Potassium (K⁺) is a critical determinant of salinity tolerance, and H₂O₂ has been recognized as an important signaling molecule that mediates many physiological responses. However, the details of how H₂O₂ signaling regulates K⁺ uptake in the root under salt stress remain elusive. In this study, salt-sensitive cucumber and salt-tolerant pumpkin which belong to the same family, Cucurbitaceae, were used to answer the above question. We show that higher salt tolerance in pumpkin was related to its superior ability for K⁺ uptake and higher H₂O₂ accumulation in the root apex. Transcriptome analysis showed that salinity induced 5816 (3005 up- and 2811 down-) and 4679 (3965 up- and 714 down-) differentially expressed genes (DEGs) in cucumber and pumpkin, respectively. DEGs encoding NADPH oxidase (respiratory burst oxidase homolog D; RBOHD), 14-3-3 protein (GRF12), plasma membrane H⁺-ATPase (AHA1), and potassium transporter (HAK5) showed higher expression in pumpkin than in cucumber under salinity stress. Treatment with the NADPH oxidase inhibitor diphenylene iodonium resulted in lower RBOHD, GRF12, AHA1, and HAK5 expression, reduced plasma membrane H⁺-ATPase activity, and lower K⁺ uptake, leading to a loss of the salinity tolerance trait in pumpkin. The opposite results were obtained when the plants were pre-treated with exogenous H₂O₂. Knocking out of RBOHD in pumpkin by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9] editing of coding sequences resulted in lower root apex H₂O₂ and K⁺ content and GRF12, AHA1, and HAK5 expression, ultimately resulting in a salt-sensitive phenotype. However, ectopic expression of pumpkin RBOHD in Arabidopsis led to the opposite effect. Taken together, this study shows that RBOHD-dependent H₂O₂ signaling in the root apex is important for pumpkin salt tolerance and suggests a novel mechanism that confers this trait, namely RBOHD-mediated transcriptional and post-translational activation of plasma membrane H⁺-ATPase operating upstream of HAK5 K⁺ uptake transporters.

Melon (Cucumis melo) is one of the top 10 fruits in the world, and its production often suffers due to soil-borne diseases. Grafting is an effective way to solve this problem. However, graft incompatibility between scion and rootstock limits the application of melon grafting. In this study, the melon was grafted onto eight Cucurbitaceae species (cucumber, pumpkin, melon, luffa, wax gourd, bottle gourd, bitter gourd, and watermelon), and graft compatibility evaluation and anatomical observation were conducted. Taking melon homo-grafted plants as control, melon grafted onto cucumber and pumpkin rootstocks was compatible, while melon grafted onto luffa, wax gourd, bottle gourd, bitter gourd, and watermelon rootstocks was incompatible based on the scion dry weight on day 42 after grafting. Meanwhile, we found that starch–iodine staining of scion stem base is an index to predict graft compatibility earlier, on day 14 after grafting. Further, microsection observations showed that there was more cell proliferation at graft junction of melon hetero-grafted combinations; vascular reconnection occurred in all graft combinations. However, excess callose deposited at graft junction resulted in the blockage of photosynthate transport, thus, leading to starch accumulation in scion stem base, and finally graft incompatibility. In addition, undegraded necrotic layer fragments were observed at graft junctions of melon grafted onto incompatible bitter gourd and watermelon rootstocks. The above results provide clues for the selection and breeding of compatible Cucurbitaceae rootstocks of melon and demonstrate that starch accumulation in scion base and callose deposition at graft junction is associated with melon graft compatibility.

Vanadium (V) is the fifth most abundant transition metal, elevated levels of V are hazardous to plants. Boron (B) is an essential micronutrient for plants and can mitigate heavy metal toxicity. However, the mechanism used by B to promote tolerance to vanadium is unknown. In this study, a combination of physiological and gene expression analysis was used to explain mechanism of B (75 µM) induced V (40 mg L^-1) stress tolerance in watermelon. V stress severely reduced root and shoot growth and increased the accumulation of ROS. B application improved tolerance to V by enhancing the expression of B transporter genes (ClaNIP5;1-1, ClaNIP5;1-2, ClaBOR4) that facilitated B uptake and transport while restricting V transport in plant tissues. At cellular level, the higher V retention in leaves was achieved by cell wall chelation, whereas, the higher V exclusion in vacuole of root cell was driven by elevated vacuolar H⁺-ATPase, H⁺-PPase activities, and transcript level of ClaVHP1;1, ClaPDR12-1 and ClaPDR12-2 genes facilitated by B application. Moreover, B application reduced tissue ROS cascade by enhancing antioxidant enzymatic activity and expression of superoxide dismutase (ClaCSD1-1, ClaCSD1-2, ClaCSD3, ClaMSD1) and catalase (ClaCAT2-1, ClaCAT2-2) genes that enhanced the defense mechanism of the V treated plants, improved root and shoot growth and tolerance index of watermelon. In conclusion, we demonstrate that ameliorative effect of B in tolerance to V of watermelon was based on B homeostasis and improved antioxidant defense system. These findings might help to increase watermelon production in V polluted soils.