Integrated Physiological , Biochemical , Anatomical and Molecular Studies Reveals Salt Stress Responsive Mechanisms Associated with Popular Rice Landraces in Eastern Part of India

The present study aims towards examining physiological, biochemical, anatomical and molecular traits in the high-yielding rice landraces of the eastern part of India at germination and seedling stage for low-to-moderate salinity stress tolerance. The germination percentages of 12 high-yielding varieties were analyzed under seven different concentrations of salt (0, 50, 100, 150, 200, 250 and 300 mM). Although, Lalat performed very well with 63.66 % germination rate at 300 mM salt stress conditions, higher levels of salinity exhibited detrimental effects on other varieties. Hence, the salt concentrations 150 mM and 250 mM were optimally selected to screen the varieties for physiological and biochemical traits. The high-yielding varieties Lalat, Vamsi and Pratiksha exhibited lowest and similar trend of reduction over control (ROC) with relative water content (RWC) for 250 mM salt concentrations. The level of proline, increased with increasing concentrations of NaCl among most of the varieties and significantly high content was found in Vamsi. The chlorophyll content was high in Lalat as well as Pratiksha and reduction over control was highest in Vamsi under 150 mM salt concentrations. Interestingly, Lalat emerged as the most salt-tolerant landrace for physiological and biochemical traits. It was compared with salt-sensitive MTU7029 for anatomical changes on adaxial surface of the leaf blade and noticed out to be with less widened and few number of stomatal pores in response to salt stress. Further, expression analysis of salt-specific genes OsHKT1;5 and OsNHX1 resulted in identification of novel salt-stress responsive alleles/motifs like W-boxes, Box-W1, E2Fb and MBS in Lalat (salt-tolerant). Thus, the study disclosed the anatomical changes in adaxial surface of leaf and association of potential cis-acting regulatory elements (CARE) in regulation of salt-stress responsiveness in these two-contrasting rice landraces in the eastern part of India. Further, the study laid a foundation to explore transcriptional gene regulation to open-up the pathway towards crop improvement for salt-stress tolerance. Article history Received: 11 July 2017 Accepted:13 September 2017


Introduction
Rice (Oryza sativa L.) is the main food for more than half of the world's population.Globally, rice production is more than 470 million metric tons where India is contributing 264.77 million tons to the rice consuming population of the world 1 .Major constraints like salinity limit the production of rice in highly cultivated regions like eastern part of India despite of ideal conditions like temperature and rainfall.
Rice plants to establish under salt stress conditions are crucial at germination and seedling stage development.Rice seed germination can determine its seedling growth and yield to some extent.In this direction, selection of high-yielding varieties for rapid and uniform germination under salt stress conditions can further contribute towards strengthening seedling vigor 2 .The high-yielding rice varieties like MTU-7029, CR Boro dhan-2, MTU-1001, Pooja, Pratiksha, BPT-5204, Sarala, Jogesh, Lalat, Swarna sub-1, Nellore sannalu, Vamsi were selected for the present study.For instance, Lalat was developed and released in India based on its best performance at field level with yield ranging from 40-45 quintals/ha and resistant for many biotic stresses 3,4 .However, salinity-stress induced differential anatomical and molecular responses for these genotypes like Lalat at tribal dominated part of the country is far from being elucidated.
Salinity stress is negatively correlated to plant growth due to osmotic stress 5 .The earlier and noticeable salt stress response is the reduction in leaf surface which shows protuberances followed by disruption of leaf blades as the stress intensifies 6 .Consequently, ruptures the stomatal glands thereby reducing photosynthetic carbon-di-oxide (CO 2 ) fixation leading to CO 2 starvation.Furthermore, induces the production of osmoprotectants like proline protects chlorophyll molecules to certain severity of salt stress 7 thereafter directing towards sodium (Na + ) accumulation.In conjunction with such studies, reports on rice cultivars tend to possess active OsHKT1;5 and OsNHX1 alleles specific for remarkable tolerance to salt stress 8,9,10 further expedited the research in identifying superior transcripts.As few novel transcripts associated with such salt-stress responsive alleles, a comprehensive study to relate physiological, biochemical, anatomical with transcriptional gene regulation analysis was performed using popular landraces.
The rice landraces used in the study are cultivated amongst tribal people at eastern part of India.Despite high-yielding and multiple resistances, these rice varieties 3 possess high potential for crop improvement and they are still understudied for various other physiological, anatomical and molecular traits.Thus, it becomes scientist's responsibility to explore genetically viable, highyielding and stress tolerant rice varieties to the tribal society.To meet the demand of coastal tribal people through agriculture, this study has been initiated to explore the presently cultivated high-yielding rice landraces by understanding the association of physiological, biochemical and anatomical traits responsiveness with that of transcriptional gene regulation for salt tolerance.

Physiological trait Analysis Germination Percentage
The seeds used were healthy and uniform.Initially, germination percentage was calculated by sowing seeds (50 numbers) of each variety separately, in sterilized blotted petriplates.The seeds were sown and grown in the sterilized blotted petriplates and around 4 ml of sterile distilled water (control) and six different concentrations of salt (NaCl) solution (50 mM, 100 mM, 150 mM, 200 mM, 250 mM and 300 mM) was added.The replications (three) were maintained for each variety with different salt concentrations.The germination experiment was carried out in a chamber with constantly maintained temperature of 30ºC and were periodically checked and watered.The germination percentage was measured on alternative days till 11th day.

Fresh/dry weight and Relative Water Content
After recording the germination rate, the seedlings of all genotypes were continued to grow till 14th day under 0 mM, 150 mM and 250 mM salt stress conditions.The fresh weight (FW) of the shoot part from each replication/variety was measured.They were transferred and immersed in sterile distilled water for 24 hrs.After the incubation time, turgor weight (TW) was recorded and dried the samples at 80°C for 3 days to measure dry weight (DW).Further, the values of fresh weight (FW), turgor weight (TW) and dry weight (DW) were used to calculate 11 relative water content (RWC) as follows, RWC = (Fresh Weight -Dry Weight)/(Turgid Weight -Dry Weight)*100

Biochemical Parameters and Analysis Chlorophyll and Carotenoids Estimation
The 14th day harvested seedlings (0.5 g) of all genotypes (control and salt-stressed) were homogenized in mortar and pestle separately by using 80 % acetone.The homogenized solution was centrifuged at 1500 -2000 rpm for 8 -10 min and the supernatant was collected.The total chlorophyll was measured at 663 nm and 645 nm and carotenoids at 520 nm 12 .It was further calculated for content of chlorophyll a, chlorophyll b and total chlorophyll as follows, Chl a = 12.7*(the value of 663 nm) -2.69*(the value of 645 nm) Chl b = 22.9*(the value of 645 nm) -4.68*(the value of 663 nm) Total Chl = 20.2*(thevalue of 645 nm) + 8.02*(the value of 663 nm) The chlorophyll stability index (CSI) was analyzed according to Murty and Majumder 13 and is expressed as a percentage.

Leaf Proline Estimation
The control and NaCl treated 14th day old seedling samples of each variety were used to study the proline content.The samples were weighed uniformly to 0.5 g and homogenized in 1 ml of 3 % aqueous sulfo-salicylic acid.It was centrifuged at 8000 rpm for 12 min to remove the debris.A ratio of 1;1 (acid Ninhydrin and glacial acetic acid) was mixed with the supernatent and the mixture was heated to 100ºC in water bath for 1 hr and then the tubes were immediately transferred to ice bath.An equal aliquot of toluene was added to the mixture and vortexed for 15 to 20 sec.Further, the chromophore phase was used to measure the absorbance at 520 nm.The readings obtained were analyzed with a basic standard curve of L-Proline 14 .

Anatomical examinations Scanning Electron Microscope
The two extreme salt responsive rice varieties i.e., Lalat (salt-tolerant) and MTU7029 (salt-sensitive) were selected.The seedlings (14th day old control and salt-treated samples/specimen) were measured 2 cm away from tip and 0.2 cm of the leaf part was cut laterally and processed separately.The leaf histology of extreme salt tolerant and sensitive rice leaf samples was observed using the Scanning electron microscope (JEOL 6510 LV Model, Japan).The leaf samples were processed to sterilize the surface and mount it in EMS Quantomix WET-SEM (QX-102) capsules on the stubs.The specimen was immersed in the QX imaging buffer for 5 to 10 minutes initially.The sample was not required to dry at critical point as it was mounted on to QX-102 capsules directly.The capsule was studded onto a metal stub with double sided carbon tape to fix it on the stage (Note: The QX-102 capsules are for imaging various wet biological samples and can be visualized directly without any coating or embedding) and the specimen was viewed under Scanning Electron Microscope (JEOL 6510 LV Model, Japan).

Molecular analysis Polymerase Chain Reaction (PCR) Based Allele Mining
The varieties Lalat (salt-tolerant) and MTU7029 (salt-sensitive) were selected for allele mining.The seedlings grown for 14 days were used to extract DNA by cetyl trimethyl ammonium bromide (CTAB) method as described 15 .The extracted DNA was quantified using nano-drop (ND-1000 spectrophotometer) and its quality was assessed on ethidium bromide (EtBr) stained 0.8% (w/v) agarose gel electrophoresis.The  16 .

Statistical Analysis
All recorded data were analyzed for analysis of variance (ANOVA) using the SPSS statistical software package version 20.0.The differences between treatments and genotypes were analyzed using Tukey's Honest Significant Difference (HSD) test.Pearson's correlation coefficients were used to draw inferences on relationships among various traits.

Biochemical Estimations Chlorophyll and Carotenoids Content variations under Salt Stress Conditions
A noticeable significant difference in chlorophyll content among all the genotypes over salinity treatment, within treatment as well as among genotypes was observed at P≤0.05 (Table 2).Total chlorophyll content decreased with increased salt concentrations.A minimum reduction over control (ROC) was significant in Lalat with 1.

Proline Accumulations and its variations
Increasing salt concentrations triggered the level of proline in leaves of all the genotypes used.
The proline content increased with increasing salt concentrations in all the genotypes except for Pooja, Sarala, Pratiksha and MTU1001.These genotypes exhibited a trend of increased proline at 150 mM NaCl over control and decreased trend at 250 mM salt stress conditions.The genotypes including Lalat, Jogesh, Vamsi, BPT 5204, MTU7029, Nellore sannalu and CR Boro dhan-2 showed a noticeable increase in proline content with increasing salt concentrations.The increase of proline in leaves of all genotypes was more than 100 % in 150 mM NaCl over control conditions.Despite of increased proline content under 150 mM salt stress conditions, Swarna sub-1 demonstrated consistency in its level of proline even at 250 mM salt treatment.A highly significant variation at P≤0.05 was observed among the treatments, genotypes and genotype over treatment for proline content at seedling stage (Table 2).Invariably, the performance of Lalat was highly significant and found to be the most salttolerant under higher concentrations of salinity for all the traits measured, whereas, the performance of MTU7029 was significantly low under salt stress conditions.Therefore, the control and 150 mM salt treatments were further short-listed to study the anatomical and molecular traits of two-contrasting genotypes more precisely.

Anatomical Changes in Rice Genotypes under Salt Stress
The cell damage on the adaxial surface of rice leaves due to salinity treatments were studied using scanning electron microscope.

Molecular Level Responses of Rice Landraces Allelic variations and Stress-Responsive Superior Cis-Acting Regulatory Elements
The primer OsHKT1;5 and OsNHX1 was used spanning both exon and intron set with amplicon sizes 1.6 kb and 1.5 kb respectively.Among these

discussion
The germination test can determine the percentage of seeds that are viable in a given sample.The degree of germination in relation with seed vigour estimates its potential field performance 17 .The seed germination is dependent on seed structure as well as the environmental factors that affect the growth of the embryo.In the present study, the germination rate in most of the genotypes was inhibited at higher concentrations of salinity stress 18,19 .Salinity stress had significant effect on all the genotypes on their germination rate.The reduction over control was high in MTU-7029, CR Boro dhan-2, MTU-1001, Pooja, Pratiksha, BPT-5204, Sarala, Jogesh, Swarna sub-1, Nellore sannalu and Vamsi except Lalat under different concentrations of salt like 0, 50 mM, 100 mM, 150 mM, 200 mM 250 mM, 300 mM.The germination rate remained invariable in Lalat but none of other genotypes survived.Hence, the study was further focused with salt treatments 0, 150 and 250 mM to concretize the structure of physiological and biochemical traits.
In this direction, few physiological traits like fresh weight (FW), dry weight (DW) and relative water content (RWC) was measured in all the genotypes.A significant positive correlation was observed between FW, DW and RWC 20,21,22,23 .Relative water content is a potential parameter to quantify the degree of osmotic stress in plants 24 .The reduction over control of RWC was significant under 150 mM NaCl stress conditions but significantly high reduction was recorded with increased salt stress conditions.A similar trend of reduction over control in RWC was prominent in most of the genotypes, indicating higher level of reduction with increased degree of stress.
Proline content in leaves of all the genotypes increased with increasing salt concentrations.A similar observation was reported in other parts of tomato plants like stem and leaves under 160 mM NaCl 25 treatments.The trend remained similar to proline content among introgression lines derived from wild types of rice 26 .In the present study, the long term exposure to NaCl stress exhibited ~six folds increase in the most salt-tolerant Lalat whereas ~five folds in the most salt-sensitive MTU7029 (Table No.: 2).Proline, an osmoprotectant gets accumulated as an important defense mechanism in higher plants.
The accumulation is at higher level under stress conditions like salinity, drought, cold, etc., and it also protects the photosynthetic pigment chlorophyll.The chlorophyll pigments are more sensitive to salinity stress in salt sensitive rice genotypes 27 due to hypersensitivity of chlorophyll-b to salinity than that of chlorophyll-a 28 .Though, some of the genotypes in the present study showed highest proline content, but their performance for other biochemical traits was poor.For instance, Vamsi indicated with highest proline content (~30 folds) under high concentrations of salinity (250 mM) but with lowest chlorophyll as well as carotenoid content.However, Lalat performed best among all the genotypes with consistency in all the traits like chlorophyll and carotenoids.It was only ~2 folds reduction under stress conditions over control.Under salt stress conditions, many rice genotypes were found to show increased proline content with increasing salinity levels 26,29,30,31 .
Salinity stress adversely influences the mechanism of stomatal conductance in plants by disrupting the glands/pores 32,33 .In the current study, an attempt of understanding the anatomy of stomatal glands under salt stress conditions resulted in the significantly affected leaf blades on the adaxial surface of rice seedlings of salt-sensitive genotype.
The stomatal glands on adaxial leaf surface were uniform throughout in both the genotypes under control conditions but the glands bulged and widened intensely in salt-sensitive MTU7029 under salt stress conditions.The effect was remarkably less in the leaf blades of salt-tolerant Lalat.The stomatal glands regulate the uptake of carbon-di-oxide for the process of photosynthesis against loss of water but under salt stress conditions, the glands increase in number and gets widened leading to dryness of leaves by inhibiting metabolic rate in plants 31 .
Diversified genetic information is one of the sources at molecular level to screen and shortlist the genotypes for various stress tolerance.Usage of trait specific markers for phenotyping would expedite the process of genotypic selection for allele mining.Several molecular and bioinformatic tools like PCR-allele mining would expedite the identification of superior alleles and nucleotide sequence motifs.Various studies have been reported on characterization of genes 34,35 , but very limited is known about the cis-acting regulatory elements and their regulation in plants involved in stress-responsive expressions in plants 36 .The genomic sequence information of Arabidopsis/rice 37,38,39 and professional bioinformatic tools have unfastened the way for advanced studies on developmental, regulation and expression of novel genes.Significant variations in PCR-based allele mining and identification of cis-acting regulatory elements were observed in both salt-tolerant Lalat and salt-sensitive MTU7029 genotypes.All the sequences obtained from allele-mining were used for identification of cis-acting regulatory elements.These elements involved in gene expression and some in regulatory mechanisms that contribute to stress-regulation in rice genotypes 40 .Many motifs are associated with complex protein domains and transcription factors coding for multiple regulatory mechanisms 41 .Interestingly, salt-tolerant Lalat exhibited regulatory elements that are associated with salt stress-responsiveness.The regulatory elements like Skn-1, CAAT and TAAT-box in OsNHX1 were common in both the genotypes that are required for endosperm expression, in promoter and enhancer regions and around -30 of transcription start respectively and the result was similar to some of the studies reported in aquatic plants 42,43 .Studies reported a salt-responsive BADH promoter from Atriplex centralasiatica and found elements like GC-motif and CAAT-box related to stress 44,45

Fig. 1 :
Fig.1: Scanning electron microscopy of adaxial leaf surface of rice seedlings

table 1 : Pearson's correlation co- efficient amongst the physiological characteristics in genotypes sown and grown for 14 days under control, 150 and 250 mM naCl at seedling stage.
and analysed to sequence.The sequences were analyzed in 'PLACE' and 'PLANT CARE' professional bioinformatics databases to identify cis-acting regulatory elements

table 2 :
Biochemical analysis (Proline, Chlorophyll and Carotenoids) in rice landraces of Eastern part of India.Each value represents mean of three replications.Means followed by different letters in the same column are significantly different at the P<0.01/P<0.05levelaccording to tukey's hSd test.Proline (µg/g FW) total Chlorophyll (mg/g FW) Carotenoids (mg/g FW)under different concentrations of salt (0, 50 mM, 100 mM, 150 mM, 200 mM 250 mM, 300 mM), to select two optimum levels of stress conditions.The rate of germination was measured on every alternative day and final germination percentage was calculated on 11th day.In all the genotypes, the germination rate decreased with increase in salinity stress concentrations.The pattern of germination rate was consistent under 50 mM salt stress among Lalat (96.33 %), Jogesh (94.66 %) and BPT 5204 (94.00 %).At 100 mM NaCl concentrations, the reduction over control (ROC) was more than 30 % among Pooja with 35 % and 38.66 % in MTU7029 and remaining all genotypes showed around 20 % ROC.The genotypes under 150 mM NaCl conditions exhibited 5 to 10 % difference reduction over 100 mM salt stress.The differences were highly significant at 250 mM salt stress conditions under which all the genotypes showed ≥50 % ROC except Lalat.A consistently similar degree of significance was noticed in Lalat with 33.66 % under 250 mM NaCl conditions.The genotype remained stable under NaCl concentration 300 mM with around ~3 % reduction over 250 mM stress conditions, whereas, all other genotypes with 100 % ROC.However, none of the genotypes survived at 300 mM NaCl stress conditions on 11th day except Lalat which showed 63.66 % germination rate.Hence, based on germination rate under seven different salt concentrations, three optimum NaCl treatments (0 mM, 150 mM and 250 mM) were selected.The physiological and biochemical level differentiations among these genotypes were further studied under 0 mM, 150 mM and 250 mM salt stress conditions.

Shoot Fresh Weight (FW) and dry Weight (dW) to Relate with Relative Water Content (RWC)
CR Boro dhan-2 with ROC of 4.3, 6.6 and 8.3 folds respectively under 250 mM stress conditions.The level of carotenoids in all the genotypes against salt stress conditions was highly significant at P≤0.01.
With the gene OsHKT1;5 in Lalat, more salt-stress specific motifs W-boxes, CAAT-box, E2Fb, G-box, GC-motif and MBS were found.The G-boxes are known to get regulated under various stresses such as UV irridation, salt stresses, hormonal imbalances, etc.49.The superior motif E2Fb is a transcription activator that is involved in cell cycle regulation or in DNA replication by stimulating cell proliferation and delays differentiation 50 .Hence, PCR-based allele mining in our study demonstrated transcriptional regulation of the stress-responsive genes and superior alleles in Lalat, confirming the genotype as salt-tolerant at molecular level.ConclusionsWe characterized the rice landraces at physiological, biochemical, anatomical and molecular level at seedling stage.Our results revealed the dynamic invariable performance of genotypes under different and high concentrations of salt.The changes in expression patterns of all genotypes demonstrated in various traits facilitated in selecting extreme salt-tolerant and salt-sensitive landraces.The physiological and biochemical traits revealed Lalat as the best performer with high proline content that in turn protected the levels of chlorophyll even under high salinity stress conditions.The anatomical structure of adaxial leaf surface also indicated less bulging and few numbers of widely opened stomatal glands in Lalat on contrary to salt-sensitive MTU7029.All the traits correlated positively with allele mining variations in salt-specific genes OsNHX1 and OsHKT1;5 among Lalat and MTU7029 genotypes.The study also disclosed the involvement of probable and potential association of cis-acting regulatory elements in expression and regulation of salt-stress responsiveness in these two-contrasting genotypes.This strategy has a constructive foundation to study the organizational mechanisms for gene regulations and transcriptional association with stress-responsive superior alleles in various rice genotypes.