Plants are exposed continuously to a variety to adversely changing environmental factors such as heat, cold, light, drought, acidity, alkalinity, oxidative damage and metal damage which affect their distribution, growth, development and productivity. These stressful conditions are associated with the losses in the productivity of many of the agriculturally important crops and therefore, also affect the economic returns of the country. Thus efforts are concerted worldwide to understand the mechanism of the stresses and studies performed to combat the problems before the damage occurs. There are several natural ways of self defense in the plants to cope with these stressful conditions: they can induce several functional or regulatory genes (Bartels and Sunkar 2005) or can undergo different physiological or biochemical changes. The accumulation of some functional substances, such as compatible solute and protective proteins, is an important element of the physiological and biochemical response to the stressful conditions (Liu et al., 2007). In addition to these response by the plants, molecules known as 'polyamines', have also been known to be an integral part of plant stress response (Bouchereau et al. 1999; Walters 2003; Alcázar et al. 2006b).
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Polyamines, putrescine, spermine and spermidne and cadaverine, are one of the widely and distributed N containing organic molecules which were discovered more than 100 years ago and hold their significance from the minutest bacteria to multicellular pants, animals and mammals. In addition to their stabilizing effects, which they confer by binding to the intracellular anions (DNA, RNA, Chromatin and proteins), they are also known to possess several regulatory functions as well (Igarasahi et al., 2000: Alcazar et al., 2006b; Kusano et al., 2008; Alcazar et al., 2010). In plants, they have been associated with regulating many physiological processes, such as organogenesis, embryogenesis, floral initiation and development, leaf senescence, fruit development and ripening, and abiotic and biotic plant stress responses (Galston and Kaur-Sawhney 1990; Kumar et al. 1997; Walden et al. 1997; Malmberg et al. 1998; Bouchereau et al. 1999; Bagni and Tassoni 2001; Alcázar et al. 2006b; Kusano et al. 2008; Alcazar et al, 2010).
Several changes in the concentrations of the polyamines in the plant cells take place while responding to the stressful conditions (Bouchereau et al. 1999; Alcázar et al. 2006b; Groppa and Benavides 2008; Alcazar et al., 2010). The importance of this process can be exemplified by the fact that the levels of Put may accounted for 1.2% of the dry matter, representing at least 20% of the nitrogen (Galston 1991) in the stressful conditions. Though the exact mechanism of the involvement of polyamines during stressful conditions is not fully understood, studies are ongoing to study the molecular aspects (Liu et al., 2007; Alcazar et al., 2010). Evaluating the complete genome sequence of the Arabidopsis has facilitated the use of global 'omic' approaches in the identification of target genes in polyamine biosynthesis and signalling pathways (Alcazar et al., 2010). The advantages of the progress made in these directions has made possible the generation of Arabidopsis transgenic plants which are resistant to various stresses (Alcazar et al., 2010). Efforts can be made towards the development of such varieties for the agriculturally important crops as well. Such studies add to the economic potential from the agricultural sector touched by the biotechnological advances and hence further research in these directions is noteworthy.
Role of Polyamines in abiotic stress
Richards and Coleman in 1952 observed the presence of a predominant unknown ninhydrin positive spot that accumulated in barley plants when exposed to potassium starvation. This compound was identified as Put. Later on it was shown that K+-defcient shoots fed with L-14C-arginine produced labelled Put in a more rapid way compared to feeding with labelled ornithine (Alcazar et al., 2010). These results suggested that decarboxylation of arginine was the main way of accumulation of Put under K+ defciency (Smith and Richards 1964). The relevance of the ADC pathway in plant responses to abiotic stress was later on established by Galston et al. at Yale University (Flores and Galston 1982). It has been observed that polyamines accumulate during various stressful conditions (see Bouchereau et al. 1999; Alcázar et al. 2006b; Groppa and Benavides 2008; Alcazar et al., 2010). These all support the fact that polyamines do play a protective role durng the streeful conditions. Several examples have been quoted by Alcazar et al (2010) in which genetic modification of the genes involved in the biosynthetic pathway have proven useful in discerning the function of the polyamines in plant responses to the abiotic stress. The different stress factors have been briefly discussed below:
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Mineral deficiency: This is one of the most common stress related factors affecting plants almost everywhere. However, studies related to this type of stress are often performed on leaves and/or seedlings, as the external symptons of deficiency become acute. The accumulation of Put in leaves of K+ deficient barley plants was first reported by Richards and Coleman and subsequent studies by others have established that specific role of Put in maintaining a cation- anion balance in plant tissues. As a result of K+ starvation, this diamine accumulation (via ADC activation), is widespread among mono- and dicotyledonous species and may well be a universal response (Bouchereau et al., 1999). The exact reason behind the increase in Put is unclear. The induced high levels of Put might be the cause of the stress injury. Put might also be beneficial for the plants. Alternatively, high levels of Put could be one of the many physiological changes induced by mineral nutrient deficiency without any special significance (Bouchereau et al., 1999). There are several other examples listing the changes in the polyamines content while responding to the mineral deficiencies (Geny et al., 197; Geny et al., 1998). However the changes differed according to the tissue and the stage of development.
Cold stress: The injury due to the cold causes alteration of the membrane structure as proposed by Raison and Lyons (Raison and Lyon, 1970) that the chilling injury involves phase transition in the molecular ordering of membrane lipids. This can cause several deleterious effects like increase in membrane permeability and alteration of the activity of membrane proteins. Cold treatment has been reported to increase the levels of Put and this correlates with the increase in the induction of Arginine Decarboxylase (ADC) genes (ADC1, ADC2 and SAMDC2) (Urano et al. 2003; Cuevas et al. 2008; Cuevas et al. 2009). On the other hand, levels of free Spd and Spm remain constant or even decrease in response to cold treatment (Alcazar et al., 2010). The absence of correlation between enhanced SAMDC2 expression and the decrease of Spm levels may be a result of increased Spm catabolism (Cuevas et al. 2008; Alcazar et al., 2010). Boucereau et al. (1999) reports that in the chilling-tolerant-cultivar, chilling induced an increase of free Absicissic acid (ABA) levels first, then ADC activity and finally free Put levels. Fluridone, an inhibitor of ABA synthesis, inhibited the increase of free ABA levels, ADC activity and free Put levels in chilled seedlings of the chilling-tolerant cultivar. These effects resulted in a lower tolerance to chilling and could be reversed by the pre-chilling treatment with ABA. All these results suggest that Put and ABA are integrated in a positive feedback loop, in which ABA and Put reciprocally promote each other's biosynthesis in response to abiotic stress (Figure 1). This highlights a novel mode of action of polyamines as regulators of ABA biosynthesis (Alcazar et al., 2010).
Thermal stress: When exposed to heat stress, plants have the ability to synthesize uncommon long chain PA's (caldine, thermine). The levels of free and bound PAs, as well as ADC and Polyamine oxidases (PAO) activities, were higher in tolerant than in sensitive cultures (Kuehn et al., 1990; Philipps et al., 1991; Roy and Ghosh 1996; Bouchereau et al., 1999). The increased activities of the transglutaminases indicated the high content of the polamines. This indicates a correlation between heat-stress tolerance, ADC, PAO and transglutaminase activities (Bouchereau et al., 1999).
Dought stress: Certain plants during water scarcity tend to accumulate putrescine (Put) which is supported by the fact that transcript profiling under these conditions induces the expression of certain genes involved in the biosynthetic pathway. The expression of some of these genes is also induced by ABA treatment (Perez-Amador et al. 2002; Urano et al. 2003; Alcazar et al., 2010). This throws light upon the fact that up- regulation of PA-biosynthetic genes and accumulation of Put under water stress are mainly ABA-dependent responses (Alcazar et al., 2010).
Salt stress: Differences in PA (Put, Spd, Spm) response under salt-stress have been reported among and within species. For example, according to Prakash et al. (1988), endogenous levels of PAs (Put, Spd and Spm) decreased in rice seedlings under NaCl stress, whereas Basu et al. (1988) reported that salinity results in accumulation of these compounds in the same material (Bouchereau et al., 1999). Santa-Cruz et al. (1997) reported that the (Spd+Spm):Put ratios increased with salinity in the salt-tolerant tomato species (Lycopersicon pennellii, Carrel D'Arcy) but not in the salt-sensitive tomato species (L. esculentum). In both species, stress treatments decreased the levels of Put and Spd. The Spm levels did not decrease with salinity in L. pennellii over the salinization period, whereas they greatly decreased in L. esculentum. The effects of different NaCl concentrations on maize embryogenic cells derived from immature embryo cultures of a salt-sensitive inbred line (cv, w64) and a resistant hybrid (cv Arizona) have also been reported where increased salt concentration remarkably decresed the growth of the calluses and showed a significant increase in the total PA (Put, Spd) content, especially caused by a rise in Put. It has been reported by Bouchereau et al .(1999) that using the inhibitors of Put synthesis, the ADC pathway in tomato plants operates in both stress and control conditions, whereas the ODC pathway is prompted only in the stress conditions. This finding is further supported by the studies of Urano et al. (2003) who concluded that the expressions of the Arginine decarboxylase 2(ADC 2) and spermine synthases (SPMS) during the 24 hour stress treatment maintained and hence increased the levels of Put and Spm. Yamaguchi et al. in 2006 also suggested the protective role of Spm polyamine when its addition suppressed the salt senstivity in Spm deficient mutants. Bouchereau et al . (1999) suggests that polyamine responses to salt stress are also ABA-dependent, since both ADC2 and SPMS are induced by ABA. Infact, Alcazar et al. (2006) reports that stress-responsive, drought responsive (DRE), low temperature-responsive (LTR) and ABA-responsive elements (ABRE and/or ABRE-related motifs) are present in the promoters of the polyamine biosynthetic genes. This also reinforces the view that in response to drought and salt treatments, the expression of some of the genes involved in polyamine biosynthesis are regulated by ABA (Alcazar et al., 2010). The study of the Arabidopsis thaliana flowers by Tassoni et al. (2010) has also supported the hypothesis that polyamine levels (mainly Spm here) increase with the increase in the salt concentration and therefore contribute to the plant tolerance during the stressful conditions.
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Osmotic stress: Osmotic treatments using sorbitol induce high levels of Put and ADC in detached oat leaves (Flores and Galston, 1984). Spd and Spm show a dramatic decrease. Bouchereau et al. (1999) reports that osmotica with widely different assimilation routes, such as sorbitol, mannitol, proline, betaine and sucrose, all induce a rise in Put. These changes are coincident with measurable signs of stress, such as wilting and protein loss. Tiburcio et al. in 1995 reported that when peeled oat leaves are incubated with sorbitol in the dark, they lose chlorophyll and senesce rapidly. Senescence could be delayed by including Spm in the incubation medium. The senescence- retarding effect of Spm was correlated with increase in the incorporation of labeled precursors into proteins, RNA and DNA. They also concluded that osmotic shock in the dark induces an activation of the pathway catalyzed by ADC. Borrell et al. (1996) have reported the regulation of ADC synthesis by Spm in osmotically-stressed oat leaves using a polyclonal antibody to oat ADC and a cDNA clone encoding oat ADC. Treatment with Spm in combination with osmotic-stress resulted in increased steady-state levels of ADC mRNA, yet the levels of ADC activity decreased. This absence of correlation has been explained by the fact that Spm inhibits processing of the ADC proenzyme, which results in increased levels of this inactive ADC form and a subsequent decrease in the ADC- processed form (Boucherau et al., 1999). They also studied that in osmotically-stressed oat leaves, degradation of cytochrome thylakoid proteins and the enzyme Rubisco can be avoided by addition of Spm to the incubation medium. Thus post-translational regulation of ADC synthesis by Spm may be important in explaining its anti-senescence properties. Interestingly, Masgrau et al. (1997) concluded that the overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic-stress in the homologous oat leaf stem (chlorosis and necrosis). Therefore, optimum levels of polyamines are necessary for the proper growth and development of the plants (Bouchereau et al., 1999). Recently, Liu et al. in 2010 have investigated the changes in the content and the form of polyamines (PAs) in the leaves of two wheat (Triticum aestivum L.) cultivar seedlings, differing in drought tolerance, under the osmotic stress by Polyethylene glycol (PEG) 6000 treatment. The results suggested that free-Spd, -Spm and PIS-bound Put (perchloric acid insoluble bound putrescine) facilitated the osmotic stress tolerance of wheat seedlings. The important roles of reactive oxygen species in the relationship between ethylene and polyamines (PA's) have also been investigated in leaves of spring wheat seedlings under root osmotic stress (Li et al., 2010).
Hypoxia: There has been lot of work done by Reggiani's grup on the role of polyamines under the hypoxia stress conditions. Reggiani et al., (1990) reported that that are many examples available where plant shoots and seedlings of different Gramineae species, when subjected to lack of oxygen, provide evidence of an association between tolerance and the capacity to accumulate Put. Species such as rice and barnyard grass which are adapted to germinate in an oxygen deprived environment, showed a greater capacity of Put accumulation than the anoxia-intolerant species (Reggiani and Bartani, 1989). This consideration supports the hypothesis for a role of Put as a protective compound against hypoxia (Reggiani and Bartani, 1990; Bouchereau et al., 1999). Reggiani et al. in 1989 has reported that Put is required for the anaerobic elongation of rice coleoptiles but it has no effect on aerobic elongation of rice coleoptiles where auxin is active. This group has also concluded that with a decrease in oxygen concentration, the conjugated Put became predominant in comparison with the free forms (80% at 0.3% oxygen) and that there is a negative correlation between Put accumulation (specially under conjugated forms) and shoot elongation (reggiani and Bartini, 1989; Bouchereau et al., 1999). On the other hand, the results of Lee et al., (1996) have indicated that increase in the activies of ADC and ODC and Put levels are essential for the elongation of Scirpus shoots grown under submergence.
Damage by Ozone: Ozone, the protective gas in the upper atmosphere is known to protect us from the harmful UV rays of the sun. But it is known to have serious effects on the vegetation. Experiments are ongoing throughout the world in this respect. According to Heagle et al. (1989) O3-stress can lead to a significant decline in net photosynthesis, cause leaf injury and accelerate senescence, even when applied at low levels. Reaction to this stress triggers many biochemical changes in the plants like increase in ABA, peroxidases, phenolic compounds, ethylene and polyamines which form a part of the plant self defense mechanism. Rowland- Bamford et al. in 1989 observed that the ADC activity in the ozone treated barley leaves increased before the damage became apparent. Many more examples have been quoted by Bouchereau et al. (1999) supporting the protective role of the polyamines during the ozone damage. Though the exact mechanism is not clear, but there can be a possibility of PA's being involved in the free radical scavenging (Bors et al., 1989). This is also supported by the fact that the levels of superoxide radical formed enzymatically with xanthine oxidase or chemically from riboflavin or pyrogallol were inhibited in vitro by Put, Spd or Spm at 10-50 mM (Drolet et al., 1986). Also, superoxide radical protection was inhibited by PAs when added to microsomal membrane preparations. These findings have been also supported by the fact that PA's tend to inhibit lipid peroxidation (Tang and Newton, 2005; Zhao and Yang, 2008). These conclusions were however disputed by the findings of Langebartels et al. (1991) as mentioned by Bouchereau et al. (1999). Leaf injury, caused by O3 in the tobacco cultivar Bel W3, could be prevented by feeding Put, Spd or Spm through the root. These exogenous treatments were correlated with a 2- to 3-fold increase in soluble conjugated Put and Spd (monocaffeoyl forms). Conjugated Put and Spd associated with cell wall and membrane fractions were increased 4- to 6-fold. When free PAs were assayed in vitro for their radical-scavenging properties, very low rate constants were found. On the other hand, PA conjugates had relatively high rate constants. It was thus concluded that free PAs could not account for the protection against O3 damage. But assuming their role in the ozone damage, it was suggested that the protective effect of exogenous free PAs was mediated by their prior conversion to conjugated forms. Consistent with this hypothesis, it was found that monocaffeoyl Put, an effective scavenger of oxyradicals, was present in the apoplastic fluid of tobacco leaves exposed to O3 (Langebartels et al., 2003). The results Navakoudis et al. (2003) also supports these findings according to which the enhanced atmospheric ozone is the accumulation of polyamines, generally observed as an increase in putrescine level, and in particular its bound form to thylakoid membranes. A study by Schraudner et al. (1990) also discovered a relationship between ethylene emission and PA biosynthesis was found in O3-treated potato and tobacco plants, the leaves of which show early senescence in response to the pollutant. In the presence of O3, all compounds of ethylene biosynthetic pathway in tobacco leaves were up-regulated. Put and Spd levels also increased, as did Ornithine Decarboxylase (ODC) activity (Bouchereau et al., 1999).
Integration of polyamines with other molecules during stress conditions
Polyamines affect several physiological processes in plants by activating the biosynthesis of signaling molecules like NO, H2O2; they affect ABA synthesis and signaling and are involved in Ca2+ homeostasis and ion channel signaling during the abiotic stress conditions Figure 1 summarizes this information.
Abscissic acid ABA) is an anti-transpirant that reduces water loss through stomatal pores on the leaf surface in response to water deficit, resulting in the redistribution and accumulation of ABA in guard cells and finally closure of the stomata (Bray 1997). Many authors (Liu et al. 2000; An et al. 2008; Alcazar et al., 2010) have reported that Put, Spd and Spm also regulate stomatal responses by reducing their aperture and inducing closure and that Put modulates ABA biosynthesis in response to abiotic stress. Thus polyamines are involved in the ABA mediated stress responses which affect the stomatal closure. Polyamines are also linked with reactive oxygen species (ROS) and NO signaling as amino oxidases during the catabolic process generate H2O2 which is a ROS (associated with plant defense and abiotic stress) and also there are evidences in which polyamines are reported to enhance the production of NO (Tun et al., 2006). NO is also known to enhance the salt stress tolerance in plants by regulating the content and proportions of the different types of free polyamines (Fan et al., 2010). According to Neill et al. (2008), both H2O2 and NO are involved in the regulation of stomatal movements in response to ABA, in such a way that NO generation depends on H2O2 production. Thus, altogether, the available data indicate that polyamines, ROS (H2O2) and NO act synergistically in promoting ABA responses in guard cells (Alcazar et al., 2010).
Polyamines are positively charged compounds, which can interact electrostatically with negatively charged proteins, including ion channels. Indeed, polyamines at their physiological
concentration block the fast-activating vacuolar (FV) cation channel in a charge-dependent manner (Spm, 4+ > Spd 3+ >> Put 2+), at both whole-cell and single-channel level, thus indicating a direct blockage of the channel by polyamines (Bruggemann et al. 1998). Aording to Alacazar et al. (2010), in response to different abiotic stresses, such as potassium deficiency, Put levels are increased drastically (reaching millimolar concentrations), whereas the levels of Spd and Spm are not significantly affected and this increase of Put may significantly reduce FV channel activity. In the similar manner, during another kind of stess, i.e., salinity, Bruggemann et al. (1988) has reported that all PA levels increase, and the enhanced Spm concentration probably blocks FV channel activity. These observations can be explained by the fact that as in animal and bacterial cells, polyamines in plants may thus modulate ion channel activities through direct binding to the channel proteins and/or their associated membrane components (Delavega and Delcour 1995; Johnson 1996; Alcazar et al., 2010). Phosphorylation and dephosphorylation of ion channel proteins are closely related to their activities. Thus, polyamines could also affect protein kinase and/or phosphatase activities to regulate ion channel functions (Bethke and Jones 1997; Michard et al. 2005; Alcazar et al., 2010). However, Zhao et al. (2007) points out that for elucidating the moleculat mechanisms underlying polyamine action, identification of ion channel structural elements and/or receptor molecules regulated by polyamines would be of great importance.
Polyamines also tend to maintain Ca2+ homeostasis. Several examples have been reported by Alcazar et al. (2010). Yamaguchi et al. (2006, 2007) proposed that the protective role of Spm against high salt and drought stress is a consequence of altered control of Ca2+ allocation through regulating Ca2+permeable channels. The increase in cytoplasmic Ca2+results in prevention of Na+/K+ entry into the cytoplasm, enhancement of Na+/K+ influx to the vacuole or suppression of Na+/K+ release from the vacuole, which in turn increases salt tolerance (Yamaguchi et al. 2006; Kusano et al. 2007; Alcazar et al., 2010). Thus polyamines have a definite role to play to main the calcium homeostasis during the stressed conditions.
Figure 1: Simplified model for the integration of polyamines with ABA, ROS (H2O2), NO, Ca2+ homeostasis and ion channel signalling in the abiotic stress response (Source : Alcazar et al., 2010).
Conclusions and Future Perspectives
Polyamines have now been considered as secondary messangers in addition to being known as vital plant regulators (Liu et al., 2007). Although the exact mechanism of action of polyamines during the stressful conditions is not known, genetic tools have been played useful; traditional quantitative trait locus (QTL) mapping (Alonso-Blanco et al. 2009) and by genome-wide association mapping (Nordborg and Weigel 2008) can be used for the identification of the genes underlying the mode of action and regulation of polyamines (Alcazar et al., 2010). Cloning of these genes would be another added advantage as these could be used in the same way as farm chemicals to alleviate or mitigate stress derived injury for crop protection. Transfer of such technology to the other crops will help in creating germplasms which would be better adapted to the harsh stressful conditions and thus contributing towards enhanced agricultural productivity.