Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cellpromoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.

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BackgroundRecurring damage to aerial organs of plants necessitates their prompt repair, particularly their vasculature. While vascular regeneration assay in aerial plant parts such as stem and inflorescence stalk are well established, those on leaf vasculature remained unexplored.Recently we established a new vascular regeneration assay in growing leaf and discovered the underlying molecular mechanism. ResultsHere we describe the detailed stepwise method of incision and the regeneration assay used for studying the leaf vascular regeneration. By using a combination of micro-surgical perturbations, brightfield microscopy and other experimental approaches, our new findings show that the regeneration efficiency decreases with aging of the leaf, and increases with the nearness of the wound towards the proximal end of the leaf. ConclusionThis easy-to-master vascular regeneration assay is an efficient and rapid method to study the mechanism of vascular regeneration in growing leaves. It can be readily adapted for other plant species and can be combined with cellular and molecular biology techniques.