AFB1-generated cGMP targets CNGC14 to trigger rapid auxin responses in plants

forthcoming

Rapid auxin responses initiate cellular changes, which are important for rapid, auxin-induced root growth inhibition and root gravitropism. Accumulating evidence shows that these rapid responses are mediated by TIR1/AFB signaling: Auxin enters the cell by influx or diffusion and activates the AFB1; within seconds, AFB1 activates a trio of tightly connected rapid responses, including PM depolarization, CNGC14-dependent cytosolic Ca2+ increase, and apoplast alkalinization, which lead to rapid root growth inhibition. However, how AFB1 mediates these rapid cellular responses via CNGC14 remains unknown. Until recently, my host lab found that TIR1/AFBs have additional Guanylate cyclase activity and that its product, cGMP, rapidly and transiently increases in roots after auxin treatment. Importantly, application of cell-permeable cGMP triggers cytosolic Ca2+ increase in a CNGC14-dependent manner and rapid root growth inhibition in Arabidopsis. These observations collectively imply a linear AFB1 - cGMP - CNGC14 - Ca2+ non-transcriptional signaling pathway, which explains well at least part of the mysterious rapid auxin responses. However, how the auxin perception product, cGMP regulates CNGC14 and orchestrates Ca2+ influx is still unclear in planta. Thus, filling this major knowledge gap is eagerly awaited and will open entirely new possibilities for a mechanism of cytosolic Ca2+ oscillation in planta. In this proposed research, I aim to test the hypothesis that AFB1-generated cGMP acts as a second messenger, targeting CNGC14 to trigger rapid auxin responses in plants. We will i) clarify the binding between cGMP and CNGC14 in vitro and in vivo; ii) test the importance of cGMP binding to CNGC14 functions in plants; iii) explore the mechanism, by which cGMP regulates CNGC14. My investigation will not only be a significant step in our understanding of non-transcriptional auxin mechanisms but will have huge implications for other rapid, Ca2+-dependent cellular responses.