A PP6-Type Phosphatase Holoenzyme Directly Regulates PIN Phosphorylation and Auxin Efflux in Arabidopsis

Abstract

The directional transport of the phytohormone auxin depends on the phosphorylation status and polar localization of PIN-FORMED (PIN) auxin efflux proteins. While PINIOD (PID) kinase is directly involved in the phosphorylation of PIN proteins, the phosphatase holoenzyme complexes that dephosphorylate PIN proteins remain elusive. Here, we demonstrate that mutations simultaneously disrupting the function of Arabidopsis thaliana FyPP1 (for Phytochrome-associated serine/threonine protein phosphatase1) and FyPP3, two homologous genes encoding the catalytic subunits of protein phosphatase6 (PP6), cause elevated accumulation of phosphorylated PIN proteins, correlating with a basal-to-apical shift in subcellular PIN localization. The changes in PIN polarity result in increased root basipetal auxin transport and severe defects, including shorter roots, fewer lateral roots, defective columella cells, root meristem collapse, abnormal cotyledons (small, cup-shaped, or fused cotyledons), and altered leaf venation. Our molecular, biochemical, and genetic data support the notion that FyPP1/3, SAL (for SAPS DOMAIN-LIKE), and PP2AA proteins (RCN1 [for ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1] or PP2AA1, PP2AA2, and PP2AA3) physically interact to form a novel PP6-type heterotrimeric holoenzyme complex. We also show that FyPP1/3, SAL, and PP2AA interact with a subset of PIN proteins and that for SAL the strength of the interaction depends on the PIN phosphorylation status. Thus, an Arabidopsis PP6-type phosphatase holoenzyme acts antagonistically with PID to direct auxin transport polarity and plant development by directly regulating PIN phosphorylation. INTRODUCTIONAuxin is a fundamental plant hormone that regulates almost every aspect of plant growth and development, including embryogenesis, organogenesis, apical dominance, tissue regeneration, and tropisms (reviewed in Bennett and Scheres, 2010;Grunewald and Friml, 2010;Peris et al., 2010). Auxin is transported from its sites of biosynthesis to its sites of action by a polarized auxin transport system. Molecular genetic studies in Arabidopsis thaliana have lead to the identification and functional characterization of several key players of the polarized auxin transport system, such as the auxin uptake carriers AUXIN RESISTANT1/LIKE AUX1 (AUX1/ LAX) (Swarup et al., 2008), the auxin efflux carriers, including PIN-FORMED (PIN) family proteins Chen et al., 1998;Müller et al., 1998;Friml et al., 2002;Petrásek et al., 2006), and P-glycoprotein auxin transporters ABCB1 (for ATP BINDING CASSETTE PROTEIN SUBFAMILY B1), ABCB4, and ABCB19 (Terasaka et al., 2005;Bouchard et al., 2006;Blakeslee et al., 2007;Lin and Wang, 2005). PIN proteins are asymmetrically targeted to the plant cell plasma membranes, resulting in distinct polar subcellular localization in a given tissue. For example, PIN1 is localized in the basal (rootward, lower) plasma membrane of stele cells and xylem cells in the vascular system, which is required for long-distance auxin flow from the shoot apex to the root tip Friml e...

Regulation of Hormonal Control, Cell Reprogramming, and Patterning during De Novo Root Organogenesis

“…Our data suggest a model in which subcellular PIN3 localization shifts from basal to apical membranes in vascular cells near the wound to redirect auxin flow backward and thus maintaining high auxin levels in the proximal petiole vasculature. Interestingly, an auxin-dependent switch in PIN3 polarization contributing to auxin-flow reversal is involved in the shoot gravitropic response (Rakusová et al, 2016), where basal-to-apical shift in PIN localization has been described to depend on phosphorylation (Dai et al, 2012). It is thus tempting to speculate that auxin-dependent phosphorylation of PIN3 would be involved in maintaining high auxin levels in the petiole base vasculature during root regeneration.…”

The dynamics of plant plasma membrane proteins: PINs and beyond

“…Together with in vitro evidence for PID-mediated phosphorylation of bacterially expressed PIN fragments and PID-dependent PIN phosphorylation in transient protoplast expression assays (Michniewicz et al, 2007), this led to models suggesting that PINs represent substrates for PID, although we are still lacking conclusive in planta evidence for a direct interaction. PID activity appears to be antagonized by a PP6-type phosphatase heterotrimeric protein complex, consisting of PHYTOCHROME-ASSOCIATED SERINE/THREONINE PROTEIN PHOSPHATASE (FyPP), SAPS DOMAIN-LIKE (SAL) and the PP2AA regulatory subunit, which was shown to interact with PIN proteins and to antagonize their phosphorylation (Michniewicz et al, 2007;Dai et al, 2012). Intriguingly, PP2AA phosphatase regulatory subunits were shown to colocalize with PIN proteins at plasma membrane domains (Michniewicz et al, 2007), consistent with a scenario in which modulation of phosphatase activity at the plasma membrane directly feeds back on PIN phosphorylation status, thereby shaping PIN sorting decisions (Fig.…”

The PP6 Phosphatase Regulates ABI5 Phosphorylation and Abscisic Acid Signaling inArabidopsis

“…In a recent study, we showed that Arabidopsis FyPP1 (for Phytochrome-associated serine/threonine protein phosphatase1) and FyPP3, two homologous catalytic subunits of PP6, physically interact with SAL (for SAPS-domain like protein) and PP2AA proteins (RCN1/PP2AA1, PP2AA2, and PP2AA3) to form a PP6-type heterotrimeric holoenzyme that directly regulates the phosphorylation status of PIN-FORMED (PIN) proteins (auxin efflux carriers) and subsequently affects auxin efflux and plant development (Dai et al, 2012a(Dai et al, , 2012b. In this study, we extend these findings by showing that FyPP also plays a critical role in regulating ABA signaling in Arabidopsis.…”

“…The PP2A holoenzyme consists of an enzymatically active catalytic subunit (PP2Ac), a 65-kD regulatory A subunit (PP2A A), and a variable regulatory B subunit (PP2AB) (Farkas et al, 2007). Although there are high sequence similarities among the C subunits of PP6 and PP2A phosphatases, they were classified as different phosphatases because PP6 requires Zn 2+ for its activity, whereas PP2A does not require any cation Wang et al, 2007;Dai et al, 2012a). PPPs are ubiquitous enzymes in all eukaryotes, but their regulatory functions are largely unknown in plants (Farkas et al, 2007).…”