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Polyamines: An Overview and Prospects in Crop Improvement
Neelam Setia and R.C. Setia
Department of Botany Punjab Agricultural University, Ludhiana-141004, India email: firstname.lastname@example.org
Polyamines are low molecular weight polycationic compounds that are ubiquitously distributed in all living organisms. The basic mechanisms of polyamine biosynthesis and metabolism coupled with identification of mutants with altered metabolism have greatly helped in understanding role of these compounds in a wide range of cellular, growth and developmental processes in plants, such as cell division, embryogenesis, flower and fruit development, post-harvest fruit management and senescence. Different polyamines have been implicated in imparting resistance/protection against various abiotic and biotic stresses. At physiological pH, polyamines being fully protonated perform various physiological functions by binding to the negative charges of nucleic acids and phospholipids, and thereby stabilize the function of nucleus and cell membranes. They are suggested as good candidates in protecting the plants against oxidative damage being induced by a wide variety of stress conditions. In this review, recent advances relevant to the molecular mechanism of polyamine action and their involvement in signal transduction are also discussed keeping in view the future prospectives of polyamine research in crop improvement.
Keywords: Polyamines, biosynthesis, metabolism, growth, embryogenesis, senescence, plant stresses, signal transduction
INTRODUCTION Polyamines are low molecular weight nitrogen-containing aliphatic compounds found in all living organisms. The major polyamines found in plant cells are putrescine, spermine and spermidine. In addition, cadaverine, a diaminopentane, is a common constituent of legumes and is present in much less concentration than putrescine. Less common polyamines like 1,3-diamine propane and homospermidine, which differ from the common polyamines in the number of methylenic moieties between amino groups, have been detected in a broad spectrum of biological systems including plants, algae, bacteria and animals (Oshima, 1983; Cohen, 1998). Also nor-spermidine and norspermine types are reported in Medicago sativa (Rodriguez-Garay et al., 1989). Many lines of research evidences, such as treatments with exogenous polyamines, quantification of their endogenous levels, 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
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shown their role in wide range of cellular, growth and developmental processes like DNA replication, transcription and translation, cell division, organogenesis, senescence, environmental stresses and infection 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 under extreme environmental conditions both in bacteria and higher plants. The ability of polyamines to enable biological systems to grow or function under extreme conditions has provided opportunities for 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 in polyamine metabolism (Galston,1983). Recent studies of Tun et al. (2006) using a fluorimetric method employing the cell-impermeable nitric oxide (NO) binding dye revealed that polyamines, spermidine and spermine induced NO biosynthesis in Arabidopsis seedlings. They observed that induction of NO synthesis was tissue specific being maximum in the elongation zone of Arabidopsis root tip and primary leaves, especially in veins and trichomes. However, a little or no effect of polyamines on NO production was observed in cotyledons in presence of polyamines. The involvement of polyamines in regulation of various plant responses in interaction with hormones suggests their role as plant growth regulator. But unlike plant growth regulators, polyamines in plants are required in millimolar concentrations, rather than in micromolar levels typical of the traditionally accepted plant hormones and play dual function, i.e., both structural and regulatory. Due to their versatile involvement in controlling various physiological, biochemical and developmental processes in plants, polyamines are now recognized as a new class of plant growth regulators (Galston and Kaur-Sawhney, 1995). This article, besides general introduction to polyamines, also presents an overview of their involvement in various growth and developmental processes with focus on recent advances in polyamine research in relation to crop improvement.
STRUCTURE, OCCURRENCE AND LOCALIZATION OF POLYAMINES Polyamines, the low molecular weight nitrogen containing aliphatic compounds, are positively charged at physiological pH due to presence of amino groups and act as polycations, with charges distributed along a flexible carbon chain. They occur in free or conjugated forms. Polyamine conjugates with phenolic acids are widespread in higher plants (Martin-Tanguy, 1985). Polyamines are also bound to some macromolecules like proteins and nucleic acids (Smith, 1985). The conjugates may act as storage forms of polyamines from which free bases may be released and transported as and when required. The level of polyamines in plant cells depends on their biosynthesis, degradation, conjugation, transport and conversion to other metabolites. Structure of H2N-(CH2)4 H2N-(CH2)3 NH2-(CH2)3 H2N-(CH2)5 polyamines NH2 Putrescine (Diamine) NH - (CH2)4- NH2 Spermidine (Triamine) NH - (CH2)4 - NH - (CH2)3 - NH2 Spermine (Tetramine) NH2 Cadaverine (Diaminopentane)
Some common polyamine-conjugates Coumaroyl putrescine Alkyl cinnamoyl putrescine Caffeoyl putrescine Caffeoyl spermidine
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Feruloyl putrescine Alkyl cinnamoyl spermine Hydroxycinnamoyl putrescine Coumaroyl agmatine Polyamines being small, soluble, diffusible molecules at cellular pH, their immobilization in the cell for localization is difficult to achieve. Several approaches used to study the localization of polyamines 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 found in cytoplasm fraction. Polyamines present in the cell wall are mainly associated with pectic polysaccharides 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-third of the total polyamines are bound to the cell wall possibly due to adsorption of protonated polyamines due to net negative charge. Vacuole is suggested as the temporary reservoir of polyamines, as it can be readily transported across the tonoplast and plasma membrane. Polyamines are also found associated with thylakoid membranes of chloroplasts and other subcellular organelles like mitochondria (Cowley and Walters, 2002).
POLYAMINE BIOSYNTHESIS AND METABOLISM A 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 ornithine decarboxylase (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 to its relative abundance in storage proteins, involvement in long distance transport, and position in plant economy with nitrogen to carbon ratio. Spermidine and spermine are synthesized by the addition of an aminopropyl group to one or both primary amino groups of putrescine by spermidine and spermine synthases, respectively. The aminopropyl moiety is derived from decarboxylated S-adenosyl methionine (SAM) which in turn is derived from methionine. SAM is also involved in ethylene biosynthesis (Evans and Malmberg, 1989; Cohen, 1998). The genes for both ADC and ODC have been 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 during cell elongation and under the conditions of stress. The activity of ADC in most of the tissues is modulated by light and hormones. In some legumes, cadaverine is synthesized from L-lysine by action of lysine decarboxylase (Smith, 1985). The level of polyamines in plant cells is regulated by both synthesis and degradation. The breakdown or catabolism of polyamines is brought about by specific amino oxidases that include diamine oxidases (DAO), highly specific for diamines, and the reaction products of DAO on putrescine are pyrroline, H2O2 and ammonium. A second class of plant enzymes, polyamine oxidases (PAO), catalyzes an analogous reaction, yielding pyrroline and aminopropylpyrroline from spermidine and spermine, 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 spermidine are recycled (Smith, 1985). The catabolic oxidation of polyamines not only regulate their concentration, but may also play other important roles, for example, use of H2O2 generated by action of polyamine oxidases in cell wall lignification.
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Methionine S-Adenosyl methionine synthase S-Adenosyl L-Methionine (SAM) SAM decarboxylase Decarboxylated SAM
Agmatine Agmatine iminohydrolase N-Carbamoyl putrescine
NCarbamoyl putrescine amidohydrolase
L-Ornithine Ornithine decarboxylase Putrescine Spermidine synthase
Spermidine Spermine synthase
Fig. 1. Polyamine biosynthetic pathway (Names of enzymes are shown in italics).
TRANSPORT Extensive 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 of polyamines is energy dependent and is a function of external pH. In plants, there are reports to suggest that polyamine transport occurs as an active bi-directional process requiring energy and like other metabolites/nutrients the translocation of polyamines is dependent on both temperature and relative humidity (Tiburcio et al., 1997). Plant cells are also equipped with efficient systems for uptake of exogenously applied polyamines. Polyamines taken up from external source or synthesized within 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 protoplasts in the presence of Ca2+ and observed its stimulation in their presence. Application of vandate, an ATPase inhibitor, strongly inhibited IAA-stimulated spermidine uptake, suggesting thereby involvement of energy-dependent mechanism in this transport. GROWTH AND DEVELOPMENTAL RESPONSES TO POLYAMINES In higher plants, polyamines have been implicated in a wide range of growth and developmental processes including cell division, embryogenesis, root development, flowering, fruit development and leaf senescence. In addition to their roles in developmental processes, polyamines also play an important role in plant stress responses. Cell Division In general, high levels of free polyamines are observed in cells undergoing division. During transition from G1 to S phase during cell division, both spermine and spermidine are required. The conversion
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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 division is known to depend on the activity of multiprotein complex, which includes cyclins. The cyclins in quiescent sugar beet cells induced by polyamines, especially putrescine and their precursors arginine and ornithine, indicates their role in controlling gene expression and cell division. Exogenous application of polyamines to protoplasts or cells in tissue culture results in a temporary or sustained increase in cell division (Evans and Malmberg, 1989). High levels of spermine were observed in primary root apices and in decapitated roots during lateral root formation using DFMO (αdifluoromethyl ornithine) and thymidine. Further ODC was localized primarily in the meristematic zones (Evans and Malmberg, 1989). The treatment of cells with inhibitors of polyamine biosynthesis results in an arrest of cell proliferation and this inhibition can be reversed by their external supplementation. Further, the observed correlations between peaks of polyamine levels in synchronizable tobacco cells and peaks of casein kinase II activity suggest that interactions with polyamines might be important in regulating kinase activity during cell cycle.
Embryogenesis The morphogenetic potential of polyamines to produce embryoids was first investigated in carrot cultures. A significant increase in ADC activity and putrescine content was observed when carrot cultures from callus medium were shifted to embryogenesis medium (Montague et al., 1978). The induction 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 more ethylene and less polyamines, possibly because of switching the fate of SAM, a common precursor of both. The evidence suggests that the morphogenetic potential of callus to produce embryoids is elicited by polyamines and inhibited by ethylene. It remains to be determined if the interaction between 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 embryogenesis and failed to show increase in polyamine content when placed in the medium without auxin (Fienberg et al., 1984). Four critical stages of embryogenesis including callus induction, cellular acquisition of morphogenetic competence, expression of embryogenic programme and development and maturation of somatic embryos have been identified during somatic embryogenesis from leaf discs of egg plant (Solanum melongena). During induction of embryogenic callus in egg plant, there were high titers of free, conjugated and total putrescine as compared with spermine and spermidine, because of 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 of embryogenesis further revealed high levels of polyamines, especially putrescine, in discs from apical region of leaf with high embryogenic capacity than from basal region of leaf with poor embryogenic capacity. Treatment of basal discs with putrescine for 4 to 7 days improved embryogenesis. A significant difference in polyamine requirement during somatic embryogenesis has been observed in two alfalfa lines. Both genotypes showed accumulation of putrescine during induction as medium with 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 the genotypes, however, embryogenesis was reduced in only one of the two genotypes. Based on the existing information, it is inferred that correlations between polyamines and their biosynthetic enzymes
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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).
Rooting Development of roots is under the control of hormonal, metabolic and environmental cues that can act on genetically controlled developmental programmes, and thus, can affect the plasticity of root architecture. 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 in root development and growth has been established in several plant systems, mostly involving use of stem/ hypocotyl segments under in vitro conditions (Coúee et al., 2004). The induction of rooting in stem cuttings of Phaseolus vulgaris with indolebutyric acid (IBA) is accompanied by increased levels of polyamines (Jarvis et al., 1985; Evans and Malmberg, 1989). Application of inhibitors of polyamine biosynthesis reduced their endogenous levels and inhibited IBA-induced rise in spermine and spermidine and checked root induction and growth. However, IBA induced increase in polyamine content failed to stimulate rooting in Vigna hypocotyls cuttings (Friedman et al., 1982). The increased level of polyamines during root and nodule growth in mungbeans is also accompanied by changes in RNA, DNA and protein contents. The induction of rooting in tobacco callus cultures is found to be inversely related to putrescine and alkaloid titers. Studies by Chriqui et al. (1986) have provided evidence that there exists a synergistic effect between auxins and ornithine during rhizogenesis in Datura innoxia leaf explants. A positive correlation has been observed between agmatine, spermidine and spermine contents and primary 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 and root growth in seedlings of Pringlia antiscorbutica, the sub-antarctic cruciferous species (Hummel et al., 2002, 2004). They demonstrated the dominance of ADC pathway in synthesis of polyamines in 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 of this polyamine in the rhizosphere has a bacteriostatic effect on root cells colonizing Pseudomonas fluorescence strain WCS 365. The colonizing ability of bacteria decreases as a result of increased uptake of putrescine (Kuiper et al., 2001). Spermidine and spermine are less effective in influencing the root colonizing ability of bacteria as compared to putrescine. Flowering Polyamines 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 found absent in young plants but accumulate progressively in apical leaves and then in large quantities in different floral parts (Martin-Tanguy, 1985). The relationship between polyamines and flowering has been observed in whole plants. Polyamine titers in relation to photoperiodic induction of flowering are well characterized in a short day plant, Xanthium strumarium and a long day plant, Sinapsis alba. Their role in flowering was established by use of polyamine enzyme biosynthesis inhibitors which prevented flowering and was resumed in absence of inhibitors or when polyamines were applied exogenously (Applewhite et al., 2000; Slocum and Galston, 1985). Cyclohexylamine (CHA), a specific inhibitor of spermidine synthesis significantly reduced number of floral primordia in Polyanthus tuberosa. Exogenous application of putrescine and silver nitrate (AgNO3) in shoot cultures
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of Cichorium intybus under in vitro conditions enhanced floral initiation and floral development. The morpohogenetic response and level of endogenous conjugated pool of polyamines diminished following 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 organs of two ecotypes and genetic line of Arabidopsis thaliana revealed that flowers had highest titers of these polyamines, with spermidine predominating (Applewhite et al., 2000). Application of appropriate inhibitors lowered spermidine titer and inhibited bolting and flowering in this species. Further, enzyme inhibitors of spermidine synthesis given shortly before the transition from short day conditions to long day conditions prevented flowering in this species. Further, the addition of spermidine in the growth medium significantly accelerated flowering in delayed flowering mutant CS 3123 of Arabidopsis. These studies have clearly demonstrated close connection between polyamines and reproductive development in A.thaliana (Applewhite et al., 2000). The induction of flowering has also 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). Acquisition of some lines of mutants in various crops, such as tobacco and petunia, further helped in implicating the 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 polyamine metabolism (Malmberg and McIndoo, 1983). Generally, high polyamine levels contribute to the pattern of abnormal stamen development. The role of polyamines in regulation of pollen germination and 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 higher level of neutral hydroxy cinnamic amides than female ones, but the latter have more hydroxyl cinnamic amides than the former (Martin-Tanguy, 1985). The concentrations of polyamines also vary in sterile and fertile organs. Studies in Chrysanthemum, tobacco, some Araceae species and tomato (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 the corresponding maintainer line. However, in certain species, higher levels of polyamines were found to be responsible for stamen sterility. Such a discrepancy could be attributed to polyamine homeostasis in a given plant species. Studies on the effect of repeated severe pruning of hazel nut trees on endogenous 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 mechanism of action of polyamines during floral development still remains unclear. Polyamines might be a part of complex mechanism involved in flowering signal. However, much more deep insight is still required to understand the mechanism of polyamine action during flowering in plants.
Fruit development Studies on polyamine titers during different stages of fruit development clearly points toward relationship between polyamines and fruit development. The level of polyamines is generally high during 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 the major one in avocado, putrescine in tomato and spermidine in opera (Liu et al., 2006). Early stages of fruit development involve active cell division and require sufficiently high level of polyamines.
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The decline in their content during later stages of fruit development may act as a signal for onset of ripening process. However, in fruits like citrus and tomato, increase in polyamine titers is also reported during maturation and ripening (Saftner and Baldi, 1990). Ethylene, a gaseous hormone playing an important role in fruit ripening, and polyamines share a common precursor, SAM for their synthesis. In one pathway, SAM releases ethylene through ACC (1-aminocyclopropane-1-carboxylic acid), and in the other it is converted into decarboxylated SAM 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 thus reflect the possibility of existence of inverse relationship between them. Comparatively higher levels of polyamines and low levels of ethylene have been observed in long-keeping tomato fruits (Dibble et al., 1988). Further, an increase in level of endogenous polyamines has been observed in vitro cultured kiwi fruit explants by blocking ethylene synthesis by using ethylene inhibitor amino ethoxy vinyl glycine. Exogenously applied polyamines have also been found effective for increasing fruit set and yield in apple (Biasi et al., 1988), delaying maturation and flesh softening in fruits and reducing fruit drop by inhibiting abscission in lot of plants. (Aziz et al., 2001; Liu et al., 2006). An immediate increase in firmness has been observed when McIntosh and Golden Delicious apples harvested 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 to fruits as spermidine and spermine at concentrations higher than 1 mM led to the development of small black spots in apples. Based on the existing information, the effects of polyamines are at variance among different plant species, polyamine type, concentration and stage of application. The promotory effects of exogenously applied polyamines could be countered by their inhibitors (Tarenghi and Martin-Tanguy, 1995), suggesting thereby their potential for post-harvest fruit management.
Stomatal Movements A stomatal aperture defined by two guard cells is responsible for gas exchange between plants and the atmosphere. Changes in guard cell turgor that instigate opening and closing of stomatal aperture are controlled by number of factors through modulation of ion channel activity and pumps (Ward et al., 1995). Among the ion channels in guard cells, the inward K+ channels are an important player in stomatal regulation. A number of studies have shown that inward K+ channel-inhibiting processes or 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 with different 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 in guard cells for induction of stomatal closure. Mechanism of stomatal regulation is one of the most studied mechanisms of plant responses to stresses. Many of the stress factors are known to elevate polyamines. Electrophysiological and molecular studies in animal systems have demonstrated the role of polyamines in modulation of ion channels by direct binding to the channel protein or membrane component (Johnson, 1996). Studies of Brüggemann et al. (1998) showed that in higher plants, polyamines block fast activating vacuolar cation channels. Among the ion channels in the guard cells, the inward K+ channels are an important
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player in stomatal regulation and factors/processes blocking inward K+ channels inhibit stomatal opening. Studies of Liu et al. (2000) in Vicia faba leaves revealed involvement of polyamines in regulation of voltage-dependent inward K+ channels in plasma membrane of guard cells. Whole-cell patch clamp analysis showed that intracellular applications of all natural polyamines including spermidine, spermine, cadaverine and putrescine induced closure of stomata by inhibiting the inward K+ current across the plasma membrane of guard cells. Further, identification of target channel at molecular level 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 molecular level, they suggested that polyamines target KATI-like inward K+ channels in guard cells and modulate stomatal movements and provide a link between stress conditions, polyamine levels and regulation of stomatal movements.
Senescence Senescence 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 by cessation of photosynthesis, disintegration of organelle structures, loss of chlorophyll, proteins, a dramatic increase in lipid oxidation, breakdown of cell wall components and disruption of cell membranes leading to loss of cell/tissue structure (Buchanan-Wollaston, 1997). Senescence, though a terminal developmental stage in the life cycle of a plant, can also be accelerated by an array of both abiotic and biotic factors (Smart, 1994). Like other plant processes, polyamines also act as antisense agents in a number of plant species by inhibiting ethylene synthesis. Both spermidine and spermine are reported to delay leaf senescence by preventing chlorophyll loss and stabilizing the thylakoid membranes, possibly by checking inhibition of RNAase and protease activities and ethylene synthesis (Lee et al., 1997; Pandey et al., 2000). While comparing ethylene production with changes in endogenous polyamine levels from control and MGBG treated petals of carnation flowers in presence of spermine during entire incubation period, it was suggested that the inhibition of senescence by 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, although cytokinins 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 have been reported where polyamines did not seem to retard senescence. The increased flower longevity and retarded senescence in cut carnation with aminotriazole was the result of inhibition of climacteric peak 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 carnations failed to increase flower longevity (Downs and Lovell, 1986). On the contrary, an increase in endogenous level of free putrescine was observed in carnation flowers during senescence, while no variation in content of free polyamines was observed in climacteric and non-climacteric carnation flowers at pre-climacteric stage. Studies of Botha and Whitehead (1992) on petunia flowers revealed that initial decline in polyamines during pre-climacteric stages was not accompanied by concomitant increase in ethylene production. This can be due to the fact that synthesis of ACC by enzyme ACC synthase is rate limiting step and not the availability of SAM in the pathway of ethylene production
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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 not result 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 biochemical levels still remains unresolved.
POLYAMINES IN STRESS MANAGEMENT Changes in polyamine levels and activities of enzymes of their biosynthesis have been observed in a 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 to environmental challenges by changing their growth and developmental patterns. Abiotic Stresses The earliest report of increase in level of polyamines dates back to 1950’s in barley plants grown under K+ deficiency conditions and this effect has been widely confirmed (Smith, 1985). Potassium deficiency resulted in a 20-fold increase in putrescine levels, a 6-fold increase in ADC activity in oat shoots (Young and Galston, 1984) and 7-fold increase in regenerating buds of tobacco (Klinguer et al., 1986). Subsequently, accumulation of polyamines under potassium deficient conditions has been reported in several plant species (Flores, 1991). Tachimoto et al. (1992) using aseptic cultures of Lemna paucicostala 6746 and Lemna gibba G3 revealed that putrescine application resulted recovery of growth under K+ deficient conditions. Similar association with putrescine/ADC activity increases has been reported for magnesium and calcium deficiency and for a surplus ammonium ions, especially in cereals. Putrescine has also been found to be a substitute for inorganic nitrogen for the growth of in vitro explants from dormant tubers of Helianthus tuberosum (Evans and Malmberg, 1989). In tobacco cell suspension culture, greater accumulation of putrescine was observed with ammonium ions than with nitrate, thus, demonstrating that polyamine biosynthesis is affected by both nitrogen source and their level (Evans and Malmberg, 1989). Since then, large number of investigations revealed an increase in level of putrescine in response to other abiotic stresses like osmotic 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 putrescine level decreases. It is quite likely that excessive putrescine accumulation may have deleterious effects on plants as evidenced by presence of chlorotic and necrotic lesions in transgenic plants overexpressing oat ADC. In a comparative study of salt tolerant and salt sensitive rice cultivars, differential accumulation of ADC mRNA during salinity stress was observed which had correlations with differences in ADC enzyme activity. Studies of Lin and Kao (1995) have shown that endogenous levels of putrescine decrease in rice seedlings under conditions of salinity (NaCl) stress. The addition of precursors of putrescine resulted in an increase in level of putrescine in NaCl treated roots and shoots, but did not allow the recovery of growth inhibition of rice seedlings. They concluded that endogenous putrescine may not play a significant role in control of NaCl inhibited growth. Putrescine accumulation has also been observed in response to osmotic stress. The accumulation of putrescine was more massive in stressed cereal leaf sections and protoplasts than in intact droughtstressed plants (Tiburcio et al., 1986). Putrescine accumulation was found to be due not only to
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activation of ADC pathway but also the inhibition of spermidine synthase activity. Further, it has also been observed that arginine and ornithine are preferentially utilized as precursors for accumulation during osmotic stress, and for proline during water stress. Accumulation of novel polyamines like caldopentamine, 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 to strong environmental constraints and exhibits a high flexibility in polyamine metabolism in comparison with other crucifers. A massive amount of polyamine agmatine is observed in roots and shoots of this species (Hummel et al., 2002). Further, comparison of mineral supply and temperature effects strongly indicated a trade-off of polyamine involvement between developmental processes and responses of plants to stresses (Hummel et al., 2004). The accumulation of putrescine reported in fruits of apple, zucchini squash, Citrus and Capsicum is correlated with chilling injury. The accumulation 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, especially putrescine, occurred in hardened plants of Phaseolus, alfalfa and wheat. The increased putrescine in alfalfa and wheat during hardening induction was found to decline rapidly on dehardening. It has been found that chilling tolerant plants accumulate polyamines in response to chilling to a much greater extent than chilling sensitive ones (Lee, 1997). Recently, Shen et al. (2000) studied the involvement of polyamines in chilling tolerance of cucumber cultivars. They observed three-fold increase 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 with spermidine to sensitive cultivars prevented chill-induced increases in content of hydrogen peroxide and generation of peroxide radicals in leaves. Presoaking treatment of seeds of different cultivars of Brassica species with polyamines reduced the chilling induced inhibition of germination and seedling growth, and prevented loss in membrane integrity, generation of peroxide radicals and onset of senescence 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 the effect of reduced oxygen availability on polyamine metabolism in a number of Poaceae species have provided evidence for association between capacity to accumulate polyamines and tolerance to anoxia (Evans and Malmberg, 1989; Kao 1997). The accumulation of free, conjugated and bound putrescine and to some extent, conjugated spermine and spermidine was reported in rice coleoptiles under conditions of oxygen deficit. In contrast, accumulation of polyamine conjugates was severely inhibited in seedling roots, which require oxygen to grow. Anoxic conditions stimulated increased ADC activity. Changes in cellular pH also affect accumulation of polyamines. At cellular pH, polyamines behave as cations and are fully protonated. They can interact with anionic macromolecules, such as DNA, RNA, phospholipids and some proteins (Schuber, 1989). Studies on the effect of low pH on polyamines in tobacco cell cultures revealed that mutant Dfr I resistant to DFMO grew significantly faster than did normal wild type tobacco cell culture under conditions of low pH and maintained higher levels of putrescine (Tabor and Tabor,1984). They suggested that high putrescine levels might have some protective functions under low pH conditions. Changes in endogenous polyamine titers observed in spruce trees under conditions of acid rain suggest that polyamines can act as
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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 and in plants grown on sewage sludge containing a high quantity of cadmium and in pea, tomato and barley plants stressed with chromium (Slocum and Flores, 1991). The physiological analysis has proved that exogenously applied polyamine diethylenetriamine reduced the toxic effects of arsenic in 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 results in rapid increase in both free and bound polyamines in different plant species. Seasonal variations are also reported to exert their influence on polyamine metabolism in different plant parts of juvenile spruce trees and needles of scotch pine (Evans and Malmberg, 1989). Polyamines also act as protectants against ozone damage (Bors et al., 1989). Exogenous application of polyamines to an ozone-sensitive tobacco cultivar provided protection to the leaves against ozone damage along with increasing free and conjugated putrescine and spermidine (Bors et al., 1989). The increase in free and conjugated polyamines was higher in leaves of ozone tolerant line than in those of ozone sensitive line. Increase in putrescine synthesis was also reported in other plant species when exposed to ozone (Kao, 1997). It has been shown that application of herbicides like paraquat, nopropamide and atrazine, increased putrescine 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 in biosynthesis of spermidine increased over ten-fold as compared to isogenic strains containing spermidine. The toxicity of paraquat is eliminated by growing the organism in a medium containing spermidine. Application of spermidine and spermine or precursors of polyamine biosynthesis resulted increase in spermine and spermidine levels as well reduction in paraquat toxicity in rice plants. It is believed that paraquat toxicity results from oxidative stress due to generation of free radicals. The reduction of herbicide toxicity by polyamines is due to increased activities of catalase and peroxidase (Kao, 1997).
Biotic Stresses In the recent past, there is growing interest in exploring possible involvement of polyamines in the defense reaction of plants to pathogen infection. Most work on polyamines in incompatible interactions between 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. In plant 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 increase in DAO activity and in some interactions of PAO. The intercellular spaces are often the first sites invaded by pathogens. DAO and PAO are predominantly localized in the cell wall, di and polyamines in the apoplast will be oxidized there and the H2O2 produced could be utilized by cell wall peroxidases in lignification (Angelini et al., 1993), and as a local trigger for programmed cell death (PCD). A 20-fold increase in content of free spermine has been observed in the intercellular spaces of tobacco leaves exhibiting hypersensitive response to TMV infection (Yamakawa et al., 1998). Similar increases in level of free and conjugated forms of polyamines have been reported in barley following inoculation with powdery mildew fungus Blumeria graminis (Cowley and Walters, 2002). Chickpea cultivar
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resistant to the fungal pathogen Ascochyta rabiei has constitutively higher activity of DAO than susceptible cultivar. Further, activities of DAO and peroxidase increased markedly following inoculation of resistant cultivar with pathogen (Angelini et al., 1993). Changes in polyamine titers in two cultivars of barley with varying sensitivity to powdery mildew, Chariat (resistant) and Golden promise (susceptible), suggest that polyamine metabolism and DAO activity in particular may be involved in the mechanism conferring resistance to powdery mildew in Chariat cultivar (Asthir et al., 2004). Work of Cowley and Walters (2002) in barley suggests that spermine accumulating in the 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 polyamine catabolism may be involved in fulfilling one or more of several roles in plant defense including triggering the hypersensitive response. Also, accumulation of spermine is suggested to act as a trigger for PR protein and/or trigger cap sage activity leading to HR (Walters, 2003). Elicitor molecules like salicylic acid associated with activation of various plant defense responses following pathogen 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 of H2 O2 .
MECHANISM OF ACTION The pace of polyamine research in the recent past is accelerating rapidly, and with advances in microchemical techniques and studies with cloned genes and range of experimental approaches, our understanding of mechanism of action of polyamines continues to expand. The identification of polyamine response elements and corresponding transacting protein factors that respond to polyamines has opened new exciting area to study their role in transcription (Wang et al., 2002). From the versatile range of effects of polyamines in plants at various levels of organizations, it is difficult to ascribe a single mechanism of their growth stimulating/protective action. In order to explain various physiological roles of polyamines at molecular level, several mechanisms have been suggested. Polyamines, being polycationic in nature, can bind to the acidic sites of biomolecules like nucleic acids, proteins and phospholipids of cellular membranes and other anionic cellular compounds (Tassoni et al., 2002). At physiological pH, polyamines are fully protonated and perform many physiological functions by binding to the negative charges of DNA and phospholipids and thereby stabilize the function of nucleus and cell membranes. These are essential for maintaining the structural integrity of the developing plant cell walls, by strengthening the links between cell components. Polyamines also play an important role in checking lipid peroxidation and inhibition of transition of ACC to ethylene by quenching of free radicals in various systems (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 damage being induced by wide variety of stress conditions (Igarashi and Kashiwagi, 2000). The exact mechanism of action of polyamines remains to be elucidated, but accumulating evidence in plant and animal systems supports the idea that besides their biophysical effects, polyamines interact with protein kinases and transcription factors. The involvement of polyamines in regulation of 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 based transduction mechanism, where inositol phospholipids are an important complex group of signals or
Polyamines: An Overview and Prospects in Crop Improvement 389
signal precursors, involved in a number of independent pathways (Meijer and Munnik, 2003). The lipid signaling cascade involves different types of enzymes. Among these, phospholipase D is a key component that hydrolyses phospholipids at terminal phosphodiester bond. Recent studies on in vivo effect of polyamines on key components of the phospholipid based signaling suggest that they may modulate the cellular signals by differentially affecting components of phospholipid cascade (Echevarria-Machado et al., 2002, 2004).
PROSPECTS OF POLYAMINE RESEARCH IN RELATION TO CROP IMPROVEMENT The wide array of effects of polyamines on various growth and developmental processes and inducing tolerance against environmental stresses as well as defense against microorganisms and insects compel the 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 bivalent regulator of cellular functions (Pignatti et al., 2004). The multifacet effects of polyamines offer a great potential for yield improvements in crop plants. Polyamines influence different developmental processes by affecting metabolism and various physiological processes under normal and stress conditions. However, the observed differences between growth stimulating/protective activities of exogenously applied putrescine, spermine and spermidine 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 polyamines offers a great potential for their use in micropropagation and production of artificial seeds. Efficient plant regeneration is a prerequisite for plant transformation. Manipulation of endogenous polyamine levels by their exogenous application may prove useful in improving regeneration and maintenance of morphogenetic capacity of cultures. The ability of polyamines to induce flowering under noninductive 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 help in improving yield and yield components in different crop plants. Numerous model systems on sexual morphogenesis in plants are currently established (Tanurdzic and Banks, 2004), and application of polyamines to these systems could also be profitable exercise for establishing their role in floral development and sex differentiation. In the area of reproductive biology, a great deal of attention has been given to their involvement in fruit development and senescence because of potential metabolic connection between polyamines and ethylene through propylamine group of SAM. The effects of exogenously applied polyamines in regulating these processes are similar to those obtained with cytokinins with exceptions of a few counter examples (Walden et al., 1997). Delayed fruit ripening and onset of senescence processes can be attributed to the significant free radical scavenging property of polyamines. Also, increase in fruit firmness and delayed fruit softening by polyamines could be ascribed to their property of inhibiting cell wall degrading enzymes and maintaining rigidity of cell walls. Polyamines and ethylene are antagonistic and exogenously applied polyamines can lower ethylene production. Their potential may be exploited for retarding the senescence and aging in plants. Future studies should be focused on the expression of senescence associated genes in polyamine mutants and examination of effect of
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their exogenous application will greatly help to further clarify the role of polyamines in these processes. Alterations in polyamine titers in response to various stresses in different plant species suggest that 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 and decamethylenediamine, are reported to protect the crops against frost damage, wilting, loss of chlorophyll and photochemical oxidation. Novel polyamines, such as thermo-spermine and caldopentamine have been found to confer thermoprotection during in vitro protein synthesis, suggesting thereby possibility of exploiting the use of such compounds against high temperature stress. The specific inhibition of polyamine biosynthesis in fungi using their biosynthesis inhibitors has 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 biosynthesis by using sense and antisense transgenic approaches. Further, genes encoding ADC or ODC, SAMDC and SAMS have also been cloned from plant and animal sources. The use of transgenic approaches has revealed the feasibility of modulating cellular polyamine contents. Generally, genetic manipulation of single steps of polyamine biosynthesis pathway (ODC or ADC) lead to their elevated levels. But thus far, polyamine induced response or developmental event has been correlated only with changes in polyamine levels and spectra. Recently, several important advances have been made in plant polyamine research. For example, most of the genes encoding polyamine biosynthetic enzymes have been isolated, and transgenic plants or mutants with changed polyamine metabolism have been created. The analysis of transgenic plants together with results from mutant studies indicates that changes in levels of polyamines can alter growth and developmental processes and improve resistance against various stresses. Further, application of advanced genomic and proteomic approaches will help to elucidate the role of polyamines in particular plant processes. The physiological and molecular mechanism of action of polyamines in improving productivity and resistance to environmental stresses, however, needs further elucidation at various levels of organizations. Thus, it would seem an opportune time to resolve whether polyamines, the small biomolecules, trigger the growth and developmental processes in plants. The new findings in this field may allow us to employ polyamines as highly promising natural substances suitable for wide application in plant protection and to boost crop production.
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