Unfolded protein response in rice (Oryza sativa L.) varieties with different level of salt stress tolerance

Plants activate the unfolded protein response as part of cellular adaptation, thereby maintaining the endoplas‐ mic reticulum homeostasis during external stresses exposure. In this study, we examined the relationship between the degree of salt tolerance and unfolded protein response‐related gene expression in India salt‐tolerant Pokkali and INPARI 35 varieties compared to the Indica salt‐sensitive counterpart IR64 and INPARI 4 varieties. Our result showed that the salt tolerance of Pokkali and INPARI 35 had been confirmed by their higher survival rate, higher chlorophyll content, lower electrolyte leakage, and lower H2O2 and malondialdehyde content under salt stress conditions. Furthermore, the expression of unfolded protein response genes was highest in INPARI 35, whereas IR64 and INPARI 4 exhibited low gene induction during endoplasmic reticulum stress conditions. Among the four examined varieties the salt toler‐ ant Pokkali surprisingly showed the lowest induction of all examined unfolded protein response‐related genes. These results indicated the possibility that unfolded protein response supports the rice plant for adapting to the saline environment.


Introduction
Soil salinity has become one of the major constraints that negatively affect rice productivity worldwide. As the gly cophyte cereal plant, rice generally exhibits low adapt ability against high concentrations of NaCl that present in the rhizosphere area. Salinity stress occurs when the concentration of sodium (Na + ) and chloride (Cl ) ions in the soil solution exceeds the tolerable limit of rice. Salt stress affects the physiology and biochemistry processes of rice by given water stress, disturbs ion homeostasis, nu tritional disorders, alteration of metabolic processes, ox idative stress, reduction of growth and cell division, and membrane disorganization (Ya'acob et al. 2017).
Rice that are grown under high salinity conditions ex perience both osmotic and ionic stresses. Osmotic stress occurs when the concentration of Na+ is high in the roots. High concentration NaCl in the soil solution leads to the decreased concentration of potassium (K + ), displacement by Na + , depolarization of cell membrane. The decrease of K+ also caused decreased turgor cell and stomata clo sure (Ragel et al. 2019). Rice has several mechanisms to respond the stress including, the accumulation of osmo protectant compounds, ion homeostasis, reactive oxygen species (ROS) detoxification, and activated several genes to maintain the balance of physiological processes of the cells (Hoang et al. 2016). A previous study reported an increased ROS production in rice plants was mediated by a specific gene in response to high extracellular NaCl con centration (Laohavisit et al. 2013).
Endoplasmic reticulum (ER) is responsible for synthe sizing half of eukaryotes protein, but its function is easily disrupted when the cells are exposed to unfavorable envi ronments. The disturbed ER function leads to the accu mulation of misfolded and unfolded protein in the lumen, which subsequently generates the condition commonly termed as ER stress. In order to reestablish ER home ostasis, plant activated unfolded protein response (UPR) by increase protein folding capacity through the up regu lation of a subset genes encoding the ERlocalized chaper one protein and degrading misfolded proteins by trigger ing the ERassociated protein degradation (ERAD) mech anism Howell 2016; Fanata et al. 2013). In rice, OsbZIP39 and OsbZIP74 transcription factors play a piv otal role in UPR activation. ER stress activates both tran scription factors through ER to nucleus relocation by pro teolytical cleavage of OsbZIP39 and OsbZIP74 mRNA unconventional splicing resulting in upregulation of sev eral ER chaperones such as BiP1, PDIL11, Calnexin, and Calreticulin (Lu et al. 2012; Takahashi et al. 2012.
Our experiment used two salt tolerant rice varieties, Pokkali and INPARI 35, altogether with IR64 and INPARI 4 as sensitive varieties to investigate the relationship be tween salinity stress tolerance and UPR induction level. The salt tolerance level and expression of ER chaperone genes in each variety have been analyzed through pheno typic, biochemical, and molecular methods. . Rice seeds were imbibed in the water for three days and twenty germinated rice seeds of each variety were planted in pots containing natural soil. The plants were grown for two weeks in a greenhouse with 28-32°C temperature under natural sunlight exposure and daily irrigated by submerging the threefourths of the pots into the water.

Salt Tolerance Analysis
Salt tolerance analysis was conducted by soaking the pots of 2weekold rice seedling into 100 mM NaCl for six days. The salt tolerance of each rice variety was man ifested by the survival rate at the six days of salt stress treatment by counting the percentage of living plants over the total population of the seedling.

Relative Electrolyte Leakage
Electrolyte leakage ratio was measured according to Ueda et al. (2013). The leaves of salt stress and nontreated seedling were cut into 0.5-1 cm length and 0.5 g of small cute leaves were gently shaken in 20 mL distilled water in flat bottomed Makarthy tube for 24 h at room temperature. Electrolyte conductivity of the water was measured using a conductivity meter (Horiba Scientific) and the result was expressed as EC1. The tubes containing mixture water and leaves were autoclaved at 120°C for 15 min, and the elec trolyte conductivity of cooled water was measured to ob tain the total electrolyte conductivity (EC2). Relative elec trolyte leakage was counted using the formula: EC1/EC2 × 100%.

Total Chlorophyll
Total chlorophyll was measured according to the method of Arnon (2018); Ma et al. (2018) with modification. Two hundred milligram of smallcut fresh leaves were placed into 15 mL conical tube containing 10 mL abso lute ethanol. The mixtures were gently agitated for 48 h at room temperature. Chlorophyll content was analyzed by measuring the OD value of ethanol solution using Hitachi U2900 spectrophotometer at 649 nm and 665 nm. Total chlorophyll was expressed as mg g 1 fresh weight.

Hydrogen peroxide content
Hidrogen peroxide content of salt stress treated rice was measured according to the method described by Velikova et al. (2000). Three hundred milligram of fresh leaves were ground and homogenized with 3 mL of 0.1% (v/v) trichloroacetic acid. The homogenate was centrifuged at 12,000 rpm for 15 min at 4°C, and the resulting super natant was transferred into a new microtube. The reaction mixtures were subsequently made by mixing 0.5 mL su pernatant, 0.5 mL of 50 mM sodium phosphate buffer (pH 7.0) and 1 mL of 1 M potassium iodide. Absorbance was measured at 390 nm and the hydrogen peroxide content was expressed as µg g 1 fresh weight.

Malondialdehyde Content
Malondialdehyde (MDA) contents were measured accord ing to the method described by Hodges et al. (1999). One hundred milligrams of fresh weight leaves from each stress treatment were ground and homogenized with 1 ml of 0.1% (v/v) trichloroacetic acid solution and centrifuged at 10,000 rpm for 5 min at 4°C. Five hundred microliter of supernatant were mixed with 500 µL 0.5% (w/v) TBA in 20% TCA. The mixtures were reacted at 100°C for 30 min and then stopped by incubating the mixture in ice for 10 min. Mixtures were subsequently centrifuged at 10.000 rpm for 10 min at 4°C and the resulting supernatants were used for MDA measurement using spectrophotometry at 532 dan 600 nm. MDA content was calculated using an ex tinction coefficient 1.55 × 10 5 mM1 cm1 and expressed as µMg 1 fresh weight.

Total RNA Isolation and qPCR Analysis
Total RNA was extracted from the leaves of the control and 5 mM DTT treated seedling using RNAprep pure kit (Tiangen) following the manufacturer's instruction. One microgram of total RNA was used for cDNA synthesis us ing a Revertra Ace qPCR RT Kit (Toyobo). The qPCR was performed using the CFX96™ RealTime PCR Detection System (BioRad) and SsoFast EvaGreen Supermix (Bio Rad) and specific primer sets (Supplementary Table 1). OsActin1 was used as internal reference gen to evaluate the transcriptional abundance of the selected UPR genes.

Statistical analysis
Each experiment of this study consisted of three biological replicates, and the statistical analysis was performed using analysis of variance (ANOVA)..

The tolerance of rice against salt stress
To investigate the level of salinity tolerance of IR 64, IN PARI 4, Pokkali, and INPARI 35 rice varieties, twoweek old seedlings were subjected to salt stress treatment by soaking the growing media with 100 mM NaCl. The tol erance level of each rice variety was represented as stress survival rate and total chlorophyll at six days of stress treatment. In our experimental condition, all seedlings of four rice varieties showed normal growth under non stress conditions and growth reduction was obviously seen in NaCl treated seedlings (Figure 1a). Both Pokkali dan INPARI 35 showed the highest survival rate at 79.17% and 75.83%, respectively, in saline conditions. IR 64 and INPARI 4 showed lower survival rates at 51.67% and 47.50%, respectively (Figure 1b). The result above clearly showed that salinity tolerance of tolerant and sensitive va rieties was significantly different.
The salt tolerance response of plants is usually shown by the higher chlorophyll content, which is related to the higher photosynthetic rate during salt stress (Solangi et al. 2016). To confirm the survival rate result, we analyzed the total chlorophyll content of seedlings under salinity and their membrane integrity using relative electrolyte leakage (REL) analysis. On the total chlorophyll content analysis, Pokkali and INPARI 35 varieties showed a slight decrease of chlorophyll content, whereas IR64 and INPARI 4 exhib ited a dramatic decrease of total chlorophyll content com pared to control (Figure 2). Furthermore, the REL analy sis result showed that Pokkali and INPARI 35 exhibited a lower electrolyte leakage value of 30.72% and 27.18%, re spectively, which statistically were not different from the control treatment (Figure 3). On the other hand, we ob served the twofold increase of electrolyte leakage in IR 64 and INPARI 4 at 70.25% and 66.33%, respectively, in dicating NaCl treatment leading to severe membrane dam age on both varieties. Therefore, these results confirm that seedlings of Pokkali and INPARI 35 showed higher vigor than IR 64 and INPARI 4 under salt stress exposure.

Oxidative stress level during salt stress
High accumulation of reactive oxygen species (ROS) dur ing salt stress has induced oxidative damage of membrane lipids. To obtain the biochemical evidence for elucidat ing the different levels of salt tolerance on four tested rice varieties, we analyzed the oxidative stress level by mea suring the H 2 O 2 content and malondialdehyde (MDA) as the product of lipid peroxidation. Our data showed that salt stress has efficiently induced the production of H  PARI 4 were increased by 29 and 39fold, respectively, whereas Pokkali and INPARI 35 showed lower H 2 O 2 in duction levels (18 and 16fold, respectively) ( Figure 4). Moreover, the MDH content in IR 64 and INPARI 4 was increased by 60.8% and 33.6%, respectively, and Pokkali and INPARI 35 contained a lower induction level of MDH at 3.8% and 3.2%, respectively ( Figure 5). These results were in accordance with the previous studies that rice tol erance varieties to salinity stress have the lowest amount of ROS than the sensitive plant (Hoang et al. 2015). There fore, these results confirm that salt stressinduced oxida  tive stress was lower in salttolerant rice varieties but higher in the saltsensitive counterparts.

Activation of unfolded protein response by ER stress
The phenotypical and biochemical results above have con firmed that Pokkali and INPARI 35 are categorized as salt tolerance varieties, whereas IR 64 and INPARI 4 are salt sensitive. To investigate whether the difference of salt tolerance in the tested rice correlates with the induction level of UPR, we analyzed the expression level of sev eral ER stressinducible genes in rice seedlings that ex perience ER stress induced by dithiothreitol (DTT). The induction levels of genes encoding ERresident chaper one such as; binding immunoglobulin protein1 (BiP1), binding immunoglobulin protein2 (BiP2),calreticulin2 (CRT2), protein disulfide isomeraselike 23 (PDIL23), and Calnexin (CNX) were analyzed by qPCR analysis. Our result showed that ER stress was efficiently induced by DTT, as shown by the upregulation of all ER chaperone genes ( Figure 6). Moreover, we found that the induction of ER chaperone genes was lower in saltsensitive IR64 and    The cell that accumulated a high concentration of Na + generates more ROS that further contribute to the dam age of cellular membrane (Ueda et al. 2013). Our re sult showed that H 2 O 2 was highly accumulated in salt sensitive IR 64 and INPARI 4 varieties, contributing to the higher lipid peroxidation activity as manifested by high MDA content in these salt sensitive varieties. These data were in accordance with the result of Abdelgawad et al. (2016) that showed the high level of H 2 O 2 , MDA and elec trolyte in maize seedling leakage as the response of salt stress treatment.
The obvious salt tolerance level in the four tested rice varieties has become the main investigation object to cor relate the salt tolerance level with the UPR. The salt sensitive IR 64 and INPARI 4 showed the moderate ac tivation of all tested genes under DTTinduced ER stress treatment. On the other hand, the salttolerant INPARI 35 showed the highest expression of BiP1, BiP2, PDIL2 3, and CNX among the tested varieties. The contrast re sults were shown by the salttolerant Pokkali where BiP2, CRT2, PDIL23, and CNX were expressed at the lowest level. BiP1 is known as the abundant ER chaperone pro tein and OsPDIL23 also encodes a protein associated with protein folding and both genes were highly expressed un der ER stress (Qian et al. 2015). It was also reported that rice overexpressed both BiP1 and CNX under ER stress conditions, but CNX expression is inversely related to BiP1 expression (Wakasa et al. 2011; Qian et al. 2015. Our result also showed that CNX was expressed inversely to that of BiP1. Interestingly, Pokkali rice showed the low est expression of CRT2 in both control and ER stress treat ment. This result was in contrast to the positive function of calreticulin where the overexpression of wheat CRT in tobacco enhanced the drought and salt tolerance level (Xi ang et al. 2015). Future investigation might be needed to deeply study the important roles CRT2 for salt tolerance, especially in other salttolerant rice varieties.

Conclusions
Our phenotypical and biochemical results confirmed that Pokkali and INPARI 35 are salt tolerant, whereas IR 64 and INPARI 4 are salt sensitive. The activation of un folded protein response through upregulation of ER chap erone genes might support the rice plant adaptation to the