Identification and functional characterization of STG5.2 conferring salt tolerance in rice.

Genetic diversity is important for developing salt-tolerant rice varieties. This research used the existing whole-exome sequences of eight Thai rice varieties, including the standard checks for tolerance and susceptibility, and the previously reported salt-tolerant genes to understand the mechanisms of salt tolerance in Thai rice, as well as the bulk-segregant analysis (BSA) to identify salt-tolerant QTLs in ‘Jao Khao’, the salt tolerant Thai rice. Salt responsive characteristics were used to classify tolerant and susceptible genotypes at the seedling stage. Phylogenetic analysis of SNPs from exome sequences showed that the Thai rice varieties were monophyletic and distantly related to 'Pokkali' and IR29. 'Lai Mahk' and 'Luang Pratahn', which were characterized as Thai salt tolerant varieties, were closely related based on either the exome SNPs or SNPs from 164 salt tolerant genes. Genomic regions conferring salt tolerance traits were identified in an F2 population of the Thai landrace salt tolerant variety, 'Jao Khao', and the salt susceptible line, IR29, via BSA. In total, 600 F2 individuals were phenotyped in the hydroponic system. The 14-day old seedlings were treated with salt stress in a stepwise manner to reach 12 dS.m-1 for 12 days. The F2 members showed a high level of distribution in the standard salt injury evaluation system (SES), cell membrane stability (CMS), relative water content (RWC), whole plant fresh weight (WFW), plant height (PH), and root length (RL). SES showed a strong negative correlation with CMS, RWC, and WFW, but it showed less of a negative correlation with PH and RL. The BSA predicted 4 QTLs harboring the putative salt-tolerant regions on chromosome 3 (CMS trait) and chromosomes 2, 6, and 12 (RWC trait). Three of the identified QTLs overlapped with some reported salt-tolerant QTLs, while the QTLs on chromosome 6 overlapped with drought-tolerant QTLs. The validation of the QTLs was carried out by designing four molecular markers. Their possible involvement in salt tolerance was assessed in an F3 population. The findings revealed that F3 individuals with the ‘Jao Khao’ allele were more tolerant to salt stress than the ones with the allele from IR29 or containing the heterozygous allele, indicating that these QTLs are important in salt tolerance. In the future, the 4 salt stress-tolerant Thai rice varieties can be employed in rice breeding. Moreover, 4 salt tolerant QTLs identified in the study will be used to breed salt tolerant rice, and the developed genetic markers can be used in the marker assisted-breeding program.

Soil salinity is one of the major abiotic stresses limiting rice growth. Hybrids outperform their parents in salt tolerance in rice, while its mechanism is not completely understood. In this study, a higher seedling survival was observed after salt treatment in an inter-subspecific hybrid rice, Zhegengyou1578 (ZGY1578), compared with its maternal japonica Zhegeng7A (ZG7A) and paternal indica Zhehui1578 (ZH1578). A total of 2584 and 3061 differentially expressed genes (DEGs) with at least twofold changes were identified between ZGY1578 and ZG7A and between ZGY1578 and ZH1578, respectively, in roots under salt stress using the RNA sequencing (RNA-Seq) approach. The expressions of a larger number of DEGs in hybrid were lower or higher than those of both parents. The DEGs associated with transcription factors, hormones, and reactive oxygen species (ROS)-related genes might be involved in the heterosis of salt tolerance. The expressions of the majority of transcription factors and ethylene-, auxin-, and gibberellin-related genes, as well as peroxidase genes, were significantly higher in the hybrid ZGY1578 compared with those of both parents. The identified genes provide valuable clues to elucidate the heterosis of salt tolerance in inter-subspecific hybrid rice.

Salinity stress progressively reduces plant growth and productivity, while plant has developed complex signaling pathways to confront salt stress. However, only a few genetic variants have been identified to mediate salt tolerance in the major crop rice, and the molecular mechanism remains poorly understood. Here, we identify ten candidate genes associated with salt-tolerance (ST) traits by performing a genome-wide association analysis in rice landraces. We characterize two ST-related genes, encoding transcriptional factor OsWRKY53 and Mitogen-activated protein Kinase Kinase OsMKK10.2, that mediate root Na+ flux and Na+ homeostasis. We further find that OsWRKY53 acts as a negative modulator regulating expression of OsMKK10.2 in promoting ion homeostasis. Furthermore, OsWRKY53 trans-represses OsHKT1;5 (high-affinity K+transporter 1;5), encoding a sodium transport protein in roots. We show that the OsWRKY53-OsMKK10.2 and OsWRKY53-OsHKT1;5 module coordinate defenses against ionic stress. The results shed light on the regulatory mechanisms underlying plant salt tolerance.

Soil salinity results in oxidative stress and heavy losses to crop production. The S-acylated protein SALT TOLERANCE RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (STRK1) phosphorylates and activates CATALASE C (CatC) to improve rice (Oryza sativa L.) salt tolerance, but the molecular mechanism underlying its S-acylation involved in salt signal transduction awaits elucidation. Here, we show that the DHHC-type zinc finger protein DHHC09 S-acylates STRK1 at Cys5, Cys10, and Cys14 and promotes salt and oxidative stress tolerance by enhancing rice H2O2-scavenging capacity. This modification determines STRK1 targeting to the plasma membrane or lipid nanodomains and is required for its function. DHHC09 promotes salt signaling from STRK1 to CatC via transphosphorylation, and its deficiency impairs salt signal transduction. Our findings demonstrate that DHHC09 S-acylates and anchors STRK1 to the plasma membrane to promote salt signaling from STRK1 to CatC, thereby regulating H2O2 homeostasis and improving salt stress tolerance in rice. Moreover, overexpression of DHHC09 in rice mitigates grain yield loss under salt stress. Together, these results shed light on the mechanism underlying the role of S-acylation in RLK/RLCK-mediated salt signal transduction and provide a strategy for breeding highly salt-tolerant rice.

BackgroundSoil salinization is a major abiotic environmental stress factor threatening crop production throughout the world. Salt stress drastically affects the growth, development, and grain yield of rice (Oryza sativa L.), and the improvement of rice tolerance to salt stress is a desirable approach for meeting increasing food demand. Receptor-like cytoplasmic kinases (RLCKs) play essential roles in plant growth, development and responses to environmental stresses. However, little is known about their functions in salt stress. Previous reports have demonstrated that overexpression of an RLCK gene SALT TOLERANCE KINASE (STK) enhances salt tolerance in rice, and that STK may regulate the expression of GST (Glutathione S-transferase) genes.ResultsThe expression of STK was rapidly induced by ABA. STK was highest expressed in the stem at the heading stage. STK was localized at the plasma membrane. Overexpression of STK in rice increased tolerance to salt stress and oxidative stress by increasing ROS scavenging ability and ABA sensitivity. In contrast, CRISPR/Cas9-mediated knockout of STK increased the sensitivity of rice to salt stress and oxidative stress. Transcriptome sequencing analysis suggested that STK increased the expression of GST genes (LOC_Os03g17480, LOC_Os10g38140 and LOC_Os10g38710) under salt stress. Reverse transcription quantitative PCR (RT-qPCR) suggested that four stress-related genes may be regulated by STK including OsABAR1, Os3BGlu6, OSBZ8 and OsSIK1.ConclusionsThese findings suggest that STK plays a positive regulatory role in salt stress tolerance by inducing antioxidant defense and associated with the ABA signaling pathway in rice.

Amino acid transporters (AATs) play important roles in plant growth and stress tolerance; however, whether the abscisic acid signaling pathway regulates their transcription in rice (Oryza sativa) under salt stress remains unclear. In this study, we report that the transcription factor OsMYB2 (MYB transcription factor 2) of the abscisic acid signaling pathway mediates the expression of the gene encoding the AAT aromatic and neutral AAT 1 (OsANT1), which positively regulates growth and salt tolerance in rice. OsANT1 was mainly expressed in the leaf blade and panicle under normal conditions and transports leucine, phenylalanine, tyrosine, and proline (Pro), positively regulating tillering and yield in rice. Nevertheless, salt stress induced the accumulation of abscisic acid and strongly increased the expression level of OsANT1 in the root, resulting in enhanced salt tolerance of rice seedlings, as evidenced by higher Pro concentration and antioxidant-like enzyme activities and lower malondialdehyde and hydrogen peroxide concentrations. Moreover, we showed that OsMYB2 interacts with the promoter of OsANT1 and promotes its expression. Overexpression of OsMYB2 also improved tillering, yield, and salt tolerance in rice. In conclusion, our results suggest that the transcription factor OsMYB2 triggers OsANT1 expression and regulates growth and salt tolerance in rice, providing insights into the role of the abscisic acid signaling pathway in the regulatory mechanism of AATs in response to salt stress.

Salinity is a significant limiting factor that adversely affects plant growth, distribution and crop yield. Ethylene responsive factors play crucial roles in plant responses to and tolerance of various abiotic stresses. Recently, we revealed that OsERF2 is involved in root growth by transcriptionally regulating hormone and sugar signaling in rice. Here, we report that OsERF2 is a direct target gene of OsEIL1 and negatively regulates salt tolerance in rice. Compared to the wild type, the gain-of-function mutant of OsERF2 (nsf2857) and the knockdown of OsERF2 via an artificial microRNA (Ami-ERF2) exhibited decreased and increased salt tolerance, respectively. The enhanced salt tolerance observed in Ami-OsERF2 lines was associated with lower accumulations of malondialdehyde and reactive oxygen species (ROS) under salt stress, while the opposite was true for nsf2857 plants, which exhibited decreased salt tolerance. At the transcriptional level, several stress-related genes encoding ROS and NAD(P)H-related oxidoreductases were downregulated in nsf2857 plants but upregulated in Ami-ERF2 plants. Furthermore, yeast one-hybrid and ChIP assays revealed that OsEIL1 can bind to the of EBS cis element present in the promoter of OsERF2 (-bp), suggesting that OsEIL1 may directly regulate the expression of OsERF2. Collectively, our findings indicate that OsERF2 is a direct downstream factor involved in the regulation of salt tolerance in rice, highlighting its potential application in the genetic improvement of tolerance to abiotic stresses in this crop.

Key messageReproductive stage salinity tolerance is most critical for rice as it determines the yield under stress. Few studies have been undertaken for this trait as phenotyping was cumbersome, but new methodology outlined in this review seeks to redress this deficiency. Sixty-three meta-QTLs, the most important genomic regions to target for enhancing salinity tolerance, are reported.Although rice has been categorized as a salt-sensitive crop, it is not equally affected throughout its growth, being most sensitive at the seedling and reproductive stages. However, a very poor correlation exists between sensitivity at these two stages, which suggests that the effects of salt are determined by different mechanisms and sets of genes (QTLs) in seedlings and during flowering. Although tolerance at the reproductive stage is arguably the more important, as it translates directly into grain yield, more than 90% of publications on the effects of salinity on rice are limited to the seedling stage. Only a few studies have been conducted on tolerance at the reproductive stage, as phenotyping is cumbersome. In this review, we list the varieties of rice released for salinity tolerance traits, those being commercially cultivated in salt-affected soils and summarize phenotyping methodologies. Since further increases in tolerance are needed to maintain future productivity, we highlight work on phenotyping for salinity tolerance at the reproductive stage. We have constructed an exhaustive list of the 935 reported QTLs for salinity tolerance in rice at the seedling and reproductive stages. We illustrate the chromosome locations of 63 meta-QTLs (with 95% confidence interval) that indicate the most important genomic regions for salt tolerance in rice. Further study of these QTLs should enhance our understanding of salt tolerance in rice and, if targeted, will have the highest probability of success for marker-assisted selections.

Salt stress is an important abiotic stress, which has seriously affected the reproductive development of rice in many parts of the world. Therefore, it is particularly important to understand the genetic mechanism of salt tolerance in rice. In this study, we preliminarily located some quantitative trait loci (QTL) for root length, bud length, and survival percent under different salinity conditions (0, 100, 200 and 400 mM NaCl), using a population of chromosome segment substitution lines (CSSLs) constructed by Nipponbare and 9311. A total of 18 QTLs were identified, which explained the phenotypic variation of 4.76–37.59%, among which 13 QTLs were detected under salt stress condition. These salt tolerance related QTLs were divided into two categories, QTL expressed both under control and salt stress conditions (qSP3, qBL8), and QTL expressed just under salt stress condition (qBL3, qRL3, qSP4, qRL5, qSP7, qSP9, qRL10, qBL11-1, qRL11-1, qRL11, qSP12). qSP3, qSP4, qRL5, qSP7, qRL10, qRL10-1, qRL11, qRL11-1, qBL11, qBL11-1 and qSP12 were reported to be related with salt stress for the first time. These QTLs identified under salt stress may be valuable genetic factors for improving salt tolerance of rice by molecular markers technology, which will help to further understand the genetic mechanism of salt tolerance of rice.

Polyamine oxidase 3 is involved in salt tolerance at the germination stage in rice

Plant growth-promoting bacteria Bacillus amyloliquefaciens NBRISN13 modulates gene expression profile of leaf and rhizosphere community in rice during salt stress

Ethylene plays important roles in plant growth, development, and stress responses. The ethylene signaling pathway has been studied extensively, mainly in Arabidopsis (Arabidopsis thaliana). However, the molecular mechanism of ethylene signaling is largely unknown in rice (Oryza sativa). Previously, we have isolated a set of rice ethylene-response mutants. Here, we characterized the mutant maohuzi6 (mhz6). Through map-based cloning, we found that MHZ6 encodes ETHYLENE INSENSITIVE3-LIKE1 (OsEIL1), a rice homolog of ETHYLENE INSENSITIVE3 (EIN3), which is the master transcriptional regulator of ethylene signaling in Arabidopsis. Disruption of MHZ6/OsEIL1 caused ethylene insensitivity mainly in roots, whereas silencing of the closely related OsEIL2 led to ethylene insensitivity mainly in coleoptiles of etiolated seedlings. This organ-specific functional divergence is different from the functional features of EIN3 and EIL1, both of which mediate the incomplete ethylene responses of Arabidopsis etiolated seedlings. In Arabidopsis, EIN3 and EIL1 play positive roles in plant salt tolerance. In rice, however, lack of MHZ6/OsEIL1 or OsEIL2 functions improves salt tolerance, whereas the overexpressing lines exhibit salt hypersensitivity at the seedling stage, indicating that MHZ6/OsEIL1 and OsEIL2 negatively regulate salt tolerance in rice. Furthermore, this negative regulation by MHZ6/OsEIL1 and OsEIL2 in salt tolerance is likely attributable in part to the direct regulation of HIGH-AFFINITY K(+) TRANSPORTER2;1 expression and Na(+) uptake in roots. Additionally, MHZ6/OsEIL1 overexpression promotes grain size and thousand-grain weight. Together, our study provides insights for the functional diversification of MHZ6/OsEIL1 and OsEIL2 in ethylene response and finds a novel mode of ethylene-regulated salt stress response that could be helpful for engineering salt-tolerant crops.

Molecular mechanisms of salinity tolerance in rice

Malate dehydrogenase (MDH) is widely present in nature and regulates plant growth and development, as well as playing essential roles, especially in abiotic stress responses. Nevertheless, there is no comprehensive knowledge to date on MDH family members in rice. In this study, a total of 12 MDH members in rice were identified through genome-wide analysis and divided into three groups on the basis of their phylogenetic relationship and protein-conserved motifs. Evolutionary analysis showed that MDH proteins from rice, maize and wheat shared a close phylogenetic relationship, and the MDH family was conserved in the long-term process of domestication. We identified two segmental duplication events involving four genes, which could be the major force driving the expansion of the OsMDH family. The expression profile, cis-regulatory elements and qRT-PCR results of these genes revealed that a few OsMDH showed high tissue specificity, almost all of which had stress response elements in the promoter region, and ten MDH members were significantly induced by salt stress. Through gene-based association analysis, we found a significant correlation between salt tolerance at the seedling stage and the genetic variation of OsMDH8.1 and OsMDH12.1. Additionally, we found that the polymorphism in the promoter region of OsMDH8.1 might be related to the salt tolerance of rice. This study aimed to provide valuable information on the functional study of the rice MDH gene family related to salt stress response and revealed that OsMDH8.1 might be an important gene for the cultivar improvement of salt tolerance in rice.

OsALY4 acts as an m5C-mRNA reader facilitating mRNA export and contributing to chilling and salt tolerance in rice.