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Turkish Journal of Botany http://journals.tubitak.gov.tr/botany/ Review Article Turk J Bot (2015) 39: 887-910 © TÜBİTAK doi:10.3906/bot-1503-27 Metabolic and molecular-genetic regulation of proline signaling and its cross-talk with major effectors mediates abiotic stress tolerance in plants Aryadeep ROYCHOUDHURY*, Aditya BANERJEE, Vikramjit LAHIRI Post-Graduate Department of Biotechnology, St. Xavier’s College (Autonomous), Kolkata, West Bengal, India Received: 17.03.2015 Accepted/Published Online: 21.09.2015 Printed: 21.12.2015 Abstract: Proline (Pro) accumulation is a common response of several plant species to combat abiotic stresses. Under stress conditions, Pro acts as an excellent compatible solute in the plant system, participating in the alleviation of stress sensitivity. Though the metabolic pathways associated with Pro are well studied, parts of its regulatory cascades are still not properly known. It has also been conjectured that epigenetic modifications regulate Pro metabolism during abiotic stress. Apart from Pro, the plant abiotic stress responses are essentially mediated by multiple effectors. Hence, proper analysis of the cross-talks of Pro with the other components of the abiotic stress response has turned out to be mandatory in order to design multistress-tolerant transgenic lines. Highlighting the relation between Pro and seed germination is also essential to understand the notion behind plant susceptibility and survival during stress. Generally, Pro has a universal mechanism to generate abiotic stress tolerance through stabilization of structural components, enzyme structures, and regulation of osmotic adjustments. The success achieved through recent transgenic approaches leading to more accumulation of Pro in the sink has also been focused on in the present review. Key words: Proline, compatible solute, epigenetic modification, cross-talk, seed germination, abiotic stress, stress tolerance 1. Introduction Considering the percentage of land area affected and loss of crop productivity, study of abiotic or environmental stress biology and its management continues to be a significant area of research in plant biotechnology. A report by the Food and Agricultural Organization in 2007 stated that only 3.5% of the global land area is free from any environmental constraints (http://www.fao.org/docrep/010/a1075e/ a1075e00.htm). Thus, knowledge for combating abiotic stresses is essential to genetically design major stresstolerant food crops. This is because their production is expected to decline in the future due to dearth of arable land, depletion of water resources, global warming, and drastic climatic alterations (Cramer et al., 2011). Globally, high salinity affects the largest count of crop production on at least 20% of irrigated land. The middle of the 21st century is forecasted to have around 50% loss in cultivable land due to increased salinity. Dehydration stress studies have also been diverted to salt stress because the response patterns in both are almost the same. Temperature in the subzero range results in the formation of ice crystals within the tissue intercellular spaces (Roychoudhury et al., 2013). Heavy metal toxicity, leaf wilting, electrolytic leakage, leaf abscission, changes in leaf area, generation * Correspondence: aryadeep.rc@gmail.com of reactive oxygen species (ROS), accumulation of free radicals disrupting cellular homeostasis by membrane lipid peroxidation, etc. are among the various adverse effects of abiotic stresses on plants. Thus, the viability of each cell is challenged under such circumstances. Abiotic stresses are multigenic in nature, being governed by multiple loci and also occurring at multiple stages. Often, the plant is simultaneously affected by multiple stresses. Therefore, designing a genetic model that is stress-tolerant is extremely difficult and challenging (YamaguchiShinozaki and Shinozaki, 2006). Stress-tolerant plants like Craterostigma plantagineum, Mesembryanthemum crystallinum, and Thellungiella halophila can be used as valuable models in which the systems biology can be studied and integrated in crop plants to enhance stress tolerance (Bartels and Sunkar, 2005). Unlike animals, plants are sessile and do not have the advantage of fleeing away from adverse sites; they must stand and resist the stress conditions. Vivid molecular responses at the biochemical and molecular level are exhibited by plants to overcome stressful conditions (Roychoudhury and Nayek, 2014; Banerjee and Roychoudhury, 2015a, 2015b). The molecular responses involve interactions and cross-talks among several related 887 ROYCHOUDHURY et al. / Turk J Bot pathways. The universal stress hormone abscisic acid (ABA) acts as the chief regulator of abiotic stress responses, including osmotic stresses. The main physiological function of ABA during stress is to promote stomatal closure, minimize transpiration, and hence conserve water in the cells. ROS and reactive nitrogen species (RNS) are almost always the earliest signals in abiotic stresses (Molassiotis and Fotopoulos, 2011). ROS and RNS aid in the activation of a coordinated network of responses, among which those for the nitrosative effects of RNS are less documented (Cramer et al., 2011). A network of longdistance signaling is triggered by plants at the molecular level for perception of stress cues. This is accompanied by transduction of signals to activate the adaptive responses and finally cellular responses. Switching-on of a broad class of stress-specific genes, generally called the osmotic stress responsive (OR) genes, activates the cellular responses. The differences in signal perception and transduction mechanisms determine the differences in the level of stress tolerance among various genotypes of a plant species (Roychoudhury et al., 2013). The root growth and osmotic adjustment (OA) aid in maximizing water uptake during dehydration stress. This, along with minimizing stomatal and cuticular water loss, results in lower osmotic stress in the plants under such conditions. The osmotic homeostasis maintenance during salt stress and prevention of ice-nuclei formation in cold stress is done by OA. Plants mediate stress signaling via mainly three approaches: 1) reestablishing cellular homeostasis, via ionic and osmotic stress signaling; 2) controlling the extent of damages and inducing repair through a detoxification mechanism; 3) signaling to overcome the stressful period and aid the plant to regenerate with new potential through coordination of cell division and tissue expansion (Roychoudhury et al., 2013). 2. Compatible solutes Compatible solutes help the plant cells to maintain optimum turgor and resume growth during ionic stress through OA. This is done by efficient water uptake by the reduction of the cytosolic osmotic potential. Such compatible solutes or osmolytes are extremely crucial for water retention during dehydration stresses, as they can maintain the hydration sphere of proteins, utilizing their high polarity and hydrophilicity (Roychoudhury and Chakraborty, 2013). Two theories, the ‘preferential exclusion model’ and ‘preferential interaction model’, have been proposed regarding the role of compatible solutes in water retention and stabilizing the protein structures. The preferential exclusion model proposes the exclusion of compatible solutes from hydration shell of proteins. This enhances protein stability and mutual interactions among proteins. The preferential interaction model basically 888 depicts the interactions occurring between proteins and compatible solutes (Bohnert and Shen, 1999). Compatible solutes are potential low-molecular-weight chaperones stabilizing macromolecular assemblies and protein folding. The reduction of the inhibitory effects of ions on stress-induced enzyme activities is regulated by such compatible solutes to increase their thermal stability. Compatible solutes also prevent the dissociation of enzyme complexes and scavenge the toxic hydroxyl radicals to preserve the membrane and hence the cellular integrity. It can be suggested that compatible solutes also stabilize the photosystem II complex (Fatemeh et al., 2012). Osmolytes have been reported to function as cryoprotectants, thereby implicating a role in protection from cold stress and freeze–thaw cycles (Wangxia et al., 2003), an indication of overlap between cold stress and salinity stress responses. Common osmolytes accumulated during stresses are major carbohydrates like sucrose, fructose, and glucose; sugar alcohols like pinitol, ononitol, and cyclitol; polyols like adonitol, sorbitol, mannitol (all straight chain compounds), and myo-inositol (cyclic polyols); complex sugars like trehalose, raffinose, and fructans; free amino acids like proline (Pro) and glycine betaine (GB); organic acids like lactate, malate, citrate, succinate, fumarate, benzoate, salicylate, malonate, and γ-amino butyric acid (GABA); free ammonia and quaternary ammonium compounds like β-alanine-betaine, Pro-betaine, and hydroxyproline-betaine; and tertiary sulfonium salts like dimethylsulfoniopropionate and choline-o-sulfate (Hayat et al., 2012; Roychoudhury and Chakraborty, 2013). Salinity induces hyperaccumulation of Na+ and Cl-, but these do not act as osmolytes as they interfere with cellular functions at high concentrations and need to be sequestered to the vacuole. Compatible solutes, however, are not sequestered to vacuoles; they remain mostly in the cytosolic and chloroplastic compartments (Shinozaki and Yamaguchi-Shinozaki, 1999) where they perform their protective functions. The low-molecular-weight aliphatic amines or polycations playing protective roles during stress are the polyamines (PAs), which constitute another major group of compatible solutes. At physiological pH, the positive charge residing on them helps in the interaction with the cell membrane. Thus, disintegration of the membrane during osmotic stress is prevented by the PAs. The potency of this action is maximum in tetramine spermine (Spm), followed by triamine spermidine (Spd) and then diamine putrescine (Put) (Roychoudhury and Das, 2014). The rate limiting step of PA biosynthesis is catalyzed by S-adenosylmethionine decarboxylase (SAMDC). The diamine oxidase (DAO) catalyzes Put catabolism, while polyamine oxidase (PAO) catalyzes Spd and Spm catabolism. Wimalasekera et al. (2011) showed ROYCHOUDHURY et al. / Turk J Bot that Arabidopsis copper amino oxidase 1 (CuAO1), responsible for the degradation of Put, was a potential contributor of ABA-induced NO production and that this activity plays a central role in most stress responses. NO is responsible for posttranslational S-nitrosylation of proteins. Under stress conditions in Citrus, 271 proteins were found to be regulated by Put, Spm, and Spd by S-nitrosylation. The first plant PA-transporter, called the PA uptake transporter 1 (OsPUT1), was characterized in all tissues of rice, except seeds and roots (Mulangi et al., 2012). Cross-talk between the PA signaling pathway and ABA signaling has been reported (Roychoudhury and Das, 2014). The maximum accumulation of Put and Spd was recorded in rice seedlings treated with exogenous ABA. Total soluble PA content in ABA-treated aromatic rice variety Gobindobhog also increased, showing its saltsusceptible nature (Roychoudhury et al., 2009). It has been hypothesized that PAs stimulate the DNA binding activity of some TFs to the recognizing abscisic acid responsive elements (ABREs) of target genes (Roychoudhury and Das, 2014). The PAs control the activity of several ion channels indirectly by regulating the plasma membrane potential. This is mediated by the activation of H+/ATPase and interaction of PAs with the 14-3-3 proteins (Garufi et al., 2007). During abiotic stresses, Spm inhibits stomatal opening and promotes closure by regulating the KAT1like voltage-dependent inward K+ channel in the guard cells of Vicia faba (Kusano et al., 2007a, 2007b). Salt tolerance involves ion homeostasis and the inhibition of ROS by certain plasma membrane ion channels like the nonspecific cation channel (NSCC), while some other cation channels are activated by ROS-induced conductance (ROSIC). Such changes often promote efflux of K+ and influx of Na+. PA regulates ion homeostasis and also balances the Ca2+ level in cells. A recent model of Ca2+ homeostasis in plants has been put forward to be as follows: 1) ROS formation, degradation, and transport; 2) PA catabolism and transport; 3) feedback loops of NSCC, ROSIC, and NADPH oxidase; and 4) PA- and ROSdependent Ca2+ pump activation (Pottosin et al., 2012). All the compatible solutes, about which we have made a brief mention above, individually hold immense potential in helping the plant system to battle and win against the environmental challenges. Each compatible solute has a unique mode of action and downstream effects. However, inappropriate and incorrect gene expression of osmolytes is often accompanied by pleiotropic effects like growth retardation and necrotic lesions due to interference with the normal pathways of primary metabolism. To avoid such a scenario, metabolic engineering must be made stress-inducible and/or tissue-specific (Garg et al., 2002). In the following sections of this review, we broadly discuss the free amino acid Pro as a representative osmolyte, with a detailed focus on its interaction with other stress regulators and its role as a potent mediator of abiotic stress responses. In spite of more than 40 years of study on the role of Pro in combating abiotic stress, bits and pieces of information remain astray. Several observations on Pro by different groups, as knit together in this review, will probably aid in opening future prospects of research in this field. 3. Pro: a general account Pro is often regarded as an imino acid due to the presence of a secondary amino group. The cyclic structure of Pro dictates restricted flexibility in conformation, for which it decides on the stability of secondary protein structures. Pro is a necessary and most common compatible solute found in diverse families of plants and bacteria experiencing abiotic stress. These properties make Pro unique among the proteinogenic amino acids (Lehmann et al., 2010). As mentioned earlier, Pro serves as a potent compatible solute during stress. Due to low molecular weight and highly soluble organic nature, Pro is nontoxic for the cell at high concentrations. High levels of Pro have been detected in plant species under stresses like high soil salinity, dehydration and water scarcity, chilling temperatures, ROS-mediated oxidative stresses, heavy metal toxicity, and ultraviolet (UV) ray exposure. Pro accumulation under abiotic stresses depends on the plant species and can be up to 80% of the cellular amino acid pool. This is mainly due to the increased synthesis of Pro, accompanied with its decreased degradation under such unfavorable conditions (Kavi Kishor et al., 2005). Salt stress in Arabidopsis triggers Pro accumulation to about 20% of the total amino acid pool (Liu and Zhu, 1997). Pro is also a stabilizer of the plasma membrane and the subcellular structures, which are necessary for the viability of the stressed cell. In such cases, Pro often acts as a protein-compatible hydrotope, alleviating cytoplasmic acidosis. The metabolic NADP+/ NADPH ratio, cellular pH, and cellular redox status are also maintained by Pro under stress conditions (Hayat et al., 2012). The members of Solanaceae can increase their Pro pool by more than two orders of magnitude when exposed to abiotic stress (Djilianov et al., 2005). The modern world is likely to face a tremendous shortage of food in coming times. Thus, it becomes the onus of agricultural biologists to carve out plans in order to solve such food scarcity problems in the future. It has been identified that several crop plants like rice that are obviously glycophytes mostly die of abiotic stresses, about which we have mentioned above. Pro as a compatible solute has shown a ray of hope in combating such stresses in crop plants through its overexpression in the appropriate tissue cells via genetic engineering. Exogenous application of Pro has also gained tremendous impetus in developing stress tolerance in 889 ROYCHOUDHURY et al. / Turk J Bot some plant species. Since the first report of the role of Pro in defeating stresses in wilting perennial rye grass (Lolium perenne), the quest to utilize this potentiality in crops has been on the rise (Szabados and Savoure, 2009). 4. Pro metabolism and transport 4.1. Pro biosynthesis 4.1.1. From glutamate Pro biosynthesis was first characterized in bacteria (Kavi Kishor et al., 2005). Here, Pro is formed from glutamate via three steps. The pathway is initiated by the conversion of phosphorylated glutamate to γ-glutamyl phosphate and next to glutamate-γ-semialdehyde (GSA) by γ-glutamyl kinase and glutamate-γ-semialdehyde dehydrogenase respectively. The Δ1 pyrroline-5-carboxylate (P5C) is subsequently formed from GSA via spontaneous cyclization. The direct catalysis of glutamate by the P5C synthetase (P5CS) yields GSA in plants and other eukaryotes. This reaction requires both reduced nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine trinucleotide phosphate (ATP). The P5C reductase (P5CR) then reduces P5C to Pro, utilizing NADPH in both prokaryotes and eukaryotes (Kavi Kishor et al., 2005; Lehmann et al., 2010). The γ-glutamyl kinase catalyzes the rate-limiting step of Pro biosynthesis in bacteria and yeast. The rate-limiting step in the case of plants is P5CS, which is regulated via the allosteric inhibitory effect of the end product of the pathway, i.e. Pro (Sekine et al., 2007). The genomes of plants like Arabidopsis, Medicago sativa, Medicago truncatula, and Oryza sativa exhibit two homologous genes encoding P5CS (Armengaud et al., 2004). With specific roles varying among plant species, P5CS paralogs were reported to have different functions during plant life and development (Kavi Kishor et al., 2005). Blockage of Pro accumulation occurred in Arabidopsis p5cs1 and p5cs2 knockout mutants, due to the nonredundant activities of the mutated enzymes (Székely et al., 2008). The observed functional specialization of the P5CS genes was accredited to the duplication of the genes after monocot and dicot divergence, as revealed through phylogenetic analysis (Turchetto-Zolet et al., 2009). The normal site of P5CS1 localization is in the cytosol of leaf mesophyll cells as seen through green fluorescent protein (GFP) fusion tracing. However, in the embryonic cells and roots, its localization occurs within organelles similar to fusiform bodies (Szabados and Savoure, 2009). Experimental evidence also shows that P5CS1-GFP gets localized in the chloroplasts when the plants are exposed to salt stress. The P5CS2-GFP in Arabidopsis was found to accumulate in the cytosol (Székely et al., 2008). Thus, it can be inferred that the housekeeping Pro biosynthesis is mediated by the P5CS2 gene. In response to abiotic stresses, Pro biosynthesis gets shifted to the chloroplast under the control of the stress- 890 induced P5CS1 gene (Szabados and Savoure, 2009). Copperinduced Pro accumulation has been reported in detached leaves. Such accumulation is mediated by abscisic acid (ABA) (Chen et al., 2001), and it was also associated with nitric oxide (NO) generation in Chlamydomonas reinhardtii (Zhang et al., 2008). This intracellular NO plays a potent role in Cu-induced Pro synthesis and downstream stress signaling. Further investigations disclosed that this effect was mainly due to the application of sodium nitroprusside, which potentially acts as a NO donor. This NO enhanced the activity of P5CS in the Cu-treated algae. This observation of Pro accumulation was not recorded when a NO scavenger instead of a NO donor was used (Zhang et al., 2008). Biochemical identification of two isoforms of P5CR protein was done in pea and spinach. It remains unclear whether the origin of the two isoforms was from one or two genes (Murahama et al., 2001; Lehmann et al., 2010). The case is more clear for Arabidopsis, as it has been determined that a single gene encodes P5CR. The P5CR accumulates in plastids in response to unfavorable conditions, while the housekeeping concentration of Pro under normal conditions is maintained by P5CR in the cytosol (Szabados and Savoure, 2009). The P5CR activity maintains the redox potential of the cell by affecting the reduction of NADPH. The localization of P5CR in the plastids also indicates the function of this enzyme in counteracting the photoinhibitory damage of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) enzyme under adverse abiotic stress conditions (Kavi Kishor et al., 2005). 4.1.2. From arginine Arginase converts arginine to ornithine, from which α-keto-δ-aminovalerate is generated by the enzyme ornithine-α-aminotransferase (αOAT). The α-keto-δaminovalerate is spontaneously cyclized to pyrroline2-carboxylate (P2C) by the catalysis of P2C reductase. This pathway has not yet been reported in plant systems (Kavi Kishor et al., 2005). However, in plants, ornithineδ-aminotransferase (δOAT) catalyzes the formation of P5C and glutamate from ornithine and α-ketoglutarate, via transamination reactions (Stranska et al., 2008). The normal Pro accumulation was observed in δoat mutants of Arabidopsis. The plants could not mobilize nitrogen from arginine or ornithine. This indicates that rather than its role in Pro biosynthesis, δOAT enzyme mainly regulates arginine degradation (Funck et al., 2008). The localization of δOAT is mainly in the mitochondria. This prevents the direct utilization of the δOAT-generated P5C by P5CR, due to the localization of the latter in the cytosol or plastids (Funck et al., 2008). More δOAT activity was observed in younger plants of Arabidopsis than older ones. In such young seedlings, the Pro content, P5CS mRNA, and δOAT activity increased under salt stress. Electroporation of Vigna aconitifolia cDNA expression library in Pro ROYCHOUDHURY et al. / Turk J Bot auxotroph mutants of Escherichia coli restored the Pro prototrophy. The isolation of the cDNA clones encoding δOAT was successful through utilization of this novel ‘trans-complementation’ (Kavi Kishor et al., 2005). 4.2. Pro catabolism and degradation In plants and all higher eukaryotes, the site of Pro catabolism is the mitochondria. FAD as a cofactor is required for the oxidation of Pro to P5C by proline dehydrogenase (PDH), bound to the inner mitochondrial membrane. This step generates NADP/NADPH cycling or redox balance. The conversion of P5C to glutamate is carried out by pyrroline-5-carboxylate dehydrogenase (P5CDH), using NAD+ as the cofactor (Tanner, 2008). Analyses in Arabidopsis and tobacco indicate the clear presence of two homologous PDH genes (Ribarits et al., 2007; Verbruggen and Hermans, 2008). Such instances have not been firmly reported for P5CDH, since this enzyme is encoded by a single copy of the corresponding gene in all plant species, though there is evidence that two copies of P5CDH are present in Nicotiana plumbaginifoila and Zea mays (Mitchell et al., 2006; Lehmann et al., 2010). A substantial level of identity was found between the cDNA clone of Arabidopsis ERD5 (early responsive to dehydration stress), localizing in the mitochondria and yeast PUT1 (proline utilization) (Kavi Kishor et al., 2005). Redox homeostasis is ultimately maintained through the cycling of Pro via its catabolic and anabolic pathways through glutamate. This is because the mitochondrial fraction had enhanced levels of ERD5 in response to Pro, added to Arabidopsis cultured cells, and this high accumulation was not detected when the cultured cells were undergoing dehydration. ERD5 was, however, upregulated when the cells were rehydrated (Kavi Kishor et al., 2005). 4.3. Transport of Pro The biosynthetic and catabolic compartmentalizations of Pro give a clear indication about the importance and intensity of Pro transport between the cytosol, chloroplasts, and mitochondria. Specific Pro transporters have been recruited by the cell for this purpose. The mitochondria of Triticum durum contains a Pro uniporter, which aids in the passage of Pro into the mitochondrial matrix. A Pro-glutamate shuttle between the cytosol and the mitochondrial matrix has also been identified in a Pro/ glutamate antiporter in the mitochondrial membrane of T. durum (Di Martino et al., 2006). The delivery of arginine and ornithine through the mitochondrial membrane is sustained by the basic amino acid (BAC) transporters (Palmieri et al., 2006). The de novo biosynthesis, along with Pro transport, is essential. This was supported by the fact that the halophyte Limonium folium accumulated Pro in the vacuoles under normal conditions. When exposed to salt stress, high amounts of Pro remained in the cytoplasm (Gagneul et al., 2007). Some transporters have also been identified in Arabidopsis and tomato pollens. Under stresses, three transporters (Pro T1, Pro T2, and AAP6) of the amino acid permease (AAP) family were expressed in Arabidopsis. The plants experiencing salt stress showed ubiquitous expression of Pro T1 in roots, stems, and flowers. Under dehydration stresses, Pro T2 was expressed with AAP6 expression restricted to the sink tissues of roots and cauline leaves (Hayat et al., 2012). 4.4. Regulatory mechanism in Pro metabolism CONSTANS (CO) is a transcriptional activator in the flowering pathway in Arabidopsis. It promotes flowering in response to long day length. P5CS2 is also another target of CONSTANS (Samach et al., 2000). P5CS2 expression is also upregulated during hypersensitive responses triggered by avirulent bacteria, salicylic acid (SA), and ROS (Fabro et al., 2004). The osmotic and salinity stresses induce higher expression of P5CS1 through ABA-dependent and ABA-insensitive 1 (ABI1)-mediated pathways and also through H2O2-mediated signaling (Verslues et al., 2007). Light promotes P5CS1 expression, while brassinosteroids inhibit the same (Szabados and Savoure, 2009). Under normal conditions, phospholipase D (PLD) functions as a negative regulator, and under salt stress, phospholipase C (PLC) and calcium function as positive regulators of Pro accumulation (Roychoudhury et al., 2013). However, the reverse effects of PLD and PLC on Pro accumulation was found in the halophyte Thellungiella halophila. The CaM4 calmodulin mediates the calcium burst and interacts with MYB2 TF in its active conformation. Activated MYB2 upregulates the transcription of P5CS1 (Ghars et al., 2008). Both ABA-dependent and ABA-independent signaling pathways can influence Pro accumulation. Calcium plays an important role in Pro accumulation through the ABA-dependent pathway (Roychoudhury et al., 2013). The TFs that elevate abiotic stress response through the ABA-dependent pathway belong to the MYC/MYB families. Transgenic lines of Glycine max overexpressing GmMYB76 showed higher expression levels of responsive to dehydration29B (rd29B), DREB2A, P5CS, rd1, early dehydration inducible10 (erd10), and cold-regulated78/ responsive to dehydration29A (cor78/rd29A). On the contrary, the expression levels of rd29B, cor6.6, cor15a, and cor78/rd29A was lowered in GmMYB92 transgenic plants, though the levels of DREB2A, rd17, and P5CS remained high. This shows the different levels of stimulated tolerance depending on the nature of the host plants (Roychoudhury et al., 2013). Several complex developmental and osmotic regulations also decide the activity level of P5CR. Plant growth regulators like indole-3-butyric acid (IBA), ABA, and kinetin when added exogenously mimicked and initiated Pro accumulation, similar to that during salt and water stress, in Guizotia abyssinica (Sarvesh et al., 1966). The benzyl aminopurine (BAP) induced the 891 ROYCHOUDHURY et al. / Turk J Bot same in M. crystallinum, while gibberellic acid (GA) did not. When NaCl and ABA or NaCl and kinetin were added, the response was additive, unlike when a combination of inducing phytohormones was administered, leading to a higher level of Pro accumulation than either individually. This is indicative of the fact that NaCl- or phytohormoneinduced Pro accumulations are the end results of different signaling pathways (Sarvesh et al., 1966). Thus, salt and growth regulators are the independent initiators of Pro accumulation during stress. The convergent effect of the hormones in upregulating Pro biosynthesis during periods of abiotic stress remains to be completely worked out, although NaCl-induced growth inhibition can be alleviated by exogenous addition of GA and ABA (Sarvesh et al., 1966). The kinetin and ABA when supplied did not show the same response. The balance between cytokinin and ABA is also of significant importance. During stress, the level of ABA rises and that of cytokinin falls. In Arabidopsis, cytokinin reduced the level of AtP5CS1 mRNA, while BAP enhanced AtP5CS2 mRNA in leaves (Hu et al., 1992). In either case, the level of expression in the root was unaffected (Figure 1). In contrast to the biosynthetic branch, Pro catabolism is accelerated under dark and stress-free conditions through PDH and P5CDH. A Pro- and hypoosmolarity-responsive element (PRE) motif ACTCAT has been identified through promoter analysis of the PDH gene. The TFs that bind to these motifs to activate PDH transcription are AtbZIP-2, AtbZIP-11, AtbZIP-44, and AtbZIP-53, all belonging to the bZIP family of TFs. A network of group-S bZIPs regulates PDH expression. This has been analyzed mainly through chromatin immunoprecipitation (Weltmeier et al., 2006). Pro can up regulate P5CDH expression, which under normal conditions is transcribed at low basal levels. The promoters of P5CDH in Arabidopsis and cereals also contain a short sequence similar to the PRE motif. The wounding and pathogen attack activates the FIS1 gene, which encodes P5CDH in flax (Linum usitatissimum) (Mitchell et al., 2006). Small interfering RNA (24 nt and 21 nt siRNA) in Arabidopsis is generated through the natural antisense overlapping of the 3’ untranslated region (UTR) of P5CDH and the salt-induced SIMILAR TO RCD ONE 5 (SRO5) genes. The transcript levels of P5CDH are reduced by the cleavage of P5CDH by the generated siRNAs during stress, thus aiding in the accumulation of Pro (Szabados and Savoure, 2009). 4.5. Changes in DNA methylation pattern governs Pro accumulation The modulation in DNA methylation induced by abiotic stress may play a functional role in plant stress tolerance (Karan et al., 2012). The metabolic processes may be subjected to regulation at the level of gene expression. DNA Figure 1. The biosynthesis of Pro under stressed conditions is responsible for the essential triggering of multiple factors. Inhibitory regulation is also achieved through phospholipase D and brassinosteroids. 892 ROYCHOUDHURY et al. / Turk J Bot methylation patterns and, more importantly, changes in the same are integral components of the epigenetic code, which is instrumental in marking transcriptional status of genes, with methylation generally being an inhibitory signal for transcription. Pro metabolism is also regulated by DNA methylation (Chan et al., 2005). The DNA methylation pattern of the three key genes involved in Pro biosynthesis was analyzed using methylation-sensitive amplification polymorphism and methylation-sensitive Southern blotting, where enhanced Pro accumulation in plants exposed to 15% (w/v) polyethylene glycol (PEG) was recorded, as opposed to control plants (Zhang et al., 2013). Many studies have demonstrated that osmotic stress induces in­creases in δ-OAT and P5CS abundance and activity. Thus, upregulation of P5CS and δ-OAT expression may contribute to the accumulation of Pro in response to osmotic and salinity stress (Verslues and Sharma, 2010). The selfed progenies (S1 and S2) of osmotically stressed plants (S0) accumulated higher concentrations of Pro in leaves under both normal and osmotic stress conditions than the unstressed control plants. In these S1 plants, P5CS and δ-OAT showed DNA demethylation in response to osmotic stress. The methylation state of the other gene, P5CR, was however unaffected. The nonrandomness of the change was validated by a housekeeping gene used as a control. The demethylation of P5CS and δ-OAT and their consequent upregulation contributed to the enhanced Pro synthesis and accumulation (Zhang et al., 2013). More significantly, most of these stress-induced epigenetic modifications are reset to the basal level once the stress is relieved, but some of the modifications may be stable, i.e. they may be carried forward as “stress memory” (Chinnusamy and Zhu, 2009) (Figure 2). This causes heritable changes in methylation pattern and consequently the phenotype in subsequent generations, thus helping the plants to cope with osmotic stress (Zhang et al., 2013). However, it cannot be confirmed whether the overexpression of the alleles undergoing stable epigenetic modifications (e.g., demethylation of P5CS) (Figure 2) to develop “stress memory” is suppressed via Figure 2. Epigenetic modifications are mainly responsible for overexpression of P5CS and δ-OAT in response to osmotic stress. P5CR expression is also responsible for overaccumulation of Pro. However, the overexpression pattern of this gene has not been found to be manipulated by epigenetic changes. On application of osmotic stress, demethylation occurs in P5CS and δ-OAT, probably leading to high promoter accessibility to the transcriptional complex and hence higher transcription rates. Upon removal of stress, basal levels of transcription are restored. However, some instances have shown that the modifications remain stable in order to develop a memory against osmotic stress. Such memory may trigger heightened responses against osmotic stress once the plant system faces it again. It is unknown how the overexpression of the alleles that undergo stable epigenetic modifications is suppressed after removal of stress. 893 ROYCHOUDHURY et al. / Turk J Bot other chromatin modifications after the stress is removed. Further studies are required for a definitive answer to this pertinent question. 5. Protective roles of Pro: cross-talks with other signaling pathways 5.1. Pro: plant–water relations, photosynthesis, and growth Stress directly affects plant–water relations, indirectly affecting photosynthesis. This is because water uptake, ascent of sap, stomatal functioning, and chlorophyll biosynthesis are altogether hampered during stress, ultimately leading to reduced leaf water potential (Roychoudhury and Chakraborty, 2013). Exogenous Pro treatment resulted in a significant increase in the leaf water potential in Vicia faba during salinity stress. Restoration of photosynthesis occurred in Olea europaea ‘Chemlali’ facing salt stress. The pattern of restoration maintained a linear relation with increasing concentration of the exogenous Pro added (Ben Ahmed et al., 2010). Exogenous Pro in comparison to other compatible solutes like GB was more efficient in alleviating NaCl-generated stress in tobacco cells. If compared in terms of distribution, the resistance due to the effect of exogenous Pro was higher in the case of stomata on the abaxial surfaces than those on the adaxial surfaces. The even more striking fact is that lower concentrations of exogenous Pro have been found to be more effective than ABA spray in increasing stomatal resistance and also in maintaining turgidity in the leaves of stressed barley and wheat (Hayat et al., 2012). Pro also plays an important role in stabilizing the mitochondrial electron transport system (ETS) and other proteins including the CO2-fixing RUBISCO, thus providing a direct link between Pro application and increased photosynthetic yield. Another means by which Pro regulates plant growth and development is by being the constituent of what are called Pro-rich proteins (PRP). A study showed that a hydroxyproline-rich protein called Sickle (Sic) is important in development and stress tolerance in Arabidopsis. The downregulation of the sic gene by miRNA-mediated posttranscriptional gene silencing produced plants with loss of function and undesirable characteristics such as dwarfism, delayed maturity and flowering, abnormal inflorescence or phyllotaxy, and serrated, sickle-shaped leaf margins. This demonstrated the importance of the Pro-regulated Sic protein for normal development in Arabidopsis. Pro also acts to promote hypersensitive response (HR) followed by programmed cell death (PCD) in incompatible plant–pathogen interactions. In such a scenario, there is an increase in ROS and Pro concentration in the infected tissue (Ayliffe et al., 2002). Interestingly, Pro plays a role in the modulation of Agrobacterium infection 894 (Haudecoeur et al., 2009). Pro acts as the antagonist of the GABA-mediated quorum-quenching mechanism that protects the plants from colonization by Agrobacterium, thus allowing successful infection and transfer of the Ti plasmid. 5.2. Cross-talk between Pro and ABA One of the major structural proteins in the plant cell wall is hydroxyproline-rich glycoproteins (HRGP). Based on domain characteristics, HRGPs can be classified into: 1) Pro-rich proteins with (Pro)3XYLys repeats; 2) extensin-type proteins with Ser(Pro)3-5 repeats; and 3) arabinogalactan proteins (AGPs) with central domains rich in (Ser/Ala/Thr) Pro repeats. The nonbranched arabinose (Ara) oligosaccharides on hydroxyproline (Hyp) primarily contain the Pro repeats. The AGPs play important roles in plant growth and development, as both membrane-bound and secreted AGPs play roles in cell division, cell expansion, apoptosis, floral abscission, pollen tube guidance, pollen incompatibility, and plant–microbe interactions (Seifert and Roberts, 2007; Tseng et al., 2013). Under abiotic stress, ABA affects the development and functions of the roots. The repetitive proline-rich proteins (RePRPs) have come up as chief examples of the cross-talk existing between ABA and structural Pro. The RePRPs in rice are ABA-responsive and root-specific proteins, with unusual PX1PX2 sequence motifs (Tseng et al., 2013). The first Pro-rich glycoprotein identified in rice was the shootspecific OsPRP1 (Akiyama and Pillai, 2003). OsPRP1 was suppressed by ABA and treatment with methyl jasmonate. The Pro content in specific rice RePRPs was found to be around 40%, with the X1 and X2 in the unique motif PX1PX2, containing polar residues like Lys, Asn, Glu, or Gln. These RePRPs are not classical AGPs as they do not bind to β-Yariv reagent and are heavily glycosylated with Ara and glucose, instead of Ara and galactose. The ZmPRP in Zea mays was found in the xylem of the root maturation region and are thought to be involved in secondary cell wall formation. WPRP1 from wheat has been found in rapidly dividing tissues in shoots. Such PRPs with the unique PX1PX2 motif have not been identified in dicots (Tseng et al., 2013). The RePRP accumulation is inhibited by stress or ABA treatment, resulting in decreased growth rate in the elongation zone. However, in order to maintain the root length, the normal functioning of the cell division zone is essential (Yamaguchi et al., 2010). Pro in this case is not free and does not play the roles of an osmolyte. Still, we have introduced this significant fact to illuminate the effects of ABA on Pro associated with structural proteins. Exogenous application of ABA triggered Pro content in the seedlings of three rice varieties, M-1-48, Nonabokra, and Gobindobhog (aromatic); however, the effect was most dramatic in the most salt-sensitive variety, Gobindobhog, ROYCHOUDHURY et al. / Turk J Bot clearly pointing to the cross-talk between ABA and Pro in determining stress tolerance (Roychoudhury et al., 2009). A possible clue of cross-talk also exists with the fact that ABA and Pro sprayed together in stressed cotton plants significantly enhanced chlorophyll content, chlorophyll stability index, leaf relative water content, and dry matter accumulation at low water potential (Gadallah, 1995). Induction of Pro synthesis by ABA and salt stress correlates with a striking activation of P5CS1 expression, whereas P5CS2 is weakly stimulated. ABA and salt stress suppresses the PDH expression in shoots and roots of light-grown plants. The reason is that the maintenance of high Pro concentration in a stressed cell is essential for its viability. However, the light-dependent induction of P5CS1 by ABA and salt stress is downregulated in dark-adapted plants. As a result, PDH activity significantly increases in such plants. The steroid hormone brassinolide regulates the inhibition of P5CS1 during dark adaptation. This indicates the presence of a cross-talk between the transduction pathway associated with the reception of light and Pro content in plants. In ABA-hypersensitive prl1 and brassinosteroiddeficient det2 mutants, enhanced Pro accumulation and P5CS1 induction have been recorded. The prl1 mutation reduces the basal level of PDH expression, whereas the det2 mutation increases the inhibitory effect of ABA over PDH (Abraham et al., 2003; Ibragimova et al., 2012). Application of 50 µM ABA to Arabidopsis wild-type, ABA-deficient aba1-1 mutant, and ABA-insensitive abi11 and abi1-2 mutant seedlings triggered the expression of AtP5CS and not AtP5CR. The similar transcript levels in the wild-type and ABA-deficient mutants indicated that the expression of either of the genes was mediated by endogenous ABA (Savoure et al., 1997). However, the ABA-treated abi1-1 mutants accumulated less Pro than the ABA-treated wild type. On exposing the aba1-1 and abi1-1 mutants to salt stress, Pro accumulation further decreased in them in comparison to the wild-type. This typically indicates the indirect role of ABA over Pro accumulation during salt adaptation (Savoure et al., 1997). Thus, the Pro biosynthetic genes are ABA-independent during stress exposure, although previous instances do show that their expression can be triggered by exogenous application of ABA. It has been suggested that the endogenous ABA levels can affect the accumulation of Pro during salt stress, and this possibly indicates posttranscriptional regulation of the biosynthesis of Pro in response to NaCl toxicity in soil (Roychoudhury and Chakraborty, 2013). Experiments with canola leaf discs (CLDs) subjected to hyperosmotic stress showed that exogenously supplied ABA downregulated PDH during poststress recovery, though the expression levels of P5CS were not relatively heightened. The ABA content in ABA-treated turgid CLDs was insufficient to determine the extent of Pro accumulation, as ABA levels were maintained low during P5CS expression in the tissues (Trotel-Aziz et al., 2003). The ABA-induced genes during abiotic stresses mainly encode proteases, chaperonins, enzymes of sugar, Pro and other compatible solute metabolism, S-adenosylmethionine decarboxylase (SAMDC) that catalyzes the rate-limiting step in PA biosynthesis, ion and water channel proteins, antioxidants, and TFs (Basu and Roychoudhury, 2014). 5.3. Cross-talk between Pro and antioxidants The ROS play important roles in protecting plants against harmful pathogens that cause biotic stress. They contribute to the formation of tracheary elements, lignifications, and other important developmental processes (Das and Roychoudhury, 2014). However, nucleic acid damage, oxidation of proteins, lipids, and degeneration of chlorophyll pigments, along with uncontrolled K+ efflux from cells, occur due to excess ROS accumulation in the plant system (Chen and Dickman, 2005). The ROS cause modifications in covalent bond, accompanied with direct oxidation of amino acids like Cys (to form disulfide bonds), Met (to form Met sulfoxide), Arg, Lys, Thr, and even Pro residues of crucial proteins (Anjum et al., 2014). Thus, it is crucial to regulate ROS generation within the compatible and optimum limits of the plant. Pro is a potent ROS scavenger (Chen and Dickman, 2005). This view was further corroborated when reduced ROS levels were documented in the roots of Arabidopsis upon exogenous treatment of Pro. The ROS-induced K+ efflux also decreased significantly in this case. The main antioxidant enzymes like catalase (CAT), peroxidase (POX), and superoxide dismutase (SOD) also had their activities enhanced in the presence of Pro, which was applied exogenously to tobacco suspension cultures facing salt stress (Hoque et al., 2007a). The ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) are the main enzymes regulating the ASCGSH cycle. Exogenous Pro treatment of tobacco cultures exposed to salt stress upregulated the activity of these enzymes, resulting in the more efficient operation of the ascorbate-glutathione cycle (Hoque et al., 2007a). Exogenously applied Pro thus acts as a ROS scavenger (Kaul et al., 2008). Tobacco cells exposed to cadmium stress and treated with Pro had enhanced activities of SOD and CAT and hence a low rate of membrane lipid peroxidation (Islam et al., 2009). Salt stress in Nicotiana tabacum ‘Xanthi’ showed ROS production in the plant system. Along with ROS, the production of intracellular ammonia, and expression of gdh-NAD, A1 (encoding the α-subunit of glutamate dehydrogenase, GDH) was also induced. During stress treatment, an increase in the immunoreactive α-polypeptide and assembly of anionic 895 ROYCHOUDHURY et al. / Turk J Bot isoenzymes were also reported. The α-GDH subunit expression is mediated by the salt stress-generated ROS signaling. As a result, production of glutamate for Pro synthesis occurs due to the anionic iso-GDHs, which also play crucial roles as antistress enzymes in detoxifying ammonia (Skopelitis et al., 2006). 5.4. Cross-talk between Pro and PAs As mentioned earlier, the stress-induced regulation of the qualitative composition and the quantitative content of low-molecular-weight organic osmolytes is one of the key strategies of plants to adapt to adverse stress conditions. The cross-talk between Pro accumulation and PA synthesis has been recently postulated to be mediated by ABA (Shevyakova et al., 2013). Such cross-talk was studied in the glycophyte Phaseolus vulgaris exposed to salt stress. In a phytotron chamber and on Jonson nutrient medium, 2-week-old seedlings were grown for 6 days, being exposed to high NaCl concentrations of 50 mM and 100 mM. For the first 3 days, the roots were daily treated with 1, 5, 10, or 50 µM ABA for 30 min. High salt stress resulted in higher endogenous ABA level and a drastic 14-fold increase in Pro concentration. The concentration of free PAs (Put, Spm, Spd, and cadaverine) was reduced with the accumulation of 1, 3-diaminopropane, which is a product formed due to the oxidation of high molecular weight PAs. The steady maintenance in plant growth, stabilization of the water and sodium balance, and induction of chlorophyll and carotenoid biosynthesis showed that the ABA treatments aided the plants to overcome 100 mM NaCl stress. The ABA treatment actually suppressed the NaCl-responsive Pro and endogenous ABA accumulations. The normal levels of Put and Spd were restored. However, in contrast to wild-type plants, the Spm and cadaverine levels increased by 4- to 5-fold with a reduction in the contents of 1,3-diaminopropane and malondialdehyde (MDA) and also the activity of SOD (Shevyakova et al., 2013; Roychoudhury and Das, 2014). Thus, it was hypothesized that since both the biosynthetic pathways of Pro and PAs utilize glutamate as the common precursor, it is this step that is regulated by ABA in mediating the cross-talk. More studies obviously need to be performed for further approval. The transgenic soybean plants overexpressing P5CR exhibited enhanced levels of PAs. This shows that even the manipulation of the Pro biosynthetic pathway can affect the accumulation of PAs, further supporting the possibility of cross-talk (Simon-Sarkadi et al., 2006). The transgenic plants had Pro levels that were about 124-fold higher in comparison to the wild-type plants during exposure to stress. These plants also had low levels of Spd and Put. This was explained by the fact that, due to the much higher rate of channeling of Arg and Glu for Pro biosynthesis, less 896 was available to be used for PA biosynthesis. The wildtype plants, however, showed increased levels of Put and Spd on exposure to stress. The Pro and PA levels were simultaneously elevated in wild-type plants exposed to drought stress because these plants had more proteins degraded and required more osmolytes to recover from the injury (Simon-Sarkadi et al., 2006). In the case of transgenic plants, the extremely high Pro content seemed to have compensated for the activity of normal levels of Pro and PAs together. The greater increase in Pro content in the transformed plants helped them to better tackle the stress conditions than the wild-type plants during stress. However, the increased PA levels also make significant contributions in combating dehydration stress by reducing the stress-induced damages. Exogenous application of Spd and Spm during salinity stress in three rice varieties, namely M-1-48, Nonabokra, and Gobindobhog, enhanced the level of Pro over salt-treated counterparts; however, the effect was the most pronounced with Spd application in the most salt-sensitive variety, Gobindobhog, probably as a measure to ward off excess damages (Roychoudhury et al., 2011). An exceptional observation showed that the increase in Put content by salicylic acid in maize did not improve its tolerance against dehydration stress (Nemeth et al., 2002; Roychoudhury and Das, 2014). For better stress responses, the gradual changes in Pro and PA concentrations in the due course of time is more crucial than their level at the end of a long period of treatment. 6. Pro regulates seed germination during abiotic stress Seed dormancy is a major factor in dictating plant fitness. This complex trait has been reported to be influenced by genetic components, tissues forming the seed, and also integrative environmental signals. Seed dormancy is absolutely essential as it delays germination unless the environment is not favorable to sustain the growth of the plant (Graeber et al., 2012). Apart from the phytohormones like ABA and gibberellins, nonenzymatic processes, chromatin factors, and dormancy-specific genes have also been found to regulate seed dormancy (Linkies and Leubner-Metzger, 2012). Heikal and Shaddad (1982) were among the first to link seed germination with Pro. They found that the ‘interaction effect’ between Pro and osmotic stress induced germination and growth in seeds under osmotic stress. Pro played roles as a major source of energy and nitrogen required during poststress metabolism and also as an osmotic regulator in seeds under stress. Exogenous application of Pro improved the growth rate and seed germination in Arabidopsis exposed to chilling temperatures (Hare et al., 2003). In a recently conducted study, Bhamburdekar and Chavan (2011) studied the pattern of Pro accumulation in germinating