Uncovering protein regulation during adventitious root formation in Platycladus orientalis cuttings

Abstract Cell totipotency and pluripotency are the cellular basis for root regeneration in Platycladus orientalis cuttings, and the regeneration of adventitious roots is a key determinant for improving stem-cutting. However, the cellular basis and physiological regulation of adventitious root formation are still ambiguous. In this research, root primordia initiation and organogenesis were histologically observed, dynamic alterations in soluble proteins were monitored, and Tandem Mass Tag protein profiling during adventitious root development was carried out. It was explicitly shown that the root primordium primarily originated from undifferentiated xylem cells for indirect (callus) rooting and from dividing cells in the cortex for direct (cortex) rooting. During the entire process of adventitious root development, the activities of peroxidase (POD) and polyphenol oxidase (PPO) peaked, and the activity of indole acetic acid oxidase (IAAO) decreased during the prophase of adventitious root formation in both the direct and indirect rooting, suggesting the positive regulation of POD and PPO and the negative regulation of IAAO during adventitious root initiation. Compared with those of indirect rooting, the relatively greater activities of POD and PPO and lower activity of IAAO were related to direct rooting and the number of adventitious roots. A total of 4265 proteins were identified from the base of the cuttings, of which 343, 236 and 37 proteins were highly expressed before treatment, in root primordia induction to adventitious root formation, and adventitious root elongation stages, respectively. Through hierarchical cluster analysis, 23 peroxidase and endogenous hormone regulatory proteins were screened and obtained; these included 10 peroxidases, 1 auxin regulatory protein, 3 ABA regulatory proteins, 2 jasmonic acid regulatory proteins, and 3 gibberellin regulatory proteins, which were highly expressed during the late cutting period. Finally, a hypothetical model of the regulatory network of the differential proteins involved in adventitious root formation in P. orientalis was constructed.