Introduction

Salt-affected soils have become a global concern because they reduce agricultural productivity. Over 800 million hectares of land worldwide are affected by salt (Munns and Tester 2008). Salt accumulation in the soil can be caused by the parent rock from which the soil is formed, the intrusion of seawater, and water irrigation (Morton et al. 2018). In addition to naturally occurring soil salinity, salinization increases owing to climate change, which acts through increased evaporation during periods of drought (Van Zelm et al. 2020). Compared to normal soils, saline soils have excessive soluble salts such as sodium, chloride, and sulfate. These minerals cause nutrient imbalance in the soil and reduce water availability. These conditions negatively affect the plant growth and development. Plants can be primarily divided into two groups based on the effect of salt on plant growth: glycophytes, which are plants sensitive to soil salinity, and halophytes, which are plants that grow naturally under high salinity and generally tolerate high salt concentrations (Reddy et al. 2017; Zhu 2001). Most crop plants, including rice, the most widely consumed grain worldwide, are glycophytes that cannot tolerate salt stress (Assaha et al. 2017). The demand for rice has increased in recent years due to the rapidly growing global population. Therefore, there is an urgent need to address salinity problems in rice plants. Characterizing the salt tolerance mechanisms is critical for further experiments to increase crop yield on salinized agricultural land (Chuamnakthong et al. 2019).

Plant responses to salt stress occur in two distinct phases: osmotic stress—a rapid response to an increase in external osmotic pressure—and ionic stress—a slower response due to the accumulation of Na+ in the leaves (Munns and Tester 2008). Osmotic stress starts immediately after the salt concentration around the roots increases to a threshold level, significantly reducing shoot growth. Osmotic stress is also known as water stress because water is unavailable to plants owing to the high solutes in the soil or medium, negatively affecting roots’ water uptake. Ionic stress begins when salt accumulates to toxic concentrations in the old leaves, leading to nutritional disorders, alteration of metabolic processes, membrane disorganization, and reduced cell division and expansion (Hasegawa et al. 2000a, b; Munns and Tester 2008; Zhu 2001).

For plants to persist in saline soil, adverse effects of Na+ and Cl− on plant metabolism must be avoided. Na+ exclusion and compartmentalization are the two mechanisms by which plants cope with ion toxicity (Jiadkong et al. 2023). The latter is the plant’s strategy to sequester Na+ and Cl− in the vacuoles and maintain a low concentration in the cytosol, where most cellular metabolism occurs, resulting in a situation known as tissue tolerance. Tissue tolerance is the capacity of the tissue to function while containing high levels of internal Na+ or Cl− (Munns et al. 2016). The main component of tissue tolerance is the compartmentalization of ions within vacuoles, which involves ion transporters in the tonoplast, particularly vacuolar Na+/H+ antiporters (Flowers and Colmer 2015). Na+/H+ antiporters catalyze the exchange of Na+ for H+ across membranes and regulate the cytoplasm’s internal pH, cell volume, and Na+ levels (Fukuda et al. 2011).

Many rice varieties and cultivars are growing worldwide, especially in Asian countries, where rice is a staple food. The two main subspecies of Asian cultivated rice are indica and japonica. Although rice is categorized as a glycophyte that is highly sensitive to salt stress, salt sensitivity varies across cultivars (Mekawy et al. 2018b; Sriskantharajah et al. 2020). Lee et al. (2003) suggested that the tolerance level of indica was higher than that of japonica rice tested. Four rice genotypes (FL478, Kamini, AC847, and IR29) showed differential salt sensitivity when subjected to 12 dS m−1 of salt stress for 7 days (Chakraborty et al. 2012). Wangsawang et al. (2018) screened japonica rice cultivars and identified a highly salt-tolerant cultivar with an effective Na+ exclusion mechanism in the leaf sheaths. These variations can be exploited to identify genes and proteins contributing to salt tolerance. Some traditional cultivars and rice landraces are more tolerant to various stresses than elite cultivars (Walia et al. 2005). These resistant genotypes are considered good genetic sources of further breeding.

Many studies on salinity tolerance mechanisms have been placed on excluding Na+ from shoots. However, studies of tissue tolerance mechanisms remain elusive. Our preliminary experiment screened japonica rice varieties and landraces for salt tolerance, with FL478 and IR29 used as salt-tolerant and salt-sensitive checks, respectively. We identified a japonica rice variety, SZK, with tissue tolerance mechanisms. Under salt stress, this variety maintained a high biomass while having high Na+ in the shoots. To understand the mechanisms of salinity tolerance in SZK, we conducted a comparative analysis to characterize the tissue tolerance mechanisms of SZK under salt stress. We compared SZK’s physiological and molecular characteristics with those of FL478, a well-known salt-tolerant indica rice variety, and Kunishi-jinjya-mai (Kunishi), a salt-sensitive japonica variety. Further analysis of the transcriptomic profiles of SZK and functional gene analysis were conducted to elucidate the molecular changes associated with salt tolerance in SZK.

Materials and Methods

Plant Materials and Growth Conditions

This study used two japonica rice varieties, Shuzenji-kokumai (SZK) and Kunishi-jinja-mai (Kunishi), and one indica variety, FL478. The seeds of SZK and Kunishi were obtained from the Hiroshima University Plant Nutritional Physiology Laboratory collection, and those of FL478 were obtained from the International Rice Research Institute. The seeds were incubated in a water bath at 60 °C for 10 min, then surface-sterilized with 0.1% benlate (fungicide) overnight at 30 °C, followed by soaking in the tap water for one more night for germination. The germinated seeds were transferred to a floating nylon mesh net. After 1 week of cultivation, Kimura B nutrient solution (0.18 mM (NH4)2SO4, 0.27 mM MgSO4·7H2O, 0.09 mM KNO3, 0.18 mM Ca(NO3)2·4H2O, and 0.09 mM KH2PO4, 20 μM NaEDTA-Fe·3H2O, 6.7 μM MnCl2·4H2O, 9.4 μM H3BO3, 0.015 μM (NH4)6Mo7O24·4H2O, 0.15 μM ZnSO4·7H2O, and 0.16 μM CuSO4·5H2O) was added (Mekawy et al. 2018a). After 2 weeks of cultivation, the four-leaf stage rice seedlings were transferred to a 180 L water container with holes. After a week, the treatment conditions were as follows: control (0 mM NaCl) and stepwise treatment (25 mM, 50 mM, 75 mM, and 100 mM for 5-day intervals). For the analysis of Na+ in time courses, salt treatment was applied at 6-day intervals, and sampling was conducted every 3 days for the treatment and every 6 days for the control. Changes in pH were monitored daily using a pH meter and adjusted to 5.0–5.5 using 2 N HCl and 2 N NaOH throughout the cultivation period. The nutrient solution was changed every 7 days, and water lost through evapotranspiration was replaced with tap water.

Physiological Parameters

After 20 days of salt stress treatment, the fresh weight (FW) of 41-day-old seedlings was measured by dissecting the roots, leaf sheaths, and leaf blades. Dry weights (DW) were obtained after oven-drying the samples at 70 °C for 72 h. The water content of the leaf blades was calculated using the following equation: (FW − DW)/FW × 100. The electrolyte leakage ratio (ELR) was determined according to the method described by Murray et al. (1989) with minor modifications. The second leaf blades from the top of the plants were cut into 5 mm lengths and placed in 50 mL propylene tubes containing 30 mL of deionized water. The initial electrical conductivity (EC1) was measured using an electrical conductivity meter (CM-31P, Kyoto Electronics, Kyoto, Japan). The samples were heated at 100 °C for 15 min to break down the tissues and release all electrolytes completely. Samples were then cooled to 25 °C, and the EC2 was measured. ELR was calculated as the conductivity ratio before heating to the conductivity after heating using the following formula: ELR (%) = (EC1/EC2) × 100.

Proline concentration was determined according to the method described by Bates et al. (1973). Fresh leaf blades (200 mg) were ground with a mortar and pestle using liquid nitrogen. The homogenated powder was mixed with 5 mL of 3% sulfosalicylic acid (w/v), followed by centrifugation at 10,000×g for 5 min at 4 °C. Next, 2 mL of the supernatant was transferred to a mixture containing 2 mL acetic acid and 2 mL acid-ninhydrin (1.25 g of ninhydrin in 30 mL of acetic acid and 10 mL 12 M phosphoric acid). The mixture was incubated at 100 °C for 1 h and cooled down by placing the tube on ice. The mixture was then added to 4 mL toluene and mixed vigorously. Finally, the chromophore concentration was determined at 520 nm using a UV spectrophotometer (UV-1850, Shimadzu Corp., Japan). The corresponding concentration of proline was determined using a standard curve of L-proline.

MDA concentrations were analyzed as described by Hodges et al. (1999). Fresh leaf blades (100 mg) were ground in liquid nitrogen and added to 3 mL of 80% (v/v) ethanol. The mixture was incubated at 25 °C for 20 min, and then centrifuged at 3,000×g for 10 min. Two sets of 1 mL of the supernatant were transferred into two glass tubes, one containing 1 mL TBA (−) solution (20% trichloro acetic acid and 0.01% butyl hydroxytoluene) and another 1 mL TBA (+) solution (20% trichloro acetic acid, 0.01% butyl hydroxytoluene, and 0.65% thiobarbituric acid). After mixing well, the mixture was incubated at 95 °C for 30 min. The reaction was stopped by placing the mixture in an ice bath, followed by centrifugation at 3,000×g for 10 min. MDA concentration was calculated from the absorbances of the samples measured spectrophotometrically (UV-1850, Shimadzu Corp., Japan) using a 155 mM−1 cm−1 extinction coefficient, according to Hodges et al. (1999).

Element Analysis

The elemental concentrations in the samples were analyzed according to Wheal et al. (2011). Dried plant samples (roots, leaf sheaths, and leaf blades) were crushed into fine powders using a crusher (Shake Master, BMS, Tokyo). The samples (100 mg) were weighed in 50 mL tubes. Subsequently, 2 mL of 5% HNO3 and 500 µL H2O2 were added, and the samples were incubated overnight at 23 °C for digestion. Afterward, the samples were heated at 80 ℃ for 30 min, the lids were loosened to release the pressure, and the temperature was increased to 125 °C for 2 h. The samples were cooled overnight, filtered through filter paper (Double Ring Quantitative Filter Paper, Grade: FAST 201), and 25 mL of Milli-Q water (Millipore, Direct-Q UV) was added. The concentrations of each element (Na+, K+, Ca, Mg, Fe, Cu, P, Mn, Zn) were measured using inductively coupled plasma optical emission spectrophotometry (SPECTROGREEN-FMD46, Hitachi).

Expression Analysis of the Genes Encoding a Na+ Transport Protein

Total RNA was extracted from the roots, leaf sheaths, and leaf blades of the control and salt stress treatments using an RNA extraction Kit Mini (Real Genomic RBC Bioscience, Taiwan) with DNase I digestion. The concentration and purity of RNA were measured using a Nanodrop Spectrophotometer (Thermo Fisher Scientific) at A260 and A280. One microgram of total RNA was reverse transcribed to cDNA using a reversed transcription 5× RT Master Mix (Toyobo Co., Ltd., Osaka, Japan). Reverse transcription was conducted at 37 °C for 15 min, and reverse transcriptase was denatured at 98 °C for 5 min to generate cDNA. Quantitative-real time polymerase chain reaction (qRT-PCR) was performed using Thunderbird Next SYBR qPCR mix (Toyobo Co., Ltd., Osaka, Japan) on an Applied Biosystems StepOne Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). qRT-PCR was conducted using the following conditions: initial denaturation at 95 °C for 30 s, followed by denaturation at 95 °C for 5 s, and the extension at 60 °C for 30 s. Forty cycles were conducted before melting curve analysis, and the melting curve was used to verify the PCR products by adjusting the temperature from 60 to 95 °C. OsUBQ5 was used as the initial control to normalize the relative expression levels of the genes. The relative abundance of the gene transcripts was calculated using the comparative 2−ΔΔCT method (Livak and Schmittgen 2001). The relative expression levels of each gene (fold change) were calculated based on that under control conditions. Details of forward and reverse primers used are listed in Table S1.

RNA Sequencing

Total RNA from the leaf sheaths and leaf blades of SZK under control and 24 h saline conditions (100 mM NaCl) was extracted using Sepasol-RNA I Super G (Nacalai Tesque Inc., Kyoto, Japan), followed by an RNA extraction Kit Mini (Real Genomic RBC Bioscience, Taiwan) with DNase I digestion. RNA quality control, cDNA library preparation, sequencing, and raw reads processing were performed on the DNBSeq platform (BGI Hongkong Tech Solution, NGS Lab) in the 150-bp paired-end mode. Low-quality, adaptor-polluted, and high-content unknown base reads were filtered, and 41–44 million clean reads were obtained from each library. Differentially expressed genes (DEGs) were identified using DESeq2 (Love et al. 2014) and RaNA-Seq (Prieto and Barrios 2020). Upregulated DEGs were tested for GO (Gene Ontology) term enrichment and KEGG pathway analysis using ShinyGO v0.80 (http://bioinformatics.sdstate.edu/go/) (Luo and Brouwer 2013; Ge et al. 2020; Kanehisa et al. 2021). The raw data of RNA seq were deposited to the NCBI Gene Expression Omnibus under the accession No. GSE266657.

Functional Characterization of Novel Genes Using Yeast Mutant

The coding sequences of the genes (OsCYP76M2, OsLAC28, and OsLOX-L2) were subcloned into yeast expression vector pYES2 using the In-Fusion Snap Assembly Master Mix (Takara Bio, Japan). The constructed vectors were transformed into the Na+-sensitive yeast strain AB11c which lacks Na+-ATPases and Na+/H+ antiporters (W303, ena1Δ::HIS3::ena4Δ, nha1Δ::LEU2, nhx1Δ::TRP1) using the LiAC/ssNDA/PEG method, as described by Gietz and Schiestl (2007). Growth spot assays on synthetic minimal glucose medium (SD) lacking uracil were performed as described previously (Marešová and Sychrová 2007).

Statistical Analysis

Statistical analyses were performed using the Statistix × software (Analytical Software, Tallahassee, FL, USA), and inferential statistics were performed using an analysis of variance at a significance level of 0.05. Multiple comparisons were performed using Tukey’s test.

Results

Plant Growth and Physiological Characterization Under Salt Stress

Salts were gradually applied at levels of 25–100 mM NaCl at 5-day intervals to the three rice varieties (FL478, SZK, Kunishi). As shown in Fig. 1A, the salt stress-treated seedlings had a smaller size than the control seedlings, particularly in the salt-sensitive Kunishi variety. Most of the leaves of Kunishi were markedly wilted and rolled, whereas only a small number of leaves of FL478 and SZK were wilted (Fig. 1A). Salt stress reduced the overall biomass of all the varieties. Dry weights of the roots, leaf sheaths, and leaf blades in Kunishi decreased significantly, whereas the reduction in FL478 was only significant in the leaf sheaths (Fig. 1B). Importantly, no significant decrease was observed in SZK. These results confirm that FL478 and SZK are salt-tolerant, whereas Kunishi is salt-sensitive.

Fig. 1
figure 1

Effect of salt stress (100 mM NaCl) on the growth of three varieties. A Representative images of 6-week-old rice seedlings under control and salt stress conditions. White bar = 10 cm. B Dry weight of the roots, leaf sheaths, and leaf blades of the three varieties. Data presented as means ± SE (n = 4). The same letters indicate no significant differences between the treatments in each variety (p < 0.05)

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To understand the physiological responses of the three varieties to salt stress, physiological parameters related to salt stress were examined (Fig. 2). High salinity resulted in osmotic imbalance, leading to a loss of water content in the leaves (Wangsawang et al. 2018). Water content of the leaf blades was measured to estimate the amount of water lost due to salt stress. The results showed that salt stress reduced the water content of all varieties (Fig. 2A). This reduction was significant in all varieties other than FL478, indicating that FL478 exhibited a greater potential to maintain tissue water than SZK and Kunishi. However, the reduction in water content in Kunishi (4.4%) was higher than in SZK (3.2%).

Fig. 2
figure 2

Effect of salt stress on the physiological parameters of the three varieties. A water content, B electrolyte leakage ratio, C proline concentration, and D malondialdehyde concentration. Data presented as means ± SE (n = 3). The same letters indicate no significant differences between the treatments in each variety (p < 0.05)

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Excess accumulations of Na+ in plant cells can disrupt cellular ion homeostasis and metabolism, including reactive oxygen species (ROS) generation, which is the primary cause of oxidative stress (Panda et al. 2019). Accumulation of ROS can damage cellular membranes and cause leakage of cellular components, such as electrolytes (Murray et al. 1989; Ueda et al. 2013). ELR was measured to examine the cell membrane stability under salt stress. As shown in Fig. 2B, ELR increased significantly in Kunishi from 14 to 49%, whereas it increased slightly from 16.5 to 26% in FL478. Interestingly, ELR in SZK remained unchanged, suggesting that SZK maintains membrane stability under salt stress. This result was verified by measuring MDA concentrations. MDA is one of the main products of cellular oxidation that represents cell oxidative damage. A significant increase in MDA was observed in Kunishi treated with salt stress, whereas the increase in FL478 and Kunishi was not substantial (Fig. 2D).

High salt accumulation in the soil inhibits water uptake and reduces turgor pressure, leading to wilting. To overcome water deficit, plants synthesize various osmoprotectants, such as glycine betaine, proline, and soluble sugar (Qureshi et al. 2013). Proline concentration was measured in the leaf blades to understand the involvement of osmoprotectants in the salt adaptation of the three varieties. As shown in Fig. 2C, significantly higher proline concentrations were observed in all three varieties after salt stress treatment. FL478 showed a tenfold increase in proline concentration relative to the control. Kunishi showed the highest increase of 16.9-fold, and SZK showed a slight increase of only 3.5-fold. These results suggested that proline accumulations may not be one of the mechanisms by which SZK adapts to salt stress.

Differences in Na+ and K+ Accumulation

Salt stress causes nutrient imbalance and leads to Na+ accumulation, which is toxic to plants by disrupting cellular processes (Van Zelm et al. 2020). As shown in Fig. 3A, the salt stress treatment significantly increased Na+ concentration in the roots and leaf sheaths of all three varieties. However, the Na+ concentration remained unchanged in the leaf blades of FL478, whereas the increase in Na+ concentration was nearly five-fold higher in SZK and Kunishi than in FL478. The K+ concentration in the roots and leaf blades of all three varieties increased significantly (Fig. 3B). On the contrary, K+ concentration decreased significantly in the leaf sheaths of SZK and Kunishi, whereas it remained unchanged in FL478. Consequently, higher Na+/K+ ratios were observed in SZK and Kunishi than in FL478 (Fig. 3C). Mg and Ca concentrations of the three varieties showed similar trends, significantly decreasing under salt-stress conditions (Figs. S1 and S2). P concentrations decreased in the roots, leaf sheaths, and leaf blades of FL478; however, it increased in SZK and Kunishi. Fe concentrations in FL478 and SZK in the roots, leaf sheaths, and leaf blades remained unchanged, whereas the concentration increased in the roots and leaf sheaths in Kunishi. SZK and Kunishi showed similar trends for other micronutrient concentrations, such as Mn, Cu, and Zn.

Fig. 3
figure 3

The difference in ion accumulation between three varieties under salt stress. A Na+ concentration, B K+ concentration, C Na+/K+ ratio. Data presented as means ± SE (n = 3). The same letters indicate no significant differences between the treatment in each variety (p < 0.05)

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Avoiding excess Na+ in the shoots is one of the well-characterized mechanisms of salt tolerance (Munns and Tester 2008). Nevertheless, SZK accumulated high Na+ in the leaf sheaths and leaf blades, indicating tissue tolerance mechanisms. Therefore, we investigated the initiation of Na+ accumulation. We conducted a time-course experiment to monitor the increase in Na+ concentration (Fig. 4). The results showed that SZK immediately started accumulating Na+ in the roots, leaf sheaths, and leaf blades after the salt-stress treatment was started (3 days after treatment) without any symptoms or injury observed. In contrast, FL478 and Kunishi started to accumulate Na+ 6 days after treatment (Fig. 4), indicating that SZK accumulates Na+ faster than FL478 and Kunishi.

Fig. 4
figure 4

The initiation of Na+ accumulation in the three varieties under salt stress analyzed by a time course in the A roots, B leaf sheaths, and C leaf blades. Data presented as means ± SE (n = 4)

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Differential Expression of Na+ Transporter Genes in Response to Salt Stress

Transcriptomic changes of genes encoding Na+ transporters were analyzed to understand the mechanisms underlying the differences in Na+ concentrations among the three varieties. In response to salt stress, FL478 showed high up-regulation of OsHKT1;5 expression in the roots (Fig. 5A). However, expression of this gene in the roots of SZK and Kunishi was much lower than in FL478. Moreover, only Kunishi showed up-regulation of OsHKT1;5 expression in the leaf sheaths and leaf blades. Salt stress also induced expression of OsSOS1 in the root, leaf blades, and leaf sheaths of FL478 by 1.3-, 1.6-, and 2.0-fold, respectively (Fig. 5B). These results suggested that OsHKT1;5 and OsSOS1 may contribute to the low Na+ concentrations in FL478 shoots.

Fig. 5
figure 5

Relative expression of genes encoding Na+ transporter in the roots, leaf sheaths, and leaf blades of the three varieties under salt stress. A OsHKT1;5, B OsSOS1. The expression of genes was evaluated based on that of control conditions. The same letters indicate no significant differences between the varieties (p < 0.05)

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A vacuolar Na+/H+ antiporter, OsNHX1, plays an essential role in the compartmentalization of excess Na+ in the cytoplasm into vacuoles (Fukuda et al. 2011) without any impact on plant growth, even when high Na+ accumulation occurs (Assaha et al. 2017). Relative expression of OsNHX1 was up-regulated in the leaf sheaths and leaf blades of SZK by 1.2- and 1.2-fold, respectively (Fig. 6A), indicating that this gene may contribute to tissue tolerance of SZK. Expression of other OsNHX genes was analyzed, and found that OsNHX2 expression in the leaf sheaths of SZK was highly upregulated to 5.1-fold (Fig. 6B). At the same time, the upregulation was only 1.5-fold in Kunishi, and the expression was repressed in FL478. OsNHX3 expression was significantly up-regulated in the roots of SZK compared to the other two varieties (Fig. 6C). OsNHX4 expression was up-regulated in the roots of SZK and Kunishi and in the leaf sheaths and leaf blades of FL478 (Fig. 6D). OsNHX5 expression was up-regulated only in Kunishi leaf sheaths (Fig. 6E).

Fig. 6
figure 6

Differences in the expression level of OsNHXs genes among the varieties under salt stress. A OsNHX1, B OsNHX2, C OsNHX3, D OsNHX4, E OsNHX5. The number of genes was evaluated based on that of control conditions. The same letters indicate no significant differences between the varieties (p < 0.05)

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Transcriptomic Analysis of SZK Under Salt Stress

Genome-wide transcriptome analysis was performed by using RNA-seq to ascertain the molecular changes associated with tissue tolerance in SZK (Tables S2, S3). Two biological replicates were used for each sample and treatment (leaf sheaths and leaf blades; control and salt stress). A principal component analysis (PCA) was performed to compare the gene expression between samples (Fig. 7A). A total of 1998 and 4623 DEGs were identified in the leaf sheaths and leaf blades of SKZ, respectively. Among them, 415 and 917 were upregulated, and 1583 and 3706 were down-regulated, respectively (Fig. 7B). A Venn Diagram of the up-regulated genes showed that 207 genes overlapped between the leaf sheaths and leaf blades. To identify the genes responsible for salt tolerance in SZK, we selected genes from 207 genes in the leaf sheaths, in which the expression was up-regulated to more than twofold and obtained 64 genes. We aligned these genes with those in the leaf blades (Fig. 7C) and found that expression of the genes encoding heat shock proteins (OsHSP), such as OsHSP90, OsHSP18, OsHSP70, OsHSP101, and OsHSP24.1, was up-regulated. Expression of the genes encoding heat shock transcription factors such as OsHsfA2a, small heat shock proteins such as OsHSP17.3 and OsHSP18, and cytosolic HSP70 (OsctHSP70) was up-regulated in both the leaf sheaths and leaf blades. Subsequently, qRT-PCR was performed to confirm the RNA-seq results. OsHSP90 expression was highly up-regulated by 7.2- and 20.4-fold in the leaf blades and leaf sheaths, respectively (Fig. 7D). Inducible expression of OsHSP90 was found to be specific to SZK, indicating that this gene may play an essential role in SZK under salt stress. Moreover, expressions of OsHsp20, OsHsp24.1, and OsHsfA2a (Fig. 7E–G) were also found to be upregulated specifically in the leaf sheaths of SZK, indicating that these genes may associated with tissue tolerance in SZK.

Fig. 7
figure 7

Transcriptomic analysis of SZK under salt stress. A Principal Component Analysis (PCA) plots for RNA-seq data in all samples. B Venn diagram of the up- and down-regulated genes in the leaf blades and leaf sheaths of SZK under salt stress. LB leaf blade, LS leaf sheath. C The heat map shows the top 64 up-regulated genes (> twofold) in the leaf sheaths alongside the leaf blades. D–G qRT-PCR confirmation of SZK-specific genes

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GO enrichment analysis revealed that upregulated DEGs in the leaf sheaths of SZK were involved primarily in response to abiotic stresses and protein folding, including chaperone-mediated protein folding, de novo post-translational protein folding, and chaperone cofactor-dependent protein folding (Fig. S3A), which manage by HSP protein. These results were verified by the KEGG pathway analysis which protein processing in the endoplasmic reticulum is the most significant pathway (Fig. S3A, C). GO terms of upregulated DEGs in the leaf blades sample were predominantly associated with response to abiotic stresses such as response to hydrogen peroxide, heat, and salt stress. KEGG pathway analysis of this sample showed significance in carotenoid biosynthesis.

Functional Analysis on Novel Genes Using the Yeast Mutant

Functional analysis of several genes was conducted to understand the molecular mechanisms underlying tissue tolerance in SZK. We selected three upregulated genes from the RNA-seq results (OsCYP76C2, OsLAC28, and OsLOX-L2) to be expressed in the Na+ sensitive yeast strain AB11c and observed their effect on yeast growth under salt stress conditions. As shown in Fig. 8, the growth of the AB11c cells transformed with empty pYES2 decreased in medium containing 150 mM NaCl. In contrast, the growth of the AB11c cells expressing OsCYP76C2, OsLAC28, or OsLOX-L2 was enhanced. These results indicate that these three genes may play a role in tissue tolerance of SZK.

Fig. 8
figure 8

Functional analysis of OsCYP76C2, OsLAC28, and OsLOX-L2 in the Na+ sensitive yeast strain AB11c. The left figure shows the growth of AB11c cells expressing OsCYP76M2 and empty vector (pYES2) under 0, 100, and 150 mM NaCl. The inoculated samples were serially diluted (tenfold) from left to right

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Discussions

For future crop improvement, measures of salt tolerance in landraces and new varieties or cultivars are necessary to help identify species or individuals within species that can be used to increase crops tolerance (Melino and Tester 2023). In a preliminary experiment, we identified a salt-tolerant japonica rice variety, SZK, with a unique mode of action in response to salt stress. Salt tolerance in rice is usually associated with low Na+ transport to the shoots and Na+/K+ discrimination (Na+ exclusion) (Assaha et al. 2017). However, our study demonstrated that SZK is considered a “tissue-tolerant” variety as it accumulates high Na+ concentrations in the shoots, almost the same as the salt-sensitive Kunishi, while maintaining a similar level of salt tolerance to FL478. In the present study, the physiological and molecular characteristics of tissue-tolerant SZK were investigated to elucidate its adaptation to salt stress by comparing it with the salt-tolerant FL478 and salt-sensitive Kunishi.

The most significant effect of salt stress is the reduction of plant biomass. Salt tolerance can be assessed by comparing the percentage of biomass production under salt stress and control conditions over a prolonged period (Munns 2002). From this comparative analysis, SZK was confirmed to be salt tolerant, similar to FL478, because the reduction in dry weight under salt stress was not significant in the roots, leaf sheaths, and leaf blades (Fig. 1B). Although SZK and FL478 were considerably tolerant of salt stress, a distinctive mechanistic difference was observed in their tolerance strategies. SZK had a physiological status similar to that of FL478 but superior to that of Kunishi. Both SZK and FL478 responded better in terms of water content (Fig. 2A), membrane integrity (Fig. 2B), and degree of oxidative damage (Fig. 2D). In terms of membrane integrity measured using ELR, SZK was better than FL478, as no increase in ELR under salt stress was observed. This result implied that cell injury caused by excessive salt may not occur in SZK. Synthesis of low-molecular-weight compounds or osmoprotectants such as proline is considered a vital mechanism in plants for the maintenance of the osmotic status of the cells, facilitating water absorption to prevent water loss, and scavenging ROS from cells under prevailing stress conditions (Qureshi et al. 2013). Our study indicated that proline concentration in SZK under salt stress was the lowest (Fig. 2C), suggesting that accumulation of proline may not be the mechanism by which SZK retains ion homeostasis, and this mechanism may not be associated with tissue tolerance in SZK.

While much of the emphasis in rice salt tolerance research has been placed on the exclusion of Na+ from the shoots (Lee et al. 2003; Mekawy et al. 2015; Senadheera et al. 2009; Ueda et al. 2013; Walia et al. 2007; Wangsawang et al. 2018), the present study revealed a tissue tolerance mechanism that employs Na+ sequestration from the cytosol, resulting in a high Na+ concentration while retaining growth under salt stress conditions. The tissue tolerant SZK accumulated high Na+ in the leaf sheaths and leaf blades, similar to the sensitive Kunishi (Fig. 3A), whereas FL478 had a low Na+ concentration in the leaf sheaths and leaf blades. Furthermore, Na+ concentration measured in time course showed that SZK accumulated Na+ in the roots, leaf sheaths, and leaf blades from the beginning of salt stress treatments (Fig. 4). On the last day of observation, Na+ concentration in SZK was higher than that in Kunishi, indicating that accumulation capacity of Na+ in SZK was superior to Kunishi. K+ concentration in the leaf sheaths of SZK decreased significantly, leading to a high Na+/K+ ratio, suggesting that SZK contradicts the concept that maintaining a low tissue Na+/K+ ratio is key to salt tolerance. Na+ exclusion in the root xylem epithelial region is considered the primary salt tolerance mechanism in conventional tolerant lines such as FL478, Nona Bokra, and Pokkali (Prusty et al. 2018). However, in our study, tissue tolerant SZK showed an opposite trend in shoot Na+ and Na+/K+ ratios compared to these varieties.

Among the three varieties, FL478 had a much higher expression of OsHKT1;5 in the roots (Fig. 5A), indicating superior Na+ exclusion activity, which did not occur in SZK and Kunishi. This result explains low Na+ concentrations in the leaf sheaths and leaf blades of FL478. At the same time, lower expression of OsHKT1;5 in SZK and Kunishi may cause elevated Na+ accumulation in the leaf sheaths and leaf blades, as reported by Wang et al. (2012) that the down-regulation of the OsHKT1;5 expression may contribute to the stimulation of Na+ accumulation in the old leaves. OsSOS1 expression was up-regulated in the roots, leaf sheaths, and leaf blades of FL478 and is likely also involved in the mechanism to exclude Na+ since OsSOS1 actively extrudes absorbed Na+ back into the extracellular space (Assaha et al. 2017; Hasegawa et al. 2000a, b). Excluding Na+ from the leaf sheaths and leaf blades so that a low Na+/K+ ratio could be achieved is the primary mechanism by which FL478 adapts to salt stress. Leaves are essential organs for carbon fixation and other primary metabolisms. Thus, protecting leaves from toxic ions is necessary for survival under salt stress. By excluding Na+ from the leaves, FL478 can ensure proper metabolism and retain growth even under salt-stress conditions.

Unlike FL478, SZK up-regulated the expression of OsNHX1 in the leaf sheaths and leaf blades and OsNHX2 in the leaf sheaths (Fig. 6A, B), indicating that Na+ compartmentation in the vacuoles might be active. This was confirmed by the high concentration of Na+ in the leaf sheaths and leaf blades of SZK. Mekawy et al. (2015) reported that increased accumulation of Na+ in a sensitive rice cultivar is likely a consequence of the activity of OsNHX1 as the antiporter that facilitates Na+ uptake into vacuoles in exchange for H+ in the cytoplasm. Excess Na+ in the cytosol can disrupt cellular processes, resulting in metabolic failure and cell death. The metabolic toxicity of Na+ is primarily due to its ability to compete with K+ for binding sites essential for cellular functions. K+ activates over 50 enzymes, and Na+ cannot substitute this role (Hasanuzzaman et al. 2018; Tester and Davenport 2003). Rather than excluding Na+, SZK actively compartmentalizes Na+ into vacuoles through OsNHX1 and OsNHX2 to sequester it from the cytosol, where most cellular processes occur. Barragán et al. (2012) reported that NHX1 and NHX2 have similar expression patterns and identical biochemical activities and that reverse genetics showed functional redundancy. SZK likely uses these two genes to regulate tissue tolerance.

At the cellular level, tissue tolerance is achieved by compartmentalization of Na+ and Cl− in the vacuoles, together with the synthesis of compatible solutes and their location within the cytoplasm to balance the osmotic pressure of the ions in the vacuoles (Munns et al. 2016). However, our results suggested that proline synthesis does not follow the compartmentalization of Na+ in SZK. Tissue tolerance is a specialized trait, generally reported to be present in salt susceptible rice varieties such as IR29 (Chakraborty et al. 2020) and in some newly identified wild rice accessions (Prusty et al. 2018). The halophytic wild relative of rice, Oryza coarctata, was also found to possess considerable tissue tolerance along with other important salt tolerance traits (Mangu et al. 2019; Mondal et al. 2022). Thus, SZK may have a unique tissue tolerance mechanism that has not been observed in other varieties.

Further, the molecular mechanisms underlying tissue tolerance in SZK was studied at the transcriptomic level using RNA-Seq analysis. The results showed that some OsHSP gene families, such as OsHSP90, OsHSP18, OsHSP70, OsHSP101, and OsHSP24.1, were up-regulated in both the leaf blades and leaf sheaths (Fig. 7B). Heat shock transcription factors, such as OsHsfA2a, small heat shock proteins, such as OsHSP17.3 and OsHSP18, and cytosolic HSP70 (OsctHSP70) were also up-regulated. Heat shock proteins (HSPs) and small heat shock proteins (sHSPs) play critical roles in preventing protein damage during high-temperature and salinity stress by associating with partially denatured proteins to form stable complexes and preventing their irreversible aggregation in an ATP-independent manner (Do et al. 2023). Interestingly, expression of OsHSP90, OsHsp20, OsHsp24.1, and OsHsfA2a were found to be up-regulated specifically in the leaf sheaths of SZK (Fig. 7C–F), indicating that these genes may associated with tissue tolerance in SZK. Furthermore, GO enrichment analysis related to biological processes and KEGG pathway analysis showed that upregulated DEGs in the leaf sheaths of SZK were predominantly associated with protein folding and refolding which involves HSP proteins (Fig. S3A–C).

HSPs function as a general chaperone that transiently binds to folding intermediates in vitro, prevents aggregation, and supports the refolding of intermediates to their native states (Liu et al. 2009; Raman and Suguna 2015; Schopf et al. 2017). Protein synthesis requires high concentrations of K+ for the binding of tRNA to ribosomes (Blaha et al. 2000; Tester and Davenport 2003) and preserve ribosome integrity (Rozov et al. 2019). Under salt stress conditions, protein synthesis was disrupted because K+ concentration in the cell is lower and cannot be substituted by Na+. Proteins that are formed incorrectly will be degraded by the proteasome or fixed by molecular chaperone. The ability of SZK to maintain a better physiological status than the salt-sensitive variety while having a high Na+ concentration may occur under the activity of HSPs, which prevents protein damage, allowing the plants to retain active cellular metabolisms and growth.

Functional analyses of several highly up-regulated genes (OsCYP76C2, OsLAC28, and OsLOX-L2) were conducted to identify novel genes responsible for tissue tolerance in SZK. The growth of the AB11c cells expressing OsCYP76C2, OsLAC28, and OsLOX-L2 was better than that of cells expressing the empty vector (Fig. 8), indicating that these three genes may play a role in SZK tissue tolerance. However, no studies have reported the function of these genes in salt tolerance in rice. CYP (cytochrome P450) belongs to the oxidoreductase class of enzymes and represents one of the largest enzyme families containing heme-thiolate as a cofactor (Pandian et al. 2020) with key roles in plant evolution and metabolic diversification (Hansen et al. 2021). Members of the CYPs family have many clans and diverse functions. CYPs are involved in plant defense against biotic and abiotic stresses. AtCYP76C2 from Arabidopsis is associated with rapid hypersensitive cell death, a defense mechanism against bacterial canker (Pseudomonas syringae) infection (Godiard et al. 1998). OsLACs encode laccase, an enzyme that oxidizes monolignols to produce higher-order lignins that are involved in plant development and stress responses, including salt stress (Liu et al. 2017). Some studies have reported an increase in lignification under salt stress (Dissanayake et al. 2023). OsLAC28 is highly expressed in the endosperm and was induced by Cu treatment (Liu et al. 2017). OsLOX-L2 encodes a lipoxygenase that catalyzes the hydroperoxidation of fatty acids (Ohta et al. 1992). OsLOX is involved in jasmonic acid (JA) biosynthesis and is a biotic defense-related enzyme (Tong et al. 2023). Although there is no information regarding the roles of OsCYP76C2, OsLAC28, and OsLOX-L2 in salt tolerance, our results suggest that these three genes may confer tissue tolerance in SZK. Further investigation of these three genes is required to understand their roles in salt tolerance, particularly tissue tolerance.

Conclusions

In the present study, we demonstrated that a japonica rice landrace SZK is a salt-tolerant variety comparable to the tolerant indica FL478 yet has a different coping mechanism for salt stress. FL478 and most salt-tolerant rice varieties employ the OsHKT1;5 functions to restrict Na+ accumulation in shoots. Conversely, SZK accumulated a high-Na+ concentration in the shoot, which was almost the same as that in the salt-sensitive japonica Kunishi, suggesting that SZK is tissue tolerant. SZK can maintain better growth at high Na+ concentrations by activating OsNHX1 and OsNHX2 to sequester Na+ into vacuoles so that toxic cytosolic Na+ can be avoided. SZK also activated OsHSPs to prevent protein degradation. OsCYP76C2, OsLAC28, and OsLOX-L2 are novel target genes that confer tissue tolerance in SZK.