Identification of a novel regulator in auxin canalization

Auxin was the first major plant hormone discovered. Auxin is found in all parts of the plant and is essential in the formation of leaf venation and vascular strands. When a plant is injured, auxin stimulates cell differentiation and regeneration in the vascular tissues. The uneven distribution of auxin in each position is important developmental information that must be tightly regulated by both metabolism and transport. Amongst, polar auxin transport is an active process in which auxin is transported from cell to cell, with asymmetry and directionality being key characteristics. Polar transport is primarily dependent on PIN-FORMED (PIN) proteins. Intriguingly, the polar localization of PINs on the plasma membrane is controlled by auxin. PIN proteins control the directionality of auxin fluxes, and auxin controls PIN protein localization as a result of auxin regulation of PIN localization. Such positive feedback loop between auxin and its own transporters gives the system self-organizing properties and is known as "auxin canalization", which is essential for the leaf veins development and the regeneration of vascular strands upon wounding. Several endogenous regulators, including auxin receptor TIR1/AFB proteins and transcription factor WRKY23, have been shown to be involved in auxin canalization. Nevertheless, given the spatial disparity, the perception and transcriptional regulation conveyed by TIR1/AFBs and WRKY23 in the nucleus are unlikely to be directly involved in the regulation of PIN repolarization at the plasma membrane. Therefore, it is an urgent task to identify direct players specifically and directly involved in the coordinated PIN polarity rearrangements during auxin canalization and beyond. In this study, we identify a novel gene regulated by auxin and WRKY23, which is named as Downstream of WRKY23 (DOW). When DOW is knocked out and becomes dysfunctional, the plant exhibits abnormal leaf venation patterns and vasculature regeneration after wounding is significantly impaired, indicating DOW as a promising candidate involved in auxin canalization. To better understand the molecular basis and mechanical role of this unknown gene, we will use multi-disciplinary approaches ranging from Plant Molecular Biology, Physiology and Genetics, to Biochemistry, Cell Biology and Proteomics, to decipher the precise molecular mechanism of DOW-dependent regulation of polar auxin transport in auxin canalization. We believe that our research will help us understand the mechanism underlying auxin canalization and will lead to a deeper understanding of self-organizing plant development, auxin transport and cell polarity mechanisms.

Auxin canalization is a self-organizing process that governs the flexible formation of vasculature by reinforcing the formation of auxin transport channels. A key prerequisite is the feedback between auxin signaling and directional auxin transport, mediated by PIN transporters. Despite the developmental importance of canalization, the molecular components linking auxin perception to the regulation of PIN auxin transporters remain poorly understood. Here, we identify TOW, a novel and essential component of auxin canalization that links intracellular auxin signaling with cell surface auxin perception. TOW is regulated downstream of TIR1/AFB-Aux/IAA-WRKY23 transcriptional auxin signaling. tow mutants exhibit defects in regeneration and de novo vasculature formation, along with impaired formation of polarized, PIN-expressing auxin channels. At the subcellular level, these mutants display disrupted auxin-induced PIN polarization and altered PIN endocytic trafficking dynamics. TOW localizes predominantly to the plasma membrane, where it interacts with receptor-like kinases involved in auxin canalization, including the TMK1 auxin co-receptor and the CAMEL-CANAR complex. TOW promotes PIN interaction with these kinases and stabilizes PINs at the cell surface. Together, our findings identify TOW as a molecular link between intracellular and cell surface auxin signaling mechanisms that converge on PIN trafficking and polarity, providing new insights into how auxin signaling regulates directional auxin transport for the self-organizing formation of vasculature during flexible plant development.