The role of dynamin related protein 2A in cytokinin regulated plant growth and development

Auxin is essential for many aspects of plant growth and development, from embryogenesis to organ senescence. Polar auxin transport leads to local auxin maxima and gradients, which are important for regulating various plant development process, including embryogenesis, organogenesis and vascular tissue formation. The polar auxin transport is mediated by auxin influx carrier AUX1/LAX proteins and efflux carrier PIN proteins. The regulation of PIN proteins is a key convergence of crosstalk between auxin and other hormones, such as cytokinin. Cytokinin promotes the degradation of PIN1 in Arabidopsis roots, preferentially the dephosphorylated PIN1, and the process is independent of transcription and translation (Marhavý et al., 2011; Marhavý et al., 2014). The internalization of PIN proteins from plasma membrane is controlled by clathrin-mediated endocytosis (Dhonukshe et al., 2007; Kitakura et al., 2011). However, whether cytokinin affects the PIN1 degradation through regulating the phosphorylation of PIN1 or trafficking components is unknown. Here, we demonstrate that dynamin related protein DRP2s are required for the cytokinin regulated trafficking of PIN1 and PIN2 in Arabidopsis root epidermal cells. Despite the reported redundancy of DRP2A and DRP2B in gametophyte development, we found that CK-mediated PIN1 degradation and –induced PIN2 membrane localization is affected in drp2a or drp2b single mutants. Interestingly, we found that cytokinin regulates the recruitment of DRP2A—but not DRP2B—to clathrin-coated pits, and affects the stability and plasma membrane localization of DRP2A. Furthermore, we studied the involvement of cytokinin receptors in the regulation of cytokinin on DRP2A. Additionally, the assembly and disassembly cycle of dynamin is mediated by its dephosphorylation and phosphotylation cycle; thus, we investigated the role of DRP2A phosphorylation in its cytokinin-regulated assembly and in cytokinin-regulated PIN trafficking. Altogether, we found that cytokinin regulated PIN trafficking in Arabidopsis root epidermal cells involves modulation of the phosphorylation of endocytosis accessory protein DRP2A.