| Drought stress is one of the most serious challenges for crop production which can reduce yield and quality of crops drastically.Cucumber（Cucumis sativus L.）,has wide leaves,high transpiration rate,and shallow root system,so high amount of water is needed for its optimum growth and development.These characteristics make cucumbers sensitive to drought stress,and long-term water deficiency condition significantly decreases fruit yield and quality of cucumbers.Plant grafting is a powerful technique which allows us to gather the desired and beneficial traits of different compatible species in one plant.By this method a root system of a sensitive species can be replaced with a tolerant rootstock which furtherly could improve drought tolerance.Pumpkin（Cucurbita moschata）is a powerful and tolerant rootstock which is able to improve stress tolerance of sensitive species such as cucumbers.Moreover,plant grafting is an ideal and simple method to study the long-distance signaling between distance organs such as root and shoot under drought stress.Long-distance movement of stress signals such as mobile mRNAs,proteins,peptides,and reactive oxygen species（ROS）between root and shoot can induce the stress modification responses in the shoot.However,the molecular mechanism of controlling effects of rootstock on scion under drought stress has remained obscure.In this study,we physically joined cucumber onto its own rootstock（homografts）and cucumber onto pumpkin rootstock（heterografts）using the grafting technique and explored the molecular and physiological mechanisms that pumpkin-rooted plants employ to improve drought tolerance in cucumber scion.We also investigated the possible roles of pumpkin-sourced mobile mRNAs,hydrogen peroxide,and ABA in the induction of stress tolerance in cucumber scion.The main research results are as follows:1.Physiological and molecular responses of grafted cucumbers to drought stress1.1 The results of physiological experiments showed that,compared to cucumber-rooted plants,drought tolerance in pumpkin-rooted plants can be achieved by an efficient osmotic adjustment method,better cell membrane integrity,increased antioxidant enzymes activities,and higher accumulation of ABA phytohormone in the leaves.Our physiological results supported the transcriptomic results.These results indicated that drought tolerance as a complex trait is achieved by changing the expression level of many stress-responsive genes that belong to various metabolic processes.1.2 In comparison with homografts,earlier and greater H2O2accumulation in the xylem below the graft union was accompanied by leaf ABA accumulation in heterografts in response to drought stress.ABA accumulation in the leaves helped heterografted cucumbers to sense and respond to drought stress earlier than homografts.The timely response of heterografts to drought stress led to maintaining higher water content in the leaves even in the late stage of drought stress.The identified mobile mRNAs（mb-mRNAs）in heterografts were mostly related to photosynthesis which would be the possible reason for improved chlorophyll content and maximum photochemical efficiency of PSII（Fv/Fm）.The existence of some stress-responsive pumpkin（rootstock）mRNAs in cucumber（scion）,such as heat shock protein（HSP70,a well-known stress-responsive gene）,led to the higher proline accumulation than homografts.The expression of the mobile and immobile stress-responsive mRNAs and timely response of heterografts to drought stress could improve drought tolerance in homografts.1.3 Transcriptome sequencing results showed that the use of pumpkin as rootstock differentially changed the expression level of a huge number of genes in response to drought stress compared to well-watered conditions.The identified DEGs in heterografts under drought stress were categorized into different stress-responsive groups,such as carbohydrate metabolism,lipid and cell wall metabolism,redox homeostasis,phytohormone,protein kinases,and transcription factors（TFs）.These DEGs subsequently triggered the corresponding modifications to induce drought tolerance.2.Genome-wide study and expression analysis of HSP70s in pumpkin rootstock under drought stress2.1 We further carried out the genome-wide study and expression analysis of the HSP70gene family of pumpkin rootstocks.We found 21 HSP70s that were classified into 5 groups.These genes were mostly localized in the cytoplasm,chloroplast,mitochondria,nucleus,and endoplasmic reticulum（ER）.We observed more similarity in closely linked subfamilies in terms of motifs,number of introns/exons,and the corresponding cellular compartments.According to the collinearity analysis,gene duplication occurred as a result of purifying selection.The results showed that the occurrence of gene duplication for all 9gene pairs was due to segmental duplication（SD）.Synteny analysis revealed a closer relationship between pumpkin and cucumber than pumpkin and Arabidopsis.Promoter analysis showed the presence of various cis-regulatory elements in the upstream region of the HSP70 genes,such as hormones and stress-responsive elements,indicating the potential role of this gene family in stress tolerance.2.2 Since stress-responsive mobile molecules translocate through vascular tissue from roots to the whole plant body,we used the xylem of grafted materials to study the expression patterns of the HSP70（potentially mobile）gene family under progressive drought stress conditions.The results indicated that all Cmo HSP70s had very low expression levels 4 days after stress（DAS）.However,the genes showed different expression patterns by progressing the drought period.However,the genes belonging to the same subgroup mostly showed a similar expression pattern in response to drought stress.These findings indicated the diverse roles of this gene family under drought stress and provided valuable information for further investigation of the function of this gene family,especially under stressful conditions. |
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