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 <i>Arabidopsis</i>. The <i>pid</i> mutants fail to initiate flowers on inflorescences, a phenotype that is mimicked by disrupting either the <i>NAKED PINS IN YUC MUTANTS</i> (<i>NPY</i>) gene family or <i>PIN FORMED 1</i> (<i>PIN1</i>). Both PID and NPY1 have been reported to positively modulate PIN-mediated polar auxin transport. Here, we show that overexpression of <i>NPY1</i> (<i>NPY1 OE</i>) completely suppressed <i>pid</i> null mutants, demonstrating that <i>NPY1</i> functions downstream of <i>PID. NPY1 OE</i> triggered phosphorylation of PIN proteins at multiple sites that are mostly different from the previously characterized phosphorylation sites regardless of the presence of <i>PID</i>. Phosphorylation of the newly identified PIN sites in <i>NPY1 OE</i> plants likely leads to the inhibition of PIN functions, as we previously showed that <i>pid</i> is suppressed by decreasing <i>PIN1</i> 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 <i>pid</i> suppression by <i>NPY1 OE</i>. Overexpression of <i>NPY1</i> that lacked the C-terminal motif (<i>NPY1∆C</i>) failed to rescue <i>pid</i>, but overexpression of <i>NPY1∆C</i> was still able to trigger phosphorylation of PIN proteins, including PIN2, which is known to cause agravitropic roots when mutated. <i>NPY1∆C</i> 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.