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.

中文翻译：

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分析嫁接黄瓜维管束形成对不同光强模式的时间响应机制：一种转录组学和代谢组学的联合方法


在植物嫁接中，砧木和接穗之间的维管束重新连接不仅建立了功能性木质部和韧皮部连接，还标志着嫁接愈合的完成。在我们之前专注于嫁接幼苗早期形态学和生理学的研究中，我们已经证明在第 1-3、4-6 和 7-9 天利用 50-100-150 μmol/（m2·s） 的光强度可优化黄瓜/南瓜嫁接愈合。为了进一步探索潜在的机制，我们研究了光强度范围：50-100-100 μmol/（m2·s） 和 50-100-150 μmol/（m2·s） 在同一时间范围内（移植后第 1-3 天、第 4-6 天和第 7-9 天），表示为 CK 和 T 治疗。最初，我们观察这些条件下血管束形成的组织学特征，然后分析超氧化物歧化酶 （SOD）、过氧化物酶 （POD）、过氧化氢酶 （CAT）、谷胱甘肽过氧化物酶 （GSH-Px） 酶活性、抗氧化系统中的抗坏血酸 （AsA） 含量，以及转录组和代谢组的测序。研究发现，与 CK 处理下的嫁接幼苗相比，T 处理下的嫁接幼苗实现了更快的维管束再连接和更短的愈合过程。在两种光照条件下，酶活性 （POD、SOD、GSH-Px） 随着时间的推移而增加，而 CAT 活性和 AsA 含量降低。值得注意的是，到第 9 天，T 处理 （T9d） 表现出比 CK 处理 （CK9d） 更高的 SOD 、 POD 和 CAT 活性。我们的研究结果表明，在嫁接后第 7-9 天增加光强度可以减轻嫁接部位的氧化损伤并有利于血管束重新连接。 转录组和代谢组分析显示，在接枝后 7-9 天，T 处理下肉桂醛在苯丙烷类生物合成途径中上调，半胱氨酸和蛋氨酸代谢途径中 L-同型半胱氨酸中 L-同型半胱氨酸上调。这些增强了抗氧化酶活性并减少对嫁接部位的氧化损伤。此外，氨基糖和核苷酸糖代谢途径中的 GAE 相关基因以及代谢物 D-木糖和十一碳烯基磷酸酯α-L-Ara4FN 在 T 处理下也上调。乙烯反应因子 （ERF） 转录因子 CsaRAP2.3 正向调节十一烯基磷酸 α-L-Ara4FN 表达，促进细胞壁合成，从而加速血管束重新连接。综上所述，在黄瓜嫁接愈合的维管束形成期，两种光强模式协调调节苯丙烷类生物合成、半胱氨酸和蛋氨酸代谢、氨基糖和核苷糖代谢以及 ERF 转录因子，促进抗逆和细胞壁合成的移植物愈合。此外，在愈伤组织形成阶段施加的光强度的基础上，在维管束形成期间增加光强度可以显着增强细胞壁合成，从而加速移植物愈合过程。尽管如此，这些研究结果对其他嫁接技术和物种的普遍性需要进一步研究。