| The annual growth cycle of grape is divided into growth period and dormancy period.Xylem sap ensures survival during the dormancy period,and its flow is correlated with the new sprout,called bleeding period.Nutrient supply before germination and resistance to various biotic and abiotic stresses depend on the role of bleeding cell sap.However,due to shorter sap bleeding period and difficult to sampling,has always been facing a difficulties in grape research.The physiological active ingredients were explored during the last decades,but the specific life activities and physiological functions not yet reported.In order to entirely understand the mechanism of xylem sap during bleeding period,this study explored the biological functions of bleeding sap through the rule of sap flow,the identification of active components and microbial community,the in vitro verification of sap function,the isolation of endophyte,and the functional analysis of chitinase gene family which is one of the most important stress resistant protein.1.The rules of sap flow in grapevine,its inner-and intra-diurnal changes with temperature,humidity,branch length were studied and their correlations were simultaneously analyzed during the bleeding period.In addition,in order to reveal the effects of sap flow on vine growth,relationship among the flows in different vine parts were studied.The results showed that the total bleeding volume ranged from 380 to 540 mL in Rosario Bianco,and the flow lasted for 35?47 d.Flow speed increased first followed by a decrease,and the peak period maintained around 10 d.Branch length greatly affected the bleeding volumes.The sap flow was stable in the longer branch,which mainly contributed to flow;whereas the shorter branch stopped flowing at the earliest time.The flow volume of the bleeding sap was stable and evenly distributed in each period.The total bleeding volume varied with bleeding period and differently distributed inner day.During early bleeding period,proportion was gradually increased in the morning and the bleeding volume was bigger in the daytime than in the evening.However,the situation was the opposite in the late bleeding period.In addition,we also observed that the branch length was directly correlated with the flow speed.The flow speed was faster in the longer branch when compared with the shorter ones.The branch position had no significant effect on the flow speed.The total flow volume was always greater in branches than trunk.To conclude,the assumed sap flow model was proposed based on the above results,which would lay foundation for the further studies on bleeding sap.2.Qualitative and quantitative detection were carried out.The results demonstrated that bleeding sap is slightly acidic in nature and pH ranges from 5.2 to 5.5.The main sugar detected are fructose,glucose,and lactose and their total concentration ranged from 420?3100 mg L-1.The contribution of major organic acids,such as oxalic acid,tartaric acid,malic acid,lactic acid,and citric acid content was 400?1400 mg L-1 and.Additionally,16 different amino acids detected in the bleeding sap,including the essential amino acids threonine and glutamic acid.Different types of mineral elements were identified and were in the order of potassium>calcium>phosphorous>sulfur>magnesium>sodium>manganese>zinc>copper>iron.Nine different hormones exhibited an increasing trend during the bleeding period with the content of ABA>IAA>JA>ZR>IPA>GA4>DHZR>GA3>BR.3.Explore the effect of bleeding cell sap on grapes.Yearly cuttings were cultured in bleeding sap,in addition with MS medium and H2O.Our results showed that the bleeding sap and MS could promote the germination of cuttings and up-regulate the dormancy related genes.Moreover,the cuttings grew stronger under the treatment of bleeding sap.However,it was confirmed via plate co-cultivation that the bleeding sap had a certain resistance to Botrytis cinerea,and there was no significant effect on the disease incidence resistance during the whole bleeding period.In order to explore the roles of grape endophytes played in grey mould resistance,Rhodotorula mucilaginosa,Cryptococcus,Rahnella aquatilis and Serratia proteamaculans were isolated and purified from grape bleeding sap by morphological observation,physiological characterization and 16S rDNA sequence analysis.In vitro antibacterial effects of the isolated endophytes were tested via plate co-cultivation and grape berry co-infection of both grey mould and the individual endophyte.The results indicated strong inhibition of grey mould's growth by Cryptococcus,Rahnella aquatilis and Serratia proteamaculans.These results are of practical interest for improving grapevine resistance to grey mould.4.Expression profile and macro transcriptome were analyzed to further understand the microbial community of xylem sap and the growth activities during bleeding period.The results showed that the RNA in xylem sap during bleeding period was little and also the growth was weak.Through DEGs analysis,metabolic pathways,biosynthesis of secondary metabolites,plant hormone signal transduction and plant pathogen interaction are the four pathways with the largest number of enriched genes,which represents that promote plant growth and development and resistant to pathogens were the main function of xylem sap.The microbial components in xylem sap are abundant,and mainly involved in disease resistance,RNA transportation,carbon source metabolism,hormone signal transduction pathways,closely related to sap transport and germination.5.Study the classification,function and its response to various hormones and stress treatment of chitinase,which was the most important resistance-associated protein in xylem sap during bleeding period.In this study,we identified 42 Chitinase genes from grape genome-wide data by bioinformatics analysis,and divided into five groups:A,B,C,D and E according to evolutionary relationships,protein and gene structure.Quantitative real-time reverse transcription PCR(qRT-PCR)and publicly available microarray data analysis revealed distinct temporal and spatial expression patterns of VvChis in various tissues,organs and developmental stages.Combining promoter action element,GO and KEGG analysis,and prediction of interaction proteins,we revealed the function of Chitinase.The interaction between Chi and MTL was confirmed by yeast two-hybrid and bimolecular fluorescence complementation.The transient expression techniques of cherry tomato and strawberry were used to analyze the disease resistance of five Chitinase genes in climacteric respiration and non-climacteric respiration fruits,and finally we found the important role of VvChi31 and VvChi17 in postharvest disease resistance respectively.The activity of chitinase is mostly likely affected by biotic and abiotic stresses.One the bases of classification of grape chitinase genes,we investigated the response of five classes of VvChis to biotic and abiotic stresses.The grape fruits and leaves were inoculated with Botrytis cinerea,along with chitin treatment were carried out to explore the effects of different treatments on the disease resistance.In addition,different concentrations of hormones,PEG,NaCl,NaOH,and at different temperature and UV exposure time were used to treat fruits.Combined with defense enzymatic activities,the response of VvChis to five abiotic stresses were analyzed.The results showed that all VvChis could respond to the infection of Botrytis cinerea.All genes in the corresponding groups of A,B and E were up-regulated followed by IAA,SA,MeJA and ETH,especially under 500 mg·L-1 ETH?50?mol·L-1 MeJA treatment.The C and D group's genes had no obvious characteristics for hormones and their concentrations,except for the consistent response to IAA 0.1.Under abiotic stress conditions,VvChis were effectively responded to low temperature,acid-base condition and higher NaCl concentration,while the response of skins and flesh to osmotic stress was different.After chitin treatment,the VvChis in group A and group B were down-regulated,and the expression of 19 family genes was considerably higher than rest of18 families.Among them,1.5%chitin treatment had the best disease resistant effects was significantly better than 1.5%chitosan.The expression of VvChi31 is positively correlated with disease resistance,which can be used as a marker gene. |
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