| Plant architecture includes a variety of factors such as plant height,internode length,leaf angle and branching,and an ideal architecture is necessary for increased yield;however,the volatility of the greenhouse environment often severely affects plant architecture.Tomato,an important vegetable crop,can lead to vigorous lateral growth and delayed flowering and fruiting in facility production due to suitable temperature,light and fertilization conditions,which in turn affects fruit yield and quality.The manual grooming raises the production cost and is not conducive to the sustainable development of the industry.It has been found that plant histone deacetylases are involved in the environmental regulation of plant architecture.However,current studies on the effects of mild high temperature on plant architecture have mainly focused on the model plant Arabidopsis thaliana hypocotyl elongation,and fewer studies have been conducted on horticultural crops.Therefore,it is necessary to investigate the mechanism of the effect of mild high temperature on lateral branch development in tomato,and to explore new directions for future genetic improvement.In this paper,we constructed overexpression and mutant materials of tomato deacetylase HDA4 by plant tissue culture and CRISPR/Cas9 technology,and explored the role of HDA4 in regulating the temperature sensitivity of tomato lateral branch development by combining transcriptome data.We also investigated the synergistic relationship between HDA4 and transcription factors PIF4 or TCP15,and explored the regulatory mechanism on downstream growth hormone synthesis gene FZY1 and negative regulator of growth hormone signal transduction IAA12,respectively.Meanwhile,the function of GSNOR,a GSNO reductase in the NO pathway,in lateral branch development was analyzed by phenotypic statistics and hormone assays,and its effect on HDA4 deacetylase activity was explored.The main findings were as follows:1.Tomato apical dominance is temperature-sensitive and the histone deacetylase HDA4 positively regulates this process.It was found that mild high temperature significantly suppressed total lateral branch length,while GUS staining and gene expression of the lateral shoot repressor BRC1 accumulated significantly under mild high temperature conditions,demonstrating that mild high temperature inhibited plant lateral bud development.Next,tomato lateral bud development temperature-sensitivity was confirmed by VIGS silencing of FZY1 in association with auxin.The global acetylation status under mild high temperature showed that mild high temperature reduced the global acetylation of histone H3,and the histone deacetylase HDA4 was the most significantly up-regulated expression in this process.After that,the growth phenotypes of OE-HDA4,hda4 and F3 generation of hybrid backfill material were counted and found that the total length of hda4 lateral branches increased significantly,the total length of OE-HDA4 lateral branches decreased significantly,while the F3 generation was in between.Meanwhile,in situ hybridization results of lateral shoots with real-time fluorescence quantification showed that BRC1 expression was significantly decreased in hda4 and significantly increased in OE-HDA4,while the F3 generation was also in between.Finally,experiments with WT and hda4 in response to mild high temperature revealed that hda4 attenuated the inhibitory effect of mild high temperature on tomato lateral branch development,confirming the involvement of HDA4 in the inhibition of lateral branch development by mild high temperature through enhancing apical dominance in tomato.2.HDA4 promotes auxin synthesis,positively regulates temperature sensitivity of lateral bud development and inhibits lateral branch development in tomato.Transcriptome KEGG enrichment analysis and heat map analysis of FPKM expression values revealed that some genes that are up-regulated in response to mild high temperatures in the wild type are not responsive in hda4,and the portion of these genes associated with plant hormone signaling is enriched in the auxin pathway,demonstrating that the absence of hda4 response to temperature is strongly associated with the reduced auxin content in the mutant.Next,screening by site-specific histone acetylation antibodies at different sites revealed that hda4 mainly targets the histone H3K9 site for deacetylation modifications.FZY1 gene expression and auxin content of apical buds in hda4 and OE-HDA4 were further determined and found to be decreased in the mutant and increased in the overexpressor,implying that HDA4 may promote growth hormone synthesis.Also,the temperature-treated group of hda4 exhibited insensitivity to the increase in FZY1 and auxin content,demonstrating that mild high temperature is dependent on HDA4 to promote growth hormone synthesis.By exogenously treating hda4 with the growth hormone analogue NAA and by VIGS silencing FZY1 in both OE-HDA4,it was demonstrated that auxin lies downstream of HDA4 regulation and inhibits lateral branch development.Subsequently,VIGS silencing was used to find that PIF4 silencing partially restored the inhibition of lateral branch development by OE-HDA4,implying that the inhibitory effect of HDA4 on lateral branch development is partially dependent on the function of PIF4.Further,growth phenotypes of two OE-PIF4 strains were counted and found that overexpression of PIF4 suppressed lateral branch development in tomato.Finally,the promotion of FZY1 expression by OE-HDA4 was confirmed to be associated with reduced acetylation of the histone H3K9 locus near the FZY1 gene body by Ch IP-q PCR using an antibody specific for acetylation of H3K9 ac and an endogenous antibody to PIF4,and that HDA4 enhanced the binding of PIF4 to the FZY1 promoter.3.HDA4 interacts with TCP15 to synergistically regulate auxin signaling,enhance lateral bud development temperature sensitivity,and inhibit lateral branch development.The reliability of HDA4 and TCP15 protein interactions was verified using four approaches: yeast two-hybrid,dual fluorescent molecular complementation,protein binding in vitro,and immunoprecipitation.The VIGS technique revealed that OE-HDA4 silencing of TCP15 partially alleviated the phenotype of restricted lateral bud development of OE-HDA4,implying that the inhibition of lateral bud development by OE-HDA4 is partially dependent on TCP15.Further experiments revealed that the tcp15 mutant had increased total lateral branch length and reduced response to mild high temperatures,demonstrating that TCP15 suppresses lateral branch development in tomato.Subsequently,transcriptome analysis revealed that many genes that were up-regulated in the WT in response to mild high temperatures were not up-regulated in tcp15,demonstrating the important role of TCP15 in temperature response.The common target gene of tcp15 and hda4,the auxin negative regulator IAA12,was screened by the Wayne plot intersection of the differentially up-regulated genes of both.Next,IAA12 was silenced in hda4 and tcp15,respectively,using VIGS technology,and it was confirmed by genetic means that IAA12 is located downstream of HDA4 and TCP15 together,promoting lateral branch development.Finally,the direct binding of TCP15 to the IAA12 promoter was confirmed by EMSA experiments using molecular biology;meanwhile,the binding of TCP15 to IAA12 was confirmed by Ch IP-q PCR and LUC experiments to be dependent on the deacetylation modification of the histone H3K9 site near the IAA12 promoter by HDA4.4.GSNOR regulates HDA4 deacetylase activity to inhibit tomato lateral branch development.First,the growth phenotype of GSNOR-deficient mutants was measured,and it was found that total lateral branch length was increased in gsnor.Subsequently,lateral branch development-related hormones were analyzed in gsnor,and it was found that the apical auxin content was significantly reduced in gsnor,accompanied by a significant down-regulation of the auxin synthesis gene FZY1 and a significant up-regulation of the negative signal transduction gene IAA12;meanwhile,the cytokinin content was significantly increased in the stem,however,there was no significant difference in the expression of cytokinin synthesis genes;in addition,there was no significant difference in the expression of strigolactone.These data suggest that the increase in total lateral branch length exhibited by gsnor may be mainly associated with auxin and cytokinin.Further,by screening the expression levels of the deacetylase gene family in gsnor and detecting the histone acetylation status,we found that the acetylation level of the H3K9 locus in gsnor was significantly increased,while the expression of HDA4 gene was significantly down-regulated,suggesting that there might be some association between the two.Next,the experiments confirmed the increased acetylation of histone proteins of HDA4 target genes in gsnor by two molecular means,H3K9ac-specific acetylation Ch IP assay and dual-luciferase LUC,suggesting that GSNOR may be regulating HDA4 activity.Finally,the experiments confirmed a decrease in total deacetylase enzyme activity in gsnor using the HDAC enzyme activity assay,again suggesting the possibility of GSNOR regulation of HDA4 enzyme activity from the side. |
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