Characterization And Functional Analysis Of GRAS Transcription Factors In Response To High Temperature In Rice

Rice is one of the major food crops in China,and it is also the staple food for more than half of the Chinese people.With the intensification of human industrial activities,large amounts of greenhouse gases were emitted,such as carbon dioxide(CO2)and methane(CH4).Therefore,the global temperature is rising.The yield and quality of rice grains will be affected if rice encounters high temperature during the reproductive stage.In addition to optimizing the field management to help rice to cope with high temperature stress,mining key heat responsive genes and using them to breeding novel rice cultivars with resistance to high temperature is another key method,which is also a hotspot in the research field.Transcription factors,also known as trans-acting factors,are a group of proteins that can directly bind to certain cis-acting elements in the promoter of target genes,and they can also interact with some other proteins to co-regulate the expression of target genes.In this study,four GRAS genes,GRAS49,GRAS52,GRAS92 and GRASP 1,in responsive to high temperature were successfully selected from the high-temperature transcriptome database of developing rice grains.The expression and functional analysis of these four GRAS genes were carried out to dissect their roles in rice quality formation and effects in response to high temperature.The detailed results of this study are as follows:1.Bioinformatic analysis indicated that the four gene encoded proteins belong to plant-specific GRAS family transcription factors.The four GRAS proteins can be subdivided into two different groups.Among thems GRAS49 has a high homology with the DELLA protein SLR1 in the GA pathway of rice.In addition,motif analysis showed that all four GRAS genes contained various phytohormone and stress related cis-acting elements.2.Spatial and temporal expression analyses indicated that GRAS49 dominantly expressed in rice seeds while GRAS92 specifically expressed in the leaves.GRAS52 and GRASP1 had a similar expression abundance in almost all detected tissues.Further analysis of their expression in response to high temperature showed that the expression patterns of all four genes were not changed,but their expression abundances were suppressed by high temperature.3.Subcelluar localization assay indicated that GRAS49,GRAS52 and GRAS92 expressed in both nucleus and cytoplasm,while GRASP 1 only expressed in the cytoplasm.Moreover,all four GRAS transcription factors have the transcriptional activation activity Further yeast-two hybrid analysis demonstrated that GRAS52 could directly interact with GRAS49,suggesting GRAS52 and GRAS49 are likely to form heterodimers to control the expression of target genes.4.The overexpression and knock-out lines of the GRAS genes were generated for evaluating their roles in modulating rice growth and development as well as their roles in high temperature response.At present,we already successfully generated the overexpression lines of GRAS92,GRAS49 and GRASP1,and now we are screening the homozygous transgenic lines.In addition,the gene-editing rice lines of the four GRAS genes were produced via the CRISPR/Cas9 method.Now a series of homozygous mutant lines were got after sequencing identification.Agronomic traits analysis indicated that the phenotypes of gras mutants were similar to that of the wild-type control,such as plant height and panicle length.Appearance analysis indicated that only gras92 mutation led to the increased grain length,while the mutation of all four GRAS genes resulted in the increment of chalkiness.As to the eating and cooking qualities,gras49 mutation strikingly increased the apparent amylose content(AAC)but decreased gel consistency(GC)of rice.The GC of grasp1 and gras92 mutants only slightly decreased.Mutation of all four GRAS genes notably affected the DSC characteristics of rice,especially the remarkably decreased Tp value.5.Based on the fact that all four GRAS genes were selected from the RNA-seq database of high-temperature treated developing seeds of rice,the homozygous gras mutants were used for analyzing the effects of each GRAS gene in mediating high temperature response of rice.In general,high temperature declined rice plant height,increased rice chalkiness,decreased its AAC.Further analysis indicated that the chalkiness of gras mutants is more serious than the wild-type control.Among them,the gras49 mutant had a most remarkable decline of AAC.Moreover,high temperature induced an increment of all DSC characteristics of rice,but no consistent changes of GC were observed in different gras mutants.The above results implied that although the expression of all four GRAS genes was suppressed by high temperature,each GRAS gene had different effects on rice agricultural traits,rice quality and the response to high temperature because they were different in amino acid composition,expression level,spatial and temporal expression pattern,subcellular localization and protein-protein interaction.Therefore,each GRAS gene may play different roles in rice quality formation and mediating rice response to high temperature.The detailed mechanisms require further analysis.