Expression And Functional Analysis Of Xyloglucan Endotransglycosylase/Hydrolase Genes In Persimmon Fruit

Persimmon?Diospyros kaki L.?fruit is quite perishable and susceptible to softening after harvest,which leads to deteriorated quality and major economic losses.Xyloglucan is a major hemicellulose molecule,which can limit the expand of cell wall and strengthen the rigidity of the cell wall by forming a skeletal network with cellulose fibrils.XTH,an important enzyme involved in xyloglucan metabolism,exhibits xyloglucan endotransglycosylase and/or xyloglucan endohydrolase activities,and plays important roles in plant growth,fruit softening anddefense response.Research on XTH is lack of deepmechanism,and there are few reports on the identification and function analysis of XTH genes in persimmon.In this study,we firstly isolated the key XTH genes from persimmon fruit by expression analyses,which suggested to play important roles in fruit growth and softening.Then,the mechanism of key XTHs action was explored at enzymatic level using recombined proteins and subcellular localization.On this basis,we explored the function of persimmon DkXTH1 and DkXTH8 genes to provide the roles of XTH in plant growth,fruit softening and defense response.The main results are listed as followings.1.Four full-length XTH sequences,designated DkXTH4-8,were isolated from‘Fupingjianshi'by RACE amplification.Similar to DkXTH1-4,the new persimmon XTH genes containedcommon features with other XTHs gained from other plants.Phylogenetic analysis revealed thatDkXTH1-8 genes were classified in group?/?.In predicted three-dimensional structures of DkXTH1 and DkXTH2,the loop 2 had five amino acids equal to PttXET16A,which shown XET activity,and less than TmNXG1,which contained strict XEH activity.2.Different tissues and‘Fupingjianshi'fruits with different developmental stages were sampled to investigate the expression patterns of persimmon XTH genes?DkXTH1-8?.QPCR analysis revealed that the expression of DkXTH5 and DkXTH1 peaked in the fruit with the first and second maximal growth rates,respectively.Likewise,DkXTH4 expression shown a maximum value along with both the first and second maximal growth rates,which suggested toparticipate in fruit growth.Furthermore,the DkXTH1 mRNA was expressed at an extremely high level in both leaf and fruit fast growing tissues,which was positively associated with XET activities.However,the expression level of DkXTH2,DkXTH3,DkXTH6 and DkXTH8was very high in mature fruit than that in immature growing stage,which should play important roles in fruit softening.In particular,the expression pattern of DkXTH8 appeared parallel torapid decline in firmness,which was notably detectable in fruits,with very little expression in other tissues,such as flower,leaf,steam andcalyces.3.The expression profiles of DkXTH1 and DkXTH8 were examined in response to multiple hormonal and cold environmental treatments.DkXTH1 shown low expression levels during persimmon fruit postharvest storage.However,it was positively up-regulated by GA₃and cold treatment,which should have important roles in maintaining cell wall integrity and delaying fruit firmness decline.In contrast,DkXTH8 exhibited its high transcript levels during persimmon fruit softening,which was positively associated with rapid decline in firmness.Expression of DkXTH8 was positively up-regulated by propylene and ABA treatments,but suppressed by GA₃ and cold treatment.4.The recombined DkXTH1 and DkXTH8 proteins were gained byprokaryotic expression.After purified using a Ni-NTA resin column,both DkXTH1 and DkXTH8 soluble proteins showed significant XET activity without any detected XEH activity.DkXTH1 and DkXTH8proteins were most active for pH at 5.5 and 6.0,respectively,witha rapid loss of activity when the pH ranged from 4 to 5.However,the XET activity of recombined DkXTH8 protein had a higher affinity for small acceptor molecules than that of recombined DkXTH1 protein.Both DkXTH1 and DkXTH8 proteins could function by targeting to the cell wall under regulation of a signal peptide.5.Overexpression of DkXTH1 enhanced tolerance to abiotic stresses in transgenic Arabidopsis plants and delayed transgenic tomato fruits softening.Transgenic Arabidopsis plants overexpressing DkXTH1showed significantly increased levels of seed germination under high salinity,ABA and drought stress compared to WT.DkXTH1-transgenic seedlings and mature plants showed significantly higher chlorophyll content and lower MDA content after the application of abiotic stress.Transgenic tomatoes presented delayed fruit softening coupled with postponed color change,a lower ethylene peak and genes expression levels,and higher firmness in comparison with the WT tomatoes during storage.Microscopic observation revealed that transgenic plants exhibited higher density of cell wall and intercellular spaces,which suggested to maintain cell wall structural integrity.6.Overexpression of DkXTH1 promoted leaves and stems growth in transgenic Arabidopsis plants and enhanced transgenic tomato fruits expansion.Broader leaves were gained in transgenic Arabidopsis and stems contained more large cells than that in WT.Transgenic tomato fruits collected at mature stage had larger diameter,resulting from the overactivity of DkXTH1 involving in cell wall assembly.7.Overexpression of DkXTH8 resulted in increased leaf senescence and faster fruit ripening and softening rates in tomato.Leaf senescence was promoted in DkXTH8-transgenic Arabidopsis,coupled with higher chlorophyll degradation,electrolyte leakage and MDA content.Meanwhile,both AtSAG12 and AtSAG13 shown higher expression levels in transgenic plants than that in WT,which expression were strictly associated with senescence.Transgenic tomatoes presented faster color change,firmness decline and destroyed cells during storage.Most importantly,transgenic plants overexpressing DkXTH8 displayed more irregular and twisted cells due to cell wall restructuring,resulting in wider interstitial spaces with less compact cells.We suggest that DkXTH8 expression causes cells to be easily destroyed,increases membrane permeability and cell peroxidation,and accelerates leaf senescence and fruit softening in transgenic plants.