Genetic Analysis Of Cold Tolerance At Seeding Stage And Functional Characterization Of OsMYB30 In Regulation Of Cold Sensitivity In Rice

Rice(Oryza sativa L.)is one of the most important food crops in the world,but the growth and productivity of rice can be severely affected by cold stress.Understanding the cold response mechanisms and exploring additional genetic resources for the cold tolerance will deepen the understanding of cold response and adaptation and help to improve cold tolerance of rice.This study was comprised of two parts.The first part was investigation of the genetic variation of 529 rice accessions under natural chilling and cold shock stress conditions at the seedling stage using genome wide association studies.The second part was characterization of the OsMYB30 which was involved in the regulation cold response.For the first part,the genetic variation of 529 rice accessions were investigated under natural chilling and cold shock stress(4°C)conditions at the seedling stage,the data of 16 traits were collected,and analyzed for the variation and correlation.We noticed that the traits under different stress conditions had no significant correlations.Then we performed genome wide association studies(GWAS)on the whole panel(529 accessions)and indica,japonica subpopulations,and focused on the GWAS results from the whole panel in the following analyses.A total of 132 loci or QTLs(Quantitative trait locus)were identified from the GWAS on the whole panel(529 accessions),12 loci were common for the data collected from both chilling and cold shock stress conditions.After that,the comparison of cold tolerance loci from GWAS in this study and genetic mapping was performed,68 loci from this study were located in or overlapped with the reported QTL.By checking our whole genome expression profiling data,102 cold stress-responsive genes(potential candidates)within 200 kb(100 kb upstream and 100 kb downstream of the Lead SNPs)for the 132 loci were chosen,these genes were up-regulated or down-regulated in different levels by cold stress which could be selected for further transgenic identification.Interestingly,the haplotype analysis of a known gene OsMYB2,and an unknown gene 07 g,revealed significantly indica-japonica differentiation and latitude tendency for the accessions belong to the four haplotypes.Thus we selected 114 chilling tolerant(CT),143 chilling sensitive(CS)accessions under natural chilling stress condition,123 cold shock insensitive(CSI)and 131 cold shock sensitive(CSS)accessions under cold shock stress condition from the panel according to their comprehensive performance.The chilling tolerance group(CT accessions),which mainly consisted of japonica accessions,had a wider latitudinal distribution than the chilling sensitivity(CS accessions)group.Accessions that were insensitive under the two stress conditions(CT overlapped with CSI accessions)had a wider latitudinal distribution than the accessions with sensitivity under the two stress conditions(CS overlapped with CSS accessions).The above results confirmed the correlation between cold adaptability and indica-japonica differentiation and latitudinal distribution.For the second part,previous studies have shown that overexpression of OsMYB30 increased cold stress sensitivity while T-DNA insertion mutant osmyb30 showed significantly increased cold tolerance compared to the wild type(WT)plant,and numbers of differentially expressed genes(DEGs)were found in gene chip after cold stress,suggesting OsMYB30 played an important role in cold response regulation.In this study,we identified that a few β(Beta)-amylase(BMY)genes were down-regulated by OsMYB30 based on the microarray data.The expression levels of BMY2/6/10 were decreased in the OsMYB30-OE plant and increased in osmyb30 mutant plant,respectively.The following results showed that OsMYB30 can bind to the promoter of BMY genes in vivo and in vitro.BMY genes encoding β-amylase degraded starch to maltose,and the β-amylase activity and maltose content were decreased in the OsMYB30-OE plant and increased in osmyb30 mutant plant,respectively,which was correlated with the expression pattern of BMY genes.For the OsMYB30-OE and WT with cold sensitivity,application of appropriate concentration of maltose had protection function for cell membrane under cold stress condition.And this protection effect on rice in cold stress was confirmed using a few other cold sensitive rice accessions.Besides this,we screened OsJAZ9 as an interaction protein of OsMYB30,and results here confirmed OsMYB30 can interact with OsJAZ9 in vivo and in vitro.OsJAZ9 showed higher expression level in the Os MYB30-OE plants and lower expression level in the osmyb30 mutant under the cold stress condition,and BMY6/10 were repressed at transcriptional level in OsJAZ9-OE plants under cold stress condition.What’s more,OsJAZ9 had a significant effect in suppressing the transcriptional activation of OsMYB30 and in the repression of BMY genes mediated by OsMYB30 based on the dual-luciferase transient assay in rice protoplasts.In conclusion,we firstly employed a genome wide association strategy with 529 accessions for rice cold tolerance at the seedling stage,and detected loci related to the two types of cold stresses(natural chilling and cold shock).The loci detected in this study may provide valuable information for the cold adaptability of rice,and the accessions classified as CT or CS would be potential genetic resources for rice improvement.On theother hand,we found an undocumented negative regulation mechanism for cold tolerance in rice,which was mediated by OsMYB30 and its interaction protein OsJAZ9 through the regulation of the BMY genes.These findings have also expanded our understanding of the MYB family in cold stress regulation in plants.