| The basis of size control in plants is an important biological question.At the cytological level,cell division and cell expansion determine the final organ size.However,the mechanistic basis for this regulation remains unclear.Fruit size and shape are important agronomic traits that are associated with crop yield.As one of the major vegetables in greenhouse production in China,Cucumber（Cucumis sativus L.）is known for its rapid fruit enlargement and its extreme diversity in fruit shape and size.These features make cucumber fruits an excellent system in which to study rapid fruit development.Therefore,the study on the morphogenesis of cucumber fruit has important theoretical and practical value.Here,the study identified a cucumber short fruit mutant（sf2）and cloned the candidate gene,Short Fruit 2（SF2）.By analyzing the function of SF2 and genome-wide target mapping of SF2,our findings revealed a complex regulatory network of fruit cell proliferation mediated by HDC1 and elucidated the patterns of HDC1-mediated regulation of gene expression.The main results are as follows:1.SF2 promotes cell proliferation of cucumber fruitWe identified recessive allelic variation in a cucumber HISTONE DEACETYLASE COMPLEX 1（HDC1）homologue,SF2,using a combination of the MutMap strategy and traditional linkage analysis.SF2 may be involved in histone deacetylation modification.The cell proliferation was repressed by the amino acid change from glycine（G）to glutamate（E）at residue 515（G515E）of SF2,resulting in a short fruit.Transgenic complementation and the knockout of SF2 showed that SF2 promotes cell proliferation during fruit development.Furthermore,SF2 protein is expressed in meristematic tissues in which rapid cell proliferation was occurring,suggesting that post-transcriptional regulation of SF2.The placenta maintain‘quasi-meristematic’activity and allow fruit cell proliferation.2.SF2 recruits HDAC complex and promotes histone deacetylationSF2 interacts with HDA19A,HDA19B,SIN3-LIKE1,SIN3-LIKE3,SAP18,and MSI1.SF2G515E shows an impaired capacity for interaction with SIN3-LIKE1 and SIN3-LIKE3,and the capacity of SF2-HDAC complex targeting to genomic regions decreased in the sf2 mutant by luciferase complementation imaging assays,Co-immunoprecipitation assay and ChIP-seq（Chromatin Immunoprecipitation sequencing）.The ChIP assays using anti-H3K9ac and anti-H3K14ac indicated that the relative histone acetylation levels increased.Accordingly,immunoblotting demonstrated that the levels of histone H3 acetylation increased in sf2 fruit,demonstrating that SF2 promotes histone deacetylation.3.Identification of the core target genes activated or repressed by SF2We obtained 321 core repressed genes and 237 core activated gene by the overlapping of genes bound by the HDAC complex,genes upregulated or downregulated in the sf2 mutant,and hyper-H3K9ac or hyper-H3K14ac genes,using RNA-seq and ChIP-seq data in WT（wild-type;406）and sf2 fruit.Furthermore,SF2 directly repressed expression of genes involved in hormone biosynthesis and signal transduction,including the negative regulators from the auxin,gibberellin（GA）and cytokinin（CK）pathways,and positive regulators from the Abscisic acid（ABA）,Jasmonate（JA）and poliamine（PA）pathways.SF2 also activated the expression of genes involved in cell cycle regulation,which is consistent with a role for SF2 in facilitating cell proliferation.Our results point to a general role for HDC1 in coordinating phytohormone signaling during cell proliferation of meristematic cells.4.SF2 core targets are involved in the regulation of fruit morphogenesisThe study related the expression patterns of SF2 core target genes in our transcriptome data during fruit development.The 236 genes（73.5%）from the core repressed gene set showed reduced expression levels during exponential cell proliferation（0 to 3 DAA）,indicating that the expression patterns of most of the core repressed genes were negatively related to cell proliferation.The 154 genes（65.5%）from the core activated gene set showed elevated expression levels during exponential cell proliferation,indicating that the expression patterns of most of the core activated genes were positively related to cell proliferation.The result is consistent with the role of SF2 in promoting cell proliferation,supporting the notion that the direct target gene networks of SF2 are involved in regulating cell proliferation during fruit development.5.SF2 regulates cell proliferation through its effect on CK and PA homeostasisThe CKX enzyme activity was significantly elevated in sf2,while the content of isopentenyladenine（iP）and dihydrozeatin（DZ）decreased.Exogenous treatment with thidiazuron（N-phenyl-N’-1,2,3-thiadiazol-5-yl urea,TDZ）,an inhibitor of CKX,partially complemented the short fruit phenotype.This is consistent with a role for SF2 in facilitating cell proliferation through modulation of CK contents via targeting of its metabolic（CKX7）.We also found that Spd levels were significantly higher in sf2 than in WT.Exogenous treatment with methyl-glyoxyl-bis guanylhydrazone（MGBG）,a competitive inhibitor of SAMDC,partially complemented the short fruit phenotype.The result indicated that SF2facilitates cell proliferation through targeting of SAMDC genes.The combined treatment of TDZ and MGBG on sf2 fruit resulted in an 80%increase in length,suggesting agonistic effects of CK and polyamines on rapid cell proliferation regulated by SF2.We revealed that SF2 controls fruit cell proliferation through directly targeting the biosynthesis and metabolism of CK and PA. |
