| Phytohormones and transcription factors are key factors that regulate the development and maturation of fruits,and epigenetic regulatory factors also play an crucial role in fleshy fruit ripening,coordinating with hormones and transcription factors.Epigenetic modifications include DNA methylation,histone post-translational modifications,chromatin remodeling,and noncoding RNAs.DNA methylation is a conservative but reversible epigenetic modification that usually involves the addition of a methyl group to the fifth carbon atom of the cytosine ring.The DNA methylation level of the whole genome decreases during the tomato fruit ripening,including promoter regions of many key ripening-related transcription factors and other mature-related genes.Methyltransferase inhibitor treatments could induce premature fruits.The up-regulated expression of the DNA demethylase gene SlDML2(DEMETER-like 2)is the key gene for the decline in the methylation level of the whole fruit genome,which affects the methylation level of the promoter of the ripening-related transcription factors,and ultimately affects the ethylene biosynthesis and fruit ripening.However,regulatory factors that directly regulating SlDML2 expression is limited.In this study,we identified a High Mobility Group A3(HMGA3)protein that directly bind to the promoter of SlDML2,then the bioinformatics,subcellular localization,transcriptional activation assay in yeast and tissue expression pattern were performed.The EMSA and the dual luciferase experiment were performed to analyze the specific binding ability of SlHMGA3 to SlDML2 promoter and SlHMGA3 activation ability.The homozygous line of slhmga3 mutants was obtained by CRISPR/Cas9 system to study the SlHMGA3 function on the fruit ripening,the expression of genes involving in ethylene biosynthesis and signaling and ethylene production.Bisulfite pyrosequencing was used to analyze the dynamic changes on the methylation levels of CNR and NOR promoters and the expression levels of key ripening transcription factors during fruit ripening.The main results of this study are as follows:1.Yeast one-hybrid screening of SlDML2 promoter.In this experiment,we used yeast one-hybrid library to identify potential proteins that bind to SlDML2 promoter.Thirty positive clones were sequenced,of which 14 clones encoded the same HMGA protein: XP004236944.Cluster analysis and multiple sequence alignment analysis of the protein showed that XP 004236944 had the highest homology with GH1-HMGA3 in Arabidopsis..GH1-HMGA3 and XP 004236944 had a globular histone H15 domain at the N-terminus and four AT-hook motifs at the C-terminus that both belonged to the family of high mobility group proteins,therefore XP 004236944 in tomato was named SlHMGA3 here.The subcellular location showed that SlHMGA3 was located in the nucleus.Transcriptional activation assay in yeast showed that SlHMGA3 had no activational activity.Tissue expression analysis showed that SlHMGA3 was widely expressed in various tissues of tomato,with relatively higher expression in reproductive organs,and the expression was increasing during the fruit ripening process,suggesting that SlHMGA3 may be involved in the fruit ripening process.2.The AT-hook motif of SlHMGA3 binds to the AT-rich region of the SlDML2 promoter and promotes the expression of SlDML2.Yeast one-hybrid interaction experiment showed that SlHMGA3 bound to SlDML2 promoter fragment containing AT-rich regions.EMSA assay showed that SlHMGA3 could bind to ATAAATAAATA nucleotide sites.Among the two domains(H15 domain and AT-hook motif)of SlHMGA3,only the AT-hook motif could bind to the SlDML2 promoter.The result of dual luciferase activation experiments using transient expression detection in tomato fruit protoplast showed that SlHMGA3 could promote SlDML2 gene expression.3.The SlHMGA3 mutation delays the initiation of ripening.By CRISPR/Cas9 editing technology,two homozygous mutant lines of slhmga3-1 and slhmga3-2 were obtained in the T3 generation.In the slhmga3-1 mutant line,two amino acids were deleted in the H15 domain of the SlHMGA3 protein,but the protein still contains one AT-hook motif;while in the slhmga3-2 mutant line,the H15 domain of the SlHMGA3 protein was incomplete,and all four AT-hook motifs were lacking.The maturation initiation time of the two mutant lines was delayed by 3 and 6 days,respectively.The fruit coloring,carotenoid accumulation and chlorophyll degradation were also delayed,which further confirmed the delay of fruit ripening.4.SlHMGA3 mutation delays the expression of SlDML2,ethylene biosynthesis and signaling genes.q PCR results showed that SlDML2 gene expression was increased and then decreased along with the fruit ripening,but the up-regulated time of SlDML2 in slhmga3 mutant fruits was significantly delayed.The up-regulation time of ethylene biosynthesis genes ACO1,ACS2 and ACS4 and the peak of ethylene production in the two mutant lines were significantly delayed.The up-regulation of ethylene signal related genes ETR3,ETR4,EIN2,EIL3 and CTR1 was also significantly delayed.These results suggest the mutation of SlHMGA3 gene affects the expression of ethylene-related genes by delaying the expression of SlDML2 gene,and then delays ethylene biosynthesis and finally fruit ripening.5.SlHMGA3 mutation affects the promoter methylation level of the key mature-related transcription factors and thus affects their expression.The bisulfite pyrosequencing method was conducted to analyze the methylation level of NOR and CNR gene promoter fragments in wild type and slhmga3 mutant fruits.The result showed that in the mutant,the methylation level of the NOR promoter region remained higher compared with that of the wild type during fruit ripening,while the down-regulation time of the CNR promoter region methylation level was significantly delayed.The q PCR result showed that the up-regulated expression of CNR,RIN,NOR,TAGL1,AP2 a,FUL1,E4 and E8 genes were delayed during fruit ripening.In addition,the expression of SlHMGA3 in ripening defect mutants Cnr,nor,rin was inhibit during tomato fruit ripening,but it had no change in gf,hp and nr mutants.These results indicate that SlHMGA3 can affect the expression level of key ripening related genes by regulating the methylation level of their promoters.In addition,CNR,NOR and RIN may have a feedback regulation on the expression of SlHMGA3.6.The SlHMGA3 protein interacts with the β isoform SlPP2A-B’’/β protein in the B’’family of the regulatory subunit of serine/threonine protein phosphatase 2A.The result of subcellular localization showed that SlPP2A-B’’/β protein was located in the nucleus,and tissue expression analysis showed that the expression level of SlPP2A-B’/β gradually decreased during the fruit ripening process.Yeast two-hybrid experiments,Split-LUC and Bi FC experiments showed that SlHMGA3 could interact with SlPP2A-B’’/β,and the interaction was in the nucleus.These results suggest that SlHMGA3 protein may be dephosphorylated by protein phosphatase SlPP2A-B’’/β,thereby affecting the activity of SlHMGA3 protein.In summary,this study obtained the novel high mobility group protein SlHMGA3 that can bind to the promoter of SlDML2 gene,and the AT-hook motif of this protein can bind to the AT-rich region in the promoter of SlDML2.After gene-editing of SlHMGA3,a stable loss-of-function mutant was obtained,which showed the phenomenon of delayed initiation of ripening.The up-regulation of SlDML2 gene was significantly delayed,resulting in a significant delay in up-regulation of the expression of ethylene-related genes and ethylene synthesis.The up-regulated expression of related transcription factors was also significantly delayed,while CNR and NOR remained at a high level and decreased with delay during fruit ripening,respectively.In this study,SlHMGA3 and protein phosphatase SlPP2A-B’/βcan interact with each other,which lays a foundation for studying the dephosphorylation of SlHMGA3 protein.These results demonstrate that SlHMGA3 affects the methylation process by binding to the SlDML2 promoter,thereby affecting fruit ripening. |
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