Function Analysis Of The Splicing Factor SFH Involved In Expression Regulation Of Heat Shock Response In Arabidopsis

Heat stress caused by elevated temperature could disturb cellular homeostasis and lead to adverse impacts on plant growth and development.Plants have evolved a series of adaptive mechanisms to cope with heat stress.The accumulation of heat shock proteins(Hsps)regulated by heat stress transcription factors(Hsfs)is assumed to play a central role in the heat stress response and in thermotolerance in plants.We have a certain understanding of Hsfs-Hsps response pathway.However,many of the key factors mediating the heat response pathways remain unkown.In order to indentify regulators involed in heat stress,we established a forward genetic method for screening mutants with altered Hsp expressions.A firefly luciferase reporter gene driven by the Hsp18.2 was introduced into Arabidopsis thaliana glabrous1 mutants(Col-0 background).Seeds from a homozygous line harboring a single copy of Hsp18.2pro:LUC transgene was used as the wild type P-LUC,and mutagenized by EMS(ethyl methanesulfonate).Then the mutants with altered luminescence expression under heat stress treatment were screened by using a hypersensitive CCD camera.Through this method,we found a mutant named hl307 show elevated luciferase expression comparing with wild type and further studied.In our previous research,it was found that hl307 mutant displayed the elevated luciferase expression comparing with wild type under the treatment with 38? for 1 hour and the mutant gene was identified by map-based cloning and next-generation sequencing.In hl307,a nucleotide change of G2140A from the start codon was found in this mutant gene.The mutational site lies in the 5' end splice site of the sixth intron,leading to the splicing change of this gene.The bioinformatics analysis showed that this gene encode a splicing factor.Therefor,the gene was designated SFH(Splicing Factor Related to Heat Stress).Our further study showed that the hl307 mutant displayed the obviously higher luciferase expression compared with the wild type under 38? treatment for half hour,1 hour,2 hours and 5 hours.In addition,hl307 is hypersensitive to heat stress.Under normal growth conditions,hl307 were early flowering and greatly reduced in size compared with P-LUC.T-DNA insertion lines of SFH gene identified from TAIR seed stocks were respectively crossed with hl307,the seedings of filial generation(F1)displayed the significantly elevated luciferase expression compared with WT.And luminescence of hl307 complementation transformants(Cmp)is between the luminescence of WT and hl307 mutant.Hence the above phenotypes observed in hl307 plants are indeed resulted from the mutation of SFH.SFH gene expresses in all tested tissues including roots,stems,leaves,flowers and siliques.Analysis of several Hsfs and Hsps expression showed that most Hsfs and Hsps expression level in hl307 mutants were lower than in P-LUC.This results provides some clues for explaining the phenotype that hl307 is hypersensitive to high temperature.Additionally,the image of the SFH-GFP fusion protein showed that SFH is localized in the nucleus.In conclusion,SFH protein can inhibit the expression of Hspl8.2 and may play important roles in pre-mRNA splicing and heat stress response.The current findings can help us to understand the mechanisms about thermotolerance in plants.