Mechanism Of Brassinosteroid Regulating Tomato Chilling Tolerance Through Abscisic Acid Synthesis And SRT1 Mediated Histone Deacetylation

Cold stress is one of the main abiotic stresses faced in the stage of growth and development,leading to an imbalance in the homeostasis of endogenous hormones in plants,and causing damage to the membrane system,photosynthetic system,and enzyme activity of plants.For warm vegetables such as tomato(Solanum lycopersicum),chilling stress encountered during growth can pose a serious threat to their fruit yield and quality.Exploring an effective way to enhance chilling tolerance is of great significance for tomato production.Brassinosteroids(BRs)are a class of hormones that have important regulatory functions in plant growth,development,and stress response.However,the specific mechanism of BR signaling regulating chilling tolerance in tomato is unclear.In this study,tomato was used as the research object,and comprehensive techniques such as molecular biology,genetics,and plant physiology were used to reveal that BR signaling enhances tomato cold tolerance by regulating abscisic acid(ABA)synthesis and histone deacetylase SRT1,and to clarify that the interaction between BZR1 and SRT1 weakens histone acetylation and transcriptional expression near the promoters of negative regulator genes MYB13 and b ZIP17,Thereby enhancing the mechanism of tomato cold tolerance.The main research results are as follows:1.BR signaling plays an important role in regulating tomato chilling tolerance.Chilling(4℃)promoted the accumulation of the main active components CS(Castosterone)and BL(Brassinolide)in BRs.Exogenous pretreatment of HBR(28 high brassinolide)can effectively improve tomato chilling tolerance;BR synthesis gene DWARF overexpressed plants(DWF-OE)and BR signaling pathway key transcription factor BZR1 overexpressed plants(BZR1-OE)showed better chilling tolerance;At the same time,the BR synthesis gene mutant dwf and BR signaling mutant bzr1 were more sensitive to chilling stress.Further research found that chilling promoted the accumulation of BZR1 protein and increased the proportion of dephosphorylated BZR1 protein.Meanwhile,it was found that chilling inhibited the protein accumulation and phosphorylation of GSK3 protein kinase BIN2,a negative regulator of BR signaling.By constructing BIN2-OE overexpression plants and using VIGS(Virus induced gene silencing)to silence BIN2 gene,it was demonstrated that BIN2 negatively regulates the protein accumulation and chilling tolerance function of BZR1.2.BR signaling enhances tomato chilling tolerance by promoting ABA synthesis.It was found that overexpression of the DWF or BZR1 gene promoted the accumulation of ABA under chilling,while the ABA content in BIN2-OE plants and dwf,bzr1 mutants were significantly reduced compared to wild-type(WT)under chilling.The expression of the key rate limiting enzyme gene NCED1(Nine cis-epoxycarotinoid dioxygenase 1)for ABA synthesis in the aforementioned tomato materials also showed a similar trend to ABA content,which suggests that BR signaling may enhance ABA accumulation at low temperatures by promoting the expression of the ABA synthesis gene NCED1.Subsequently,transcriptional regulation related experiments were used to demonstrate that BZR1 can directly bind to NCED1 promoter.We constructed overexpression materials of DWF-OE and BZR1-OE with NCED1 mutant notabilis(not)as the background,clarifying that under ABA synthesis deletion conditions,DWF-OE and BZR1-OE no longer promote ABA accumulation,and the ability to improve tomato chilling tolerance was significantly weakened,proving that the regulation of chilling tolerance by BR and BZR1 mainly relies on ABA synthesis.3.Transcription factor BZR1 interacts with histone deacetylase SRT1 to synergistically regulate tomato chilling tolerance.The interaction between BZR1 and histone deacetylase SRT1 was proved by yeast two hybrid(Y2H),bimolecular fluorescence complementation(Bi FC)and immuno coprecipitation(co-IP)experiments.Low temperature induced an increase in SRT1 gene expression level and protein accumulation.We constructed SRT1-OE transgenic overexpression materials and srt1 mutants,and identified the important role of SRT1 in tomato chilling tolerance.Subsequently,SRT1-OE/bzr1 and BZR1-OE/srt1 double mutants were constructed,demonstrating the interdependence between BZR1 and SRT1 in regulating chilling tolerance.By utilizing multiple histone acetylation site specific antibodies,it was determined that SRT1 mainly inhibits the acetylation of histone H3K9 site.Further research has found that the inhibitory ability of SRT1 on H3K9 site acetylation level is significantly reduced under the background of bzr1 mutant.Therefore,the interaction between BZR1 and SRT1 promotes tomato chilling tolerance by regulating the acetylation modification of histone H3K9.4.The interaction between BZR1 and SRT1 inhibits the expression of MYB13 and b ZIP17 genes,thereby regulating tomato chilling tolerance.Through transcriptome analysis,198 genes were found to be jointly inhibited by BZR1 and SRT1 at low temperature,including MYB13 and b ZIP17.Further research has found that MYB13 and b ZIP17 serve as negative regulatory factors for low temperature,and the H3K9 Ac and transcriptional levels of MYB13 and b ZIP17 genes are significantly inhibited by BZR1 and SRT1,and BZR1 and SRT1 are interdependent during this regulatory process.Through transcriptional regulation experiments,it was verified that BZR1 directly binds to the MYB13 and b ZIP17 promoters,and it was found that SRT1 does not affect the binding of BZR1 to the two genes,but enhances the transcriptional inhibition of BZR1 on MYB13 and b ZIP17.5.BR regulates SRT1 phosphorylation and protein stability through BIN2.By constructing DWF-OE/SRT1-OE and dwf/SRT1-OE double mutants,it was found that an increase in endogenous BRs content enhances the protein accumulation of SRT1 under chilling.Silencing BIN2 gene using VIGS can significantly enhance the accumulation of SRT1 protein,and exogenous treatment of BIN2 inhibitor Bikinin also has a similar effect.Subsequently,protein interaction experiments were conducted to demonstrate the interaction between BIN2 and SRT1.Further research found that BR signaling inhibited the serine phosphorylation(p Ser)level of SRT1 protein,while silencing BIN2 and exogenous Bikinin treatment resulted in a decrease in serine phosphorylation level of SRT1 protein.Through further experimental analysis,it was found that the serine at 317 th of SRT1 protein was the main site regulated by BIN2 phosphorylation,and the point mutation of serine at 317 th effectively prevented BIN2 from regulating the stability of SRT1 protein.