Grafting with pumpkin rootstock is commonly used not only to improve the quality of cucumber fruits but also to confer biotic or abiotic stress tolerance. However, the molecular mechanism of grafted cucumbers to drought stress and the possible roles of mobile mRNAs to improve stress tolerance have remained obscure. Hence, we conducted transcriptome sequencing and combined it with morpho-physiological experiments to compare the response of homografts (cucumber as scion and rootstock) (C) and heterografts (cucumber as scion and pumpkin as rootstock) (P) to drought stress. After applying drought stress, homografts and heterografts expressed 2960 and 3088 genes in response to drought stress, respectively. The identified DEGs in heterografts under drought stress were categorized into different stress-responsive groups, such as carbohydrate metabolism (involved in osmotic adjustment by sugar accumulation), lipid and cell wall metabolism (involved in cell membrane integrity by a reduction in lipid peroxidation), redox homeostasis (increased antioxidant enzymes activities), phytohormone (increased ABA content), protein kinases and transcription factors (TFs) using MapMan software. Earlier and greater H2O2 accumulation in xylem below the graft union was accompanied by leaf ABA accumulation in heterografts in response to drought stress. Greater leaf ABA helped heterografted cucumbers to sense and respond to drought stress earlier than homografts. The timely response of heterografts to drought stress led to maintain 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 pumpkin-rooted plants.

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Heat shock protein 70s (HSP70s) are highly conserved proteins that are involved in stress responses. These chaperones play pivotal roles in protein folding, removing the extra amounts of oxidized proteins, preventing protein denaturation, and improving the antioxidant system activities. This conserved family has been characterized in several crops under drought stress conditions. However, there is no study on HSP70s in pumpkin (Cucurbita moschata). Therefore, we performed a comprehensive analysis of this gene family, including phylogenetic relationship, motif and gene structure analysis, gene duplication, collinearity, and promoter analysis. In this research, we found 21 HSP70s that were classified into five groups (from A to E). These genes were mostly localized in the cytoplasm, chloroplast, mitochondria, nucleus, and endoplasmic reticulum (ER). We could observe more similarity in closely linked subfamilies in terms of motifs, the number of introns/exons, and the corresponding cellular compartments. According to the collinearity analysis, gene duplication had occurred as a result of purifying selection. The results showed that the occurrence of gene duplication for all nine gene 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 up-stream region of the HSP70 genes, such as hormones and stress-responsive elements, indicating a potential role of this gene family in stress tolerance. We furtherly performed the gene expression analysis of the HSP70s in pumpkin under progressive drought stress. Pumpkin is widely used as a rootstock to improve stress tolerance, as well as fruit quality of cucumber scion. 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. The results indicated that all CmoHSP70s had very low expression levels at 4 days after stress (DAS). However, the genes showed different expression patterns by progressing he drought period. For example, the expression of CmoHSP70-4 (in subgroup E) and CmoHSP70-14 (in subgroup C) sharply increased at 6 and 11 DAS, respectively. However, the expression of all genes belonging to subgroup A did not change significantly 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 on the function of this gene family, especially under stressful conditions.

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