissues ismodulated by light and hormones. In some legumes, cadaverine is synthesized from L-lysine byaction of lysine decarboxylase (Smith, 1985).

The level of polyamines in plant cells is regulated by both synthesis and degradation. Thebreakdown or catabolism of polyamines is brought about by specific amino oxidases that includediamine oxidases (DAO), highly specific for diamines, and the reaction products of DAO on putrescineare pyrroline, H2O2 and ammonium. A second class of plant enzymes, polyamine oxidases (PAO),catalyzes an analogous reaction, yielding pyrroline and aminopropylpyrroline from spermidine andspermine, respectively, which are further catabolized to form b-alanine and succinic acid, respectively,and are incorporated into Krebs cycle. Thus, carbon and nitrogen from putrescine and spermidineare recycled (Smith, 1985). The catabolic oxidation of polyamines not only regulate theirconcentration, but may also play other important roles, for example, use of H2O2 generated byaction of polyamine oxidases in cell wall lignification.


L-Arginine

L-Ornithine

Putrescine

Spermidine

Spermine

Methionine

S-AdenosylL-Methionine (SAM)

Decarboxylated SAM

Agmatine

N-Carbamoylputrescine

Argininedecarboxylase

Arginase S-Adenosylmethionine synthase

Agmatineiminohydrolase

Ornithinedecarboxylase

Spermidinesynthase

Sperminesynthase

SAMdecarboxylase

N-Carbamoylputrescine

amidohydrolase

Fig. 1. Polyamine biosynthetic pathway (Names of enzymes are shown in italics).

TRANSPORTExtensive studies have been carried out on the uptake of polyamines by yeast and bacterial cells,and their transporters have been isolated and cloned (Cohen, 1998). In bacteria, rate of uptake ofpolyamines is energy dependent and is a function of external pH. In plants, there are reports tosuggest that polyamine transport occurs as an active bi-directional process requiring energy and likeother metabolites/nutrients the translocation of polyamines is dependent on both temperature andrelative humidity (Tiburcio et al., 1997). Plant cells are also equipped with efficient systems foruptake of exogenously applied polyamines. Polyamines taken up from external source or synthesizedwithin different plant parts are transported via xylem and/or phloem to other parts of the plant.Kanchanapoom et al. (1991) studied the effect of IAA on uptake of spermidine in carrot protoplastsin the presence of Ca2+ and observed its stimulation in their presence. Application of vandate, anATPase inhibitor, strongly inhibited IAA-stimulated spermidine uptake, suggesting therebyinvolvement of energy-dependent mechanism in this transport.

GROWTH AND DEVELOPMENTAL RESPONSES TO POLYAMINESIn higher plants, polyamines have been implicated in a wide range of growth and developmentalprocesses including cell division, embryogenesis, root development, flowering, fruit developmentand leaf senescence. In addition to their roles in developmental processes, polyamines also play animportant role in plant stress responses.

Cell DivisionIn general, high levels of free polyamines are observed in cells undergoing division. During transitionfrom G1 to S phase during cell division, both spermine and spermidine are required. The conversion


of putrescine to these two amines appears to be important in controlling the rate of cell division(Galston and Kaur-Sawhney, 1995). The transition of cells from G2 to mitosis during cell divisionis known to depend on the activity of multiprotein complex, which includes cyclins. The cyclins inquiescent sugar beet cells induced by polyamines, especially putrescine and their precursors arginineand ornithine, indicates their role in controlling gene expression and cell division. Exogenousapplication of polyamines to protoplasts or cells in tissue culture results in a temporary or sustainedincrease in cell division (Evans and Malmberg, 1989). High levels of spermine were observed inprimary root apices and in decapitated roots during lateral root formation using DFMO (α-difluoromethyl ornithine) and thymidine. Further ODC was localized primarily in the meristematiczones (Evans and Malmberg, 1989). The treatment of cells with inhibitors of polyamine biosynthesisresults in an arrest of cell proliferation and this inhibition can be reversed by their externalsupplementation. Further, the observed correlations between peaks of polyamine levels insynchronizable tobacco cells and peaks of casein kinase II activity suggest that interactions withpolyamines might be important in regulating kinase activity during cell cycle.

EmbryogenesisThe morphogenetic potential of polyamines to produce embryoids was first investigated in carrotcultures. A significant increase in ADC activity and putrescine content was observed when carrotcultures from callus medium were shifted to embryogenesis medium (Montague et al., 1978). Theinduction of embryogenesis elicited by polyamines was inhibited by polyamine inhibitors and ethylene.Further, callus cells grown in the presence of auxins were not embryogenic and produce a moreethylene and less polyamines, possibly because of switching the fate of SAM, a common precursorof both. The evidence suggests that the morphogenetic potential of callus to produce embryoids iselicited by polyamines and inhibited by ethylene. It remains to be determined if the interactionbetween ethylene and polyamines occurs at the level of their biosynthesis or signaling pathways.Carrot cell mutant (WOO1C) with high internal levels of auxin did not go through embryogenesisand failed to show increase in polyamine content when placed in the medium without auxin (Fienberget al., 1984).

Four critical stages of embryogenesis including callus induction, cellular acquisition ofmorphogenetic competence, expression of embryogenic programme and development and maturationof somatic embryos have been identified during somatic embryogenesis from leaf discs of eggplant (Solanum melongena). During induction of embryogenic callus in egg plant, there were hightiters of free, conjugated and total putrescine as compared with spermine and spermidine, becauseof high activity of ADC, which is a prerequisite for cell division leading to callus formation(Rodriguez et al., 1999). Studies on changes in ADC and polyamine titers during critical stages ofembryogenesis further revealed high levels of polyamines, especially putrescine, in discs from apicalregion of leaf with high embryogenic capacity than from basal region of leaf with poor embryogeniccapacity. Treatment of basal discs with putrescine for 4 to 7 days improved embryogenesis. Asignificant difference in polyamine requirement during somatic embryogenesis has been observed intwo alfalfa lines. Both genotypes showed accumulation of putrescine during induction as mediumwith auxin and exhibited sharp fall in putrescine content upon transfer to the medium without auxin(Bais and Ravishankar, 2002). Use of polyamine inhibitors reduced polyamine content in both thegenotypes, however, embryogenesis was reduced in only one of the two genotypes. Based on theexisting information, it is inferred that correlations between polyamines and their biosynthetic enzymes


and cellular growth processes, especially embryogenesis are not universal and are species specific(Evans and Malmberg, 1989; Galston et al., 1997; Bais and Ravishankar, 2002).

RootingDevelopment of roots is under the control of hormonal, metabolic and environmental cues that canact on genetically controlled developmental programmes, and thus, can affect the plasticity of rootarchitecture. In addition to the involvement of five classical hormones, some other growth regulators,such as polyamines are reported to modulate root developmental pattern. The role of polyamines inroot development and growth has been established in several plant systems, mostly involving use ofstem/ hypocotyl segments under in vitro conditions (Coúee et al., 2004). The induction of rooting instem cuttings of Phaseolus vulgaris with indolebutyric acid (IBA) is accompanied by increasedlevels of polyamines (Jarvis et al., 1985; Evans and Malmberg, 1989). Application of inhibitors ofpolyamine biosynthesis reduced their endogenous levels and inhibited IBA-induced rise in spermineand spermidine and checked root induction and growth. However, IBA induced increase in polyaminecontent failed to stimulate rooting in Vigna hypocotyls cuttings (Friedman et al., 1982). The increasedlevel of polyamines during root and nodule growth in mungbeans is also accompanied by changesin RNA, DNA and protein contents.

The induction of rooting in tobacco callus cultures is found to be inversely related to putrescineand alkaloid titers. Studies by Chriqui et al. (1986) have provided evidence that there exists asynergistic effect between auxins and ornithine during rhizogenesis in Datura innoxia leaf explants.A positive correlation has been observed between agmatine, spermidine and spermine contents andprimary root growth, whereas putrescine level showed neutral or negative effects on this trait.Similarly, positive correlation has also been observed between spermidine and spermine content androot growth in seedlings of Pringlia antiscorbutica, the sub-antarctic cruciferous species (Hummelet al., 2002, 2004). They demonstrated the dominance of ADC pathway in synthesis of polyaminesin these seedlings by modulation of polyamine metabolism with the use of inhibitors.

Putrescine is an important component of root exudates in tomato. The increased availability ofthis polyamine in the rhizosphere has a bacteriostatic effect on root cells colonizing Pseudomonasfluorescence strain WCS 365. The colonizing ability of bacteria decreases as a result of increaseduptake of putrescine (Kuiper et al., 2001). Spermidine and spermine are less effective in influencingthe root colonizing ability of bacteria as compared to putrescine.

FloweringPolyamines are reported to influence floral development in a wide range of crop species. In general,hydroxy cinnamoyl acid amides, formed by coupling of phenolic acids and polyamines are foundabsent in young plants but accumulate progressively in apical leaves and then in large quantities indifferent floral parts (Martin-Tanguy, 1985). The relationship between polyamines and floweringhas been observed in whole plants. Polyamine titers in relation to photoperiodic induction of floweringare well characterized in a short day plant, Xanthium strumarium and a long day plant, Sinapsisalba. Their role in flowering was established by use of polyamine enzyme biosynthesis inhibitorswhich prevented flowering and was resumed in absence of inhibitors or when polyamines wereapplied exogenously (Applewhite et al., 2000; Slocum and Galston, 1985). Cyclohexylamine (CHA),a specific inhibitor of spermidine synthesis significantly reduced number of floral primordia inPolyanthus tuberosa. Exogenous application of putrescine and silver nitrate (AgNO3) in shoot cultures


of Cichorium intybus under in vitro conditions enhanced floral initiation and floral development.The morpohogenetic response and level of endogenous conjugated pool of polyamines diminishedfollowing treatments with polyamine biosynthesis inhibitors DFMA (α-difluoro methyl arginine)and DFMO (Bais et al., 2000).

Measurement of titers of endogenous spermidine and putrescine extracted from various organsof two ecotypes and genetic line of Arabidopsis thaliana revealed that flowers had highest titers ofthese polyamines, with spermidine predominating (Applewhite et al., 2000). Application of appropriateinhibitors lowered spermidine titer and inhibited bolting and flowering in this species. Further, enzymeinhibitors of spermidine synthesis given shortly before the transition from short day conditions tolong day conditions prevented flowering in this species. Further, the addition of spermidine in thegrowth medium significantly accelerated flowering in delayed flowering mutant CS 3123 ofArabidopsis. These studies have clearly demonstrated close connection between polyamines andreproductive development in A.thaliana (Applewhite et al., 2000). The induction of flowering hasalso been reported in seedlings of morning glory by using polyamines, especially putrescine,cadavarine and related compounds like unnatural 1,6-diaminohexane (Wada et al., 1994). Acquisitionof some lines of mutants in various crops, such as tobacco and petunia, further helped in implicatingthe role of polyamines in floral development (Walden et al., 1997).

Petunia mutants with abnormal polyamine titers exhibit irregular development of the floral organs.Aberrant morphology of anthers and ovules was observed in tobacco mutants deficient in polyaminemetabolism (Malmberg and McIndoo, 1983). Generally, high polyamine levels contribute to thepattern of abnormal stamen development. The role of polyamines in regulation of pollen germinationand tube growth is also well documented (Song et al., 1999). Apart from floral development,polyamines also play role in sex expression and control of fertility. The male flowers have higherlevel of neutral hydroxy cinnamic amides than female ones, but the latter have more hydroxylcinnamic amides than the former (Martin-Tanguy, 1985). The concentrations of polyamines alsovary in sterile and fertile organs. Studies in Chrysanthemum, tobacco, some Araceae species andtomato (Aribaud and Martin-Tanguy, 1994; Malmberg, 1980; Liu et al., 2006; Rastogi and Sawhney,1990a,b) revealed that their sterile lines had low level of polyamines than fertile lines or thecorresponding maintainer line. However, in certain species, higher levels of polyamines were foundto be responsible for stamen sterility. Such a discrepancy could be attributed to polyamine homeostasisin a given plant species. Studies on the effect of repeated severe pruning of hazel nut trees onendogenous polyamine content revealed high levels of spermidine and spermine during flowering,whereas reverse trend is linked to the onset of dormancy (Rey et al., 1994). The underlying mechanismof action of polyamines during floral development still remains unclear. Polyamines might be a partof complex mechanism involved in flowering signal. However, much more deep insight is stillrequired to understand the mechanism of polyamine action during flowering in plants.

Fruit developmentStudies on polyamine titers during different stages of fruit development clearly points towardrelationship between polyamines and fruit development. The level of polyamines is generally highduring early stages of fruit development, and declines during later stages of development and ripening.However, the behaviour of individual polyamines varies with species, with spermine being themajor one in avocado, putrescine in tomato and spermidine in opera (Liu et al., 2006). Early stagesof fruit development involve active cell division and require sufficiently high level of polyamines.


The decline in their content during later stages of fruit development may act as a signal for onset ofripening process. However, in fruits like citrus and tomato, increase in polyamine titers is alsoreported during maturation and ripening (Saftner and Baldi, 1990).

Ethylene, a gaseous hormone playing an important role in fruit ripening, and polyamines sharea common precursor, SAM for their synthesis. In one pathway, SAM releases ethylene throughACC (1-aminocyclopropane-1-carboxylic acid), and in the other it is converted into decarboxylatedSAM which serves as a donor of the aminopropyl group for spermidine and spermine production.Possibly, they compete with each other during fruit development and ripening processes, and thusreflect the possibility of existence of inverse relationship between them. Comparatively higher levelsof polyamines and low levels of ethylene have been observed in long-keeping tomato fruits (Dibbleet al., 1988). Further, an increase in level of endogenous polyamines has been observed in vitrocultured kiwi fruit explants by blocking ethylene synthesis by using ethylene inhibitor amino ethoxyvinyl glycine. Exogenously applied polyamines have also been found effective for increasing fruitset and yield in apple (Biasi et al., 1988), delaying maturation and flesh softening in fruits andreducing fruit drop by inhibiting abscission in lot of plants. (Aziz et al., 2001; Liu et al., 2006). Animmediate increase in firmness has been observed when McIntosh and Golden Delicious applesharvested at optimum commercial maturity were infiltered with polyamines (Kramer et al., 1991).However, they did not observe any visible effect of polyamine application on ethylene production.These workers pointed out that high concentrations of polyamines may cause chemical injury tofruits as spermidine and spermine at concentrations higher than 1 mM led to the development ofsmall black spots in apples. Based on the existing information, the effects of polyamines are atvariance among different plant species, polyamine type, concentration and stage of application. Thepromotory effects of exogenously applied polyamines could be countered by their inhibitors (Tarenghiand Martin-Tanguy, 1995), suggesting thereby their potential for post-harvest fruit management.

Stomatal MovementsA stomatal aperture defined by two guard cells is responsible for gas exchange between plants andthe atmosphere. Changes in guard cell turgor that instigate opening and closing of stomatal apertureare controlled by number of factors through modulation of ion channel activity and pumps (Ward etal., 1995). Among the ion channels in guard cells, the inward K+ channels are an important playerin stomatal regulation. A number of studies have shown that inward K+ channel-inhibiting processesor factors often inhibit stomatal opening. Such factors include ABA, Ca2+ levels and polyamines(Liu et al., 2000). Polyamines, like ABA, have been shown to induce stomatal closure, but withdifferent mechanism, whereas ABA elicit turgor loss by activating anion channels and outward K+

channels (Pei et al., 1997). On the contrary, polyamines do not affect inward K+ or anion channels,suggesting thereby possibility of some other polyamine targets in addition to inward K+ channels inguard cells for induction of stomatal closure.

Mechanism of stomatal regulation is one of the most studied mechanisms of plant responses tostresses. Many of the stress factors are known to elevate polyamines. Electrophysiological andmolecular studies in animal systems have demonstrated the role of polyamines in modulation of ionchannels by direct binding to the channel protein or membrane component (Johnson, 1996). Studiesof Brüggemann et al. (1998) showed that in higher plants, polyamines block fast activating vacuolarcation channels. Among the ion channels in the guard cells, the inward K+ channels are an important


player in stomatal regulation and factors/processes blocking inward K+ channels inhibit stomatalopening.

Studies of Liu et al. (2000) in Vicia faba leaves revealed involvement of polyamines in regulationof voltage-dependent inward K+ channels in plasma membrane of guard cells. Whole-cell patchclamp analysis showed that intracellular applications of all natural polyamines including spermidine,spermine, cadaverine and putrescine induced closure of stomata by inhibiting the inward K+ currentacross the plasma membrane of guard cells. Further, identification of target channel at molecularlevel revealed that spermidine induced closure of stomata occurred due to inhibition of inward K+

current carried by KATI channel.Single channel recording analysis indicated that regulation of K+ channels by polyamines requires

unknown cytoplasmic factors (Liu et al., 2000). In an effort to identify the target channel at molecularlevel, they suggested that polyamines target KATI-like inward K+ channels in guard cells andmodulate stomatal movements and provide a link between stress conditions, polyamine levels andregulation of stomatal movements.

SenescenceSenescence is a highly ordered and genetically regulated process, which can be viewed at cell,tissue, organ or whole plant level. The process is largely an oxidative, mainly characterized bycessation of photosynthesis, disintegration of organelle structures, loss of chlorophyll, proteins, adramatic increase in lipid oxidation, breakdown of cell wall components and disruption of cellmembranes leading to loss of cell/tissue structure (Buchanan-Wollaston, 1997). Senescence, thougha terminal developmental stage in the life cycle of a plant, can also be accelerated by an array ofboth abiotic and biotic factors (Smart, 1994). Like other plant processes, polyamines also act asantisense agents in a number of plant species by inhibiting ethylene synthesis. Both spermidine andspermine are reported to delay leaf senescence by preventing chlorophyll loss and stabilizing thethylakoid membranes, possibly by checking inhibition of RNAase and protease activities and ethylenesynthesis (Lee et al., 1997; Pandey et al., 2000). While comparing ethylene production with changesin endogenous polyamine levels from control and MGBG treated petals of carnation flowers inpresence of spermine during entire incubation period, it was suggested that the inhibition of senescenceby polyamines is related to their ability to suppress ethylene production (Serrano et al., 1991).Further, the effects of exogenous polyamine applications are similar to those of cytokinin, althoughcytokinins are applied at 0.1 mM and polyamines are applied at roughly 1-10 mM concentrations.

The antisenescence behaviour of polyamines is not universal, as several counter examples havebeen reported where polyamines did not seem to retard senescence. The increased flower longevityand retarded senescence in cut carnation with aminotriazole was the result of inhibition of climactericpeak of ethylene production, but the treatment had no effect on levels of polyamines (Serrano et al.,1991). Also, the addition of 10 mM putrescine and spermine to culture solutions of cut carnationsfailed to increase flower longevity (Downs and Lovell, 1986). On the contrary, an increase inendogenous level of free putrescine was observed in carnation flowers during senescence, while novariation in content of free polyamines was observed in climacteric and non-climacteric carnationflowers at pre-climacteric stage. Studies of Botha and Whitehead (1992) on petunia flowers revealedthat initial decline in polyamines during pre-climacteric stages was not accompanied by concomitantincrease in ethylene production. This can be due to the fact that synthesis of ACC by enzyme ACCsynthase is rate limiting step and not the availability of SAM in the pathway of ethylene production


during pre-climacteric stage. This is further supported by the use of D-arginine and MGBG(Methylglyoxal-bis-guanylhydrazone), inhibitors of putrescine synthesis, respectively, which did notresult in stimulation of ethylene synthesis during pre-climacteric phase of petunia flowers. However,the degree of relationship between polyamines and ethylene at both physiological and biochemicallevels still remains unresolved.

POLYAMINES IN STRESS MANAGEMENTChanges in polyamine levels and activities of enzymes of their biosynthesis have been observed ina variety of plant species in response to various stresses (Martin-Tanguy, 1985; Kao, 1997; Walters,2003). These reports indicate that polyamines are intricately involved in the plant’s responses toenvironmental challenges by changing their growth and developmental patterns.

Abiotic StressesThe earliest report of increase in level of polyamines dates back to 1950’s in barley plants grownunder K+ deficiency conditions and this effect has been widely confirmed (Smith, 1985). Potassiumdeficiency resulted in a 20-fold increase in putrescine levels, a 6-fold increase in ADC activity inoat shoots (Young and Galston, 1984) and 7-fold increase in regenerating buds of tobacco (Klingueret al., 1986). Subsequently, accumulation of polyamines under potassium deficient conditions hasbeen reported in several plant species (Flores, 1991). Tachimoto et al. (1992) using aseptic culturesof Lemna paucicostala 6746 and Lemna gibba G3 revealed that putrescine application resultedrecovery of growth under K+ deficient conditions. Similar association with putrescine/ADC activityincreases has been reported for magnesium and calcium deficiency and for a surplus ammoniumions, especially in cereals. Putrescine has also been found to be a substitute for inorganic nitrogenfor the growth of in vitro explants from dormant tubers of Helianthus tuberosum (Evans andMalmberg, 1989). In tobacco cell suspension culture, greater accumulation of putrescine was observedwith ammonium ions than with nitrate, thus, demonstrating that polyamine biosynthesis is affectedby both nitrogen source and their level (Evans and Malmberg, 1989). Since then, large number ofinvestigations revealed an increase in level of putrescine in response to other abiotic stresses likeosmotic stress, acid stress, temperature stress and atmospheric pollutants (Young and Galston, 1984;Kao, 1997). However, the trend of increase in putrescine content is not common in all the plants.For instance, in certain species, the levels of spermine and spermidine increase, whereas putrescinelevel decreases. It is quite likely that excessive putrescine accumulation may have deleterious effectson plants as evidenced by presence of chlorotic and necrotic lesions in transgenic plantsoverexpressing oat ADC.

In a comparative study of salt tolerant and salt sensitive rice cultivars, differential accumulationof ADC mRNA during salinity stress was observed which had correlations with differences in ADCenzyme activity. Studies of Lin and Kao (1995) have shown that endogenous levels of putrescinedecrease in rice seedlings under conditions of salinity (NaCl) stress. The addition of precursors ofputrescine resulted in an increase in level of putrescine in NaCl treated roots and shoots, but did notallow the recovery of growth inhibition of rice seedlings. They concluded that endogenous putrescinemay not play a significant role in control of NaCl inhibited growth.

Putrescine accumulation has also been observed in response to osmotic stress. The accumulationof putrescine was more massive in stressed cereal leaf sections and protoplasts than in intact drought-stressed plants (Tiburcio et al., 1986). Putrescine accumulation was found to be due not only to


activation of ADC pathway but also the inhibition of spermidine synthase activity. Further, it hasalso been observed that arginine and ornithine are preferentially utilized as precursors for accumulationduring osmotic stress, and for proline during water stress. Accumulation of novel polyamines likecaldopentamine, previously reported in thermophilic bacteria grown at high temperatures over 50ºC,have also been reported in shoot meristems of alfalfa subjected to drought stress (Oshima, 1983).

Temperature stress has also been reported to induce changes in polyamine patterns in plants.Pringlea antisorbutica, unique endemic cruciferous species from the subarctic zone, is subjected tostrong environmental constraints and exhibits a high flexibility in polyamine metabolism in comparisonwith other crucifers. A massive amount of polyamine agmatine is observed in roots and shoots ofthis species (Hummel et al., 2002). Further, comparison of mineral supply and temperature effectsstrongly indicated a trade-off of polyamine involvement between developmental processes andresponses of plants to stresses (Hummel et al., 2004). The accumulation of putrescine reported infruits of apple, zucchini squash, Citrus and Capsicum is correlated with chilling injury. Theaccumulation of putrescine in these species may either be a cause of or result of chilling injury(Flores and Galston, 1982; Slocum et al., 1984). A large increase in polyamine levels, especiallyputrescine, occurred in hardened plants of Phaseolus, alfalfa and wheat. The increased putrescine inalfalfa and wheat during hardening induction was found to decline rapidly on dehardening. It hasbeen found that chilling tolerant plants accumulate polyamines in response to chilling to a muchgreater extent than chilling sensitive ones (Lee, 1997). Recently, Shen et al. (2000) studied theinvolvement of polyamines in chilling tolerance of cucumber cultivars. They observed three-foldincrease of free spermidine in leaves during chilling and again during rewarming in resistant cultivars.No increase in these polyamines occurred in chilling sensitive cultivar Suyo during either period.The activity profile of biosynthetic enzymes appears to mediate these variations. Pretreatment withspermidine to sensitive cultivars prevented chill-induced increases in content of hydrogen peroxideand generation of peroxide radicals in leaves. Presoaking treatment of seeds of different cultivars ofBrassica species with polyamines reduced the chilling induced inhibition of germination and seedlinggrowth, and prevented loss in membrane integrity, generation of peroxide radicals and onset ofsenescence in leaves exposed to chilling stress under in vitro conditions (Setia and Setia Unpublished).

Reduced availability of oxygen is also reported to affect polyamine metabolism. Studies on theeffect of reduced oxygen availability on polyamine metabolism in a number of Poaceae specieshave provided evidence for association between capacity to accumulate polyamines and tolerance toanoxia (Evans and Malmberg, 1989; Kao 1997). The accumulation of free, conjugated and boundputrescine and to some extent, conjugated spermine and spermidine was reported in rice coleoptilesunder conditions of oxygen deficit. In contrast, accumulation of polyamine conjugates was severelyinhibited in seedling roots, which require oxygen to grow. Anoxic conditions stimulated increasedADC activity.

Changes in cellular pH also affect accumulation of polyamines. At cellular pH, polyaminesbehave as cations and are fully protonated. They can interact with anionic macromolecules, such asDNA, RNA, phospholipids and some proteins (Schuber, 1989). Studies on the effect of low pH onpolyamines in tobacco cell cultures revealed that mutant Dfr I resistant to DFMO grew significantlyfaster than did normal wild type tobacco cell culture under conditions of low pH and maintainedhigher levels of putrescine (Tabor and Tabor,1984). They suggested that high putrescine levelsmight have some protective functions under low pH conditions. Changes in endogenous polyaminetiters observed in spruce trees under conditions of acid rain suggest that polyamines can act as


biochemical indicators of acid rain damage. Weak acids are reported to increase polyamine levels,while weak bases reduce their levels (Kao, 1997).

Putrescine accumulation has also been reported in pea and oat plants exposed to cadmium andin plants grown on sewage sludge containing a high quantity of cadmium and in pea, tomato andbarley plants stressed with chromium (Slocum and Flores, 1991). The physiological analysis hasproved that exogenously applied polyamine diethylenetriamine reduced the toxic effects of arsenicin maize plants by increasing the leaf gas exchange, plastid pigment content and soluble proteins(Stoeva et al., 2005). Air pollutants, fumigation with sulfur dioxide and UV-B exposure also resultsin rapid increase in both free and bound polyamines in different plant species. Seasonal variationsare also reported to exert their influence on polyamine metabolism in different plant parts of juvenilespruce trees and needles of scotch pine (Evans and Malmberg, 1989). Polyamines also act asprotectants against ozone damage (Bors et al., 1989). Exogenous application of polyamines to anozone-sensitive tobacco cultivar provided protection to the leaves against ozone damage along withincreasing free and conjugated putrescine and spermidine (Bors et al., 1989). The increase in freeand conjugated polyamines was higher in leaves of ozone tolerant line than in those of ozonesensitive line. Increase in putrescine synthesis was also reported in other plant species when exposedto ozone (Kao, 1997).

It has been shown that application of herbicides like paraquat, nopropamide and atrazine, increasedputrescine level but had little effect on levels of spermidine and spermine in different plant species(Preston et al., 1992; Zheleva et al., 1994). The toxicity of paraquat in strains of E.coli defective inbiosynthesis of spermidine increased over ten-fold as compared to isogenic strains containingspermidine. The toxicity of paraquat is eliminated by growing the organism in a medium containingspermidine. Application of spermidine and spermine or precursors of polyamine biosynthesis resultedincrease in spermine and spermidine levels as well reduction in paraquat toxicity in rice plants. It isbelieved that paraquat toxicity results from oxidative stress due to generation of free radicals. Thereduction of herbicide toxicity by polyamines is due to increased activities of catalase and peroxidase(Kao, 1997).

Biotic StressesIn the recent past, there is growing interest in exploring possible involvement of polyamines in thedefense reaction of plants to pathogen infection. Most work on polyamines in incompatible interactionsbetween plants and pathogens has been focused on polyamines conjugated to phenolic compounds,especially hydroxy cinnamic acid amides, and changes in free polyamines and their catabolism. Inplant defense, the polyamines have proposed roles in structural defense as an antimicrobial compound,a signal molecule in the induction of systemic acquired resistance, and as an inducer of PR proteins(Chen et al., 1993; Peng and Kuc, 1992). A common feature of host-pathogen interactions is increasein DAO activity and in some interactions of PAO. The intercellular spaces are often the first sitesinvaded by pathogens. DAO and PAO are predominantly localized in the cell wall, di and polyaminesin the apoplast will be oxidized there and the H2O2 produced could be utilized by cell wall peroxidasesin lignification (Angelini et al., 1993), and as a local trigger for programmed cell death (PCD). A20-fold increase in content of free spermine has been observed in the intercellular spaces of tobaccoleaves exhibiting hypersensitive response to TMV infection (Yamakawa et al., 1998). Similar increasesin level of free and conjugated forms of polyamines have been reported in barley following inoculationwith powdery mildew fungus Blumeria graminis (Cowley and Walters, 2002). Chickpea cultivar


resistant to the fungal pathogen Ascochyta rabiei has constitutively higher activity of DAO thansusceptible cultivar. Further, activities of DAO and peroxidase increased markedly followinginoculation of resistant cultivar with pathogen (Angelini et al., 1993). Changes in polyamine titersin two cultivars of barley with varying sensitivity to powdery mildew, Chariat (resistant) and Goldenpromise (susceptible), suggest that polyamine metabolism and DAO activity in particular may beinvolved in the mechanism conferring resistance to powdery mildew in Chariat cultivar (Asthir etal., 2004). Work of Cowley and Walters (2002) in barley suggests that spermine accumulating inthe apoplast could be oxidized by PAO to yield H2O2, which may be involved in resistance response.It is proposed that at least in some host-pathogen interactions, H2O2 accumulation following polyaminecatabolism may be involved in fulfilling one or more of several roles in plant defense includingtriggering the hypersensitive response. Also, accumulation of spermine is suggested to act as atrigger for PR protein and/or trigger cap sage activity leading to HR (Walters, 2003). Elicitormolecules like salicylic acid associated with activation of various plant defense responses followingpathogen attack have also been shown to increase polyamine content in plant tissues (Nemeth et al.,2002). Changes in activity of these enzymes suggest a role for these enzymes in the production ofH2O2.

MECHANISM OF ACTIONThe pace of polyamine research in the recent past is accelerating rapidly, and with advances inmicrochemical techniques and studies with cloned genes and range of experimental approaches, ourunderstanding of mechanism of action of polyamines continues to expand. The identification ofpolyamine response elements and corresponding transacting protein factors that respond to polyamineshas opened new exciting area to study their role in transcription (Wang et al., 2002). From theversatile range of effects of polyamines in plants at various levels of organizations, it is difficult toascribe a single mechanism of their growth stimulating/protective action.

In order to explain various physiological roles of polyamines at molecular level, severalmechanisms have been suggested. Polyamines, being polycationic in nature, can bind to the acidicsites of biomolecules like nucleic acids, proteins and phospholipids of cellular membranes and otheranionic cellular compounds (Tassoni et al., 2002). At physiological pH, polyamines are fullyprotonated and perform many physiological functions by binding to the negative charges of DNAand phospholipids and thereby stabilize the function of nucleus and cell membranes. These areessential for maintaining the structural integrity of the developing plant cell walls, by strengtheningthe links between cell components. Polyamines also play an important role in checking lipidperoxidation and inhibition of transition of ACC to ethylene by quenching of free radicals in varioussystems (Drolet et al., 1986). Also due to their high affinity for binding to the biological membranes,polyamines are suggested as good candidates in protecting plant cells against oxidative damagebeing induced by wide variety of stress conditions (Igarashi and Kashiwagi, 2000).

The exact mechanism of action of polyamines remains to be elucidated, but accumulating evidencein plant and animal systems supports the idea that besides their biophysical effects, polyaminesinteract with protein kinases and transcription factors. The involvement of polyamines in regulationof protein kinases in both nuclear and cytoplasmic functions has been reported in Ranunculus petioles(Bachrach et al., 2001). Differential effects of spermine on phosphatidylinositol-3-kinase and”phosphatidylinositol” l5-kinase suggest that polyamines may be linked to phospholipid basedtransduction mechanism, where inositol phospholipids are an important complex group of signals or


signal precursors, involved in a number of independent pathways (Meijer and Munnik, 2003). Thelipid signaling cascade involves different types of enzymes. Among these, phospholipase D is a keycomponent that hydrolyses phospholipids at terminal phosphodiester bond. Recent studies on in vivoeffect of polyamines on key components of the phospholipid based signaling suggest that they maymodulate the cellular signals by differentially affecting components of phospholipid cascade(Echevarria-Machado et al., 2002, 2004).

PROSPECTS OF POLYAMINE RESEARCH IN RELATIONTO CROP IMPROVEMENTThe wide array of effects of polyamines on various growth and developmental processes and inducingtolerance against environmental stresses as well as defense against microorganisms and insects compelthe plant scientists to consider them as candidates for active plant growth regulators. Recently,polyamines have also been implicated in cell death (apoptosis) and, thus, can be classified as bivalentregulator of cellular functions (Pignatti et al., 2004). The multifacet effects of polyamines offer agreat potential for yield improvements in crop plants.

Polyamines influence different developmental processes by affecting metabolism and variousphysiological processes under normal and stress conditions. However, the observed differencesbetween growth stimulating/protective activities of exogenously applied putrescine, spermine andspermidine might be due to variations in their chemical structure, uptake, transport and metabolism,as well influence on their endogenous levels. But it is still ambiguous with respect to cause- and –effect relationship between polyamine titers and physiological effects.

The in vitro promotion of various developmental events, such as cell division, callus growth,somatic embryogenesis, root and shoot formation, protoplast regeneration by application of polyaminesoffers a great potential for their use in micropropagation and production of artificial seeds. Efficientplant regeneration is a prerequisite for plant transformation. Manipulation of endogenous polyaminelevels by their exogenous application may prove useful in improving regeneration and maintenanceof morphogenetic capacity of cultures. The ability of polyamines to induce flowering under non-inductive conditions or in non-flowering genotypes may also find practical application in agriculture.Further, the effectiveness of polyamines to improve pollination and ovule longevity can greatly helpin improving yield and yield components in different crop plants. Numerous model systems onsexual morphogenesis in plants are currently established (Tanurdzic and Banks, 2004), and applicationof polyamines to these systems could also be profitable exercise for establishing their role in floraldevelopment and sex differentiation.

In the area of reproductive biology, a great deal of attention has been given to their involvementin fruit development and senescence because of potential metabolic connection between polyaminesand ethylene through propylamine group of SAM. The effects of exogenously applied polyaminesin regulating these processes are similar to those obtained with cytokinins with exceptions of a fewcounter examples (Walden et al., 1997). Delayed fruit ripening and onset of senescence processescan be attributed to the significant free radical scavenging property of polyamines. Also, increase infruit firmness and delayed fruit softening by polyamines could be ascribed to their property ofinhibiting cell wall degrading enzymes and maintaining rigidity of cell walls. Polyamines and ethyleneare antagonistic and exogenously applied polyamines can lower ethylene production. Their potentialmay be exploited for retarding the senescence and aging in plants. Future studies should be focusedon the expression of senescence associated genes in polyamine mutants and examination of effect of


their exogenous application will greatly help to further clarify the role of polyamines in theseprocesses.

Alterations in polyamine titers in response to various stresses in different plant species suggestthat they may be involved in protecting the crop plants against abiotic stresses, especially drought,salinity, low pH, anoxia and extreme temperatures. Long chain alkylenediamines, such as octa anddecamethylenediamine, are reported to protect the crops against frost damage, wilting, loss ofchlorophyll and photochemical oxidation. Novel polyamines, such as thermo-spermine andcaldopentamine have been found to confer thermoprotection during in vitro protein synthesis,suggesting thereby possibility of exploiting the use of such compounds against high temperaturestress. The specific inhibition of polyamine biosynthesis in fungi using their biosynthesis inhibitorshas emerged as a useful and promising approach for controlling fungal diseases in plants.

Attempts have been made in the recent past to find means to manipulate polyamine biosynthesisby using sense and antisense transgenic approaches. Further, genes encoding ADC or ODC, SAMDCand SAMS have also been cloned from plant and animal sources. The use of transgenic approacheshas revealed the feasibility of modulating cellular polyamine contents. Generally, genetic manipulationof single steps of polyamine biosynthesis pathway (ODC or ADC) lead to their elevated levels. Butthus far, polyamine induced response or developmental event has been correlated only with changesin polyamine levels and spectra. Recently, several important advances have been made in plantpolyamine research. For example, most of the genes encoding polyamine biosynthetic enzymeshave been isolated, and transgenic plants or mutants with changed polyamine metabolism have beencreated. The analysis of transgenic plants together with results from mutant studies indicates thatchanges in levels of polyamines can alter growth and developmental processes and improve resistanceagainst various stresses. Further, application of advanced genomic and proteomic approaches willhelp to elucidate the role of polyamines in particular plant processes.

The physiological and molecular mechanism of action of polyamines in improving productivityand resistance to environmental stresses, however, needs further elucidation at various levels oforganizations. Thus, it would seem an opportune time to resolve whether polyamines, the smallbiomolecules, trigger the growth and developmental processes in plants. The new findings in thisfield may allow us to employ polyamines as highly promising natural substances suitable for wideapplication in plant protection and to boost crop production.

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Polyamines: An Overview and Prospects in Crop Improvement

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