An increase of NPY1 expression leads to inhibitory phosphorylation of PIN-FORMED (PIN) proteins and suppression of pinoid (pid) null mutants.

The PINOID (PID) protein kinase is required for flower initiation in Arabidopsis. The pid mutants fail to initiate flowers on inflorescences, a phenotype that is mimicked by disrupting either the NAKED PINS IN YUC MUTANTS (NPY) gene family or PIN FORMED 1 (PIN1). Both PID and NPY1 have been reported to positively modulate PIN-mediated polar auxin transport. Here, we show that overexpression of NPY1 (NPY1 OE) completely suppressed pid null mutants, demonstrating that NPY1 functions downstream of PID. NPY1 OE triggered phosphorylation of PIN proteins at multiple sites that are mostly different from the previously characterized phosphorylation sites regardless of the presence of PID. Phosphorylation of the newly identified PIN sites in NPY1 OE plants likely leads to the inhibition of PIN functions, as we previously showed that pid is suppressed by decreasing PIN1 gene dosage or decreasing PIN1 activity. Furthermore, we show that the Ser/Thr rich C-terminal motif in NPY1 is phosphorylated and is required for pid suppression by NPY1 OE. Overexpression of NPY1 that lacked the C-terminal motif (NPY1ΔC) failed to rescue pid, but overexpression of NPY1ΔC was still able to trigger phosphorylation of PIN proteins including PIN2, which is known to cause agravitropic roots when mutated. NPY1ΔC overexpression plants displayed a complete loss of root gravitropic response, likely caused by PIN2 phosphorylation. Our results suggest a pathway for auxin mediated-flower initiation, in which PID regulates NPY1 accumulation and/or activity, and subsequently, NPY1 triggers phosphorylation of PIN proteins and inhibition of PIN functions.