Mechanism Analysis On Grafting Affinity Between Cucumber And Pumpkin Based On Proteomics And Transcriptomics

Cucumber(Cucumis sativus L.)is one of the most popular cultivation conditions,and is sensitive to various biotic and abiotic stress.In the present study,the effectiveness of grafting on excellent resistance rootstock can increase cucumber stress tolerance,the technology of graft breeding is widely used to practical production.The excellent resistance rootstocks of cucumber is not enough and graft incompatibility limits the effective use of superior rootstocks.Study of the mechanism of affinity on cucumber almost rest on the aspects of physiology and biochemistry,however reports on aspects of molecular biology are rare.'Dongyangshenli'(Cucurbita moschata D.)and 'Heizinangua'(Cucurbita ficifolia Bouche),two pumpkin rootstocks with significantly different compatibility,were acquired through screening of resistant rootstocks according to graft survival rate and plants quality.And subsequently molecule mechanism of graft affinity was demonstrated from two aspects of proteomics and transcriptomics used 'Jinchun No.4' as scion,'Dongyangshenli' and'Heizinangua' as rootstock,non-grafted and self-grafted cucumber seedlings as control.The main results are as follows:1.The effect of grafting with 15 resistant pumpkin rootstocks on cucumber survival rate and plants quality were studied used ' Jinchun No.4' as scion.Finally,a comprehensive evaluation on cucumber plants quality of grafting affinity was given according to subordinate function values analysis.The results indicates that the survival rate of cucumber plants grafted with 'Dongyangshenli' was obviously lower than the other combinations and the quality of cucumber plants grafted with 'Heizinangua' performed best and 'Dongyangshenli' grafted combination performed worst.The comprehensive evaluation suggests that 'Heizinangua' rootstock perform better compatibility and 'Dongyangshenli'rootstock perform worse compatibility when grafted with cucumber scion.2.Using 'Jinchun No.4' as scion,'Dongyangshenli' and 'Heizinangua' as rootstock,non-grafted cucumber seedlings as control,the effects of compatibility or incompatibility on histological aspects,antioxidant enzyme activities,phenylpropanoid contents,and chlorophyll fluorescence were studied.The results showed that compatible graft combinations present a stronger resistance to the oxidative damage resulting from grafting and had relatively weak phenylpropanoid metabolisms.The results also indicated that the chlorophyll fluorescence levels of incompatible combinations were lower,except compared with the original fluorescence.Finally,a necrotic layer existed earlier in compatible graft combinations.These differences at the morphological,physiological,and cellular levels may govern compatibility and incompatibility.3.Differences in leaf proteomes of graft-compatible and graft-incompatible cucumber pumpkin combinations were investigated using two-dimensional gel electrophoresis(2-DE)and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry(MALDI-TOF/TOF MS).Cucumber seedlings 'Jinchun No.4' were used as the scions and two pumpkin cultivars,'Dongyangshenli' and 'Heizinangua',were used as the rootstocks.Non-grafted and self-grafted cucumber seedlings served as control groups.An average of approximately 500 detectable spots were observed on each 2-DE gel.A total of 50 proteins were differentially expressed in response to self-grafting,compatible-rootstock grafting,and incompatible-rootstock grafting and were all successfully identified by MALDI-TOF/TOF MS.The regulation of Calvin cycle,photosynthetic apparatus,glycolytic pathway,energy metabolism,protein biosynthesis and degradation,and reactive oxygen metabolism will probably contribute to intensify the biomass and photosynthetic capacity in graftcompatible combinations.The improved physiological and growth characteristics of compatible rootstock grafting plants are the result of the higher expressions of proteins involved in photosynthesis,carbohydrate and energy metabolism,and protein metabolism.At the same time,the compatiblerootstock grafting regulation of stress defense,amino acid metabolism,and other metabolic functions also plays important roles in improvement of plant growth.4.The effect of different pumpkin rootstocks grafting on transcriptome of cucumber leaves was studied by the high-throughput sequencing technologies.Finally,we found 223 DEGs related to graft affinity.Compared with incompatible graft combinations,82 DEGs were up-regulated and 141 DEGs were down-regulated in leaves of compatible graft combinations.Such DEGs were involved in processes such as metabolic process,protein metabolism,transport,transcriptional regulation,oxidation-reduction,defense response and signal transduction.Moreover,compatible rootstock grafting may strengthen the cell proliferation ability by increasing expression of DEGs involved in carbohydrate metabolism and cell wall,then promoting plant growth.Incompatible rootstock grafting may respond to stress caused by graft incompatibility via increasing expression of DEGs related to oxidation-reduction,defense response and kinase.