PID/WAG-mediated phosphorylation of the Arabidopsis PIN3 auxin transporter mediates polarity switches during gravitropism

Abstract

Intercellular distribution of the plant hormone auxin largely depends on the polar subcellular distribution of the plasma membrane PIN-FORMED (PIN) auxin transporters. PIN polarity switches in response to different developmental and environmental signals have been shown to redirect auxin fluxes mediating certain developmental responses. PIN phosphorylation at different sites and by different kinases is crucial for PIN function. Here we investigate the role of PIN phosphorylation during gravitropic response. Loss- and gain-of-function mutants in PINOID and related kinases but not in D6PK kinase as well as mutations mimicking constitutive dephosphorylated or phosphorylated status of two clusters of predicted phosphorylation sites partially disrupted PIN3 phosphorylation and caused defects in gravitropic bending in roots and hypocotyls. In particular, they impacted PIN3 polarity rearrangements in response to gravity and during feed-back regulation by auxin itself. Thus PIN phosphorylation, besides regulating transport activity and apical-basal targeting, is also important for the rapid polarity switches in response to environmental and endogenous signals.

A lipid code-dependent phosphoswitch directs PIN-mediated auxin efflux in Arabidopsis development

“…Based on the scenario that PDK1 acts as a master regulator of AGC kinases in different organisms 21 , and the crucial roles of AGC kinases in regulating auxin transport 30, 43 , we speculate that PDK1 might function in auxin pathway through regulating specific AGC kinases. Of the 39 AGC members, PID/WAGs 6–8, 44 , D6PKs 9 and PAX 11 have been reported to regulate either the polarity (by PID/WAGs) or the activity (by all of these) of PIN proteins respectively through phosphorylation, and hence modulate auxin fluxes. Therefore, we firstly measured the basipetal auxin transport in intact etiolated hypocotyls with [ 3 H]-IAA.…”

“…PINs are phosphorylated at multiple serine/threonine residues by various protein kinases, including PINOID (PID)/WAVY ROOT GROWTHs (WAGs) 6–8 , D6 PROTEIN KINASEs (D6PK/D6PKL1∼3, hereafter as D6PKs) 9, 10 , PROTEIN KINASE ASSOCIATED WITH BRX (PAX) 11 , MITOGEN PROTEIN KINASE3/6 (MPK3/6) 12 and CDPK-RELATED KINASE5 (CRK5) 13 , and dephosphorylated by Protein Phosphatase 2A (PP2A) 8, 14 , Protein Phosphatase 6 (PP6) 15 and PP1 16 . PID-dependent phosphorylation and PP2A-mediated dephosphorylation antagonize with each other 6, 14 , acting as a binary switch determining the apical-basal targeting of PIN proteins.…”

Antagonistic and auxin-dependent phosphoregulation of columella PIN proteins controls lateral root gravitropic setpoint angle in Arabidopsis

“…These studies showed that phosphorylation of specific serine (S) residues in the cytoplasmic loops of these membrane proteins induces localisation to the shootward (upper) plasma membrane, while their dephosphorylation causes PINs to localise to the rootward (lower) plasma membrane. To begin to explore the role of phosphorylation in the subcellular distribution of PINs within the lateral root columella we analysed lateral root GSA of PIN3:YFP phosphovariant lines in which known PID/WAG-or D6PK-targeted serine residues were mutated to either a nonphosphosphorytable alanine (A) (Grones et al, 2018;Zourelidou et al, 2014) or to the phosphomimic amino acid aspartic acid (D) (Grones et al, 2018). Previous studies have shown that phosphorylation at the specific residues of S316, S317 and S321 for PID/WAG and S215 and S283 (annotated as S4 and S5) for D6PKs can affect the gravity-induced repolarization of PIN3 in the root and hypocotyl respectively (Grones et al, 2018;Zourelidou et al, 2014).…”

“…To begin to explore the role of phosphorylation in the subcellular distribution of PINs within the lateral root columella we analysed lateral root GSA of PIN3:YFP phosphovariant lines in which known PID/WAG-or D6PK-targeted serine residues were mutated to either a nonphosphosphorytable alanine (A) (Grones et al, 2018;Zourelidou et al, 2014) or to the phosphomimic amino acid aspartic acid (D) (Grones et al, 2018). Previous studies have shown that phosphorylation at the specific residues of S316, S317 and S321 for PID/WAG and S215 and S283 (annotated as S4 and S5) for D6PKs can affect the gravity-induced repolarization of PIN3 in the root and hypocotyl respectively (Grones et al, 2018;Zourelidou et al, 2014). We found that lines in which the S316, S317 and S321 PID/WAG sites of PIN3:YFP were mutated to alanine (PIN3S>A:YFP) had more vertical lateral root GSAs, while the mutation of those same residues to aspartic acid (PIN3S>D:YFP) induced lateral roots to grow at a more horizontal GSA than control PIN3:GFP plants (Fig.…”

“…All laser power and gain settings were consistent across images. Briefly, PIN:GFP markers were imaged using a series of Z stacks and fluorescence intensity across external membranes was quantified using ImageJ as described in Grones et al, 2018. Each experiment was performed three times with at least 6 roots for each experiment.…”