Comparison of the genetic basis of salt tolerance at germination, seedling, and reproductive stages in an introgression line population of rice.

BACKGROUND: Salinity is a major limitation for rice farming due to climate change. Since salt stress adversely impact rice plants at germination, seedling, and reproductive stages resulting in poor crop establishment and reduced grain yield, enhancing salt tolerance at these vulnerable growth stages will enhance rice productivity in salinity prone areas. METHODS AND RESULTS: An introgression line (ILs) population from a cross between a high yielding cultivar 'Cheniere' and a salt tolerant donor 'TCCP' was evaluated to map quantitative trait loci (QTLs) for traits associated with salt tolerance at germination, seedling, and reproductive stages. Using a genotyping-by-sequencing based high density SNP linkage map, a total of 7, 16, and 30 QTLs were identified for five germination traits, seven seedling traits, and ten reproductive traits, respectively. There was overlapping of QTLs for some traits at different stages indicating the pleiotropic effects of these QTLs or clustering of linked genes. Candidate genes identified for salt tolerance were OsSDIR1 and SERF for the seedling stage, WRKY55 and OsUBC for the reproductive stage, and MYB family transcription factors for all three stages. Gene ontology analysis revealed significant GO terms related to nucleotide binding, protein binding, protein kinase activity, antiporter activity, active transmembrane transporter activity, calcium-binding protein, and F- box protein interaction domain containing protein. CONCLUSIONS: The colocalized QTLs for traits at different growth stages would be helpful to improve multiple traits simultaneously using marker-assisted selection. The salt tolerant ILs have the potential to be released as varieties or as pre-breeding lines for developing salt tolerant rice varieties.

Integration of QTL Mapping and Whole Genome Sequencing Identifies Candidate Genes for Alkalinity Tolerance in Rice (Oryza sativa).

Soil alkalinity is an important stressor that impairs crop growth and development, resulting in reduced crop productivity. Unlike salinity stress, research efforts to understand the mechanism of plant adaptation to alkaline stress is limited in rice, a major staple food for the world population. We evaluated a population of 193 recombinant inbred lines (RIL) developed from a cross between Cocodrie and N22 under alkaline stress at the seedling stage. Using a linkage map consisting of 4849 SNP markers, 42 additive QTLs were identified. There were seven genomic regions where two or more QTLs for multiple traits colocalized. Three important QTL clusters were targeted, and several candidate genes were identified based on high impact variants using whole genome sequences (WGS) of both parents and differential expression in response to alkalinity stress. These genes included two expressed protein genes, the glucan endo-1,3-beta-glucosidase precursor, F-box domain-containing proteins, double-stranded RNA-binding motif-containing protein, aquaporin protein, receptor kinase-like protein, semialdehyde hydrogenase, and NAD-binding domain-containing protein genes. Tolerance to alkaline stress in Cocodrie was most likely due to the low Na+/K+ ratio resulting from reduced accumulation of Na+ ions and higher accumulation of K+ in roots and shoots. Our study demonstrated the utility of integrating QTL mapping with WGS to identify the candidate genes in the QTL regions. The QTLs and candidate genes originating from the tolerant parent Cocodrie should be targeted for introgression to improve alkalinity tolerance in rice and to elucidate the molecular basis of alkali tolerance.

A Negative Regulator in Response to Salinity in Rice: Oryza sativa Salt-, ABA- and Drought-Induced RING Finger Protein 1 (OsSADR1).

RING (Really Interesting New Gene) finger proteins play crucial roles in abiotic stress responses in plants. We report the RING finger E3 ligase gene, an Oryza sativa salt, ABA and drought stress-induced RING finger protein 1 gene (OsSADR1). We demonstrated that although OsSAR1 possesses E3 ligase activity, a single amino acid substitution (OsSADR1C168A) in the RING domain resulted in no E3 ligase activity, suggesting that the activity of most E3s is specified by the RING domain. Additional assays substantiated that OsSADR1 interacts with three substrates-no E3 ligase acti and OsPIRIN, and mediates their proteolysis via the 26S proteasome pathway. For OsSADR1, approximately 62% of the transient signals were in the cytosol and 38% in the nucleus. However, transiently expressed OsSADR1 was primarily expressed in the nucleus (70%) in 200 mM salt-treated rice protoplasts. The two nucleus-localized proteins (OsSNAC2 and OsGRAS44) interacted with OsSADR1 in the cytosol and nucleus. Heterogeneous overexpression of OsSADR1 in Arabidopsis resulted in sensitive phenotypes for salt- and mannitol-responsive seed germination and seedling growth. With ABA, OsSADR1 overexpression in plants produced highly tolerant phenotypes, with morphological changes in root length and stomatal closure. The ABA-tolerant transgenic plants also showed hypersensitivity phenotypes under severe water deficit conditions. Taken together, OsSADR1 may act as a regulator in abiotic stress responses by modulating target protein levels.

The microtubule-associated RING finger protein 1 (OsMAR1) acts as a negative regulator for salt-stress response through the regulation of OCPI2 (O. sativa chymotrypsin protease inhibitor 2).

MAIN CONCLUSION: Our results suggest that a rice E3 ligase, OsMAR1, physically interacts with a cytosolic protein OCPI2 and may play an important role under salinity stress. Salt is an important abiotic stressor that negatively affects plant growth phases and alters development. Herein, we found that a rice gene, OsMAR1 (Oryza sativa microtubule-associated RING finger protein 1), encoding the RING E3 ligase was highly expressed in response to high salinity, water deficit, and ABA treatment. Fluorescence signals of its recombinant proteins were clearly associated with the microtubules in rice protoplasts. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) showed that OsMAR1 interacted with a cytosolic protein OCPI2 (O. sativa chymotrypsin protease inhibitor 2) and led to its degradation via the 26S proteasome. Heterogeneous overexpression of OsMAR1 in Arabidopsis showed retarded root growth compared with that of control plants, and then led to hypersensitivity phenotypes under high salinity stress. Taken together, OsMAR1 negatively regulates the salt-stress response via the regulation of the OCPI2 protein in rice.

Oryza sativa salt-induced RING E3 ligase 2 (OsSIRP2) acts as a positive regulator of transketolase in plant response to salinity and osmotic stress.

MAIN CONCLUSION: A rice gene (OsSIRP2) encoding the RING Ub E3 ligase was highly induced under salinity stress and physically interacted with a transketolase (OsTKL1). Overexpression of OsSIRP2 conferred salinity and osmotic stress tolerance in plants. The RING E3 ligases play a vital role in post transitional modification through ubiquitination-mediated protein degradation that mediate plants responses during abiotic stresses and signal transduction. In this study, we report an Oryza sativa salt induced Really Interesting New Gene (RING) finger protein 2 gene (OsSIRP2) and elucidate its role under salinity and osmotic stress. The transcript levels of OsSIRP2 in rice leaves were induced in response to different abiotic stresses, such as salt, drought, heat, and abscisic acid (ABA) exposure. In vitro ubiquitination revealed that the OsSIRP2 protein formed poly-ubiquitin products, whereas a single amino acid substitution in OsSIRP2 (OsSIRP2C149A) in the RING domain did not form ubiquitinated substrates, supporting the hypothesis that E3 ligase activity requires the functional RING domain. Using the yeast two-hybrid (Y2H) assay, O. sativa transketolase 1 (OsTKL1) was identified as an interacting partner. OsSIRP2 was localized in the nucleus, whereas its interacting partner (OsTKL1) was localized in the cytosol and plastids in the rice protoplasts. Fluorescence signals between OsSIRP2 and OsTKL1 were observed in the cytosol. The pull-down assay confirmed the physical interaction between OsSIRP2 and OsTKL1. In vitro ubiquitination assay and in vitro protein degradation assay revealed that OsSIRP2 ubiquitinates OsTKL1 and enhances the degradation of OsTKL1 through the 26S proteasomal pathway. Heterogeneous overexpression of OsSIRP2 resulted in conferring tolerance against salinity and osmotic stress. Overall, our findings suggest that OsSIRP2 may be associated with plant responses to abiotic stresses and act as a positive regulator of salt and osmotic stress tolerance.

Molecular characterization of rice arsenic-induced RING finger E3 ligase 2 (OsAIR2) and its heterogeneous overexpression in Arabidopsis thaliana.

Arsenic (As) accumulation adversely affects the growth and productivity of plants and poses a serious threat to human health and food security. In this study, we identified one As-responsive Really Interesting New Gene (RING) E3 ubiquitin ligase gene from rice root tissues during As stress. We named it Oryza sativa As-Induced RING E3 ligase 2 (OsAIR2). Expression of OsAIR2 was induced under various abiotic stress conditions, including heat, salt, drought and As exposure. Results of an in vitro ubiquitination assay showed that OsAIR2 possesses an E3 ligase activity. Within the cell, OsAIR2 was found to be localized to the Golgi apparatus. Using yeast two-hybrid (Y2H) assay, the 3-ketoacyl-CoA thiolase (KAT) protein was identified as an interaction partner. We found that the O. sativa KAT1 (OsKAT1) is localized to the cytosol and peroxisomes. Moreover, in vitro pull-down assay verified the physical interaction between OsAIR2 and OsKAT1. Interestingly, in vitro ubiquitination assay and in vivo proteasomal degradation assay revealed that OsAIR2 ubiquitinates OsKAT1 and promotes the degradation of OsKAT1 via the 26S proteasome degradation pathway. Heterogeneous overexpression of OsAIR2 in Arabidopsis improved the seed germination and increased the root length under arsenate stress conditions. Therefore, these results suggest that OsAIR2 may be associated with the plant response to As stress and acts as a positive regulator of As stress tolerance.

Genome-Wide Association Study Identified Candidate Genes for Alkalinity Tolerance in Rice.

Alkalinity stress is a major hindrance to enhancing rice production globally due to its damaging effect on plants' growth and development compared with salinity stress. However, understanding of the physiological and molecular mechanisms of alkalinity tolerance is limited. Therefore, a panel of indica and japonica rice genotypes was evaluated for alkalinity tolerance at the seedling stage in a genome-wide association study to identify tolerant genotypes and candidate genes. Principal component analysis revealed that traits such as alkalinity tolerance score, shoot dry weight, and shoot fresh weight had the highest contribution to variations in tolerance, while shoot Na+ concentration, shoot Na+:K+ ratio, and root-to-shoot ratio had moderate contributions. Phenotypic clustering and population structure analysis grouped the genotypes into five subgroups. Several salt-susceptible genotypes such as IR29, Cocodrie, and Cheniere placed in the highly tolerant cluster suggesting different underlying tolerance mechanisms for salinity and alkalinity tolerance. Twenty-nine significant SNPs associated with alkalinity tolerance were identified. In addition to three alkalinity tolerance QTLs, qSNK4, qSNC9, and qSKC10, which co-localized with the earlier reported QTLs, a novel QTL, qSNC7, was identified. Six candidate genes that were differentially expressed between tolerant and susceptible genotypes were selected: LOC_Os04g50090 (Helix-loop-helix DNA-binding protein), LOC_Os08g23440 (amino acid permease family protein), LOC_Os09g32972 (MYB protein), LOC_Os08g25480 (Cytochrome P450), LOC_Os08g25390 (Bifunctional homoserine dehydrogenase), and LOC_Os09g38340 (C2H2 zinc finger protein). The genomic and genetic resources such as tolerant genotypes and candidate genes would be valuable for investigating the alkalinity tolerance mechanisms and for marker-assisted pyramiding of the favorable alleles for improving alkalinity tolerance at the seedling stage in rice.

Genetic Dissection of Alkalinity Tolerance at the Seedling Stage in Rice (Oryza sativa) Using a High-Resolution Linkage Map.

Although both salinity and alkalinity result from accumulation of soluble salts in soil, high pH and ionic imbalance make alkaline stress more harmful to plants. This study aimed to provide molecular insights into the alkalinity tolerance using a recombinant inbred line (RIL) population developed from a cross between Cocodrie and Dular with contrasting response to alkalinity stress. Forty-six additive QTLs for nine morpho-physiological traits were mapped on to a linkage map of 4679 SNPs under alkalinity stress at the seedling stage and seven major-effect QTLs were for alkalinity tolerance scoring, Na+ and K+ concentrations and Na+:K+ ratio. The candidate genes were identified based on the comparison of the impacts of variants of genes present in five QTL intervals using the whole genome sequences of both parents. Differential expression of no apical meristem protein, cysteine protease precursor, retrotransposon protein, OsWAK28, MYB transcription factor, protein kinase, ubiquitin-carboxyl protein, and NAD binding protein genes in parents indicated their role in response to alkali stress. Our study suggests that the genetic basis of tolerance to alkalinity stress is most likely different from that of salinity stress. Introgression and validation of the QTLs and genes can be useful for improving alkalinity tolerance in rice at the seedling stage and advancing understanding of the molecular genetic basis of alkalinity stress adaptation.

Genome-wide transcriptome profiling of genes associated with arsenate toxicity in an arsenic-tolerant rice mutant.

The presence of arsenic (As) in polluted environments, such as ground water, affects the accumulation of As in rice grains and causes a serious threat to human health. However, the precise molecular regulations related to As toxicity and tolerance in rice remain largely unknown. In the present study, we developed an arsenic-tolerant type 1 (ATT1) rice mutant by γ-irradiation mutagenesis and performed genome-wide transcriptome analysis for the characterization of As-responsive genes. Toxicity inhibited transcriptional regulation of putative genes involved in photosynthesis, mitochondrial electron transport, and lipid biosynthesis metabolism in wild-type (WT) and ATT1 rice mutant. However, many cysteine biosynthesis-related genes were significantly upregulated in both plants. We also attempted to elucidate the putative genes associated with As tolerance by comparing transcriptomes and identified ATT1-specific transcriptional regulation of genes involved in stress and RNA-protein synthesis. This analysis identified 50 genes that had DNA polymorphisms in upstream regions that differed from those in the exon regions, which suggested that genetic variations in the upstream regions might enhance As tolerance in the mutants. Therefore, the expression profiles of the genes evaluated in this study may improve understanding of the functional roles of As-related genes in response to As tolerance mechanisms and could potentially be used in molecular breeding to limit As accumulation in rice grains.