Functional Analysis Of SAPH,a Gene Involved In Expression Regulation Of Heat Shock Response In Arabidopsis Thaliana

The stress of high temperature seriously affect the growth and development of plants.While plants evolve numbers of mechanisms to acclimate high temperature stress during the long-term evolution.For example,heat shock proteins(HSPs)play very important roles.It is kown that the expression of Hsps is induced by high temperature.In our previous research,we established a forward genetic method through which we use the firefly luciferase reporter gene(LUC)driven by the promoter of Hsp18.2 to screening mutants with altered LUC expression during heat treatment.Between these mutants,we found a mutant hl1401 exihibits significant enhanced of LUC activity compared to wild type P-LUC.We identified this mutant gene SAPH via map-based cloning and found that it plays an important role in heat stress tolerance of Arabidopsis thaliana.In this paper,using genetic transformation analysis,RNA sequencing and qRT-PCR technology,we further studied the expression and biological function of SAPH.The results showed:(1)The phenotype of mutant hl1401:The LUC activity was measured by CCD imaging system after treated with 38? for different time.It was found that mutant hl1401 displayed significant difference of LUC chemiluminescence compared to wild type P-LUC after heat shock for 2 hours.Under normal growth conditions,the plant size of mutant hl1401 is smaller than that of wild type P-LUC.The number of green,etiolation and albino seedlings were calculated after high temperature treatment and the results showed that the thermotolerance of mutant hl1401 was enhanced.The transgenic complementary plants were obtained via inflorescence infection using Agrobacterium tumefaciens.It was found that the LUC activity and plant size phenotype of the complementary transgenic lines were similar to wild type P-LUC.Therefore,the above phenotypes of hl 1401 are indeed resulted from the mutation of SAPH.(2)The relative expression of SAPH:The expression level of SAPH at the transcriptional level was up-regulated by heat shock.The results of GUS staining showed that SAPH constitutivly expressed in roots,leaves,stems,siliques and flowers.Additionally,subcellular localization by expression of SAPH-GFP fusion protein showed that SAPH was localized in nucleolus.(3)The results of RNA sequencing:Using Gene Ontology annotations to analysis the results of RNA sequencing,it was found that genes which had changed in mutant hl1401 after heat shock are mainly enriched in some biological processes,including responses to bioltic and abiotic stresses,cellular responses to hormone stimulus and so on.The expression level of most Hsfs and Hsps in mutant hl1401 and wild type P-LUC via qRT-PCR technology analysis were consistent with the results of RNA sequencing.In conclusion,SAPH is a negative regulator of heat stress tolerance of Arabidopsis thaliana,and may influence plant thermotolerance via regulating the expression of heat stress response genes.This paper can offer some important clues for the further study of the expression regulation mechanism of heat shock genes as well as plant thermotolerance.