Spatial and temporal alterations of multiple hormones during the graft union formation process in walnut (Juglans regia)

Plant grafting involves naturally or intentionally merging distinct plant parts to form a single organism. Although it is a common horticultural practice used to improve the yield, quality, and resistance of horticultural crops and study the long-distance transport of molecules, the underlying mechanism of graft union formation (GUF) remains poorly understood. In this study, we optimized the in situ analysis method for plant hormones and explored the spatial and temporal distribution of endogenous hormones (IAA, ABA, and ZR) during walnut GUF. The results demonstrated that changes in endogenous IAA and ZR levels in the graft union were consistent, with an increase during the rapid proliferation phase of callus tissue and enrichment in the cambium and cells of the grafting interface. As callus tissue entered the differentiation stage, endogenous IAA and ZR levels rapidly decreased and were mainly distributed in the callus tissue. Conversely, the level and distribution of endogenous ABA showed no significant changes during the rapid proliferation phase of callus tissue. However, they increased rapidly after entering the differentiation stage, mainly in the scion cambium and callus tissue. We also observed "S"-shaped, “M”-shaped, and “N”-shaped trends in the dynamic changes of IAA/ABA, IAA/ZR, and ZR/ABA ratios, respectively. Based on these results, we propose a spatial and temporal distribution model of endogenous hormones during walnut GUF. This provides a foundation for further investigation into the molecular mechanisms of hormone-mediated GUF in walnut and other woody plants.