Analysis And Functional Characterization Of Differential Genes In Stone Cell Development Between Pyrus Bretschneideri Cv. Dangshan Su And Pyrus Bretschneideri Cv. Lianglizaosu

As a characteristic,famous and superior fruit tree of Anhui Province,Pyrus bretschneideri cv.Dangshan Su is the largest diploid pear variety in China,but the inherent defects in the high content of large-diameter stone cell clusters in the fruit have significant negative effects on texture quality and flavor.In addition,the large-diameter and high content of stone cell clusters are also the common problems of some P.pyrifolia and P.ussuriensis cultivars.Therefore,reducing the size and content of the stone cell clusters of pear fruit is an urgent problem to be solved in the pear industry in China.Studying the molecular mechanism of pear stone cell development and lignification will provide theoretical basis and technical methods for regulating the formation of pear stone cell cluster and improving pear quality.In this study,Pyrus bretschneideri cv.Dangshan Su and Pyrus bretschneideri cv.Lianglizaosu(low-stone cell content bud sport of ’Dangshan Su’)were used as materials.The differences in stone cell development and lignification between the two pear varieties were analyzed by histochemical staining and ultramicroscopic observation.Through whole-genome resequencing and transcriptome sequencing,genes with sequence variation and transcriptional difference were identified.On the basis of this,combined with bioinformatics methods,candidate genes related to the development and lignification of pear stone cell clusters were screened.Finally,the function of key candidate members from the CAD(cinnamyl alcohol dehydrogenase),LIM transcription factor,and DIR(Dirigent)families were investigated.These findings are intended to further reveal the function and mechanism of PbCAD,PbLIM and PbDIR genes in the development and lignification of pear stone cells,and to explore the mechanism of pear stone cell formation.The main findings are as follows:1.The results of Wiesner staining showed that the distribution and density of stone cell clusters in the fruit of ’Dangshan Su’ were higher than that of ’Lianglizaosu’.Transmission electron microscopy showed that the stratification of lignin and cellulose micro fibrils in the mature stone cells of‘Dangshan Su’was more pronounced than that of‘Lianglizaosu’.In addition,a large number of vesicles(presumed to be autophagic vacuoles and transport vesicles)are formed during the formation of pear stone cells,and it is believed that the development of pear stone cells may be a process of co-operative lignification.2.Based on the bioinformatics and transcriptome sequencing analysis of different developmental stages of‘Dangshan Su’and‘Lianglizaosu’fruits,the expression profiles of 16 lignin metabolism-related gene families at different developmental stages were systematically studied.A comprehensive analysis of the expression profiles of each gene and the stone cells and lignin content of pear fruit revealed that there were 43 genes involved in the synthesis and transport of lignin during the formation of pear stone cells.In addition,transcription factors such as MYBs,NACs,and LIMs are also involved in the regulation of this metabolic process.The differential expression of these genes may be one of the reasons for the difference in the size and content of the stone cell clusters of the two pear varieties.3.Whole-genome resequencing showed that the genes for non-synonymous mutation and frameshift mutation in the coding sequence between ’Dangshan Su’ and ’Lianglizaosu’genomes involved biological processes such as phenylpropanoid biosynthesis,plant hormone signal transduction,and starch/sucrose metabolism.In addition,SNP,Indel or SV mutations were found in both coding and non-coding regions of genes(PbPAL,PbC4H,PbF5H,PbCAD,PbUGT,and PbPRX)involved in the synthesis,transport and polymerization of monolignols between two pear varieties.These mutations may lead to changes in the transcriptional level and catalytic function of lignin metabolism-related genes,resulting in differences in stone cell formation between the two pear varieties.4.PbCAD2 has higher transcription levels in pear fruits,while PbCAD3 is mainly expressed in buds.The lignin content,secondary cell wall thickness and lignification degree of the inflorescence stems of PbCAD2-and PbCAD3-overexpressing transgenic Arabidopsis were higher than those of wild-type Arabidopsis(WT).The ratio of conifer aldehyde/coniferyl alcohol content and the ratio of sinapoyl aldehyde/sinapyl alcohol content in PbCAD2-and PbCAD3-overexpressing transgenic Arabidopsis were lower than those in WT.This indicates that PbCAD2 and PbCAD3 have functions to promote lignin synthesis and secondary cell wall development.In addition,there was no significant difference in the driving activities of the PbCAD2 and PbCAD3 promoters(pPbCAD2 and pPbCAD3)in tobacco leaves.pPbCAD2 and pPbCAD3 can drive GUS constitutive expression in different parts of Arabidopsis seedlings.With the development,pPbCAD2 and pPbCAD3 mainly drive GUS specifically expressed in the xylem,vein and root vascular bundle of Arabidopsis,and also have driving activity in siliques and floral organs.5.We identified 14,14,9,12 and 12 members of the LIM family from the pear,grape,strawberry,peach and mei genomes,respectively.Analysis of tissue specificity and temporal expression patterns shows that the PbLIMs in the WLIM1 and WLIM2 subclades are widely expressed in different tissues,whereas the LIMs of the δLIM2 subfamily(PLIM2 and PLIM2-like subclades)are mainly expressed in flowers.The transcription of 14 PbLIMs was regulated by SA,ABA and MeJA.Transcriptome sequencing showed that Pb WLIM1a and Pb WLIM1b had higher transcription levels in pear fruit,and the expression levels were significantly different in the fruits of‘Dangshan Su’and‘Lianglizaosu’at 55 days after flowering.Through phylogenetic analysis,protein structure prediction and temporal-spatial expression patterns,it was suggested that PbWLIM1a and PbWLIM1b play key roles in lignin biosynthesis and stone cell development in pear fruit.6.Electrophoretic mobility shift assays showed that both PbWLIMla and PbWLIM1b can recognize and interact with conserved PAL-box elements.PbWLIMla binds to the PAL-box in the PbCAD2 and PbCAD3 promoters,while PbWLIM1b binds primarily to the PAL-box in the PbCAD2 promoter.This indicates that PbWLIMla and PbWLIM1b can regulate PbCAD2 and PbCAD3 transcription by binding to PAL-box.Subcellular localization analysis indicated that PbWLIMla and PbWLIM1b were localized to chloroplasts in tobacco.Overexpression of PbWLIM1a or PbWLIM1b in Arabidopsis can increase the expression levels of lignin synthesis,transport,and polymerization-related genes to varying degrees.7.35,29,26 and 14 DIRs were identified from the pear,peach,mei and grape genomes,respectively.Among them,35 PbDIRs were divided into four subfamilies(DIR-a,-b/d,-e,and-g)and irregularly distributed among 10 chromosomes.Temporal expression analysis showed that the expression changes of seven PbDIRs(DIR-a subfamily:PbDIR4 and PbDIR5;DIR-b/d subfamily:PbDIR11;DIR-g subfamily:PbDIR19;DIR-e subfamily:PbDIR23,25 and 26)in fruits were consistent with the changes of fruit lignin and stone cells contents.In addition,the subfamily of PbDIRs in fruits showed significant responses after treatment with ABA,SA,and MeJA.In vitro coniferyl alcohol coupling experiments demonstrated that PbDIR4 could direct conversion of coniferyl alcohol into(+)-pinoresinol.According to the protein tertiary structures,key amino acid residues,coupling properties and expression patterns analysis found that PbDIR4 might be involved in the polymerization of lignin monomers during the development of pear fruit cells.