A Study on the Accumulation of Proline- An Osmoprotectant Amino Acid under Salt Stress in Some Native Rice Cultivars of North Kerala, India

Salinity is a major yield reducing factor in coastal as well as arid irrigated rice production systems. Salt induced abiotic stress is an acute problem, interrupting the metabolic processes of plants, resulting in reduced growth and productivity. Proline is an amino acid which increases in plants under abiotic stress. Salinity stress affects the metabolism of plants leading to severe crop damage and loss of productivity. Oxidative stress is one consequence of salinity that may be responsible for much of the damage. We inspected the immediate accumulation of proline in salinity induced stress in seven native rice (Oryza sativa L.) cultivars. Among the seven, five namely Orthadian, Chovvarian, Kuttusan, Kuthiru and Orkazhama were collected from a saline rice tract of the region and two namely Kunhutty and Veliyan from a non-saline rice tract of the region. The plants were subjected to different levels of salt stress ranging from 0 to 200 mM of NaCl and the concentration of proline in the leaf samples was inspected. Results showed that proline concentration increased in all the cultivars studied in relation to increase in salt stress and it was progressive along with increase in stress. This property was shown by all the cultivars irrespective of the fact whether they evolved in a saline habitat or a non-saline habitat. However, proline accumulation was higher in the cultivars collected from the saline tract when compared to the cultivars collected from the non-saline tract.


Introduction
Rice is one of the world's most important cereal crops with exceptional agricultural and economic importance and is the staple food of more than 50% population worldwide. Asian farmers produce more than 90% of the total rice, with two countries, India and China, growing more than half of the total crop [1]. Various abiotic stresses including high or low temperatures, water scarcity, high salinity and heavy metals exert drastic antagonistic effects on plant metabolism and thereby plant growth, development and productivity. Salinity and drought are the two major abiotic constraints in rice production, affecting plant growth and productivity globally [2,3]. In Asia alone, 21.5 million ha of land area is thought to be salt affected, with India having 8.6 million ha of such area which constitutes a major part of problem soils in India [4]. Yield of rice, especially Asian rice (Oryza sativa L.), is highly susceptible to salinity [5]. In India and especially in coastal rice fields, soil salinity is a major stress that reduces rice productivity [6]. Salinity is detrimental to the various processes of plants such as seed germination, seedling growth and vigour, vegetative growth, flowering and fruit set and ultimately it results in diminished yield and quality [7]. Salinity is known to affect plant growth and metabolism through its osmotic effects, through specific toxic effects of ions, by disturbing membrane integrity and function thereby interfering with internal solute balance and uptake of essential nutrients [8,9]. However, improvement in salt tolerance of crop plants remains elusive, since salinity affects almost every aspect of the physiology and biochemistry of plants at both whole plant and cellular levels [10,11].
Osmotic adjustment is a mechanism to maintain water relations under osmotic stress. In order to survive and cope up with the conditions of salt stress, plants show a number of physiological and biochemical adaptations. It involves the accumulation of a range of osmotically active molecules/ions including soluble sugars, sugar alcohols, proline, proline betaine, glycine betaine, glycerol, mannitol, sorbitol, organic acids, calcium, potassium, chloride ions, abscisic acid and osmotin [12][13][14]. These compounds are small uncharged molecules highly soluble in water at physiological pH, which can therefore accumulate at high concentration in the cytosol of plant cells without causing any damage to cellular 16 A Study on the Accumulation of Proline-An Osmoprotectant Amino Acid under Salt Stress in Some Native Rice Cultivars of North Kerala, India structures, because they are generally excluded from the hydration sphere of macromolecules [15]. Compatible osmolytes, once accumulated, are thought to reduce the cellular water potential below external values, driving or preserving water in the cell, thus maintaining turgor pressure high enough to sustain growth. In rice, accumulation of proline is reported to be more in salt tolerant than in salt sensitive cultivars and has been implicated to confer tolerance to salinity stress.
Salt stress is caused by excessive accumulation of salt in the soil, either directly because of salinization, or indirectly because of water loss. As a consequence, the soil water potential progressively decreases, hampering and eventually halting the gradient of water flow from roots to apical shoot. The resulting osmotic stress may cause stomatal closure, reduced photosynthesis rate and growth inhibition. Another consequence of osmotic stress is the production of ROS and the accumulation of toxic ions such as Na + or Cl − within the cell, causing severe damage to membrane structures, proteins, nucleic acids and lipids [16]. Rapid accumulation of free proline is a typical response to salt stress. When exposed to drought or a high salt content in the soil (both leading to water stress), many plants accumulate high amounts of proline, in some cases several times the sum of all the other amino acids [17]. Proline has been found to protect cell membranes against salt injury [18]. Sultana et al. [19] have suggested that proline accumulation in both salinized leaves and grains of rice plants is implicated in osmotic adjustment to salinity. In contrast, Lutts et al. [20] have argued that proline accumulated in salt-stressed calluses had a negligible effect on osmotic adjustment and did not play a role in salt resistance in rice callus cultures.
For many years, proline has been known to be involved in the response to a number of environmental stresses, particularly salt and drought stress. The accumulation of proline upon osmotic stress is well documented in a large number of different plant species [21][22][23][24][25][26]. However, a general agreement on the precise role of proline in the response of plants to stress is still lacking and several hypotheses have been proposed on the significance of the accumulation of proline caused by stress [27]. Among the plant compatible osmolytes (or plant protectants), proline is considered of major importance, as it has been reported to accumulate in a large number of species in response to stresses such as excess salinity, drought, cold, nutrient deficiency, heavy metals, pathogen infections and high acidity [9,13]. In addition, high concentrations of proline have been observed in halophytic plants grown in saline environments [28,29,27] in the root apical region of plants growing at low water potentials [30] in suspension cultures of plant cells adapted to water or NaCl stress [31,32] and in flowers and fruits of a large number of species under normal physiological conditions [33,34].
Salt stress responses of plants may depend upon salt type, concentration and genotype. Therefore, screening for salt-stress tolerant genotypes in important crops such as rice will help in ensuring future crop production. In addition, studying differential responses of genotypes with contrasting stress tolerances will help reveal the underlying salt stress tolerance mechanisms [2]. The present investigation was aimed to study the effects of NaCl stress towards proline accumulation in seven rice cultivars, five of them collected from a traditional saline rice tract and two from a traditional non-saline rice tract of Kerala State of India.

Germination of Seeds and Transfer to the Experiment Site
The experiment was conducted in the experimental rainout poly house of Department of Botany Enough number of good caryopses were taken from single plant in the case of each cultivar and washed in running tap water to remove infected and unfilled grains and dust particles. The seeds were soaked in distilled water and allowed to germinate in 10cm diameter Petri dishes covered with lid under room temperature. The water was changed every day. The seeds started to germinate from the third day onwards. On the 10 th day, required numbers of the germinated seedlings were transferred to coloured plastic pots of 25cm diameter filled with paddy soil mixed with enriched compost in 3:1 ratio. Two seedlings were initially planted per pot and after establishment of the seedlings the smaller among the two was removed. The plants were maintained in the experimental poly house under wetland conditions, always maintaining 3cm of water above the soil level. The soil was fertilized with 1g N: P: K =18: 18: 18 per pot at fortnightly intervals starting from the 30 th day. Weeding was done manually whenever required. Plants were grown in plastic pots of 25cm diameter in Randomized Block Design with three replications.

Plant Materials and Treatments
The experimental treatment was started from the 45 th day onwards starting from 10mM (0.91dSm -1 ) to 200mM (18.26dSm -1 ) aqueous solution of sodium chloride as detailed in Table 1.

Estimation of Proline
Estimation of proline was done according to the procedure adopted by Bates et al. [35]. Fully expanded rice leaves from the experimental plants were sampled. Purified proline (Himedia, India) was used to standardize the procedure for quantifying sample values. Acid-ninhydrin was prepared by warming 1.25g ninhydrin (Himedia, India) in 30ml glacial acetic acid (Merck) and 20ml 6M phosphoric acid (Merck, India), with agitation, until getting dissolved. Kept cool (stored at 4°C), the reagent remains stable for 24 hours. Approximately 0.5g of plant material was ground in a mortar with liquid nitrogen; homogenized in 10ml of 3% aqueous sulfosalicylic acid (w/v) (Himedia, India) and the homogenate filtered using Whatman #2 filter paper. 2ml of the filtrate was reacted with acid-ninhydrin and 2ml of glacial acetic acid (Merck, India) in a test tube, incubated for 1 hour at 100°C in a boiling water bath and the reaction terminated in an ice bath. The reaction mixture was extracted with 4ml toluene (Merck, India) mixed vigorously with a test tube stirrer for 15-20 seconds. The chromophore containing toluene was aspirated from the aqueous phase, warmed to room temperature and the absorbance read spectrophotometrically (Thermo Scientific, USA) at 520nm using toluene for a blank. The proline concentration was determined from a standard graph and calculated in µg/g on the basis of fresh weight of the leaf samples [36].

Statistical Analysis
Each experiment was done in replicates of three and data expressed as mean ± standard error (SE) and significance of variation calculated at P≥ 0.05 using analysis of variance.

Results
Free proline content in the experimental plants was determined on the 90 th day, and the results are presented in Table 2 and Figs. 1 and 2. Free proline content was significantly elevated in the salt stressed plants over control plants in all the rice cultivars under study. Proline content increased in proportion to increase in salt stress applied. Rapid accumulation of free proline is a typical response to salt stress and similar responses have been observed by earlier workers in rice [37].  Both the rice cultivars collected from the non-saline tract showed significant and gradual increase in proline content when salt stress was progressively applied. Earlier workers have also reported high accumulation of proline in salt sensitive rice under salt stress [38]. In consensus with this, our results show that the salt sensitive cultivars accumulate high amounts of proline in relation to increase of salinity stress. Among the two cultivars collected from the non-saline rice tract, increase in proline content was 115.88% in Kunhutty and 100.04% in Veliyan when exposed to 200mM NaCl.
In the case of the cultivars collected from the saline rice tract, increase in proline content varied from 107.01% to 139.42% under a salt stress of 200mM NaCl. The cultivar Kuthiru showed the highest quantum of accumulation of proline followed by Kuttusan, Orthadiyan, Orkazhama and Chovvarian. Capability of these cultivars to maintain comparatively good levels of growth and yield performance under salt stress has been demonstrated by earlier studies [39]. It is interesting to note that while the cultivars from the saline rice tract showed significantly higher levels of accumulation of proline from the salt treatment level of 30mM onwards, the cultivars from the non-saline rice tract showed significantly higher levels of accumulation of proline from the treatment level of 50mM only.
When exposed to high salt content in soil, many plants accumulate high amounts of proline, in some cases several times the sum of all other amino acids [40]. Proline is a known osmoprotectant and it plays an important role in osmotic balancing, protection of sub-cellular structures and enzymes and in increasing cellular osmolarity (turgor pressure) that provides the turgor necessary for cell expansion under stress conditions [41,28].

Discussion
Soil salinity is one of the major factors limiting crop production. Salt stress results in a wide variety of physiological and biochemical changes in plants. Accumulation of low molecular weight solutes such as proline, commonly referred to as compatible solutes is one such phenomenon [42]. By lowering water potentials, the accumulation of compatible osmolytes involved in osmoregulation allows additional water to be taken up from the environment, thus buffering the immediate effect of water shortages within the organism. High levels of proline enable the plant to maintain low water potentials. In addition to acting as osmoprotectant [43], proline also serves as a sink for energy to regulate redox potentials [44,45], as a hydroxy radical scavenger [46], as a solute that protects macromolecules against denaturation [47], as means of reducing acidity in the cell [48] and acts as a storage compound and nitrogen source for rapid growth after stress [49]. Proline alleviates NaCl stress induced enhancement in oxygenase as well as carboxylase activities of Rubisco [50]. Matysik et al. [41] noticed that proline protects plants against free-radical induced damage by quenching of singlet oxygen. Proline accumulation is one of the adaptations of plants to salinity and water deficit [51,52,53]. Some workers did not observe any appreciable increase in free proline content due to stress [54,55] and some others consider enhanced proline level merely a stress effect, rather than a cause of stress tolerance [56].
Proline accumulation in leaf tissues has been suggested to result from a decrease in proline degradation, an increase in proline biosynthesis, a decrease in protein synthesis or proline utilization and hydrolysis of protein [57]. The higher accumulation of proline could be due to enhanced activities of ornithine aminotransferase (OAT) and pyrroline-5-carboxylate reductase (P5CR), the enzymes involved in proline biosynthesis [58], as well as due to inhibition of proline oxidase and proline dehydrogenase (PDH), proline catabolizing enzymes [59]. It has been reported that proline dehydrogenase also functions as pyrroline-5-carboxylate reductase (proline synthesizing enzyme) and catalyses the reaction with the same reactants and co-enzymes, but operating in an opposite direction [60]. Studying the effects of salt stress on enzyme activities involved in proline metabolism could provide valuable information on the physiological significance of its accumulation. KaviKishor et al. [61] reported that there was a surge in proline content in plants under salt stress. In many plants proline accumulates in excess of protein synthesis. Proline catabolism is repressed under osmotic stress, but once the stress is withdrawn, proline is oxidised to Δ 1 -pyrroline-5-carboxylate (P5C) by proline dehydrogenase (PDH), also known as proline oxidase, the first enzyme in the proline degradation pathway. P5C is then converted back to glutamate by the enzyme P5Cdehydrogenase (P5CDH). Thus, both PDH and P5CDH form two important enzymes in the degradation of proline to glutamate in higher plants.
The present study has shown that proline concentration increases in all the cultivars of rice studied in relation to increase in salt stress and the increase is progressive along with increase in stress. This property was shown by all the cultivars irrespective of the fact whether they evolved in a saline habitat or a non-saline habitat. However, proline accumulation was higher in salt tolerant rice cultivars when compared to sensitive cultivars. The accumulation of compatible solutes may help to maintain the relatively high water content necessary for plant growth and cellular functions. Plants respond to a variety of stresses by accumulating certain specific metabolites, the most conspicuous being amino acids in general and proline in particular. Less than 5% of the total pool of free amino acids in plants under stress free conditions is provided by proline. In many plants under various forms of stress, the concentration increases up to 80% of the amino acid pool [41]. Abiotic stresses like drought and salinity cause significant crop loss worldwide [62,63]. They affect plants through different mechanisms, causing osmotic stress and ionic imbalance, in addition to inducing oxidative stress [64]. The exact role of proline in inducing stress tolerance has to 20 A Study on the Accumulation of Proline-An Osmoprotectant Amino Acid under Salt Stress in Some Native Rice Cultivars of North Kerala, India be further investigated in terms of the mechanisms involved.

Conclusions
Salinity is a major yield reducing factor in rice production systems. Salinity induces abiotic stress interrupting the metabolic processes of plants, resulting in reduced growth and productivity. Production and accumulation of some compounds in higher concentration is a phenomenon usually associated with salt stress response in plants. Proline is an amino acid which increases in plants under abiotic stress. The accumulation of proline under salinity induced stress in seven native rice (Oryza sativa L.) cultivars including five namely Orthadian, Chovvarian, Kuttusan, Kuthiru and Orkazhama collected from a saline rice tract of the study region and two namely Kunhutty and Veliyan from a non-saline rice tract of the region were studied presently. The plants were subjected to different levels of salt stress ranging from 0 to 200 mM of NaCl and the concentration of proline in the leaf samples was inspected. Proline concentration increased in all the cultivars studied in relation to increase in salt stress and it was progressive along with increase in stress. This property was shown by both the groups of cultivars irrespective of the fact whether they were collected from the saline habitat or the non-saline habitat. However, proline accumulation was higher in the first group of cultivars when compared to the second group of cultivars.