ROS function during the early phase of vascular regeneration in Arabidopsis

When vascular tissues are damaged, plants induce site-specific formation of vascular stem cells that proliferate and differentiate into xylem and phloem, restoring vascular continuity. In addition, reactive oxygen species (ROS) are rapidly generated at wound sites in response to damage. However, the role of ROS during vascular regeneration remains largely unknown. Here, we investigated the role of ROS using an Arabidopsis inflorescence stem incision model and the vascular cell induction culture system (VISUAL). During tissue reunion of incised inflorescence stems, scavenging ROS with potassium iodide (KI) suppressed the proliferation of cambial cell layers. Consistently, the expression of cambium- and xylem-related genes, including TDR/PXY and VND7, was significantly reduced by KI treatment. Furthermore, mutation in respiratory burst oxidase homolog D (RBOHD), which encodes an ROS-producing enzyme, suppressed the proliferation of cambial cell layers. In VISUAL, KI treatment inhibited ectopic xylem formation and reduced the expression of vascular stem cell-related genes. Temporal analyses further indicated that ROS functions during the early phase of induction, as the expression of dedifferentiation-related genes such as WAK1 and ACS6 decreased under KI treatment. Notably, the expression of Arabidopsis NAC domain-containing protein 071 (ANAC071) and ANAC096 was not suppressed by KI in incised stems, and estradiol-induced expression of ANAC071 failed to rescue vascular differentiation in VISUAL under KI treatment. These findings demonstrated that ROS act prior to ANAC function during the early phase of vascular regeneration.