Improving salinity tolerance in transplanted aman rice ( Oryza sativa L.) by exogenous application of proline

Japan Abstract Salinity is the major factor reducing crop yield in coastal areas of Bangladesh. Proline (Pro) application with suitable crop varieties having higher yield potential could contribute to the improvement of crop production in saline areas. The main objective of this study was to investigate the mitigation of adverse effects of salinity in aman rice by exogenously applied Pro. The experiment was carried out at the farmer ’ s field of Batiaghata, Khulna. Characteristically, the soil was silty clay loam having pH 6.7, EC 4.6 dS m –1 , CEC 23 meq/100 g soil, organic matter 0.71%. Rice ( Oryza sativa L.) variety BR23 was used as plant material. The experiment was laid out in a randomized complete block design with three replications. There were different treatment combinations namely control (no Pro), 25 mM Pro at seedling stage, 25 mM Pro at vegetative stage, 25 mM Pro at seedling and vegetative stages, 50 mM Pro at seedling stage, 50 mM Pro at vegetative stage, 50 mM Pro at seedling and vegetative stages, 100 mM Pro at seedling stage, 100 mM Pro at vegetative stages, and 100 mM Pro at seedling and vegetative stages. Recommended doses of N, P, K, S and Zn fertilizers were applied to the all experimental plots. Thirty-day-old seedlings were transplanted in the experimental plots. Proline solutions were sprayed over plant leaves with the help of sprayer as per treatments. Salinity caused significant reductions in growth and yield of BR23 by decreasing plant height, number of effective tillers, panicle length, filled grains panicle -1 and 1000-grain weight. On the other hand, exogenous application of Pro showed a significant increase in growth and yield of BR23 under saline conditions. Results also revealed that growth and yield of rice did not increase proportionally with the increasing doses of Pro. Proline application resulted in significant increases in K + /Na + and nutrient uptake by rice under salinity. The present study suggests that exogenous application of Pro confers tolerance to salinity in aman rice by increasing K + /Na + ratio and nutrient


Introduction
Growth and yield reduction of crops are the serious issue in salinity prone areas of the world. About 20% of world's cultivated areas and nearly half of the world's irrigated lands are affected by salinity (Mali et al., 2012). Salinity effects are more conspicuous in arid and semiarid regions where limited rainfall, high evapotranspiration and high temperature associated with poor water and soil management (Azevedo et al., 2006;Jaleel et al., 2008). Agricultural productivity is severely affected by soil salinity, and the damaging effect of salt accumulation in agricultural soils has become an important environmental concern (Jaleel et al., 2007).
The world population is increasing rapidly and may reach 6 to 9.3 billion by the year 2050 whereas the crop production is decreasing rapidly because of the negative impact of various environmental stresses; therefore, it is now very important to develop stress-tolerant varieties to cope with this upcoming problem of food security. In addition, increased salinity of arable land is expected to have devastating global effects, resulting in up to 50% land losses by the middle of the twenty-first century (Mahajan and Tuteja, 2005).
Salinity causes unfavorable environment and hydrological situation that restrict the normal crop production throughout the year (Haque et al., 2014). The factors which contribute significantly to the development of saline soil are tidal flooding during wet season (June-October), direct inundation by saline water, and upward or lateral movement of saline ground water during dry season (November-May). Rice is mainly grown in salinity affected areas of Bangladesh but the average yield is very low due to lack of salt-tolerant high yielding variety and inappropriate management practices. In Bangladesh, out of 2.85 million hectares of the coastal and off-shore areas, about 1.06 million hectares are affected by salinity (SRDI, 2010).
Plants possess different defense mechanisms in order to cope with stress; one of them is associated with accumulation of osmoprotectants. Proline is one of the major osmoprotectant osmolytes, which is synthesized ARTICLE INFO by many plants in response to stress including salinity, thereby maintaining the osmotic status of the cell to ameliorate the abiotic stress (Chinnusamy et al., 2005;Burritt, 2012;Hossain et al., 2014). Proline plays roles in scavenging free radicals, stabilizing subcellular structures, and buffering celluler redox potential under stresses (Hoque et al., 2008;Banu et al., 2009;Hossain et al., 2014). Exogenous application of Pro also provides osmoprotection and facilitates the growth of salinitystress plants. Proline can protect cells against salinityinduced oxidative stress by up-regulating activities of various antioxidant enzyme activities (Hoque et al., 2007;Bhusan et al., 2016).
Exogenous application of Pro already gained considerable attention in mitigating the adverse effect of salt stress (Ashraf and Foolad, 2007;Hoque et al., 2007). There are increasing evidences that Pro application effectively regulates osmotic potential and plays a vital role in sustaining plant growth under osmotic stress (Ali et al., 2007;Ashraf and Foolad, 2007;Hoque et al., 2007). Exogenous Pro enhanced the antioxidant enzymatic activities viz. ascorbate peroxidase, peroxidase and catalase during both stress and recovery period in rice (Nounjan et al., 2012;Bhusan et al., 2016). Little information is available on the beneficial roles of exogenous Pro in modulating salt stress tolerance in rice at various phases of plant growth. Therefore, the present study aimed to investigate the mitigation of the adverse effects of salinity in aman rice by exogenously applied Pro at both seedling and vegetative stages.

Experimental site and soils
The field experiment was carried out at the farmer's field of Batiaghata, Khulna belongs to the Agroecological Zone of the Ganges Tidal Floodplain (AEZ 13). Characteristically, the soil was silty clay loam having pH 6.7, EC 4.6 dS m -1 , CEC 23 meq/100 g soil and organic matter 0.71%. The experimental area is included into the tropical monsoon climate. There are three monsoon periods appear in this region. The monsoon period lasts from May to October. About 88% of the total rainfall is observed in this time. Hailstone also occurs during this time. Sometimes storms locally called Kalboishakhi are observed.

Plant materials and treatments
The experiment was laid out in a randomized complete block design with three replications. Rice variety BR23 was used as plant material. There were ten treatment combinations with different concentrations of Pro at seedling and vegetative stages like as T 0 = Control (no Pro), T 1 = 25 mM Pro at seedling stage, T 2 = 25 mM Pro at vegetative stage, T 3 = 25 mM Pro at seedling and vegetative stages, T 4 = 50 mM Pro at seedling stage, T 5 = 50 mM Pro at vegetative stage, T 6 = 50 mM Pro at seedling and vegetative stages, T 7 = 100 mM Pro at seedling stage, T 8 = 100 mM Pro at vegetative stage, and T 9 = 100 mM Pro at seedling and vegetative stages. Recommended doses of TSP, MoP, gypsum, and zinc sulphate were applied to all the experimental plots (4.0 m × 2.5 m) during final land preparation. Recommended dose of urea was applied in three splits. Thirty-day-old rice seedlings were transplanted in the experimental plots. Three seedlings per hill were placed at a spacing of 25 cm × 20 cm. Proline solutions were sprayed over plant leaves with the help of sprayer. The volume of the spray was 25 ml per plant at both seedling and vegetative stages. Tween-20 was used as a sticky substance which helps Pro solution's droplet, maintaining a close contact with plant leaves. Other intercultural operations were done when necessary. Maturity of crop was determined when about 90% grains became golden yellow. The crop was harvested at full maturity.

Chemical analysis of plant samples
The representative grain and straw samples were dried in an oven at 65 0 C for about 24 hours before they were ground by a grinding machine. The prepared samples were stored in paper bags and finally kept into desiccators until analysis. The N, P, K, S and Na contents from grain and straw samples were determined following standard method as described by Khanam et al. (2001).

Statistical analysis
Data were analyzed statistically using analysis of variance with the help of software package MSTAT-C. The significant differences between mean values were compared by Duncan's Multiple Range Test. Differences at P≤0.05 were considered significant.

Results and Discussion
Growth and yield components of rice Salinity caused a significant decrease in plant height whereas Pro application significantly increased plant height (Table 1). Salinity significantly decreased effective tillers hill -1 of BR23. All treatments except 100 mM Pro at seedling stage increased effective tillers per hill over control. It was observed that there were no significant variations in panicle length among the treatments that exposed varying concentration of Pro application such as 25 mM, 50 mM, 100 mM Pro at seedling stage, vegetative stage, and both seedling and vegetative stages. Salinity caused a drastic decrease in filled grains per panicle of BR23. Exogenous application of Pro at different growth stages significantly increased filled grains per panicle under salinity conditions. Table 1 also shows that salinity significantly reduced the 1000grain weight of BR23. All the Pro treatments increased 1000-grain weight over control. Some of the Pro treatments contributed to the significant increase of 1000-grain weight under saline condition (Table 1).  Deivanai et al. (2011) also showed that Pro application increased plant height in two Malaysian rice cultivars (MR220 and MR232). Papon et al. (2015) showed that Pro application increased number of filled grains per panicle under saline conditions. Recently it is also reported by Bhusan et al. (2016) that exogenous Pro application increased growth of rice.
Grain and straw yields of rice Plant exposed with salinity significantly decreased grain yield of BR23 rice (Table 2). Foliar application of Pro over plant leaves significantly increased grain yield under salinity condition. All the treatments increased grain yield over control. Straw yield of BR23 was drastically reduced due to salinity. All the Pro treatments significantly increased straw yield over control (Table 2).

Table 2. Effect of exogenous proline on the grain and straw yields of T. Aman rice (BR23) under salinity conditions
Treatment Grain yield (kg/ha) Straw yield (kg/ha) T 0 Control 4672c 5063e T 1 25 mM proline at seedling stage 5438a 5863abc T 2 25 mM proline at vegetative stage 4980ab 5929ab T 3 25 mM proline at seedling and vegetative stages 5363ab 6329a T 4 50 mM proline at seedling stage 5016ab 5929ab T 5 50 mM proline at vegetative stage 4859bc 5130de T 6 50 mM proline at seedling and vegetative stages 5361ab 5862abc T 7 100 mM proline at seedling stage 4924ab 5663bcd T 8 100 mM proline at vegetative stage 4907b 5163de T 9 10 mM proline at seedling and vegetative stages 4941ab 5297de SE (±) 80.188 136.486 CV (%) 5.37 5.56 Same letter in a column represents insignificant difference at p<0.05. SE = Standard errors of means CV = Co-efficient of variation Miah et al. (1992) on two rice varieties found that salinity decreased straw yield of rice at 2.4, 6.0 and 11.8 dSm -1 conditions. It has been demonstrated that salt stress reduced grain and straw yields of rice (Bhusan et al., 2016). The protective mechanisms of Pro have been increasingly reported in the literature in plants against various stresses. It has been reported that exogenous Pro application increased grain and straw yields of rice (Papon et al., 2015;Bhusan et al., 2016).

Nutrient content and uptake
N content and uptake: There were significant variations in rice grain N content due to application of Pro. Most of the Pro treatments increased N content over control (Table 3). There was also significant variation in straw N content due to application of Pro. Most of the Pro treatments resulted in lower straw N content than control (Table 3). There were significant variations in rice total N uptake with application of Pro. All the treatments increased total N uptake over control (Table 3). P content and uptake: There were significant variations in rice grain P content due to exogenously applied Pro. Most of the Pro treatments increased P content over control (Table 4). Significant variations in rice straw P content were also observed in response to Pro application. All the treatments increased P content over control (Table 4). There were significant variations in rice total P uptake with application of Pro. All the treatments increased total P uptake over control (Table 4).  S content and uptake: Significant variation in grain S content was observed due to different treatments. Surprisingly, most of the Pro treatments did not result in increase in grain S content (Table 5). There were no significant variations in straw S content with application of Pro. Most of the treatments were found to be lower straw S content than control. Moreover, all the treatments except 25 mM Pro at seedling and vegetative stages were also found to be lower S uptake by straw than control (Table 5). There were significant variations in total S uptake due to different treatment combinations. In some cases, exogenous Pro increased total S uptake but not remarkably (Table 5).
Nutrients such as N, P, K and S play essential roles in plant metabolism. There are evidences that Pro minimizes the adverse effects of various stresses on plants by affecting the uptake and accumulation of inorganic nutrients (Ali et al., 2008). Similar to our results, Abd El-Samad et al. (2011) showed that application of Pro increased NPK nutrient uptake in rice plants.

K + /Na + ratio in grain
Potassium and sodium ratio in rice grain significantly decreased due to salinity while application of Pro significantly ameliorated K + /Na + ratio. All the treatments increased K + /Na + ratio over control. The K + /Na + ratio was found to be highest at 50 mM Pro application at seedling and vegetative stages (Table 6).

K + /Na + ratio in straw
The K + /Na + ratio in rice straw significantly decreased due to salinity but application of Pro significantly increased the K + /Na + ratio. All the treatments increased K + /Na + ratio over control. The K + /Na + ratio was found to be highest at 25 mM Pro application at vegetative stage (Table 6).  Salt stress disturbs cytoplasmic K + /Na + homeostasis, causing an increase in Na + to K + ratio in the cytosol (Zhu, 2003). It has been reported that salt stress causes increased uptake of Na + and Cl -, and decreased uptake of essential cations particularly K + (Khan et al., 2003). Proline has an added advantage under soil salinity as it may lower down Na uptake by plants and increase K uptake, thereby protecting crops from the detrimental effects of Na. Kaya et al. (2013) showed that sodium concentrations were higher in the tissues of plants grown under saline conditions while compatible solute treatments significantly reduced sodium concentration in the plant tissues. Abd El-Samad et al. (2011) showed that application of Pro increased K + /Na + ratio in rice plants. Nounjan et al. (2012) on Thai aromatic rice (cv. KDML105; salt-sensitive) also found the similar result due to exogenous application of Pro.

Conclusion
Physiological stress due to salinity is the main cause reducing crop yields in coastal saline areas of Bangladesh. Exogenous application of Pro with suitable crop varieties having higher yield potential could contribute to the improvement of crop production in saline condition. Our experiment shows that salinity reduced growth and yield of rice. On the other hand, exogenous Pro increased growth and yield contributing characters of BR-23, resulting higher grain and straw yields of rice. The increased growth and yield of rice by Pro were accompanied with higher K + /Na + ratio and uptake of nutrients. It can be concluded from the present study that Pro confers tolerance to salinity in aman rice due to increasing nutrient uptake, maintaining higher K + /Na + ratio and probably enhancing antioxidant defense systems. However, further studies are required to find out the beneficial roles of exogenous Pro at reproductive stage under saline conditions.