Analyzing the temporal response mechanisms of the vascular bundles formation of grafted cucumber to different light intensity modes: A joint transcriptomic and metabolomic approach

Abstract

In plant grafting, the reconnection of vascular bundles between the rootstock and scion not only establishes functional xylem and phloem connections, but also signifies the completion of graft healing. In our previous studies focused on early-stage morphology and physiology of grafted seedlings, we have demonstrated that utilizing light intensities of 50-100-150 μmol/(m2·s) during days 1-3, 4-6, and 7-9 optimizes cucumber/pumpkin graft healing. To further explore the underlying mechanisms, we investigate light intensity regimes: 50-100-100 μmol/(m2·s) and 50-100-150 μmol/(m2·s) during the same time frames (days 1-3, 4-6, and 7-9 after grafting), denoted as CK and T treatments. Initially, we observe histological characteristics of vascular bundle formation under these conditions, followed by the analyses of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione peroxidase (GSH-Px) enzyme activities, ascorbic acid (AsA) content in the antioxidant system, and the sequencing of transcriptome and metabolome. It is found that grafted seedlings under the T treatment achieve faster vascular bundle reconnection and shorter healing process compared to those under the CK treatment. Enzyme activities (POD, SOD, GSH-Px) increase over time under both light regimes, while CAT activity and AsA content decrease. Notably, by the 9th day, the T treatment (T9d) exhibits higher SOD, POD and CAT activities than the CK treatment (CK9d). Our findings indicate that increasing light intensity during the days 7-9 after grafting can mitigate oxidative damage at the grafting site and benefit vascular bundle reconnection. Transcriptome and metabolome analyses show the up-regulations of Cinnamaldehyde in the phenylpropanoid biosynthesis pathway, the PHGDH gene and L-Homocysteine in the cysteine and methionine metabolism pathway under T treatment in the days 7-9 after grafting. These enhance the antioxidant enzyme activities and reduce the oxidative damage to the grafting site. Additionally, GAE-related genes, and metabolites D-Xylose and Undecaprenyl phosphatealpha-L-Ara4FN in the amino sugar and nucleotide sugar metabolism pathways are also up-regulated under T treatment. The ethylene responsive factor (ERF) transcription factor CsaRAP2.3 positively regulates Undecaprenyl phosphatealpha-L-Ara4FN expression, promoting cell wall synthesis and therefore accelerating vascular bundle reconnection. In summary, during the vascular bundle formation period of cucumber graft healing, both light intensity modes coordinate regulation of phenylpropanoid biosynthesis, cysteine and methionine metabolism, amino sugar and nucleoside sugar metabolism, and ERF transcription factors, promoting graft healing from both stress resistance and cell wall synthesis. Furthermore, building upon the light intensity applied during the callus formation phase, increasing the light intensity during the vascular bundle formation period can significantly enhance cell wall synthesis, thus expediting the graft healing process. Nonetheless, the generalizability of these research findings to other grafting techniques and species requires further investigation.