Transcriptional repressor RST1 controls salt tolerance and grain yield in rice by regulating gene expression of asparagine synthetase

Significance

Improving grain yield and stress resistance is an important goal of rice breeding. In this study, we revealed that a rice gene, RST1, is associated with grain yield in both normal and saline soils. RST1 encodes the AUXIN RESPONSE FACTOR 18 protein, which negatively regulates nitrogen metabolism by transcriptional repression of OsAS1. Mutation of RST1 promotes nitrogen utilization and reduces NH₄⁺ accumulation induced by salt stress, thus improving the ability to survive under saline conditions and, consequently, increasing grain yield. Our results provide insight into the molecular mechanism, by which RST1 regulates nitrogen metabolism and salt tolerance, and highlight a promising candidate gene for use in breeding programs designed to develop rice cultivars with high yield and stress resistance.

Abstract

Salt stress impairs nutrient metabolism in plant cells, leading to growth and yield penalties. However, the mechanism by which plants alter their nutrient metabolism processes in response to salt stress remains elusive. In this study, we identified and characterized the rice (Oryza sativa) rice salt tolerant 1 (rst1) mutant, which displayed improved salt tolerance and grain yield. Map-based cloning revealed that the gene RST1 encoded an auxin response factor (OsARF18). Molecular analyses showed that RST1 directly repressed the expression of the gene encoding asparagine synthetase 1 (OsAS1). Loss of RST1 function increased the expression of OsAS1 and improved nitrogen (N) utilization by promoting asparagine production and avoiding excess ammonium (NH₄⁺) accumulation. RST1 was undergoing directional selection during domestication. The superior haplotype RST1^{Hap III} decreased its transcriptional repression activity and contributed to salt tolerance and grain weight. Together, our findings unravel a synergistic regulator of growth and salt tolerance associated with N metabolism and provide a new strategy for the development of tolerant cultivars.

Salinity is one of the most challenging environmental conditions that limit crop growth and yield (1). Rice (Oryza sativa) is a glycophyte, and its yield is reduced by 12% per unit (mS·cm⁻¹) when soil electrical conductivity is beyond three units (1). Knowledge of the critical mechanisms of salt tolerance in plants (including rice) is advancing, including salt transport and compartmentation, signaling of salt stress, and transcriptional regulation (2–4). However, little progress has been made in developing salt-tolerant varieties of rice and other crops, because limited genes are available for genomics-based crop breeding (3, 5).

Salt stress affects plant performance in several ways, including osmotic stress, ion toxicity, nutritional deficiency, and Na⁺/K⁺ imbalance (2, 3). High salinity affects nutrient absorption by impairing native soil physico-chemistry and the function of uptake systems (6). Nitrogen (N) is an essential macronutrient for plant growth and basic metabolic processes (7). Up to date, great advance has been made toward to the understanding in N transport, sensing/signaling, and the regulatory networks in plants (7–10). Relatively, the N metabolism in plants and its function in stress response are still much less known (2). Plants acquire inorganic N from soil, mainly in the form of NH₄⁺ and nitrate (NO₃⁻). NO₃⁻ is reduced to NH₄⁺, which is assimilated into organic composition during the glutamine synthetase-catalyzed conversion of glutamate (Glu) into glutamine (Gln) (7). Asparagine synthetase (AS, EC6.3.5.4) catalyzes the adenosine triphosphate (ATP)-dependent transfer of a Gln-amide group to the 2-position of aspartate (Asp), forming asparagine (Asn) and Glu (11, 12). In this reaction, NH₄⁺ can be used as an amide donor, despite its lower catalytic efficiency compared with Gln. Asn, together with Gln, Glu, and Asp, synthesize other amino acids and nitrogenous compounds. Under stress conditions (salt and cold), the NH₄⁺ metabolism is altered, which results in toxic NH₄⁺ accumulation in plant tissues. Concomitantly, stress-induced AS2 expression increases to antagonize the NH₄⁺ accumulation in Arabidopsis (13). Although it has early proposed that salt stress affects protein synthesis, how plants (crops) change metabolisms to adapt the stressed environments remains largely unknown.

Large-scale screening of a mutant population generated by ethyl methanesulfonate-induced mutagenesis isolated a mutant, rice salt tolerant 1 (rst1), which showed the enhanced tolerance to salt stress (14). In this study, we characterized the RST1 gene, which encodes an auxin response factor 18 (OsARF18) that directly represses OsAS1 expression. Loss of RST1 function improved Asn synthesis and reduced excess NH₄⁺ accumulation in shoots, thus reduced yield loss in saline soils and improved grain yield even in normal conditions. The results elucidate the regulation mechanisms of N metabolism in salt response and provide a powerful gene for precision breeding of salt-tolerant rice for sustainable agriculture.

Results

RST1 Encodes OsARF18, a Predicted Auxin Response Factor.

To explore salt tolerance mechanisms in rice, we screened an ethyl methanesulfonate-mutagenized Nipponbare (Nipp) rice population for mutants displaying altered salt response. A salt-tolerant mutant, rst1, was isolated; the RST1 locus was mapped to a 270.4-kb interval on the long arm of chromosome 6 (14). Fine mapping narrowed the RST1 locus to a 45-kb region between markers IM29432 and IM29477, which contained five open reading frames (Fig. 1A). Sequencing of the entire candidate region revealed that the rst1 mutant carries a single nucleotide substitution (G to A) in the first exon of the OsARF18 gene (Os06g0685700), which causes an amino acid change from Trp (TGG) to a stop codon (TGA) at the 330th amino acid residue (Fig. 1B and SI Appendix, Fig. S1). This change ultimately results in the deletion of part of the repression domain (RD), as well as the whole Phox and Bem1 (PB1) domain(15), of the OsARF18 protein (Fig. 1B).

Fig. 1.

Map-based cloning of RST1 and genetic complementation. (A) Fine mapping of the RST1 locus. The RST1 locus was mapped to a 45-kb region between markers IM29432 and IM29477. (B) Schematic representation of RST1 gene structure. The white boxes, filled black boxes, and black lines indicate untranslated regions, exons, and introns, respectively. A single nucleotide mutation from G to A in the first exon results in a premature termination codon. The DBD, RD, and PB1 are indicated as RST1 functional domains. (C) Salt tolerance of Nipp, rst1 and complementation lines C13 and C21. Fourteen-day-old seedlings were treated with 140 mM NaCl for 12 d, followed by recovery for 7 d under normal conditions. (D) Survival rates of seedlings in C. Data are means ± SD (n = 3). (E and F) Na⁺ (E) and K⁺ (F) contents in Nipp, rst1 and complementation lines (C13 and C21) subjected to salt stress. Data are means ± SD (n = 4). Each replicate contains 40 to 48 plants in D, eight plants in E and F. (Scale bars represent 5 cm in C.) Different letters represent a significant difference at P < 0.05 determined by Tukey’s honest significant difference (HSD) test.

Physiological analysis revealed that complementation with genomic DNA of OsARF18 (C13 and C21) in rst1 recovered completely to wild-type (WT) phenotypes with respect to survival rate, Na⁺ and K⁺ content in seedlings (Fig. 1 C–F and SI Appendix, Fig. S1). Two T-DNA insertion mutants of the OsARF18 gene, osarf18-1 (PFG_3A-09474.L) and osarf18-2 (PFG_1B-15238.L), showed salt tolerant (SI Appendix, Fig. S2); the OsARF18-overexpressed lines OE2 and OE4 exhibited salt-sensitive phenotypes (SI Appendix, Fig. S3). Together, these results demonstrate that OsARF18 corresponds to the RST1 gene and acts as a negative regulator of salt tolerance in rice.

RST1 Is a Transcriptional Repressor Controlling OsAS1 Expression.

To investigate RST1 expression patterns, qRT-PCR analysis was performed. RST1 transcripts were detected with higher expression in above-ground tissues than roots (Fig. 2A). Histochemical β-glucuronidase (GUS) staining showed that RST1 was expressed in the exodermis, sclerenchyma, endodermis, and central cylinder of the primary root elongation zone, and most live cells in leaf blade and sheath (Fig. 2 B–D and SI Appendix, Fig. S4). RST1 expression was transiently induced by salt treatment in shoots and roots (Fig. 2E); it was also sustainably induced by drought and abscisic acid, but not by auxin or other hormones (SI Appendix, Fig. S5).

Fig. 2.

Molecular characterization of RST1. (A) RST1 expression levels in various organs revealed by qRT-PCR. R, root; S, shoot; SB, stem base; LS, leaf sheath; LB, leaf blades; N I to III, node I to III; IN I to III, internode I to III; FLS, flag leaf sheath; FLB, flag leaf blades; LJ, lamina joint; SP, spikelet. Data are means ± SD (n = 3 or 4). (B–D) RST1 promoter-GUS expression patterns. Cross section of the primary root elongation zone (B), leaf blade (C), and leaf sheath (D) at the rice seedling stage. cc, central cylinder; co, cortex; en, endodermis; ep, epidermis; ex, exodermis; ls, leaf sheath; me, mesophyll; ms, mestome sheath; ph, phloem; sc, sclerenchyma; vbs, vascular bundle sheath; xy, xylem. (E) RST1 expression levels in 14-d-old seedlings treated with 140 mM NaCl for indicated time. Data are means ± SD (n = 3). (F) Subcellular localization of RST1. RST1-GFP fusion protein was co-localized with DAPI in leaf epidermal cells of tobacco leaves (Upper row) and root cells of proRST1::RST1-GFP transgenic rice plants (Lower row). (G–I) Transcriptional activity analysis of RST1 using the DLR assay system. Schematic representation of reporter and effectors are shown in G. A representative image of a tobacco leaf 48 h after infiltration is shown in H. (I) Measurement of relative luciferase activity (LUC/REN) in H. Data are means ± SD (n = 5). Each replicate contains >3 plants in A and six plants in E. (Scale bars represent 50 μm in B and C, 500 μm in D, and 40 μm in F.) In I, different letters indicate significant differences (P < 0.05, Tukey’s HSD test).

Next, the green fluorescent protein (GFP)-tagged RST1 was transiently expressed in tobacco (Nicotiana benthamiana) leaves. The RST1-GFP signal was detected in the nuclei, which was identical with the pattern in transgenic rice expressing RST1-GFP under the control of its native promoter (Fig. 2F). These results suggest that RST1 encodes a nuclear protein.

Dual luciferase reporter (DLR) assay was used to investigate the transcriptional regulation activity of RST1 (Fig. 2 G–I). VP16 and GD1 were used as the transcriptional activator and repressor control, respectively (Fig. 2G). RST1 expression resulted in lower firefly luciferase/Renilla luciferase (LUC/REN) activity than did the empty GAL4BD vector, which was consistent with the GD1 repressor results (Fig. 2 H and I). However, transcriptional activity of the truncated RST1^1–329 protein (an rst1 mutant that stops translation at the 330th residue) did not significantly differ from the activity of the GAL4BD vector (Fig. 2 H and I). These results suggest that RST1 is a transcriptional repressor and the rst1 mutation led to loss of the function.

To dissect potential RST1-regulated genes, we analyzed RNA sequencing profiles generated from WT and rst1 seedlings with or without NaCl treatment. We identified 670 differentially expressed genes, among which 41 and 58 genes were upregulated under the control and NaCl treatments, respectively, while 141 and 430 genes were downregulated (both rst1 vs. WT) (SI Appendix, Fig. S6A). Notably, four N transport and metabolism genes (OsNiR, OsGOGAT2, OsAS1, and OsNRT2;2) were included among the 27 overlapping genes that were upregulated under both control and salt stress conditions (SI Appendix, Fig. S6B and Dataset S1). However, only OsAS1 expression was upregulated in osarf18 (rst1) and downregulated in OsARF18-OE lines (Fig. 3A and SI Appendix, Figs. S7 and S8). Furthermore, OsAS1 was preferentially expressed in roots at the seedling stage and in internodes and spikelets at the heading stage, which showed overlapped expression pattern with RST1 (Fig. 2 B–D and SI Appendix, Fig. S9). Therefore, we selected OsAS1 as a potential candidate gene regulated by RST1 for further analysis.

Fig. 3.

RST1 negatively regulates salt tolerance by repressing OsAS1 expression. (A) Relative expression of OsAS1 in roots. Fourteen-day-old seedlings were treated with or without 140 mM NaCl for 3 h. OsUBQ5 was used as an internal control. Data are means ± SD (n = 3). (B) Transient expression of RST1 in tobacco leaves represses OsAS1 promoter activity. Data are means ± SD (n = 4). (C) Schematic diagram of OsAS1 promoter. Blue vertical lines indicate AuxREs on both sense and antisense strands. Grey horizontal lines represent the PCR fragments (F1 to F8) for the ChIP assays in D. Black horizontal lines represent the probes (P1 to P3) for the EMSA assays in E. (D) ChIP-qPCR assays indicate that RST1 binds to OsAS1 promoter in vivo. The data show relative enrichment of DNA precipitated with Flag antibody to those treated with IgG (set to 1). The fragments in an exon of OsAS1 and promoter of Ubi (OsUBQ5) were used as controls. Data are means ± SD (n = 3). (E) EMSA analysis of RST1 N-terminus binding to AuxREs in OsAS1 promoter. Purified His-RST1^1–272 protein (5 μg) was incubated with 50 fmol biotin-labeled probes. For the competition test, non-labeled probes with different concentrations (25 or 100 times) were added. (F) Generation of rst1 as1 double mutants by CRISPR/Cas9 approach. The sgRNA-targeting site and protospacer adjacent motif (PAM) are indicated with green and red colors, respectively. (G) Salt tolerance phenotypes of Nipp, rst1, cr-as1, and rst1 as1 double mutant lines. Fourteen-day-old seedlings were treated with 130 mM NaCl for 10 d, and then recovered for 7 d under normal conditions. (H) Survival rates of recovered seedlings in G. Data are means ± SD (n = 3). (I) AS inhibitor enhances salt sensitivity. Fourteen-day-old seedlings were treated with 140 mM NaCl (with or without 10 μM albizziin) for 10 d, and then recovered for 7 d under normal conditions. (J) Survival rates of seedlings in I. Data are means ± SD (n = 3). Each replicate contains six plants in A, 40 to 48 plants in H and J. (Scale bars represent 5 cm in G and I.) In B, H, and J, different letters represent a significant difference at P < 0.05 determined by Tukey’s HSD test. In D, ** denotes P < 0.01, two-tailed Student’s t test.

RST1-FLAG significantly reduced proAS1::LUC activity expressed in tobacco leaves, while RST1^1–329-FLAG did not (Fig. 3B), suggesting that RST1-FLAG inhibits OsAS1 promoter activity in vivo and the truncation mutation interfered with the inhibitory effect of RST1. RST1 contains a conserved DNA-binding domain (DBD) at the N-terminus that can bind to auxin-responsive elements (AuxREs, TGTCNN) in target genes (15, 16). Promoter sequence analysis identified 12 putative AuxREs within the 2744-bp promoter region of OsAS1, among which we designated eight fragments as F1 to F8, each containing at least one AuxRE (Fig. 3C). Chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis showed that three fragments (F4, F5, and F8) at the OsAS1 promoter were significantly bound by RST1 (Fig. 3D). These results were further supported by the findings in an electrophoresis mobility shift assay (EMSA) that used biotin-labeled probes (P1 to P3) containing AuxRE motifs in the F4, F5, and F8 fragments (Fig. 3E). These data suggest that OsAS1 is a direct target regulated by RST1.

To characterize the function of OsAS1 in salt tolerance, Tos-17 (osas1-1), T-DNA (osas1-2) insertion mutants, and a CRISPR-Cas 9 mutant (cr-as1) were generated, respectively (SI Appendix, Fig. S10 A–C). Phenotype analysis showed that all osas1 mutants exhibited reduced survival rates compared with the WT (SI Appendix, Fig. S10 D–G). Two gene-edited lines (rst1 cr-as1-1 and rst1 cr-as1-2) produced using the CRISPR-Cas9 system in the rst1 background showed C base deletion or CT base insertion at the eighth exon of OsAS1 (Fig. 3F); these changes resulted in a frame shift and truncation of the OsAS1 protein (SI Appendix, Fig. S11A). The rst1 cr-as1 double mutation partially reversed the salt-tolerant phenotype of rst1 (Fig. 3 G and H), suggesting that 1) OsAS1 functions as one of the main downstream factors of RST1 in the salt tolerance pathway; 2) CRISPR-derived mutations might not be null and truncated/mutated OsAS1 could somewhat function. To answer the later possibility, WT and truncated/mutated OsAS1 proteins were expressed in Escherichia coli cells (SI Appendix, Fig. S11 A and B). The results showed that the truncated OsAS1 proteins AS1^+A, AS1^−C, and AS1^+CT, which were from cr-as1 or rst1 cr-as1-1 (rst1 cr-as1-2), respectively, completely lost their activities (SI Appendix, Fig. S11C). The E. coli cells expressing WT OsAS1 proteins, but not the truncated/mutated OsAS1 proteins, accumulated more Asn as compared with those with the vector only (SI Appendix, Fig. S11E). Furthermore, the OsAS1 activity and Asn accumulation in cells expressing OsAS1 were inhibited by albizziin, an AS inhibitor (12), in a dose-dependent manner (SI Appendix, Fig. S11 D and E). Finally, albizziin enhanced salt susceptibility in both WT and mutants (rst1 and osas1-1) to varying degrees; these findings further supported OsAS1 involvement in salt tolerance (Fig. 3 I and J and SI Appendix, Fig. S12). Together, these results indicate that RST1 negatively regulates salt tolerance at least partially through the repression of OsAS1 expression.

Loss of RST1 Function Enhances Stress Tolerance by Improving Nitrogen Use and Na⁺/K⁺ homeostasis.

AS transfers a Gln-amide group to the 2-position of Asp to generate Asn and Glu (Fig. 4A) (17). OsAS1 is also responsible for the synthesis of Asn coupled with NH₄⁺ assimilation in rice roots (18). Therefore, we investigated whether RST1 is involved in Asn synthesis via OsAS1. Under normal conditions, Asn contents decreased, while Asp and Gln, the substrates of AS, increased in roots and shoots of the osas1-1 as compared with WT (Fig. 4B and SI Appendix, Fig. S13). When WT plants were subjected to salt stress, Asn contents increased dramatically in roots and shoots, but the salt-enhanced Asn production was prevented in osas1-1 (Fig. 4B). In contrast to osas1-1, the rst1 mutant displayed a higher Asn accumulation in roots and shoots, with or without salt stress (Fig. 4B). Moreover, Asn was the most abundant one after salt stress (Datasets S2 and S3). These evidences suggest that RST1 negatively regulates Asn production via OsAS1 in response to salt stress.

Fig. 4.

RST1/OsAS1-mediated nitrogen metabolism and salt tolerance. (A) Schematic diagram of metabolic pathway for NH₄⁺ assimilation in plants. AS uses Gln (or NH₄⁺) as amide donor. (B) the contents of Asn, Glu, Asp, and Gln in roots (R) and shoots (SH). Fourteen-day-old seedlings grown under normal nitrogen level (1.25 mM NH₄NO₃) were treated with (+) or without (−) 140 mM NaCl for 7 d and then were sampled for amino acid analysis. Data are means ± SD (n = 3 or 4). (C, D, F, and G) The concentrations of amino acid (C), NH₄⁺ (D), Na⁺ (F), and K⁺ (G) in xylem sap. The seedlings were transferred to fresh hydroponic solution in the presence (+) or absence (−) of 25 mM NaCl and grown for 7 d, and then used for xylem sap collection. Data are means ± SD (n = 4 or 5). (E, H, and I) The contents of NH₄⁺ (E), Na⁺ (H), and K⁺ (I) in shoots of rice seedlings. Rice growth conditions were the same as described in B. Data are means ± SD (n = 4). (J) Effects of different N nutrition and salt stress on shoot NH₄⁺ content and survival rate. Fourteen-day-old seedlings grown in nutrition solution with different forms of N (2.5 mM total N) were treated with NaCl (60 to 140 mM) for 12 d, and sampled for shoot NH₄⁺ content analysis. After recovery for 7 d, survival rate of seedlings was calculated. Data are means ± SD, n = 4 for NH₄⁺ content, 3 for survival rate. (K) The phenotypes of seedlings grown under different forms of nitrogen source. Fourteen-day-old seedlings grown in nutrition solution with different forms of nitrogen (2.5 mM total N) were treated with NaCl (Upper, 120 mM; Lower, 140 mM) for indicated days. Each replicate (n) contains 10 plants in B, 30 plants in (C, D, F, and G), and five plants in (E, H, and I). In J, each replicate contains five plants for shoot NH₄⁺ content and 40 to 48 plants for survival rate. In B, *, ** denotes P < 0.05 and P < 0.01 compared with Nipp, respectively, two-tailed Student’s t test. In C–I, different letters represent a significant difference at P < 0.05 determined by Tukey’s HSD test.

To investigate whether salt-induced Asn accumulation in shoots is ascribed to the transport from roots, we carried out amino acid composition analyses in the xylem. The results revealed that Asn and Gln are the main forms of N transported from roots to shoots through the xylem (Fig. 4C and Dataset S4), which was consistent with previous findings (19). Asn dramatically decreased in the xylem sap of cr-as1 and significantly increased in that of rst1, as compared with WT. In contrast, the Gln concentration showed an opposite pattern (Fig. 4C). These results thus suggest that RST1 regulates Asn and Gln balance under normal conditions, likely via OsAS1; this conclusion is also supported by shoot amino acid composition results (Fig. 4B). Seedlings treated with 25 mM NaCl showed an approximate 80 to 90% reduction of four amino acids in the xylem sap of three genotypes (Fig. 4C). Mutation of osas1 led to further decreases in Asn and Glu under salt treatment. These results suggest that the salt-induced Asn (Gln) accumulation in shoots could be due to de novo synthesis and/or decrease in phloem-mediated Asn (Gln) transport, rather than transport from roots.

In addition, NH₄⁺ concentration in xylem saps increased in WT by NaCl treatment, and it was higher in cr-as1, but not changed in rst1 under stress (Fig. 4D). Salt stress induced a dramatic NH₄⁺ accumulation in shoots but not in roots of WT. A higher NH₄⁺ content was detected in the cr-as1 shoots, whereas much lower NH₄⁺ content in rst1 shoots compared with WT after salt treatment (Fig. 4E and SI Appendix, Fig. S14A). We also measured Na⁺ and K⁺ concentration in xylem saps. Na⁺ concentration in xylem saps increased in WT by NaCl treatment, and it was higher in cr-as1, but much lower in rst1 under stress (Fig. 4F). Salt treatment led to reduced K⁺ concentration in xylem saps in a similar degree among three genotypes (Fig. 4G). Under salt stress, a higher Na⁺ content was detected in the cr-as1 shoots, whereas much lower Na⁺ content in rst1 shoots compared with WT, which showed a similar pattern with that of NH₄⁺ (Fig. 4H vs. Fig. 4E). However, K⁺ content was lower in the cr-as1 shoots and higher in rst1 shoots compared with WT (Fig. 4I). In roots, salt treatment led to Na⁺ increase and K⁺ decrease, with no difference among three genotypes (SI Appendix, Fig. S14 B and C).

NH₄⁺ is more favorable for anaerobic paddy-field rice roots than NO₃⁻ (7). However, the effects of N forms on salt tolerance vary depending on the plant species (20). We found that NH₄⁺-fed WT seedlings were more sensitive to salt stress than NO₃⁻-fed ones with decreased shoot fresh weight, chlorophyll content, and survival rate (Fig. 4J and SI Appendix, Fig. S15). NH₄⁺ content was significantly increased in shoots with applied NaCl concentration increase, accompanying by the decrease in survival rate (Fig. 4J and SI Appendix, Fig. S15). These results indicated that salt stress induced NH₄⁺ accumulation in rice shoots, which led to salt sensitivity, especially under NH₄⁺ as exogenous N resource. Further phenotypic analysis indicated that RST1 and OsAS1 could regulate the salt tolerance of rice under different N nutrition (Fig. 4K and SI Appendix, Fig. S16).

Together, these results suggest that RST1-OsAS1 is a key module involved in regulating rice N metabolism and NH₄⁺ concentration, and Na⁺/K⁺ homeostasis in rice shoots in response to salt stress.

Natural Variations in RST1 Coding Sequences Control Rice Salt Tolerance and Grain Weight.

To uncover the contribution of natural variations in RST1 underlying salt tolerance across O. sativa, we analyzed the genomic sequences of this gene in 321 rice accessions obtained from the 3,000 Rice Genomes Project (3K RGP) (Dataset S5) (21, 22). Twenty-one single nucleotide polymorphisms (SNPs) including seven non-synonymous SNPs were identified (Dataset S6). Four non-synonymous SNPs (G1743C, A1830C, G1986C, and G2102A) showed significant associations with shoot Na⁺ content under salt stress, while the other three SNPs (G41A, C1677T, and C1763A) did not (Fig. 5A). These four SNPs caused amino-acid substitutions conferring Gly⁵³⁰ to Arg⁵³⁰, Asp⁵⁵⁹ to Ala⁵⁵⁹, Gly⁶¹¹ to Ala⁶¹¹, and Gly⁶⁵⁰ to Ser⁶⁵⁰, respectively (Dataset S6, shown with red colors), in which Gly⁶¹¹ and Gly⁶⁵⁰ residues are located near or in the conserved PB1 domain, respectively (SI Appendix, Fig. S17). Based on the four non-synonymous SNPs, three haplotypes of RST1 were detected in 2,913 accessions of the 3K RGP (Fig. 5B). Transcriptional activity analysis demonstrated that RST1^{Hap I} and RST1^{Hap III} had the strongest and weakest repression activity, respectively (Fig. 5C). Na⁺ content and Na⁺/K⁺ ratio in shoots were significantly different among the accessions with different RST1 haplotypes. RST1^{Hap I} accessions showed the highest shoot Na⁺ content and Na⁺/K⁺ ratio, while RST1^{Hap III} accessions showed the lowest of these values (Fig. 5 D and E). In addition, we also found that 1,000-grain weight increased from RST1^{Hap I} to RST1^{Hap III} (Fig. 5F). Taken together, these results suggest that RST1^{Hap III} improved both salt tolerance and grain weight through decreased transcriptional activity.

Fig. 5.

Natural variation and association analysis of RST1. (A) Association testing of shoot Na⁺ content in 321 rice accessions with 21 variant sites in the 3.5 kb genomic region. Among the seven non-synonymous SNPs in the RST1 coding region, four SNPs (+1743, +1830, +1986, and +2102) significantly associated with shoot Na⁺ content are indicated by red dots, and the others are indicated by yellow dots. (B) Haplotypes of four non-synonymous SNPs in the RST1 coding region from 2913 rice accessions. (C) Comparison of transcriptional activity of proteins encoded by different alleles of RST1. Data are means ± SD (n = 5). (D and E) Shoot Na⁺ content (D) and Na⁺/K⁺ (E) of rice accessions carrying different RST1 haplotypes under NaCl stress. (F) Grain weight of rice accessions carrying different RST1 haplotypes. (G) Nucleotide diversity and selection analyses in RST1 and flanking regions. The X-axis denotes a 32-kb region centered on RST1 and the Y-axis indicates average nucleotide diversity (π) or Tajima’s D values. (H) Phylogenetic relationship of 2,757 cultivated and 326 wild rice accessions based on RST1 gene sequence. Accessions within different subpopulations are shown in outer cycle with different colors. Haplotypes I, II, and III are shown in inner cycle with yellow, red, and blue lines, respectively. In C, different letters represent a significant difference at P < 0.05 determined by Tukey’s HSD test. In D–F, the dashed bars within violin plots represent 25th percentiles, medians, and 75th percentiles, P values are calculated by two-tailed Student’s t tests.

We analyzed sequence variations of this gene and its flanking regions in 2,759 cultivated rice and 42 O. rufipogon accessions (22, 23). The nucleotide diversity value (π) in the RST1 coding region was much lower than its flanking regions in both cultivated and wild populations (Fig. 5G). In addition, the nucleotide diversity of RST1 in cultivated rice populations (especially XI and GJ) was lower than that in the O. rufipogon population (Fig. 5G). Furthermore, the Tajima’s D value at the region of RST1 was negative (Fig. 5G). Taken together, these results indicate that the RST1 locus could have been subjected to directional selection during domestication.

Phylogenetic analysis showed that 94.5% (777/822) GJ accessions formed RST1^{Hap III} cluster, and 89.6% (1558/1739) XI accessions formed RST1^{Hap II} cluster, Aus, and the rest of XI accessions were grouped into RST1^{Hap I} cluster (Fig. 5 B and H). Based on the four SNPs, the sequences of the 416 O. rufipogon accessions were classified into five haplotypes, with 68, 145, and 177 accessions in the RST1^{Hap I}, RST1^{Hap II}, and RST1^{Hap III} categories, respectively (Dataset S7). GJ accessions were mainly originated from Or-III and Or-II wild rice, and XI accessions were derived from Or-I wild rice (Fig. 5H). These results suggest that RST1 alleles already existed in different populations of wild rice, and they were independently inherited into different cultivars (24).

The Plants Carrying rst1 Allele Display Salt Tolerance in Saline Soils and Increased Grain Yield in Normal Soils.

Rice is sensitive to salt stress at both the seedling and reproductive stages (1). Therefore, we conducted field experiments to evaluate the role of RST1 in rice growth and yield in the field. We transplanted 1-mo-old WT and rst1 seedlings to normal paddy field (NF), the saline pool (SP), and saline field (SF). In normal field, rst1 increased the plant height, grain length, grain width and 1,000-grain weight of rice, and finally increased the grain yield by 10.5 % (Fig. 6 A–J). The vegetative and reproductive growth of WT and rst1 were severely inhibited in both SP and SF (Fig. 6A). Yield component traits (e.g., tiller number, grain number per panicle, seed setting rate, and 1,000-grain weight) of both rst1 and WT decreased under salt stress (Fig. 6 A–J). However, the tiller number, seed setting rate, seed size, and 1,000-grain weight of rst1 were significantly higher than those of WT in saline soil (Fig. 6 D and F–I). Finally, in SP and SF, the grain yield of rst1 was 1.7 times and 5.5 times that of WT, respectively (Fig. 6J). The results were repeatable across 2 y of trials (Fig. 6 and SI Appendix, Fig. S18). These results suggest that the loss of RST1 increases grain yield in normal field and reduces yield loss in saline soils.

Fig. 6.

Field trials for rst1 tolerance to salt stress. (A) Phenotypes of Nipp and rst1 grown under various conditions. NF, normal field; SP, saline pool; SF, saline field. The salt contents in soils before ploughing are shown in brackets. (B) Grain yields per plant. (C) Plant height. (D–J) Analysis of yield-related traits. Tiller number (D), grain number per panicle (E), seed setting rate (F), grain length (G), grain width (H), 1,000-grain weight (I), and grain yield per plant (J). (K) A proposed working model of RST1 in the regulation of rice growth and salt tolerance. RST1 binds to the promoter of OsAS1 to repress its expression. The mutation in rst1 results in the loss of its transcriptional repression activity and upregulation of OsAS1, which accelerates the assimilation of NH₄⁺ to Asn, thus improving N utilization. Together, mutation of RST1 enhances yield under normal condition and reduces yield loss under salt stress. The data in A–J are from year 2020, and those from 2019 are shown in SI Appendix, Fig. S18. (Scale bars represent 5 cm in B.) Data are means ± SD, n = 12, 20, 20 replicates for NF, SP, and SF, respectively, in C–F and J, n = 20 replicates for NF, SP, and SF in G and H, n = 12 replicates for NF, SP, and SF in I. Different letters represent a significant difference at P < 0.05 determined by Tukey’s HSD test.

Discussion

The world’s farmlands are increasingly affected by soil salinization. Therefore, breeding salt-tolerant crops are urgently needed for sustainable agriculture. Although a large number of genes associated with salt tolerance have been identified in the past decades, the molecular mechanism underlying N metabolism and salt tolerance is poorly understood. In this study, we have presented multiple lines of evidence demonstrating that RST1/OsARF18 negatively regulates plant growth and salt tolerance by transcriptional repression of OsAS1.

Members of the ARF family act as activators and repressors (15, 16). Several ARF repressors, such as AtARF2, OsARF4, OsARF18, and BnARF18, were reported to negatively regulate seed size and weight (25–29). Natural variation in ARF18 simultaneously affects seed weight and silique length in rapeseed. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that N metabolism and carbohydrate (C) metabolism pathways were affected in the silique wall (27). Transgenic plants with alterations in levels of enzymes that catalyze rate-limiting steps in the metabolisms usually display the increased stress tolerance with the reduced growth. One explanation for this phenomenon is that uncontrolled accumulation of the metabolites beneficial for the tolerance perturbs other metabolic pathways necessary for essential processes such as protein synthesis and cell wall synthesis (30). In this report, loss-of-function mutation of RST1 gene increased salt tolerance with improved yield performance under normal growth conditions, probably ascribed to its ability to balance the N metabolism and the growth by precisely regulating OsAS1 expression. OsAS1 has been reported to be involved in N uptake and assimilation, N remobilization to grain, and grain yield (31). The results in this work regarding growth and grain yield regulated by OsAS1 are consistent with previous findings (31) (SI Appendix, Fig. S19). More importantly, OsAS1 is involved in salt tolerance, which was negatively regulated by RST1.

Mutation in the regulatory gene (RST1) of nitrogen metabolism caused a reduction in Na⁺ but an increase in K⁺ accumulation in shoots (Figs. 1 E and F and 5D), which is consistent with previous findings that enhancement of N uptake and assimilation improve the resistance to drought and salinity (2). Secondly, multiple members of MADS-box transcription factor family are involved in the regulation of nitrate responses (32). One MADS-box TF, AGL16, has been found to bind the promoter of HKT1;1, suggesting that N singling and metabolism may regulate HKT1;1 expression and Na⁺ transport (33). Thus, the improved nitrogen metabolism regulates Na⁺/K⁺ homeostasis. On the other hand, mutation of transporters controlling Na⁺ efflux or K⁺ uptake results in Na⁺ accumulation or K⁺ deficiency, leading ionic imbalance in plant cells exposed to salt stress (2, 34). Furthermore, the excessive intracellular Na⁺ accumulation in turn damages the enzymes in nitrogen and carbohydrate metabolisms, disrupting amino acid contents and ratios (30, 35) (Fig. 4 and SI Appendix, Fig. S13). Thus, control of Na⁺/K⁺ homeostasis and improvement of nitrogen metabolism affect reciprocally and are both essential for plant growth in salt stress environments.

The disruption in the N metabolism also leads to NH₄⁺ accumulation and induces ammonia toxification (36, 37). NH₄⁺ was accumulated in rice shoots by salt stress especially under NH₄⁺ apply (Fig. 4 E and J). Free NH₄⁺ accumulation in tissues induces the deficiency in other cations (36), ROS generation (37), and impairment in the N-glycosylation of proteins (38) and in auxin homeostasis (39). The rst1 mutant, which had higher expression of OsAS1 (Fig. 3A and SI Appendix, Fig. S8) and lower content of NH₄⁺ (Fig. 4E), showed the higher K⁺ content in shoots under salt stress (Fig. 4I). By contrast, cr-as1 mutant accumulated higher NH₄⁺ and lower K⁺ contents in shoots under salt stress (Fig. 4 E and I). This supports the notion about the interaction between NH₄⁺ and K⁺. By contrast, stress-induced AS and glutamate dehydrogenase activities antagonize the NH₄⁺ accumulation process (13, 37, 40). As the product of AS, Asn has a central role in N storage and transport in plants because of its stability and high N:C ratio (2:4 for Asn, 1:4 for Asp, 2:5 for Gln, and 1:5 for Glu) (12, 41) (Fig. 4A). Asn has been implicated in N recycling and flow in vegetative cells in response to abiotic stresses. In particular, in the energy-limited conditions (e.g., salinity), GS and GOGAT are inhibited, while AS is activated (42). Such conditions favor N assimilation into Asn, which is rich in nitrogen and stable for long-distance transport or long-term storage (42). Therefore, the upregulation of OsAS1 mediated by rst1 can improve simultaneously salt tolerance and grain yield in rice under salt stress conditions.

It should be noticed that, although it was repressed by RST1, OsAS1 expression was induced by salt stress especially in the shoots of WT (Fig. 3A and SI Appendix, Fig. S8), implying that other factors (e.g., homologs of Arabidopsis bZIP1, which regulates sugar signaling via regulating AtAS1 expression) might activate OsAS1 expression (43–45). In this process, RST1 would act as a “brake.” On the other hand, OsARF18 was recently reported to be involved in IAA-mediated reproductive organ development in rice via regulating OsSUT1 expression (46). These suggest that, besides OsAS1, RST1 may be also involved in salt response via other pathways, whereas RST1-OsAS1 module uniquely regulates N metabolism and NH₄⁺ concentration in rice in response to salt stress.

The nucleotide diversity of RST1 region is drastically decreased, and it has been undergoing directional selection during domestication. Three haplotypes were detected of the RST1. RST1^{Hap III} showed the lowest shoot Na⁺ content and the Na⁺/K⁺ ratio, and its 1,000-grain weight was significantly higher than RST1^{Hap I} and RST1^{Hap II} accessions (Fig. 5 D–F). RST1^{Hap III} accessions were predominantly GJ varieties (Fig. 5B). In high latitude areas, such as in Japan, Korea, and GJ growing areas of China, there are high-proportioned RST1^{Hap III} accessions (SI Appendix, Fig. S20A), whereas Aus and XI accessions mainly carried the RST1^{Hap I} and RST1^{Hap II} alleles, the major of them are planted in southeast and south Asia (SI Appendix, Fig. S20A). Saline soils (with electrical conductivity more than 4 mS·cm⁻¹) can be generally found in arid regions, estuaries, and coastal fringes, which significantly reduces the yield of most crops (30). The distribution of RST1 haplotypes is irrelevant with soil EC (SI Appendix, Fig. S20 A and B). We speculate that the domestication of RST1 may be due to geographical origins of wild rice (24). Therefore, breeding with the superior allele of RST1, by replacing RST1^{Hap I} or RST1^{Hap II} with RST1^{Hap III} in cultivars through marker-assisted selection, may be provided more high-yield and salt-tolerant cultivars.

The RST1 protein is highly conserved in plants, particularly in gramineous crops such as Zea mays (79% identity, ZmARF19/D9HNU6), Sorghum bicolor (85% identity, C5Z8A5), and Setaria italica (87% identity, K3XVN3), as indicated by protein phylogenetic analysis and amino acid sequence alignment (SI Appendix, Figs. S17 and S21). These analyses suggest that RST1 and its orthologs offer valuable targets for marker-assisted selection or gene editing to improve salt tolerance and grain yield in crops.

In summary, we discovered that the gene RST1/OsARF18 controls rice development and salt tolerance. Loss of RST1 improves N metabolism and reduces NH₄⁺ accumulation, thereby both increasing plant growth and yield under normal condition and reducing yield loss under salt stress (Fig. 6K). This discovery establishes the regulatory mechanism underlying N metabolism and stress tolerance, as well as a basis for new rice breeding strategies to develop stress-tolerant crops that can support sustainable food security.

Materials and Methods

Details are provided in SI Appendix, Materials and Methods.

Plant Materials and Growth Conditions.

The WT rice varieties used in this study were Nipp, Dongjin, and Hwayoung. The rst1 mutant was previously identified (14). The T-DNA insertion lines osarf18 and osas1-2 were obtained from the Korea Rice Mutant Center. The cr-as1 and rst1 cr-as1 double mutants were produced using CRISPR-Cas9 approach. Primers used in this study are listed in Dataset S8.

Rice seedlings were hydroponically cultured in a growth room with the control of illumination, temperature, and humidity, and the growth condition and media composition are described in SI Appendix, Materials and Methods. For field experiments, rice plants were grown in experimental fields with or without salinity, or saline pools, and the details are described in SI Appendix, Materials and Methods.

Gene Mapping and Expression Analysis.

Gene mapping, genetic transformation, GUS staining, RNA extraction and RT-qPCR, and RNA-sequencing are described in SI Appendix, Materials and Methods.

Transcriptional Regulation Assay.

The procedures of ChIP-qPCR, EMSA, and DLR are described in SI Appendix, Materials and Methods.

Protein Analysis.

The AS activity assay was performed as previously described (47). Protein subcellular localization and immunoblotting assays are described in SI Appendix, Materials and Methods.

Physiological Analysis.

Salt tolerance determination, ion measurement, amino acid analysis, and agronomic trait analysis are described in SI Appendix, Materials and Methods.

Genetic Analysis.

Population genetic and phylogenetic analysis, and geographical distribution analysis of rice varieties, are described in SI Appendix, Materials and Methods.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Dataset S03 (XLSX)

Dataset S04 (XLS)

Dataset S05 (XLSX)

Dataset S06 (XLSX)

Dataset S07 (XLSX)

Dataset S08 (XLSX)

Dataset S09 (XLSX)

Data, Materials, and Software Availability

Raw RNA-seq data were deposited at the National Center for Biotechnology Information Sequence Read Archive under BioProject accession PRJNA812782 (https://www.ncbi.nlm.nih.gov/sra/PRJNA812782) (48). All other study data are included in the article and/or the SI Appendix.