VPS34 DN and RNAi seedlings
Phosphoinositide 3-kinases (PI3Ks) are evolutionarily conserved central regulators of cell signaling and intracellular trafficking. Although the single PI3K of Arabidopsis AtVPS34, has long been recognized as essential, its function at the organism level remains unclear. We generated stable AtVPS34 RNAi, dominant-negative (DN) and overexpression (OE) lines, which displayed pleiotropic defects in organogenesis, cell expansion, and polarity establishment. Transcriptome sequencing results show that VPS34 is involved in the response to light and auxin signaling. Further investigation revealed that the cell elongation of DN and RNAi lines slowed down, and they exhibited auxin-deficient phenotypes in the roots. Expression of the DR5rev:GFP auxin response reporter was weaker at the VPS34 RNAi root tip, accompanied by significant reduction in abundance AUX1, PIN1, and PIN2. Mechanistically, VPS34 regulates both endocytic uptake and exocytic recycling of PIN2, with RNAi lines showing delayed BFA compartment formation and PIN2 relocalization. Furthermore, knockdown of VPS34 significantly affects the function of MVB. Conversely, when VPS34 is overexpressed, the expression of RABG3F, a functional protein of MVB, is enhanced, leading to abnormal vacuole morphology. This ultimately results in impaired PIN2 transport and reduced auxin content at the root tip. Our results indicate that the plant PI3K is a critical regulator of polar auxin transport and auxin-mediated organogenesis and development.