Functional Study Of Tomato Transcription Factors VAHOX1 And SlMBP22 In Reproductive Development And Drought Stress Response

HD-Zip and MADS-box transcription factors play an important role in regulating plant growth,development and stress response.At present,the research on the function of HD-Zip and MADS-box transcription factors has made great progress.However,the molecular mechanism of the functions of HD-Zip and class Bsister(B_s)MADS-box genes in tomato has not been clearly clarified.Tomato is not only an important cash crop,but also the best model plant for studying fruit development and ripening.Based on bioinformatics analysis,we selected the HD-Zip family gene VAHOX1 and B_s-class MADS-box gene SlMBP22 as the target genes in this study.We used tomato transformation,real-time quantitative PCR(qRT-PCR),dual-luciferase reporter assay,yeast hybridization system and bimolecular fluorescence complementation(Bi FC)assays to explore the functions of these two genes in tomato growth and development as well as abiotic stress response.The main results are as follows:(1)The subcellular localization results showed that VAHOX1 was preferentially localized to the nucleus.Transcriptional activation assay demonstrated that VAHOX1possesses a transcriptional activation function and that the C-terminus of VAHOX1 is critical to its transcriptional activation activity in yeast cells.Tissue expression pattern analysis showed that VAHOX1 was expressed in all tissues,and was highly expressed in fruits.The results of hormone-responsive expression patterns showed that the expression of VAHOX1 could be induced by ethylene.It was speculated that VAHOX1might participate in ethylene-mediated fruit ripening.(2)RNAi repression of VAHOX1(VAHOX1-RNAi)and overexpression(VAHOX1-OE)transgenic plants were obtained by transgenic technology.The silencing of VAHOX1 resulted in accelerated fruit ripening,increased total carotenoid content and ethylene production,enhanced sensitivity to ethylene,and shortened the shelf life of fruits.Conversely,VAHOX1 overexpression in tomato had the opposite effect,that is,delayed fruit ripening,reduced total carotenoid content and ethylene production,and weakened the sensitivity to ethylene.RNA-Seq results showed that altering VAHOX1expression affected the transcript accumulation of a series of genes involved in ethylene biosynthesis and signal transduction and cell wall modification.Additionally,a dual-luciferase reporter assay,histochemical analysis of GUS activity and a yeast one-hybrid(Y1H)assay revealed that VAHOX1 could bind the promoter of AP2a and promote its expression.These results indicate that VAHOX1 negatively regulates tomato fruit ripening by mediating ethylene signaling pathway.(3)Abiotic stress response analysis of VAHOX1 showed that dehydration stress could strongly induce the expression of VAHOX1.A mannitol-simulated drought stress test and the soil drought stress treatment assay were conducted to investigate the drought tolerance of VAHOX1-RNAi and VAHOX1-OE transgenic plants.According to the analysis of phenotype,physiological parameters and the transcript levels of the plant stress-related genes of tomato plants,our results showed that the overexpression of VAHOX1 resulted in reduced resistance to drought stress.Conversely,the silencing of VAHOX1 could enhance the tolerance to drought stress.Therefore,VAHOX1 negatively regulates drought stress response.(4)Tissue expression pattern analysis showed that SlMBP22 was highly expressed in flowers,especially in pistils.Overexpression of SlMBP22(SlMBP22-OE)resulted in considerable alterations in floral morphology and size,and affected the expression levels of several floral homeotic genes.Further analysis by yeast two-hybrid and bimolecular fluorescence complementation assays revealed that SlMBP22 forms dimers with MC,TM5 and TM29,respectively.The defect in female reproductive development was responsible for the infertility phenotype observed in the strong overexpression transgenic plants.Transgenic fruits with mild overexpression exhibited reduced size.Analysis of tissue sections and gene expression showed that overexpression of SlMBP22 affected flower and fruit size by inhibiting cell expansion.Additionally,SlMBP22 overexpression in tomato led to increased proanthocyanidin(PA)accumulation,resulting in dark brown seed coat and affecting seed dormancy.These results indicate that SlMBP22 plays an important role in tomato reproductive development.(5)SlMBP22-OE plants displayed reduced plant height by affecting auxin and/or gibberellin signalling.SlMBP22 was induced by dehydration treatment.A mannitol-simulated drought stress test and the soil drought stress treatment assay were conducted to investigate the drought tolerance of SlMBP22-OE transgenic plants.According to the analysis of phenotype,physiological parameters and the transcripts of the plant stress-related genes of tomato plants,our results showed that SlMBP22-OE plants were more tolerant to drought stress than wild-type(WT).RNA-Seq results showed that the transcript levels of a series of genes related to hormone signalling and stress responses were altered.In conclusion,this study preliminarily explored and analyzed the function and mechanism of VAHOX1 and SlMBP22 genes in tomato reproductive development and drought stress response.These results expand our understanding of HD-Zip and MADS-box transcription factors involved in tomato fruit development and ripening as well as stress response,and provide a theoretical basis for further improving the complex regulatory network of fruit development and ripening and cultivating new tomato varieties with stress resistance.