Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport

Fang Huang; Marcelo Kemel Zago; Lindy Abas; Arnoud van Marion; Carlos Samuel Galván-Ampudia; Remko Offringa

doi:10.1105/tpc.109.072678

Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport

Huang, Fang; Kemel Zago, Marcelo; Abas, Lindy; van Marion, Arnoud; Galván-Ampudia, Carlos Samuel; Offringa, Remko 2010-04-20 00:00:00 The Plant Cell, Vol. 22: 1129–1142, April 2010, www.plantcell.org ã 2010 American Society of Plant Biologists Phosphorylation of Conserved PIN Motifs Directs Arabidopsis W OA PIN1 Polarity and Auxin Transport a a,1 b a a,2 Fang Huang, Marcelo Kemel Zago, Lindy Abas, Arnoud van Marion, Carlos Samuel Galva´ n-Ampudia, and a,3 Remko Offringa Department of Molecular and Developmental Genetics, Institute of Biology, Leiden University, 2333 EB Leiden, The Netherlands Institute for Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences (BOKU Wien), A-1190 Vienna, Austria Polar cell-to-cell transport of auxin by plasma membrane–localized PIN-FORMED (PIN) auxin efﬂux carriers generates auxin gradients that provide positional information for various plant developmental processes. The apical-basal polar localization of the PIN proteins that determines the direction of auxin ﬂow is controlled by reversible phosphorylation of the PIN hydrophilic loop (PINHL). Here, we identiﬁed three evolutionarily conserved TPRXS(N/S) motifs within the PIN1HL and proved that the central Ser residues were phosphorylated by the PINOID (PID) kinase. Loss-of-phosphorylation PIN1:green ﬂuorescent protein (GFP) (Ser to Ala) induced inﬂorescence defects, correlating with their basal localization in the shoot apex, and induced internalization of PIN1:GFP during embryogenesis, leading to strong embryo defects. Conversely, phosphomimic PIN1:GFP (Ser to Glu) showed apical localization in the shoot apex but did not rescue pin1 inﬂorescence defects. Both loss-of-phosphorylation and phosphomimic PIN1:GFP proteins were insensitive to PID overexpression. The basal localization of loss-of-phosphorylation PIN1:GFP increased auxin accumulation in the root tips, partially rescuing PID overexpression-induced root collapse. Collectively, our data indicate that reversible phosphorylation of the conserved Ser residues in the PIN1HL by PID (and possibly by other AGC kinases) is required and sufﬁcient for proper PIN1 localization and is thus essential for generating the differential auxin distribution that directs plant development. INTRODUCTION and develops pin-shaped inﬂorescences (Ga¨ lweiler et al., 1998). The PIN family proteins can be classiﬁed into two groups: (1) the The plant hormone auxin plays a central role in almost all aspects PIN1-type proteins (PIN1, 2, 3, 4, and 7) that are plasma mem- of plant development. Unidirectional cell-to-cell transport of brane (PM) localized and (2) the PIN5-type proteins (PIN5, 6, and auxin generates maxima and minima that are instrumental for 8) that localize to the endoplasmatic reticulum (ER) and seem to tropic growth responses, tissue patterning, and organ initiation be involved in the regulation of auxin homeostasis (Mravec et al., (Sabatini et al., 1999; Friml et al., 2002b, 2003; Benkova et al., 2009). The PIN1-type proteins have redundant functions, and a 2003; Sorefan et al., 2009). The polar auxin ﬂow is accomplished loss-of-function mutation in one PIN gene is sometimes com- by the concerted action of three families of membrane proteins, pensated for by the ectopic expression of other PINs (Blilou et al., the AUXIN RESISTANT1/LIKE AUX1 (AUX1/LAX) inﬂux carriers 2005; Vieten et al., 2005). As a result, only mutants in multiple PIN (reviewed in Parry et al., 2001), the PIN-FORMED (PIN) efﬂux genes show more pronounced phenotypes in embryogenesis, carriers (reviewed in Paponov et al., 2005), and the P-GLYCO- root patterning, and lateral root initiation (Benkova et al., 2003; PROTEIN (PGP/ABCB) transporters (reviewed in Geisler and Friml et al., 2003; Blilou et al., 2005). Murphy, 2006). Until now, the role of the PIN auxin efﬂux carriers The PIN1-type proteins determine the direction of cell-to-cell in polar auxin transport is most well established. The Arabidopsis auxin transport through their asymmetric subcellular localization thaliana genome encodes eight PIN proteins, named after the at the PM (Wis´ niewska et al., 2006), which is dependent not only pin-formed/pin1 mutant that is defective in polar auxin transport on tissue-speciﬁc factors, but also on the PIN protein sequence (Wisniewska et al., 2006). During speciﬁc developmental pro- cesses, dynamic changes in PIN polarity have been observed Current address: Escola Proﬁssional UNIPACS, Rua Guilherme Lahm, (Benkova et al., 2003; Friml et al., 2003; Heisler et al., 2005), and 960, Taquara, RS, Brazil. Current address: Swammerdam Institute for Life Sciences, University PIN polarity has also been shown to be modulated by environ- of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Nether- mental cues (Friml et al., 2002b; Harrison and Masson, 2008) and lands. auxin itself (Paciorek et al., 2005; Sauer et al., 2006). Many Address correspondence to [email protected]. research efforts have focused on what determines PIN polarity The author responsible for distribution of materials integral to the ﬁndings presented in this article in accordance with the policy described and, thus, what translates upstream developmental and envi- in the Instructions for Authors (www.plantcell.org) is: Remko Offringa ronmental signals into changes in plant architecture by regulat- ([email protected]). ing PIN polarity. The current model is that newly synthesized Online version contains Web-only data. OA PINs arrive at the PM in a nonpolar fashion and that PIN polarity Open Access articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.109.072678 is established and regulated by subsequent endocytosis, 1130 The Plant Cell transcytosis, and recycling back to the PM (Geldner et al., 2001; of-phosphorylation and phosphomimic PIN1:GFP proteins in Dhonukshe et al., 2008; Kleine-Vehn et al., 2008). root tips was insensitive to PID overexpression using P35S:PID, GNOM is a GDP/GTP exchange factor on ADP-ribosylation leading to opposite effects on P35S:PID-induced root collapse. factor G protein that has been shown to be involved in the Our data indicate that the regulation of PIN1 polar localization recycling of PIN proteins to the basal (root apex facing) side of through reversible phosphorylation of three conserved Ser res- the PM (Geldner et al., 2003). GNOM is a molecular target of the idues in the PIN1HL by PID and possibly other AGC kinases is an fungal toxin brefeldin A (BFA), which inhibits protein trafﬁcking essential mechanism for aspects of plant development that are and thus interferes with basal PIN1 recycling, leading to PIN1 directed by differential auxin distribution. accumulation into so-called BFA compartments (Geldner et al., 2001). Loss of function of GNOM results in severe embryo defects due to disturbance of PIN1 polarity establishment during RESULTS embryogenesis (Mayer et al., 1993; Steinmann et al., 1999). Another important molecular determinant in PIN polar target- Ser Residues in Three Conserved Motifs in the PIN1HL Are ing is the PINOID (PID) protein Ser/Thr kinase. PID was initially Phosphorylated by PID identiﬁed through the Arabidopsis pid loss-of-function mutants that phenocopy pin1 mutants (Bennett et al., 1995; Christensen The previous observations that the PIN1HL is efﬁciently phos- et al., 2000). Both PID loss- and gain-of-function mutant pheno- phorylated by PID in vitro (Michniewicz et al., 2007) prompted us to types already indicated a role of PID as a regulator of auxin map PID phosphorylation targets in the PIN1HL. Analysis of the transport (Benjamins et al., 2001). More recently, PID was shown PIN1 amino acid sequence using the NetPhos program (Blom to act as a binary switch in the apical-basal polar targeting of PIN et al., 1999) identiﬁed 23 putative phosphosites, 20 of which are proteins (Friml et al., 2004). In root cells, PID overexpression located in the PIN1HL (see Supplemental Figure 1 online). Twelve induces a PIN polarity shift from the basal to the apical (shoot synthetic peptides comprising 17 putative phosphosites were apex facing) side of the cells, leading to agravitropic root growth tested in in vitro phosphorylation assays, and six were found to be and collapse of the primary root meristem, due to depletion of the highly phosphorylated by PID (see Supplemental Figure 2 online). organizing auxin maximum. By contrast, in the inﬂorescence Since the PID-dependent basal-to-apical switch in PIN polarity meristem, pid loss of function induces an apical-to-basal shift in is not restricted to PIN1, but is also observed for PIN2 and PIN4 PIN1 polarity, which drains the auxin maxima that are necessary (Friml et al., 2004), we aligned the amino acid sequences of six for organ initiation, thus resulting in pin-like inﬂorescences (Friml PIN proteins (PIN1, 2, 3, 4, 6, and 7) in which a clear hydrophilic et al., 2004). loop (HL) can be identiﬁed. Eleven of the NetPhos predicted Ser In animal systems, modiﬁcation of cargo proteins by phos- (S) and Thr (T) residues showed conservation among the six phorylation is an important mechanism to regulate their polar Arabidopsis PIN proteins (indicated with an asterisk in Figure 1), delivery to the PM. For example, in mammalian epithelial cells, ﬁve of which appeared to be fully conserved (labeled in black and phosphorylation of the immunoglobulin receptor at a single Ser with an asterisk in Figure 1). Interestingly, these ﬁve residues residue has been shown to result in its accumulation at the apical were located in the highly phosphorylated peptides 2, 6, 8, and cell membrane (Casanova et al., 1990). Protein phosphorylation 12 (see Supplemental Figure 2 online), and two of them (Thr-227 and dephosphorylation have also been implicated in the regula- and Ser-290) were recently reported to be modiﬁed by phos- tion of polar auxin transport in plant systems. In tobacco (Nico- phorylation in vivo (Benschop et al., 2007). We noted that S290 tiana tabacum) suspension cells, the protein kinase inhibitors was located in a TPRXSN motif that was conserved among the staurosporine and K252a were found to inhibit auxin efﬂux six PIN proteins (Figure 1) and resembled the consensus phos- (Delbarre et al., 1998), and genetic and pharmacological inhibi- phorylation site of the animal AGC kinase, protein kinase A (PKA). tion of phosphatase activity in Arabidopsis led to defects in auxin Therefore, we ﬁrst tested whether Ser-290 is a PID phosphory- transport (Garbers et al., 1996; Rashotte et al., 2001). More lation target by replacing this Ser with Ala in a HIS-tagged short recent ﬁndings revealed that PIN polar localization is determined version of the PIN1HL (PIN1HLsv) (see Supplemental Figure by reversible phosphorylation of the large central PIN hydro- 1 online) and incubating wild type or mutant proteins with HIS- philic loop (PINHL) through the antagonistic action of the PID tagged PID in an in vitro phosphorylation reaction. Clear PID kinase and PP2A phosphatases (Michniewicz et al., 2007). This autophosphorylation and PID-dependent phosphorylation of indicated that the machinery of phosphorylation-regulated PM HIS-PIN1HLsv was detected (see Supplemental Figure 3A on- protein polar localization is also operational in plants. line). The S290A substitution reduced PIN1 phosphorylation by To further elucidate molecular mechanisms of PIN polar local- PID to a background level (see Supplemental Figure 3A online), ization regulated by PID phosphorylation, we set out to identify indicating that Ser-290 is a PID phosphorylation target. Two the PID phosphorylation targets in the PINHL. Here, we show that additional TPRXS(N/S) motifs were identiﬁed upstream of Ser- the central Ser residues in three conserved TPRXS(N/S) motifs 290 (Figure 1), and the Ser residue (Ser-252) of the second motif within the PIN1HL are phosphorylated by PID. Inactivation of was also shown to be modiﬁed by phosphorylation in vivo these phosphosites (nonphosphorylatable or phosphomimic (Benschop et al., 2007). For convenience in our experiments, forms) in a complementing PIN1:GFP (for green ﬂuorescent we refer to the Ser residues at positions 231, 252, and 290 as S1, protein) construct induced auxin-regulated defects in embryo S2, and S3, respectively (Figure 1). and inﬂorescence development that correlated with changes in We next tested the effect of Ser-to-Ala substitution (S1A, PIN1:GFP polar localization. Moreover, the localization of loss- S2A, S3A, or combinations) on PID phosphorylation using a Conserved Serines Direct PIN1 Polarity 1131 Figure 1. Alignment of the Amino Acid Sequences of the HL of Six Arabidopsis PIN Proteins. Conserved Ser or Thr residues predicted by NetPhos to be phosphorylated are indicated by asterisks. Residues that are conserved in all six PINHLs are blocked with black (Ser and Thr residues) or light gray (other amino acids). Residues that are conserved in four or ﬁve of the six PINHLs are blocked with dark gray. The three TPRXS(N/S) motifs are boxed, and the Ser residues at positions 231, 252, and 290 within these motifs are renumbered to 1, 2, and 3, respectively. glutathione S-transferase (GST)-tagged version of the full- quences of PIN1 proteins from ﬁve different plant species length PIN1HL. Under our experimental conditions, the GST- (Xu et al., 2005; Carraro et al., 2006) showed that the three PIN1HL was unstable, showing a reproducible pattern of identiﬁed motifs are highly conserved, even in the moss Phys- degradation bands. A single S1A or S3A substitution led to a comitrella patens (see Supplemental Figure 4 online), suggesting 40 or 20% reduction, respectively, double S1,2A and S1,3A their functional conservation throughout the evolution of land substitutions led to a 50% reduction, and triple S1,2,3A sub- plants. stitutions led to an 80% reduction of phosphorylation by PID compared with the wild-type PIN1HL (Figure 2B). These data Loss of PIN1 Phosphorylation at the Conserved Ser indicated that the central Ser residues within the three highly Residues Induces Dominant Embryo and conserved TPRXS(N/S) motifs are targets for PID phosphory- Flower Phenotypes lation in vitro. At the same time, the PIN1HL S1,3A double substitution To investigate the biological signiﬁcance of the PIN1 phosphor- construct was used to test all other Ser and Thr residues located ylation in planta, various mutant constructs were generated from in the highly phosphorylated peptides 2 (T227), 6 (T286), 8 (S377 PPIN1 (PIN promoter):PIN1:GFP (hereafter referred to as PIN1: and S380), 11 (T458 and S459), and 12 (S479) (see Supplemental GFP), in which one, two, or all three Ser residues in the encoded Figure 2 online). Based on the relative intensities of the phos- PIN1:GFP proteins were replaced by Ala (A), a nonphosphory- phorylated bands, substitution of these amino acids with Ala latable residue, or by Glu (E) to mimic phosphorylation. The residues had no clear effect on PID phosphorylation (see Sup- resulting constructs PIN1:GFP S1A(E), PIN1:GFP S3A(E), PIN1: plemental Figures 3B and 3C online), indicating that these GFP S1,3A(E), and PIN1:GFP S1,2,3A(E) were transformed into residues are not phosphorylated by PID. Arabidopsis Columbia (Col) wild-type plants, and GFP positive, BLAST analysis of the Arabidopsis protein database (http:// single locus insertion lines were selected for analysis. A previ- www.ncbi.nlm.nih.gov/protein/) showed that TPRXS(N/S) is a ously described PIN1:GFP line (Benkova et al., 2003) was used PIN-speciﬁc motif. Strikingly, alignment of amino acid se- as the control. 1132 The Plant Cell the weaker lines (e.g., PIN1:GFP S1A#14: 12.5%, n = 654) (Figure 3N), indicating that the severity of the cotyledon phenotypes corresponded to the level of mutant PIN1:GFP protein expres- sion. The stronger lines PIN1:GFP S1A#15 and PIN1:GFP S1,3A#10 developed ﬂowers with an increased number of petals and a decreased number of stamens and carpels (Figure 4B, Table 1), mimicking pid mutant ﬂoral defects (Figure 4A, Table 1) (Bennett et al., 1995). To exclude other possible reasons for the observed dominant phenotypes, such as cosuppression, the protein levels of the transgene and the endogenous PIN1 gene were quantiﬁed by protein gel blot analysis. All trangenic lines showed endoge- nous PIN1 expression similar to that of the wild type, and the mutant PIN1:GFP expression levels of the strong lines were comparable to that of the PIN1:GFP controlline(seeSupple- mental Figure 5B online). These results showed similar protein expression of both endogenous PIN1 and PIN1:GFP, suggest- ing that the dominant developmental defects observed can be attributed to the expression of the mutant PIN1:GFP proteins that outcompete endogenous PIN1, possibly by differential localization at the PM. For the PIN1:GFP S1,2,3A triple substitution lines, the post- embryo defects were more severe. No homozygous progeny could be obtained, and approximately one-fourth of the seeds from heterozygous plants (25.6%, n = 156 for line #23) failed to germinate, indicating that the homozygous progeny are embryo lethal. Among the germinating seedlings, we occasionally (9.6%, Figure 2. Ser Residues in Three Conserved Motifs within the PIN1HL Are n = 156 for line #23) observed strong patterning defects (indi- Phosphorylated by PID in Vitro. cated as “others” in Figures 3N and 3O), such as seedlings (A) In vitro assay of phosphorylation by GST-PID kinase using wild-type without a root (Figures 3G and 3H) or ball-shaped structures GST-PIN1HL and mutant protein substrates in which the indicated Ser without any discernible apical-basal axis that stopped growing residues (S1, S2, or S3) were replaced with Ala residues (A). The after germination (Figures 3I and 3J). positions of GST-PID and the full-length GST-PIN1HL are indicated in the autoradiograph (top panel) and the Coomassie-stained gel (bottom panel). Autophosphorylation of GST-PID can be observed in the top Reversible Phosphorylation of the Conserved Ser Residues panel. Under our experimental conditions, Escherichia coli–puriﬁed GST- Is Necessary and Sufﬁcient for Proper PIN1 Localization and PIN1HL was not stable, resulting in a reproducible pattern of degradation Plant Development bands. The Coomassie blue–stained gel was used as a control for protein loading. To further test the functionality of the loss- or gain-of-phos- (B) Quantitative assessment of the in vitro phosphorylation assay in (A). phorylation PIN1:GFP proteins, the PIN1:GFP and PIN1:GFP The phosphorylation intensity is expressed as the percentage of phos- S/A(E) mutant lines were crossed with the pin1 loss-of-function phorylation relative to the wild-type GST-PIN1HL protein. Numbers were mutant, and where possible, double homozygous plants were corrected for protein loading based on analysis of the Coomassie blue– selected for analysis. The pin1 mutant has aberrant cotyledon stained blot. numbers (Figures 3C, 3N, and 3O) and pin-formed inﬂores- cences with no ﬂowers or only a few defective ﬂowers (Okada The PIN1:GFP S/E and PIN1:GFP S3A plants showed largely et al., 1991). In our analyses, PIN1:GFP, as well as PIN1:GFP normal development at seedling and ﬂowering stage. By con- S1E, PIN1:GFP S3E, and PIN1:GFP S1,3E, complemented pin1 trast, other mutants exhibited a range of dominant defects. The cotyledon and inﬂorescence defects (Figures 3O, 4C, and 4D). PIN1:GFP S1A and PIN1:GFP S1,3A mutants showed cotyledon PIN1:GFP S1A and PIN1:GFP S3A partially rescued pin1 de- number defects, reﬂected by seedlings having one, three, or four fects, reﬂected by a reduced frequency of pin1 cotyledon defects cotyledons (Figures 3D to 3F), with the three-cotyledon pheno- from 17.6% (n = 318) to 2% (n = 402) and 12.1% (n = 348), type characteristic of the pid loss-of-function mutant (Figure 3B) respectively (Figure 3O), and by the observations that no pin- predominating. For these two mutant constructs, two lines each formed inﬂorescences were produced (Figures 4F and 4G). By were selected for detailed analyses, one with a lower PIN1:GFP contrast, PIN1:GFP S1,3A and PIN1:GFP S1,2,3A(E) lines expression level (PIN1:GFP S1A#14 and PIN1:GFP S1,3A#12) showed an enhanced frequency (23.9% n = 351, 32.7% n = and one with a higher PIN1:GFP expression level (PIN1:GFP 110, and 71.2% n = 66, respectively; Figure 3O) and severity of S1A#15 and PIN1:GFP S1,3A#10) (see Supplemental Figure 5A seedling defects, such as cup-shaped cotyledons (3.5%; Figure online). Notably, the stronger lines showed higher frequencies of 3K) or no cotyledons (2%; Figure 3L), phenotypes typical for cotyledon defects (e.g., PIN1:GFP S1A#15: 21.7%, n = 757) than the pin1 pid double mutant (Furutani et al., 2004) but not for Conserved Serines Direct PIN1 Polarity 1133 Figure 3. Seedling Phenotypes Induced by Manipulation of the Conserved Ser Residues in PIN1:GFP. (A) to (F) Cotyledon number defects observed in pid-14 (B), pin1 (C),and PIN1:GFP S/A ([D] to [F]) mutant seedlings compared with a wild-type seedling (A). +/ (G) to (J) Progeny from PIN1:GFP S1,2,3A#23 plants showing severe patterning defects, such as seedling without a root ([G] and [H]), with reduced cotyledons (H), or oblong structures ([I] and [J]). (K) to (L) Cotyledon defects observed in pin1 PIN1:GFP S1,3A#10 mutant seedlings showing a cup-shaped cotyledon (K) or no cotyledons (L). +/ +/ (M) Progeny from pin1 PIN1:GFP S1,2,3A#23 plants showing callus-like hypocotyl and cotyledons lacking a primary root. Bars in (A) to (M) = 10 mm. (N) and (O) Quantitative analysis of seedling defects induced by expression of the phosphomutant PIN1:GFP versions in the wild-type background (N) and in pin1 mutant background (O). The number of seedlings scored per mutant line is indicated. The legends in (O) are also used for (N). “Others” represents phenotypes other than cotyledon number defects, such as seedling without root, oblong structures, or callus-like seedlings. the pid or pin1 single mutant (Figures 3N and 3O). Around 5% 2004), in auxin transport inhibitor-induced pin-shaped inﬂores- +/2 +/2 of the progeny from pin1 PIN1:GFP S1,2,3A (double het- cence apices, PIN1:GFP was localized apically in the epidermis erozygous) plants lacked a primary root and developed callus- (Figure 5A). In a comparable region of the pin-shaped inﬂores- like hypocotyls and cotyledons (Figure 3M). At the ﬂowering cence apex, the phosphomimic PIN1:GFP S1,2,3E protein also stage, PIN1:GFP S1,3A and PIN1:GFP S1,2,3A(E) mutants could showed apical localization (Figure 5B), whereas the PIN1:GFP not complement pin1 defects and produced pin-like inﬂores- S1,3A and PIN1:GFP S1,2,3A proteins were targeted to the basal cences, with pin1 PIN1:GFP S1,2,3E occasionally forming ﬂowers side (Figures 5C and 5D), similar to PIN1 localization in pid producing seeds (Figure 4E). The pin1 PIN1:GFP S1,3A inﬂores- mutant (Friml et al., 2004). cences were branched and formed sterile ﬂowers with fused Collectively, these data indicated that PIN1 loss of phosphor- +/2 petals (Figure 4H), whereas pin1 PIN1:GFP S1,2,3A plants ylation results in its basal localization, whereas phosphomim- only produced a single needle-shaped inﬂorescence (Figure 4I). icking induces apical targeting of PIN1 in the shoot apical Next, we examined whether these mutant defects correlated meristem, both leading to failure to complement the pin1 mutant with changes in the subcellular localization of the mutant PIN1: phenotypes. These observations are consistent with the identi- GFP proteins. Consistent with previous observations (Friml et al., ﬁed role for PID as a binary switch in PIN1 basal-apical polar 1134 The Plant Cell Figure 4. Inﬂorescence and Flower Defects Observed after Expression of Phosphomutant PIN1:GFP Proteins. (A) and (B) Flowers of the pid-14 loss-of-function mutant (A) and the PIN1:GFP S1,3A#10 mutant (B) show similar defects. (C) to (I) Complementation analysis of pin1 loss-of-function mutant inﬂorescence and ﬂower defects. PIN1:GFP (C) and PIN1:GFP S1,3E#18 (D) fully rescued pin-shaped inﬂorescence defects, whereas PIN1:GFP S1A#15 (F) and PIN1:GFP S3A#6 (G) only partially rescued pin-shaped inﬂorescence defects. PIN1:GFP S1,2,3E#8 (E), PIN1:GFP S1,3A#10 (H), and PIN1:GFP S1,2,3A#23 (I) did not rescue pin1 inﬂorescence defects. Insets show details of ﬂower morphology and pin-like inﬂorescences. Bars in whole-plant photographs = 5 cm. localization in the shoot apex, suggesting that the identiﬁed Ser (5%). These embryo phenotypes resembled those of mutants residues are PID related. with defects in auxin transport (Friml et al., 2003) and gnom mutants (Mayer et al., 1993). Examination of the subcellular localization of PIN1:GFP Strong Embryo Defects Are Induced by PIN1:GFP S1,2,3A during embryogenesis showed that PIN1:GFP S1,2,3A S1,2,3A Mislocalization polarity failed to establish properly (n = 43). Endogenous PIN1 (or wild-type PIN1:GFP) proteins localize at the basal side of the The embryo and seedling lethality observed in the progeny of +/2 +/2 pin1 PIN1:GFP S1,2,3A plants led us to study the early em- provascular cells (Figure 6C), generating an auxin maximum that bryo development. Compared with pin1 PIN1:GFP embryos that deﬁnes the hypophyseal cell group (Figure 6F) and at the apical showed stereotypic patterns of cell divisions (Figure 6A), ;30% side of the epidermal cells from triangular stage on (Figure 6C +/2 +/2 (n = 86) of the embryos from pin1 PIN1:GFP S1,2,3A plants and inset), generating strong auxin activity at tips of developing exhibited a range of developmental aberrations at different cotyledons (Steinmann et al., 1999; Friml et al., 2003). In embryos +/2 +/2 stages (Figure 6B). In 15% of the embryos, the basal tier and from pin1 PIN1:GFP S1,2,3A plants with a largely wild-type suspensor cells showed defective cell divisions. The most severe morphology (65%, n = 43), the basal PIN1:GFP localization in the cases were characterized by embryos with globular structures provascular cells was lost (Figure 6D, star), causing a reduction that lacked a deﬁned apical-basal axis and bilateral symmetry of the auxin reporter PDR5:GFP signal in the hypophysis (Figure Conserved Serines Direct PIN1 Polarity 1135 Table 1. Quantitative Analysis of Dominant pid-Like Floral Organ Defects Induced by PIN1:GFP S/A Floral Organ Numbers Genotype Sepal Petal Stamen Carpel Total No. of Flowers Col 4.00 4.00 5.80 6 0.40 2.00 50 pid-14 2.80 6 1.20 8.35 6 1.42 1.10 6 1.07 0.00 20 PIN1:GFPS1A#15 4.11 6 0.68 5.53 6 0.63 4.91 6 0.85 1.52 6 0.57 45 PIN1:GFPS1,3A#10 4.08 6 0.68 5.71 6 1.09 4.15 6 0.87 1.83 6 0.33 52 Numbers are means derived from analyses of ﬂowers from at least ﬁve plants for each genotype. Standard deviations are indicated. In the case of organ fusion in the same whorl, a fused ﬂoral organ is counted as one in that whorl. In the case of organ fusion between different whorls, fused organs are counted as one organ in the each whorl. 6G). As a comparison, the basal localization of PIN1:GFP S1,3A can be mimicked by manipulation of these Ser residues. For in the provascular cells was not changed (see Supplemental additional conﬁrmation that our identiﬁed phosphoserines are Figure 6A online). In embryos exhibiting strong defects, PIN1: targets of PID activity in vivo, we crossed the PIN1:GFP, PIN1: GFP S1,2,3A polarity was dramatically disrupted, and abundant GFP S1,3A, and PIN1:GFP S1,2,3E lines with the strong PID intracellular signal was observed (Figure 6E). The cells with the overexpression line P35S:PID#21 (Benjamins et al., 2001). The ﬂuorescent signal at the PM showed no polarity or randomized PIN1:GFP S1,2,3A line could not be used for this purpose, as it polarity (see Supplemental Figures 6B and 6C online), and a clear could only be maintained in the heterozygous state, which PDR5:GFP maximum was not detected (Figure 6H). precluded an equivalent comparison of the root meristem col- These data implied that phosphorylation of the three Ser lapse frequency. residues is required for auxin-related embryo development by In line with our previous observations (Friml et al., 2004), PID regulating PIN1 PM localization. overexpression induced a clear basal-to-apical shift of PIN1:GFP localization in root stele cells (Figures 7A and 7B). By contrast, PIN1:GFP S1,3A showed basal localization in both wild-type and PIN1 Phosphorylation at the Conserved Ser Residues Is P35S:PID backgrounds (Figures 7C and 7D). Simultaneous im- RelatedtoPID Activity munolocalization showed the basal-to-apical polarity shift of Above, we showed that the conserved Ser residues are phos- PIN2 in the cortex and PIN4 in the root meristem (Figure 7M), phorylated by PID in vitro and that PID regulation of PIN1 polarity demonstrating that PID overexpression was sufﬁcient to induce and the resulting inﬂorescence development (Friml et al., 2004) PIN polarity shifts and that the S1,3A substitutions rendered Figure 5. Subcellular Localization of Wild-Type and Phosphomutant PIN1:GFP Proteins in Epidermal Cells of the Inﬂorescence Apex. Confocal laser scanning microscopy of pin-formed inﬂorescence apices of pin1 mutant plants expressing wild-type PIN1:GFP (naphthylphthalamic acid treated) (A), PIN1:GFP S1,2,3E (B), PIN1:GFP S1,3A (C), and PIN1:GFP S1,2,3A (D). The white dashed boxes in the overview images (left) indicate the position of the zoomed-in images (right). The polarity of PIN1:GFP in the epidermal cells of the inﬂorescence apex is indicated with arrows. PIN1:GFP +/ S1,2,3A#23 indicates that the plant is heterozygous for the transgene. 1136 The Plant Cell PIN1:GFP insensitive to that. On the other hand, the PIN1:GFP S1,2,3E protein exhibited apical localization in some cell ﬁles and apolar localization in others in both wild-type and P35S:PID backgrounds (Figures 7E and 7F), further indicating that the polar localization of the phosphorylation mutant PIN1:GFP proteins occurred independent of PID overexpression. Consistent with the PIN1 function of mediating auxin transport to root tips, PIN1: GFP S1,3A induced an enhancement of auxin reporter PDR5: GFP signal compared with the control PIN1:GFP roots (Figures 7G and 7I; see Supplemental Figure 7 online; Student’s t test, P < 0.05), consistent with the basal localization of PIN1:GFP S1,3A protein. By contrast, the PDR5:GFP signal was signiﬁcantly reduced in PIN1:GFP S1,2,3E root tips (Figure 7K; see Supple- mental Figure 7 online; Student’s t test, P < 0.05), in line with its preferably apical localization. In the P35S:PID background, basally accumulated PIN1:GFP S1,3A resulted in higher auxin accumulation compared with PIN1:GFP (Figures 7J and 7H; see Supplemental Figure 7 online; Student’s t test, P < 0.05) and as a result signiﬁcantly reduced the P35S:PID-induced root collapse frequency (Figure 7N). By con- trast, PIN1:GFP S1,2,3E had no signiﬁcant effect on P35S:PID- induced root collapse (Figure 7N). This might be due to the already maximal effect of PID overexpression on PIN apicaliza- tion and auxin depletion, as no signiﬁcant reduction of auxin accumulation was detected (Figures 7B and 7F; see Supple- mental Figure 7 online). The genetic interactions between phos- phomutants and PID conﬁrmed that our identiﬁed Ser residues are phosphotargets of PID. Together, these results linked phosphorylation of the con- served Ser residues to polar PIN1 driven differential auxin dis- tribution in roots and further demonstrated that the conserved phosphoserines are key determinants in modulating plant archi- tecture by instructing PIN1 polarity. DISCUSSION Previously, it has been shown that the PID kinase and PP2A phosphatases antagonistically regulate PIN polarity by reversible phosphorylation of the PINHL (Michniewicz et al., 2007). How- Figure 6. Embryo Defects Induced by PIN1:GFP S1,2,3A Mislocalization Are Due to Disturbed Auxin Distribution. ever, no functional evidence was provided to support the im- portance of this phosphorylation in planta. In this study, we (A) and (B) Differential interference contrast microscopy images of identiﬁed Ser residues centrally located in three conserved +/ +/ embryos from wild-type (A) and pin1 PIN1:GFP S1,2,3A#23 (B) TPRXS(N/S) motifs within the PIN1HL that are phosphorylated plants. Text at the bottom of each image indicates the developmental by PID in vitro. Subsequent in planta analyses of loss-of- stage of the embryo. For the defective embryos in (B), the developmental stage was based on a rough estimate of the cell number. Bars = 10 mm. phosphorylation and phosphomimic PIN1:GFP mutants proved (C) to (E) Confocal laser scanning microscopy images of pin1 PIN1:GFP that reversible phosphorylation of all three residues is required heart-stage embryos (C) and wild-type looking (D) and defective looking and sufﬁcient for proper PIN1 polar localization and auxin- +/ +/ (E) embryos from pin1 PIN1:GFP S1,2,3A#23 plants from the same regulated plant development. developmental stage. Insets in (C) and (D) represent confocal scans through the epidermal cell layer of cotyledon primordia. White arrows in (C) and (D) indicate the PIN1:GFP polarity, and a star in (D) indicates the Ser Resides in the Conserved TPRXS(N/S) Motifs Are Crucial absence of basally localized PIN1:GFP S1,2,3A protein. Phosphorylation Targets in the PIN1HL (F) to (H) Confocal laser scanning microscopy images of PDR5:GFP +/ auxin distribution in embryos from pin1 PIN1:GFP (F) and pin1 PIN1: Several Ser and Thr residues within the PIN1HL have been +/ GFP S1,2,3A#23 ([G] and [H]) plants, showing a reduced (G) or identiﬁed as phosphorylation substrates in vivo, and not surpris- mislocalized (H) auxin maximum. ingly, S2 and S3 are among them (Nu¨ hse et al., 2004; Benschop et al., 2007). Two other phosphorylation targets in the PIN1HL identiﬁed by mass spectrometry analysis are Ser-337 and Conserved Serines Direct PIN1 Polarity 1137 Figure 7. PIN1:GFP Polarity Changes Induced by Manipulation of Phosphoserines Correlate with Changes in the Auxin Maximum in the Root Tip. (A) to (F) Confocal laser scanning microscopy of primary roots of 5-d-old seedlings expressing PIN1:GFP ([A] and [B]), PIN1:GFP S1,3A#10 ([C] and [D]), and PIN1:GFP S1,2,3E#8 ([E] and [F]) in the wild-type ([A], [C],and [E])or P35S:PID ([B], [D],and [F]) background. The white dashed boxes in the overview images (top) indicate the position of the zoomed-in images (bottom), in which the PIN1:GFP polarity is indicated by arrows. The seedlings are homozygous for the indicated T-DNA constructs. Bars = 5 mm. (G) to (L) Confocal laser scanning microscopy of PDR5:GFP signals in 3-d-old seedling root tips expressing PIN1:GFP ([G] and [H]), PIN1:GFP S1,3A#10 ([I] and [J]), and PIN1:GFP S1,2,3E#8 ([K] and [L]) in the wild-type ([G], [I],and [K])or P35S:PID ([H], [J],and [L]) background. The seedlings are heterozygous for the PDR5:GFP reporter. Bar = 50 mm. (M) PIN2 and PIN4 immunolocalization in 3-d-old P35S:PID PIN1:GFP S1,3A#10 seedling roots. The arrows indicate the apical PIN2 and PIN4 localization induced by PID overexpression. Bars = 5 mm. (N) Quantiﬁcation of the effects of wild-type, loss-of-phosphorylation, or phosphomimic PIN1:GFP expression on the PID overexpression-induced root meristem collapse phenotype. Percentages are based on scoring 153, 144, 179, and 98 seedlings at 4, 6, and 8 d after germination. 1138 The Plant Cell Thr-340 in the MFSPNTG sequence (Benschop et al., 2007; In the epidermis of the shoot apex, loss-of-phosphorylation Michniewicz et al., 2007). Recent functional analysis of these res- PIN1:GFP S1,3A and PIN1:GFP S1,2,3A proteins were targeted idues in planta has shown that their phosphorylation states are to the basal side (Figures 5C and 5D), whereas the phospho- important for PIN1 polarity (Zhang et al., 2010). However, these mimic PIN1:GFP S1,2,3E protein was apicalized (Figure 5B). This residues are not directly phosphorylated by PID (Zhang et al., pattern is consistent with the binary switch mode, which pro- 2010), suggesting that other protein kinases could coordinately poses that no or low kinase activity (in the pid mutant) results in regulate PIN polarity with PID by phosphorylating Ser-337 and PIN1 localization at the basal membrane and that above thresh- Thr-340. Ser-337 could be a target of mitogen-activated protein old kinase activity (in the wild type) directs PIN1 apical localiza- kinases (Benschop et al., 2007), as mitogen-activated protein tion (Friml et al., 2004). Despite its apical localization, PIN1:GFP kinases preferably phosphorylate Ser or Thr residues followed by S1,2,3E could not complement pin1 inﬂorescence defects (Fig- a Pro in both plant and animal systems (Pearson et al., 2001; Liu ure 4E), a result that is seemingly contradictory to the observa- and Zhang, 2004). tion that in the shoot apex of wild-type plants, PIN1 is apically The TPRXS(N/S) motifs in the HL are highly conserved among localized. However, PIN1 polarity and the resulting auxin maxima six Arabidopsis PIN proteins (Figure 1) and among PIN1 homo- in the shoot apex have been reported to be highly dynamic logs from other land plant species (see Supplemental Figure 4 (Heisler et al., 2005), and a constitutively apical-localized PIN1: online), suggesting functional conservation of the motifs. Inter- GFP S1,2,3E would obviously interfere with auxin-mediated estingly, PID orthologs have been identiﬁed in maize (Zea mays) organ initiation. In addition, the shoot defects could also be and rice (Oryza sativa; McSteen et al., 2007; Morita and Kyozuka, attributable to PIN1:GFP S1,2,3E apical localization in the vas- 2007), and it has been shown that the maize ortholog BARREN cular tissues where PIN1 polarity is normally basal. INFLORESCENCE2 phosphorylates Zm PIN1a, the maize ortho- In seedling roots, loss-of-phosphorylation PIN1:GFP S1,3A log of Arabidopsis PIN1 in vitro, and that it regulates the subcel- was localized on the basal membrane of root stele cells, in both lular localization of Zm PIN1a in vivo (Skirpan et al., 2009). Further the wild type and P35S:PID background (Figures 7C and 7D), research is needed, however, to establish whether this functional indicating that the protein is unresponsive to PID activity. Even conservation extends to the conserved TPRXS(N/S) motifs in all though endogenous PIN2 and PIN4 in the same roots underwent PIN1-type proteins in Arabidopsis and in other plant species. the basal-to-apical polarity shift induced by PID overexpression For the Arabidopsis PIN1-type proteins (PIN1, 2, 3, 4, and 7), (Figure 7M), the root collapse was delayed (Figure 7N). Previ- the predicted protein structure consists of two sets of ﬁve ously, we have shown that pin2 and pin4 loss-of-function mu- transmembrane domains that are linked by a HL (Ga¨ lweiler tants delay P35S:PID-mediated root meristem collapse (Friml et al., 1998; Mu¨ ller et al., 1998). On the other hand, for the PIN5- et al., 2004). It is therefore not surprising that the basally localized type proteins (PIN5, 6, and 8), two sets of ﬁve and four trans- phosphorylation-deﬁcient PIN1 is able to reduce the frequency membrane domains, respectively, are predicted (Mravec et al., of root meristem collapse. 2009). PIN5 and PIN8 clearly lack a large central HL (Mravec By contrast, PIN1:GFP S1,2,3E was preferably targeted to the et al., 2009), but our alignment suggests that a shorter HL is apical membrane of root stele cells (Figure 7E). It has been shown present in PIN6 and that it contains two TPRXS(N/S) motifs that apical localized PIN proteins are more resistant to BFA- (Figure 1). Immunohistochemical analyses and studies using induced internalization (Kleine-Vehn et al., 2008). Consistently, reporter fusion proteins have shown that PIN1, 2, 3, 4, and 7 PIN1:GFP S1,2,3E was more resistant to BFA treatment than are localized at the PM (Ga¨ lweiler et al., 1998; Mu¨ ller et al., 1998; wild-type PIN1:GFP (Kleine-Vehn et al., 2009). This, together with Friml et al., 2002a, 2002b, 2003), whereas PIN5, 6, and 8 are the reduction of the PDR5:GFP signal in root tips compared with ER localized (Mravec et al., 2009). This suggests that for ER- that in PIN1:GFP roots (Figures 7K and 7G; see Supplemental localized PIN proteins, there has been no selective advantage Figure 7 online; Student’s t test, P < 0.05), strongly support that to maintain PINHL-located polarity determinants. Alternatively, PIN1:GFP S1,2,3E predominantly localizes to the apical mem- the loss of phosphorylation motifs may have been crucial for brane. The apical localization of phosphomimic PIN1:GFP did allowing the diversiﬁcation of PIN proteins function from the PM not induce root meristem collapse on its own. This can be to the ER. explained by the fact that the PID overexpression-induced root meristem collapse is caused by the basal-to-apical polarity change of three PIN proteins (PIN1, PIN2, and PIN4), of which Phosphorylation of the Conserved Ser Residues Directs PIN2 and PIN4 are crucial players (Friml et al., 2004). PIN1 Polar Localization and Auxin-Regulated Moreover, PIN1:GFP S1,2,3E apicalization was not complete, Plant Development as in certain cell ﬁles, apolar PIN1:GFP S1,2,3E localization was In wild-type plants, PIN1 proteins are basally localized in (pro) also detected (Figures 7E and 7F). Possibly, Glu is not a perfect vascular tissues in embryos, leaves, and roots and are apically phosphomimic in these cell ﬁles. Alternatively, there could still be localized in the epidermis of shoot apices and embryos additional PID phosphorylation targets in the PIN1HL, as the (Ga¨ lweiler et al., 1998; Benkova et al., 2003; Friml et al., 2003, S1,2,3A mutations did not completely abolish PID phosphory- 2004; Reinhardt et al., 2003). Our analyses of the subcellular lation of the PIN1HL in vitro. Nonetheless, the basal localization localization of mutant PIN1:GFP proteins in different tissues of loss-of-phosphorylation PIN1 and the apical localization showed that manipulation of the phosphoserines leads to of phosphomimic PIN1, together with their opposite effects on changes in PIN1 polarity, most (but not all) of which are consis- PID overexpression-induced root collapse, indicated that our tent with the PID binary switch function (Friml et al., 2004). identiﬁed phosphoserines are functional targets of PID. Conserved Serines Direct PIN1 Polarity 1139 During embryogenesis, phosphorylation of the conserved Ser complete loss of phosphorylation leads to not only PID-related residues seems to play a role in the maintenance of PIN1 PM morphological and cellular defects, but also defects never ob- localization rather than polarity alteration, as a complete loss of served in PID-regulated processes. This leads us to hypothesize phosphorylation induces PIN1:GFP intracellular accumulation that the three phosphoserines identiﬁed here are not only targets (Figures 6D and 6E). This mislocalized PIN1:GFP S1,2,3A inter- of PID, but also of other AGC3 kinases, and might explain why pid fered with auxin accumulation (Figures 6G and 6H), resulting in loss of function does not lead to apical-to-basal PIN2 polarity strong embryo defects (Figure 6B), similar to embryo defects changes in the root (Sukumar et al., 2009) or why the strong of gnom and pp2aa1,3 loss-of-function (Mayer et al., 1993; embryo defects induced by PIN1:GFP S1,2,3A are not observed Michniewicz et al., 2007) or PID gain-of-function (RPS5A>>PID) in pid mutants. Further research is needed to validate this (Friml et al., 2004) mutants. The common reason for the embryo hypothesis. defects in these different mutants is that the basal localization of Our data, together with the conclusion that PIN proteins and PIN1 in the provascular cells is lost, disturbing auxin accumula- PID-like kinases coevolved during the transition of plants from tion in the basal tier of the embryo (Steinmann et al., 1999; Friml water to land (Galva´ n-Ampudia and Offringa, 2007), as well as the et al., 2004; Michniewicz et al., 2007; Figures 6G and 6H), which demonstrated functional relationship between PID and PINs leads to auxin-regulated embryo defects. The enhanced intra- (Friml et al., 2004), lead us to propose that phosphorylation of the cellular accumulation of loss-of-phosphorylation PIN1 (PIN1: conserved Ser residues plays an important role in PIN-dependent GFP S1,2,3A) is in line with the observation of PIN2 accumulation auxin transport throughout the evolution of plants. However, in endomembrane structures in pid-9 loss-of-function mutant many questions still need to be answered. Functional analysis roots (Sukumar et al., 2009), which suggests that low levels of concerning phosphorylation of the three Ser residues in other PID activity result in intracellular accumulation of PIN proteins. PIN proteins and the regulation of PIN proteins by other AGC Loss-of-phosphorylation-induced intracellular PIN1 accumula- kinases will be the next challenges for the coming years. tion might be a result of reduced recycling to the PM, increased endocytosis, or reduced sorting from endosomes to the vacu- METHODS oles. Further research is needed to distinguish between these possibilities. Plant Material, Growth Conditions, and Phenotypic Analysis For all experiments, Arabidopsis thaliana of ecotype Col-0 was used. AGC Kinases and PIN Proteins: A Stable Marriage in Construction of PIN1:GFP (to produce PIN1:GFP fusion proteins) and Plant Evolution P35S:PID (to overexpress PID) and the corresponding Arabidopsis lines were described previously (Benjamins et al., 2001; Benkova et al., 2003). PID belongs to the plant-speciﬁc AGCVIII family of protein The loss-of-function alleles pid-14 (SALK_049736) and pin1 (SALK_047613) kinases, which are plant orthologs of the mammalian cAMP- were obtained from the Nottingham Arabidopsis Stock Centre. dependent PKA, cGMP-dependent protein kinase G, and protein Seedlings were grown on MA medium (Masson and Paszkowski, 1992) kinase C (Bo¨ gre et al., 2003). Among the three identiﬁed Ser at 218C and a 16-h-light/8-h-dark photoperiod. One-week-old seedlings residues, two (S2 and S3) are recognized by NetPhos as PKA were transferred to MA medium supplemented with 50 mM naphthylph- phosphorylation targets (Blom et al., 1999), corroborating previ- thalamic acid (Pfaltz and Bauer), an auxin transport inhibitor, to induce pin ous suggestions that the plant AGCVIII kinases might have been inﬂorescences. The number of seedlings with root meristem collapse was derived from the same ancestral kinase as animal PKAs (Bo¨ gre counted 4, 6, and 8 d after germination. Plants were grown on a mixture of et al., 2003; Galva´ n-Ampudia and Offringa, 2007). 9:1 substrate soil and sand (Holland Potgrond) at 218C, a 16-h photope- riod, and 70% relative humidity. In Arabidopsis, PID groups together with 22 other AGCVIII protein kinases (Bo¨ gre et al., 2003; Galva´ n-Ampudia and Offringa, 2007), of which the blue light receptors PHOT1 and PHOT2, the DNA Constructs, Sequence Alignment, and Plant Transformation root growth regulators WAG1 and WAG2, and the D6 protein Molecular cloning, DNA sequence analysis, and DNA and protein se- kinases (D6PKs) have also been shown to be involved in auxin quence alignments were performed using the Vector NTI 10 software transport-regulated plant development (Sakai et al., 2001; (Invitrogen). For the in silico prediction of putative phosphorylation sites, Santner and Watson, 2006; Zourelidou et al., 2009). Previously, we used the NetPhos software (Blom et al., 1999). The pET-PIN1HLsv it was shown that the PIN1HL is also phosphorylated by D6PKs (Galweiler et al., 1998) and pGEX-PID (Benjamins et al., 2003) constructs (Zourelidou et al., 2009). Although the D6PK genes showed a have been described before. The pET-PID fusion construct was gener- genetic interaction with PIN1, the D6PK protein had no effect on ated by cloning the PID cDNA into the pET16B (Novagen) derivative pET16H, which was kindly provided by Johan Memelink. The pGEX- PIN1 polarity regulation (Friml et al., 2004; Michniewicz et al., PIN1HL fusion was generated by cloning the PIN1HL SmaI/SalI fragment 2007; Zourelidou et al., 2009). Further investigation into the from pACT2-PIN1HL into the corresponding restriction sites of plasmid possible D6PK phosphorylation targets in PINs should provide pGEX-KG (Guan and Dixon, 1991). For construct pGreen0229 PPIN1: insight into the differential action of the distinct regulatory path- PIN1:GFP,the PIN1 gene was ampliﬁed from Col-0 DNA using prim- ways by PID and D6PKs. PID, together with WAG1, WAG2, and ers PIN1F 59-CGAATTCATTATTCCATTGGCGTTGTC-39 and PIN1R AGC3-4, groups to the AGC3 clade within the AGCVIII family 59-CAGGTACCCACTTCTTATTTTGGTGAGA-39, and the fragment was (Galva´ n-Ampudia and Offringa, 2007). The localization of all four digested with EcoRI and KpnI, and cloned into the corresponding sites of kinases at the plasma membrane (Galva´ n-Ampudia and Offringa, pGreen0229. Subsequently, the BstAPI fragment in this genomic clone 2007), and their genetic interactions (Cheng et al., 2008), suggest was exchanged for the BstAPI fragment containing the PIN1:GFP trans- functional redundancy among the AGC3 kinases. In our studies, lational fusion from pBIN-PPIN1:PIN1:GFP (Friml et al., 2003). The 1140 The Plant Cell Quickchange XL site-directed mutagenesis kit (Stratagene) was used to Membrane Protein Extraction and Protein Gel Blot Analysis generate mutant constructs. Oligonucleotides used for mutagenesis are For the protein gel blot analysis, shoots from 7-d-old seedlings (grown on listed in Supplemental Table 1 online. Arabidopsis plants were transformed 1% agar medium containing 0.53 Gambourg-B5 and 1% sucrose, with a by the ﬂoral dip method as described (Clough and Bent, 1998). 16-h-light/8-h-dark photoperiod) were used to extract the membrane protein fraction. For the PIN1:GFP S1,2,3A mutant, shoots from 3-week- Protein Puriﬁcation and in Vitro Phosphorylation Assays old heterozygote seedlings selected on antibiotic plates were used. Membrane protein extraction and protein gel blot analyses were de- Protein puriﬁcation and in vitro phosphorylation assays were performed scribed recently (Abas and Luschnig, 2010). as described before (Benjamins et al., 2003; Michniewicz et al., 2007), with the following speciﬁcations. GST/HIS-tagged full-length PID and different mutant versions of GST/HIS-tagged PIN1HL proteins were used Immunolocalization, Microscopy, and Signal Analysis in in vitro phosphorylation assays. Cultures of Escherichia coli strain Whole-mount immunolocalization was performed as described (Friml Rosetta (Novagen) containing the constructs were grown at 378Cto et al., 2004), using rabbit anti-PIN2 (dilution 1:200; Abas et al., 2006) and OD = 0.6 in 50 mL LC supplemented with 100 mg/mL carbenicillin, 30 rabbit anti-PIN4 (dilution 1:200; Friml et al., 2004) as the primary antibody mg/mL chloramphenicol, and 25 mg/mL kanamycin. The cultures were and an anti-rabbit Alexa 488 conjugate as the secondary antibody then induced for 4 h with 1 mM isopropyl b-D-1-thiogalactopyranoside at (dilution 1:200; Molecular Probes). 308C, after which cells were harvested by centrifugation (20 min at 4000 PIN1:GFP signal in shoots and embryos and DR5:GFP signal in roots rpm, 48C in tabletop centrifuge) and frozen in liquid nitrogen. Precipitated (used in Figures 7G to 7L) were visualized in water without ﬁxation. PIN1: cells were resuspended in 2 mL extraction buffer (EB; 13 PBS, 2 mM GFP signal in 3-d-old seedling roots (used in Figures 7A to 7F) was EDTA, and 2 mM DTT, pH 8.0) supplemented with 0.1% Tween 20 and 0.1 visualized with ﬁxation and permeation steps as described (Friml et al., mM of the protease inhibitors phenylmethanesulfonyl ﬂuoride, leupeptin, 2004). Signals were detected with confocal laser scanning microscopy and aprotinin (Sigma-Aldrich) and sonicated for 2 min on ice. From this (Zeiss LSM 5 confocal microscope). The images were processed by point on, all steps were performed at 48C. Eppendorf tubes containing the ImageJ software (http://rsb.info.nih.gov/ij/) and assembled in Adobe sonicated cells were centrifugated at full speed (14,000 rpm) for 20 min, Photoshop CS2. PDR5:GFP signal intensity was measured by ImageJ, and the supernatants were transferred to 15-mL tubes containing 100 mL and error bars were obtained based on the measurement of three to six preequilibrated glutathione sepharose resin from GE Healthcare (pre- seedling roots per line. The Y value is the average DR5 signal intensity of equilibration performed with three washes of EB). Resin-containing each line relative to that in wild-type PIN1:GFP line. mixtures were incubated with gentle agitation for 1 h, subsequently Embryo development was analyzed by differential interference con- centrifuged at 500 relative centrifugal force for 3 min, and the precipitated trast microscopy (Zeiss Axioplan2) on cleared ovules (1-h treatment in the resin was washed three times with 20 resin volumes of EB. Then, three clearing solution of chloral hydrate:H O:glycerol = 8:3:1). resin volumes of glutathione elution buffer (10 mM reduced glutathione and 50 mM Tris-HCl, pH 8.0) were added to the glutathione sepharose Accession Numbers resin, and the mixture was agitated for 10 min at room temperature with gentle agitation. The resin was subsequently centrifuged for 3 min at 500 Sequence data from this article can be found in The Arabidopsis In- RCF, and the supernatant containing the desired protein was transferred formation Resource (http://www.Arabidopsis.org/) or GenBank/EMBL to a new tube; this process was repeated twice more. The solutions con- databases under the following accession numbers: Arabidopsis taining the proteins were diluted 1000-fold in Tris buffer (25 mM Tris-HCl, PIN1 (gi:15219501), Arabidopsis PIN2 (gi:42558886), Arabidopsis PIN3 pH 7.5, and 1 mM DTT) and concentrated to a workable volume (usually (gi:42558887), Arabidopsis PIN4 (gi:42558871), Arabidopsis PIN6 50 mL) using Vivaspin microconcentrators with a 10-kD cutoff and a (gi:42558888), Arabidopsis PIN7 (gi:42558877), barrel medic (Medicago maximum capacity of 600 mL (Vivascience). Glycerol was added as preser- truncatula) PIN1 (gi:25986771), rice (Oryza sativa) PIN1 (gi:75251559), vative to a ﬁnal concentration of 10%, and samples were stored at 2808C. moss (Physcomitrella patens) PIN1 (gi:55859521), and maize (Zea mays) Approximately 1 mg of each puriﬁed GST/HIS-tagged protein (PID and PIN1c (gi:171850415). Arabidopsis T-DNA insertion mutants representing substrates) was added to a 20 mL kinase reaction mix, containing 13 the loss-of-function alleles are SALK_049736 (pid-14) and SALK_047613 kinase buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, and 5 mM MgCl )and (pin1). Nottingham Arabidopsis Stock Centre identiﬁcation numbers for 13 labeled ATP solution (100 mM MgCl /ATP and 1 mCi [g- P]ATP). the transgenic Arabidopsis lines PIN1:GFP and P35S:PID are N9362 and Reactions were incubated at 308C for 30 min and stopped by addition N9867, respectively. of 5 mLof 53 protein loading buffer (310 mM Tris-HCl, pH 6.8, 10% SDS, 50% glycerol, 750 mM b-mercaptoethanol, and 0.125% bromophenol Supplemental Data blue) and boiling for 5 min. Reactions were subsequently separated over 10% acrylamide gels, which were washed three times for 30 min with The following materials are available in the online version of this article. Kinase Gel wash buffer (5% trichoroacetic acid and 1% Na H P O ), 2 2 2 7 Supplemental Figure 1. Amino Acid Sequence of the Arabidopsis stained with Coomassie Brilliant Blue, and dried. Autoradiography was PIN1 Protein. performed for 24 to 48 h at 2808C using Fuji Super RX x-ray ﬁlms and Supplemental Figure 2. Relative in Vitro Phosphorylation Intensities intensiﬁer screens. The relative intensity of phosphorylation bands was of Peptides Containing Putative Phosphorylation Sites in the PIN1HL analyzed using ImageJ software (http://rsbweb.nih.gov/ij/). (Boxed) by PID. For the peptide assays, 1 mg of puriﬁed PID was incubated with 4 nmol mer of 9 biotinilated peptides (Pepscan) in a phosphorylation reaction as Supplemental Figure 3. In Vitro Phosphorylation Assays of Predicted described above. Reaction processing, spotting, and washing of the Phosphorylation Targets in the PIN1HL by PID. SAM Biotin Capture Membrane (Promega) were performed according to Supplemental Figure 4. Alignment of the Amino Acid Sequences of the protocol of the manufacturer. Following washing, the membranes the PIN1 Proteins from Five Plant Species Showing the Evolutionary were sealed in plastic wrap and exposed to x-ray ﬁlms for 24 to 48 h at Conservation of the Three TPRXS(N/S) Motifs. 2808C using intensiﬁer screens. The phosphorylation intensities of the peptides were determined by densitometry analysis of the autoradio- Supplemental Figure 5. Quantiﬁcation of Protein Expression by graphs using the ImageQuant software (Molecular Dynamics). Western Blot Analysis Using PIN1-Speciﬁc Antibody. Conserved Serines Direct PIN1 Polarity 1141 Supplemental Figure 6. Confocal Laser Scanning Microscopy Im- Blom, N., Gammeltoft, S., and Brunak, S. (1999). Sequence and ages of Mutated PIN1:GFP Localization. structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 294: 1351–1362. Supplemental Figure 7. Quantiﬁcation of Auxin Reporter PDR5:GFP Bo¨ gre, L., Okre´ sz, L., Henriques, R., and Anthony, R.G. (2003). Expression in Root Tips. Growth signalling pathways in Arabidopsis and the AGC protein Supplemental Table 1. Oligonucleotides Used in Site-Directed Mu- kinases. Trends Plant Sci. 8: 424–431. tagenesis. Carraro, N., Forestan, C., Canova, S., Traas, J., and Varotto, S. (2006). 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Publisher: Oxford University Press
Copyright: © 2010 American Society of Plant Biologists
ISSN: 1040-4651
eISSN: 1532-298X
DOI: 10.1105/tpc.109.072678
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Abstract

The Plant Cell, Vol. 22: 1129–1142, April 2010, www.plantcell.org ã 2010 American Society of Plant Biologists Phosphorylation of Conserved PIN Motifs Directs Arabidopsis W OA PIN1 Polarity and Auxin Transport a a,1 b a a,2 Fang Huang, Marcelo Kemel Zago, Lindy Abas, Arnoud van Marion, Carlos Samuel Galva´ n-Ampudia, and a,3 Remko Offringa Department of Molecular and Developmental Genetics, Institute of Biology, Leiden University, 2333 EB Leiden, The Netherlands Institute for Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences (BOKU Wien), A-1190 Vienna, Austria Polar cell-to-cell transport of auxin by plasma membrane–localized PIN-FORMED (PIN) auxin efﬂux carriers generates auxin gradients that provide positional information for various plant developmental processes. The apical-basal polar localization of the PIN proteins that determines the direction of auxin ﬂow is controlled by reversible phosphorylation of the PIN hydrophilic loop (PINHL). Here, we identiﬁed three evolutionarily conserved TPRXS(N/S) motifs within the PIN1HL and proved that the central Ser residues were phosphorylated by the PINOID (PID) kinase. Loss-of-phosphorylation PIN1:green ﬂuorescent protein (GFP) (Ser to Ala) induced inﬂorescence defects, correlating with their basal localization in the shoot apex, and induced internalization of PIN1:GFP during embryogenesis, leading to strong embryo defects. Conversely, phosphomimic PIN1:GFP (Ser to Glu) showed apical localization in the shoot apex but did not rescue pin1 inﬂorescence defects. Both loss-of-phosphorylation and phosphomimic PIN1:GFP proteins were insensitive to PID overexpression. The basal localization of loss-of-phosphorylation PIN1:GFP increased auxin accumulation in the root tips, partially rescuing PID overexpression-induced root collapse. Collectively, our data indicate that reversible phosphorylation of the conserved Ser residues in the PIN1HL by PID (and possibly by other AGC kinases) is required and sufﬁcient for proper PIN1 localization and is thus essential for generating the differential auxin distribution that directs plant development. INTRODUCTION and develops pin-shaped inﬂorescences (Ga¨ lweiler et al., 1998). The PIN family proteins can be classiﬁed into two groups: (1) the The plant hormone auxin plays a central role in almost all aspects PIN1-type proteins (PIN1, 2, 3, 4, and 7) that are plasma mem- of plant development. Unidirectional cell-to-cell transport of brane (PM) localized and (2) the PIN5-type proteins (PIN5, 6, and auxin generates maxima and minima that are instrumental for 8) that localize to the endoplasmatic reticulum (ER) and seem to tropic growth responses, tissue patterning, and organ initiation be involved in the regulation of auxin homeostasis (Mravec et al., (Sabatini et al., 1999; Friml et al., 2002b, 2003; Benkova et al., 2009). The PIN1-type proteins have redundant functions, and a 2003; Sorefan et al., 2009). The polar auxin ﬂow is accomplished loss-of-function mutation in one PIN gene is sometimes com- by the concerted action of three families of membrane proteins, pensated for by the ectopic expression of other PINs (Blilou et al., the AUXIN RESISTANT1/LIKE AUX1 (AUX1/LAX) inﬂux carriers 2005; Vieten et al., 2005). As a result, only mutants in multiple PIN (reviewed in Parry et al., 2001), the PIN-FORMED (PIN) efﬂux genes show more pronounced phenotypes in embryogenesis, carriers (reviewed in Paponov et al., 2005), and the P-GLYCO- root patterning, and lateral root initiation (Benkova et al., 2003; PROTEIN (PGP/ABCB) transporters (reviewed in Geisler and Friml et al., 2003; Blilou et al., 2005). Murphy, 2006). Until now, the role of the PIN auxin efﬂux carriers The PIN1-type proteins determine the direction of cell-to-cell in polar auxin transport is most well established. The Arabidopsis auxin transport through their asymmetric subcellular localization thaliana genome encodes eight PIN proteins, named after the at the PM (Wis´ niewska et al., 2006), which is dependent not only pin-formed/pin1 mutant that is defective in polar auxin transport on tissue-speciﬁc factors, but also on the PIN protein sequence (Wisniewska et al., 2006). During speciﬁc developmental pro- cesses, dynamic changes in PIN polarity have been observed Current address: Escola Proﬁssional UNIPACS, Rua Guilherme Lahm, (Benkova et al., 2003; Friml et al., 2003; Heisler et al., 2005), and 960, Taquara, RS, Brazil. Current address: Swammerdam Institute for Life Sciences, University PIN polarity has also been shown to be modulated by environ- of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Nether- mental cues (Friml et al., 2002b; Harrison and Masson, 2008) and lands. auxin itself (Paciorek et al., 2005; Sauer et al., 2006). Many Address correspondence to [email protected]. research efforts have focused on what determines PIN polarity The author responsible for distribution of materials integral to the ﬁndings presented in this article in accordance with the policy described and, thus, what translates upstream developmental and envi- in the Instructions for Authors (www.plantcell.org) is: Remko Offringa ronmental signals into changes in plant architecture by regulat- ([email protected]). ing PIN polarity. The current model is that newly synthesized Online version contains Web-only data. OA PINs arrive at the PM in a nonpolar fashion and that PIN polarity Open Access articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.109.072678 is established and regulated by subsequent endocytosis, 1130 The Plant Cell transcytosis, and recycling back to the PM (Geldner et al., 2001; of-phosphorylation and phosphomimic PIN1:GFP proteins in Dhonukshe et al., 2008; Kleine-Vehn et al., 2008). root tips was insensitive to PID overexpression using P35S:PID, GNOM is a GDP/GTP exchange factor on ADP-ribosylation leading to opposite effects on P35S:PID-induced root collapse. factor G protein that has been shown to be involved in the Our data indicate that the regulation of PIN1 polar localization recycling of PIN proteins to the basal (root apex facing) side of through reversible phosphorylation of three conserved Ser res- the PM (Geldner et al., 2003). GNOM is a molecular target of the idues in the PIN1HL by PID and possibly other AGC kinases is an fungal toxin brefeldin A (BFA), which inhibits protein trafﬁcking essential mechanism for aspects of plant development that are and thus interferes with basal PIN1 recycling, leading to PIN1 directed by differential auxin distribution. accumulation into so-called BFA compartments (Geldner et al., 2001). Loss of function of GNOM results in severe embryo defects due to disturbance of PIN1 polarity establishment during RESULTS embryogenesis (Mayer et al., 1993; Steinmann et al., 1999). Another important molecular determinant in PIN polar target- Ser Residues in Three Conserved Motifs in the PIN1HL Are ing is the PINOID (PID) protein Ser/Thr kinase. PID was initially Phosphorylated by PID identiﬁed through the Arabidopsis pid loss-of-function mutants that phenocopy pin1 mutants (Bennett et al., 1995; Christensen The previous observations that the PIN1HL is efﬁciently phos- et al., 2000). Both PID loss- and gain-of-function mutant pheno- phorylated by PID in vitro (Michniewicz et al., 2007) prompted us to types already indicated a role of PID as a regulator of auxin map PID phosphorylation targets in the PIN1HL. Analysis of the transport (Benjamins et al., 2001). More recently, PID was shown PIN1 amino acid sequence using the NetPhos program (Blom to act as a binary switch in the apical-basal polar targeting of PIN et al., 1999) identiﬁed 23 putative phosphosites, 20 of which are proteins (Friml et al., 2004). In root cells, PID overexpression located in the PIN1HL (see Supplemental Figure 1 online). Twelve induces a PIN polarity shift from the basal to the apical (shoot synthetic peptides comprising 17 putative phosphosites were apex facing) side of the cells, leading to agravitropic root growth tested in in vitro phosphorylation assays, and six were found to be and collapse of the primary root meristem, due to depletion of the highly phosphorylated by PID (see Supplemental Figure 2 online). organizing auxin maximum. By contrast, in the inﬂorescence Since the PID-dependent basal-to-apical switch in PIN polarity meristem, pid loss of function induces an apical-to-basal shift in is not restricted to PIN1, but is also observed for PIN2 and PIN4 PIN1 polarity, which drains the auxin maxima that are necessary (Friml et al., 2004), we aligned the amino acid sequences of six for organ initiation, thus resulting in pin-like inﬂorescences (Friml PIN proteins (PIN1, 2, 3, 4, 6, and 7) in which a clear hydrophilic et al., 2004). loop (HL) can be identiﬁed. Eleven of the NetPhos predicted Ser In animal systems, modiﬁcation of cargo proteins by phos- (S) and Thr (T) residues showed conservation among the six phorylation is an important mechanism to regulate their polar Arabidopsis PIN proteins (indicated with an asterisk in Figure 1), delivery to the PM. For example, in mammalian epithelial cells, ﬁve of which appeared to be fully conserved (labeled in black and phosphorylation of the immunoglobulin receptor at a single Ser with an asterisk in Figure 1). Interestingly, these ﬁve residues residue has been shown to result in its accumulation at the apical were located in the highly phosphorylated peptides 2, 6, 8, and cell membrane (Casanova et al., 1990). Protein phosphorylation 12 (see Supplemental Figure 2 online), and two of them (Thr-227 and dephosphorylation have also been implicated in the regula- and Ser-290) were recently reported to be modiﬁed by phos- tion of polar auxin transport in plant systems. In tobacco (Nico- phorylation in vivo (Benschop et al., 2007). We noted that S290 tiana tabacum) suspension cells, the protein kinase inhibitors was located in a TPRXSN motif that was conserved among the staurosporine and K252a were found to inhibit auxin efﬂux six PIN proteins (Figure 1) and resembled the consensus phos- (Delbarre et al., 1998), and genetic and pharmacological inhibi- phorylation site of the animal AGC kinase, protein kinase A (PKA). tion of phosphatase activity in Arabidopsis led to defects in auxin Therefore, we ﬁrst tested whether Ser-290 is a PID phosphory- transport (Garbers et al., 1996; Rashotte et al., 2001). More lation target by replacing this Ser with Ala in a HIS-tagged short recent ﬁndings revealed that PIN polar localization is determined version of the PIN1HL (PIN1HLsv) (see Supplemental Figure by reversible phosphorylation of the large central PIN hydro- 1 online) and incubating wild type or mutant proteins with HIS- philic loop (PINHL) through the antagonistic action of the PID tagged PID in an in vitro phosphorylation reaction. Clear PID kinase and PP2A phosphatases (Michniewicz et al., 2007). This autophosphorylation and PID-dependent phosphorylation of indicated that the machinery of phosphorylation-regulated PM HIS-PIN1HLsv was detected (see Supplemental Figure 3A on- protein polar localization is also operational in plants. line). The S290A substitution reduced PIN1 phosphorylation by To further elucidate molecular mechanisms of PIN polar local- PID to a background level (see Supplemental Figure 3A online), ization regulated by PID phosphorylation, we set out to identify indicating that Ser-290 is a PID phosphorylation target. Two the PID phosphorylation targets in the PINHL. Here, we show that additional TPRXS(N/S) motifs were identiﬁed upstream of Ser- the central Ser residues in three conserved TPRXS(N/S) motifs 290 (Figure 1), and the Ser residue (Ser-252) of the second motif within the PIN1HL are phosphorylated by PID. Inactivation of was also shown to be modiﬁed by phosphorylation in vivo these phosphosites (nonphosphorylatable or phosphomimic (Benschop et al., 2007). For convenience in our experiments, forms) in a complementing PIN1:GFP (for green ﬂuorescent we refer to the Ser residues at positions 231, 252, and 290 as S1, protein) construct induced auxin-regulated defects in embryo S2, and S3, respectively (Figure 1). and inﬂorescence development that correlated with changes in We next tested the effect of Ser-to-Ala substitution (S1A, PIN1:GFP polar localization. Moreover, the localization of loss- S2A, S3A, or combinations) on PID phosphorylation using a Conserved Serines Direct PIN1 Polarity 1131 Figure 1. Alignment of the Amino Acid Sequences of the HL of Six Arabidopsis PIN Proteins. Conserved Ser or Thr residues predicted by NetPhos to be phosphorylated are indicated by asterisks. Residues that are conserved in all six PINHLs are blocked with black (Ser and Thr residues) or light gray (other amino acids). Residues that are conserved in four or ﬁve of the six PINHLs are blocked with dark gray. The three TPRXS(N/S) motifs are boxed, and the Ser residues at positions 231, 252, and 290 within these motifs are renumbered to 1, 2, and 3, respectively. glutathione S-transferase (GST)-tagged version of the full- quences of PIN1 proteins from ﬁve different plant species length PIN1HL. Under our experimental conditions, the GST- (Xu et al., 2005; Carraro et al., 2006) showed that the three PIN1HL was unstable, showing a reproducible pattern of identiﬁed motifs are highly conserved, even in the moss Phys- degradation bands. A single S1A or S3A substitution led to a comitrella patens (see Supplemental Figure 4 online), suggesting 40 or 20% reduction, respectively, double S1,2A and S1,3A their functional conservation throughout the evolution of land substitutions led to a 50% reduction, and triple S1,2,3A sub- plants. stitutions led to an 80% reduction of phosphorylation by PID compared with the wild-type PIN1HL (Figure 2B). These data Loss of PIN1 Phosphorylation at the Conserved Ser indicated that the central Ser residues within the three highly Residues Induces Dominant Embryo and conserved TPRXS(N/S) motifs are targets for PID phosphory- Flower Phenotypes lation in vitro. At the same time, the PIN1HL S1,3A double substitution To investigate the biological signiﬁcance of the PIN1 phosphor- construct was used to test all other Ser and Thr residues located ylation in planta, various mutant constructs were generated from in the highly phosphorylated peptides 2 (T227), 6 (T286), 8 (S377 PPIN1 (PIN promoter):PIN1:GFP (hereafter referred to as PIN1: and S380), 11 (T458 and S459), and 12 (S479) (see Supplemental GFP), in which one, two, or all three Ser residues in the encoded Figure 2 online). Based on the relative intensities of the phos- PIN1:GFP proteins were replaced by Ala (A), a nonphosphory- phorylated bands, substitution of these amino acids with Ala latable residue, or by Glu (E) to mimic phosphorylation. The residues had no clear effect on PID phosphorylation (see Sup- resulting constructs PIN1:GFP S1A(E), PIN1:GFP S3A(E), PIN1: plemental Figures 3B and 3C online), indicating that these GFP S1,3A(E), and PIN1:GFP S1,2,3A(E) were transformed into residues are not phosphorylated by PID. Arabidopsis Columbia (Col) wild-type plants, and GFP positive, BLAST analysis of the Arabidopsis protein database (http:// single locus insertion lines were selected for analysis. A previ- www.ncbi.nlm.nih.gov/protein/) showed that TPRXS(N/S) is a ously described PIN1:GFP line (Benkova et al., 2003) was used PIN-speciﬁc motif. Strikingly, alignment of amino acid se- as the control. 1132 The Plant Cell the weaker lines (e.g., PIN1:GFP S1A#14: 12.5%, n = 654) (Figure 3N), indicating that the severity of the cotyledon phenotypes corresponded to the level of mutant PIN1:GFP protein expres- sion. The stronger lines PIN1:GFP S1A#15 and PIN1:GFP S1,3A#10 developed ﬂowers with an increased number of petals and a decreased number of stamens and carpels (Figure 4B, Table 1), mimicking pid mutant ﬂoral defects (Figure 4A, Table 1) (Bennett et al., 1995). To exclude other possible reasons for the observed dominant phenotypes, such as cosuppression, the protein levels of the transgene and the endogenous PIN1 gene were quantiﬁed by protein gel blot analysis. All trangenic lines showed endoge- nous PIN1 expression similar to that of the wild type, and the mutant PIN1:GFP expression levels of the strong lines were comparable to that of the PIN1:GFP controlline(seeSupple- mental Figure 5B online). These results showed similar protein expression of both endogenous PIN1 and PIN1:GFP, suggest- ing that the dominant developmental defects observed can be attributed to the expression of the mutant PIN1:GFP proteins that outcompete endogenous PIN1, possibly by differential localization at the PM. For the PIN1:GFP S1,2,3A triple substitution lines, the post- embryo defects were more severe. No homozygous progeny could be obtained, and approximately one-fourth of the seeds from heterozygous plants (25.6%, n = 156 for line #23) failed to germinate, indicating that the homozygous progeny are embryo lethal. Among the germinating seedlings, we occasionally (9.6%, Figure 2. Ser Residues in Three Conserved Motifs within the PIN1HL Are n = 156 for line #23) observed strong patterning defects (indi- Phosphorylated by PID in Vitro. cated as “others” in Figures 3N and 3O), such as seedlings (A) In vitro assay of phosphorylation by GST-PID kinase using wild-type without a root (Figures 3G and 3H) or ball-shaped structures GST-PIN1HL and mutant protein substrates in which the indicated Ser without any discernible apical-basal axis that stopped growing residues (S1, S2, or S3) were replaced with Ala residues (A). The after germination (Figures 3I and 3J). positions of GST-PID and the full-length GST-PIN1HL are indicated in the autoradiograph (top panel) and the Coomassie-stained gel (bottom panel). Autophosphorylation of GST-PID can be observed in the top Reversible Phosphorylation of the Conserved Ser Residues panel. Under our experimental conditions, Escherichia coli–puriﬁed GST- Is Necessary and Sufﬁcient for Proper PIN1 Localization and PIN1HL was not stable, resulting in a reproducible pattern of degradation Plant Development bands. The Coomassie blue–stained gel was used as a control for protein loading. To further test the functionality of the loss- or gain-of-phos- (B) Quantitative assessment of the in vitro phosphorylation assay in (A). phorylation PIN1:GFP proteins, the PIN1:GFP and PIN1:GFP The phosphorylation intensity is expressed as the percentage of phos- S/A(E) mutant lines were crossed with the pin1 loss-of-function phorylation relative to the wild-type GST-PIN1HL protein. Numbers were mutant, and where possible, double homozygous plants were corrected for protein loading based on analysis of the Coomassie blue– selected for analysis. The pin1 mutant has aberrant cotyledon stained blot. numbers (Figures 3C, 3N, and 3O) and pin-formed inﬂores- cences with no ﬂowers or only a few defective ﬂowers (Okada The PIN1:GFP S/E and PIN1:GFP S3A plants showed largely et al., 1991). In our analyses, PIN1:GFP, as well as PIN1:GFP normal development at seedling and ﬂowering stage. By con- S1E, PIN1:GFP S3E, and PIN1:GFP S1,3E, complemented pin1 trast, other mutants exhibited a range of dominant defects. The cotyledon and inﬂorescence defects (Figures 3O, 4C, and 4D). PIN1:GFP S1A and PIN1:GFP S1,3A mutants showed cotyledon PIN1:GFP S1A and PIN1:GFP S3A partially rescued pin1 de- number defects, reﬂected by seedlings having one, three, or four fects, reﬂected by a reduced frequency of pin1 cotyledon defects cotyledons (Figures 3D to 3F), with the three-cotyledon pheno- from 17.6% (n = 318) to 2% (n = 402) and 12.1% (n = 348), type characteristic of the pid loss-of-function mutant (Figure 3B) respectively (Figure 3O), and by the observations that no pin- predominating. For these two mutant constructs, two lines each formed inﬂorescences were produced (Figures 4F and 4G). By were selected for detailed analyses, one with a lower PIN1:GFP contrast, PIN1:GFP S1,3A and PIN1:GFP S1,2,3A(E) lines expression level (PIN1:GFP S1A#14 and PIN1:GFP S1,3A#12) showed an enhanced frequency (23.9% n = 351, 32.7% n = and one with a higher PIN1:GFP expression level (PIN1:GFP 110, and 71.2% n = 66, respectively; Figure 3O) and severity of S1A#15 and PIN1:GFP S1,3A#10) (see Supplemental Figure 5A seedling defects, such as cup-shaped cotyledons (3.5%; Figure online). Notably, the stronger lines showed higher frequencies of 3K) or no cotyledons (2%; Figure 3L), phenotypes typical for cotyledon defects (e.g., PIN1:GFP S1A#15: 21.7%, n = 757) than the pin1 pid double mutant (Furutani et al., 2004) but not for Conserved Serines Direct PIN1 Polarity 1133 Figure 3. Seedling Phenotypes Induced by Manipulation of the Conserved Ser Residues in PIN1:GFP. (A) to (F) Cotyledon number defects observed in pid-14 (B), pin1 (C),and PIN1:GFP S/A ([D] to [F]) mutant seedlings compared with a wild-type seedling (A). +/ (G) to (J) Progeny from PIN1:GFP S1,2,3A#23 plants showing severe patterning defects, such as seedling without a root ([G] and [H]), with reduced cotyledons (H), or oblong structures ([I] and [J]). (K) to (L) Cotyledon defects observed in pin1 PIN1:GFP S1,3A#10 mutant seedlings showing a cup-shaped cotyledon (K) or no cotyledons (L). +/ +/ (M) Progeny from pin1 PIN1:GFP S1,2,3A#23 plants showing callus-like hypocotyl and cotyledons lacking a primary root. Bars in (A) to (M) = 10 mm. (N) and (O) Quantitative analysis of seedling defects induced by expression of the phosphomutant PIN1:GFP versions in the wild-type background (N) and in pin1 mutant background (O). The number of seedlings scored per mutant line is indicated. The legends in (O) are also used for (N). “Others” represents phenotypes other than cotyledon number defects, such as seedling without root, oblong structures, or callus-like seedlings. the pid or pin1 single mutant (Figures 3N and 3O). Around 5% 2004), in auxin transport inhibitor-induced pin-shaped inﬂores- +/2 +/2 of the progeny from pin1 PIN1:GFP S1,2,3A (double het- cence apices, PIN1:GFP was localized apically in the epidermis erozygous) plants lacked a primary root and developed callus- (Figure 5A). In a comparable region of the pin-shaped inﬂores- like hypocotyls and cotyledons (Figure 3M). At the ﬂowering cence apex, the phosphomimic PIN1:GFP S1,2,3E protein also stage, PIN1:GFP S1,3A and PIN1:GFP S1,2,3A(E) mutants could showed apical localization (Figure 5B), whereas the PIN1:GFP not complement pin1 defects and produced pin-like inﬂores- S1,3A and PIN1:GFP S1,2,3A proteins were targeted to the basal cences, with pin1 PIN1:GFP S1,2,3E occasionally forming ﬂowers side (Figures 5C and 5D), similar to PIN1 localization in pid producing seeds (Figure 4E). The pin1 PIN1:GFP S1,3A inﬂores- mutant (Friml et al., 2004). cences were branched and formed sterile ﬂowers with fused Collectively, these data indicated that PIN1 loss of phosphor- +/2 petals (Figure 4H), whereas pin1 PIN1:GFP S1,2,3A plants ylation results in its basal localization, whereas phosphomim- only produced a single needle-shaped inﬂorescence (Figure 4I). icking induces apical targeting of PIN1 in the shoot apical Next, we examined whether these mutant defects correlated meristem, both leading to failure to complement the pin1 mutant with changes in the subcellular localization of the mutant PIN1: phenotypes. These observations are consistent with the identi- GFP proteins. Consistent with previous observations (Friml et al., ﬁed role for PID as a binary switch in PIN1 basal-apical polar 1134 The Plant Cell Figure 4. Inﬂorescence and Flower Defects Observed after Expression of Phosphomutant PIN1:GFP Proteins. (A) and (B) Flowers of the pid-14 loss-of-function mutant (A) and the PIN1:GFP S1,3A#10 mutant (B) show similar defects. (C) to (I) Complementation analysis of pin1 loss-of-function mutant inﬂorescence and ﬂower defects. PIN1:GFP (C) and PIN1:GFP S1,3E#18 (D) fully rescued pin-shaped inﬂorescence defects, whereas PIN1:GFP S1A#15 (F) and PIN1:GFP S3A#6 (G) only partially rescued pin-shaped inﬂorescence defects. PIN1:GFP S1,2,3E#8 (E), PIN1:GFP S1,3A#10 (H), and PIN1:GFP S1,2,3A#23 (I) did not rescue pin1 inﬂorescence defects. Insets show details of ﬂower morphology and pin-like inﬂorescences. Bars in whole-plant photographs = 5 cm. localization in the shoot apex, suggesting that the identiﬁed Ser (5%). These embryo phenotypes resembled those of mutants residues are PID related. with defects in auxin transport (Friml et al., 2003) and gnom mutants (Mayer et al., 1993). Examination of the subcellular localization of PIN1:GFP Strong Embryo Defects Are Induced by PIN1:GFP S1,2,3A during embryogenesis showed that PIN1:GFP S1,2,3A S1,2,3A Mislocalization polarity failed to establish properly (n = 43). Endogenous PIN1 (or wild-type PIN1:GFP) proteins localize at the basal side of the The embryo and seedling lethality observed in the progeny of +/2 +/2 pin1 PIN1:GFP S1,2,3A plants led us to study the early em- provascular cells (Figure 6C), generating an auxin maximum that bryo development. Compared with pin1 PIN1:GFP embryos that deﬁnes the hypophyseal cell group (Figure 6F) and at the apical showed stereotypic patterns of cell divisions (Figure 6A), ;30% side of the epidermal cells from triangular stage on (Figure 6C +/2 +/2 (n = 86) of the embryos from pin1 PIN1:GFP S1,2,3A plants and inset), generating strong auxin activity at tips of developing exhibited a range of developmental aberrations at different cotyledons (Steinmann et al., 1999; Friml et al., 2003). In embryos +/2 +/2 stages (Figure 6B). In 15% of the embryos, the basal tier and from pin1 PIN1:GFP S1,2,3A plants with a largely wild-type suspensor cells showed defective cell divisions. The most severe morphology (65%, n = 43), the basal PIN1:GFP localization in the cases were characterized by embryos with globular structures provascular cells was lost (Figure 6D, star), causing a reduction that lacked a deﬁned apical-basal axis and bilateral symmetry of the auxin reporter PDR5:GFP signal in the hypophysis (Figure Conserved Serines Direct PIN1 Polarity 1135 Table 1. Quantitative Analysis of Dominant pid-Like Floral Organ Defects Induced by PIN1:GFP S/A Floral Organ Numbers Genotype Sepal Petal Stamen Carpel Total No. of Flowers Col 4.00 4.00 5.80 6 0.40 2.00 50 pid-14 2.80 6 1.20 8.35 6 1.42 1.10 6 1.07 0.00 20 PIN1:GFPS1A#15 4.11 6 0.68 5.53 6 0.63 4.91 6 0.85 1.52 6 0.57 45 PIN1:GFPS1,3A#10 4.08 6 0.68 5.71 6 1.09 4.15 6 0.87 1.83 6 0.33 52 Numbers are means derived from analyses of ﬂowers from at least ﬁve plants for each genotype. Standard deviations are indicated. In the case of organ fusion in the same whorl, a fused ﬂoral organ is counted as one in that whorl. In the case of organ fusion between different whorls, fused organs are counted as one organ in the each whorl. 6G). As a comparison, the basal localization of PIN1:GFP S1,3A can be mimicked by manipulation of these Ser residues. For in the provascular cells was not changed (see Supplemental additional conﬁrmation that our identiﬁed phosphoserines are Figure 6A online). In embryos exhibiting strong defects, PIN1: targets of PID activity in vivo, we crossed the PIN1:GFP, PIN1: GFP S1,2,3A polarity was dramatically disrupted, and abundant GFP S1,3A, and PIN1:GFP S1,2,3E lines with the strong PID intracellular signal was observed (Figure 6E). The cells with the overexpression line P35S:PID#21 (Benjamins et al., 2001). The ﬂuorescent signal at the PM showed no polarity or randomized PIN1:GFP S1,2,3A line could not be used for this purpose, as it polarity (see Supplemental Figures 6B and 6C online), and a clear could only be maintained in the heterozygous state, which PDR5:GFP maximum was not detected (Figure 6H). precluded an equivalent comparison of the root meristem col- These data implied that phosphorylation of the three Ser lapse frequency. residues is required for auxin-related embryo development by In line with our previous observations (Friml et al., 2004), PID regulating PIN1 PM localization. overexpression induced a clear basal-to-apical shift of PIN1:GFP localization in root stele cells (Figures 7A and 7B). By contrast, PIN1:GFP S1,3A showed basal localization in both wild-type and PIN1 Phosphorylation at the Conserved Ser Residues Is P35S:PID backgrounds (Figures 7C and 7D). Simultaneous im- RelatedtoPID Activity munolocalization showed the basal-to-apical polarity shift of Above, we showed that the conserved Ser residues are phos- PIN2 in the cortex and PIN4 in the root meristem (Figure 7M), phorylated by PID in vitro and that PID regulation of PIN1 polarity demonstrating that PID overexpression was sufﬁcient to induce and the resulting inﬂorescence development (Friml et al., 2004) PIN polarity shifts and that the S1,3A substitutions rendered Figure 5. Subcellular Localization of Wild-Type and Phosphomutant PIN1:GFP Proteins in Epidermal Cells of the Inﬂorescence Apex. Confocal laser scanning microscopy of pin-formed inﬂorescence apices of pin1 mutant plants expressing wild-type PIN1:GFP (naphthylphthalamic acid treated) (A), PIN1:GFP S1,2,3E (B), PIN1:GFP S1,3A (C), and PIN1:GFP S1,2,3A (D). The white dashed boxes in the overview images (left) indicate the position of the zoomed-in images (right). The polarity of PIN1:GFP in the epidermal cells of the inﬂorescence apex is indicated with arrows. PIN1:GFP +/ S1,2,3A#23 indicates that the plant is heterozygous for the transgene. 1136 The Plant Cell PIN1:GFP insensitive to that. On the other hand, the PIN1:GFP S1,2,3E protein exhibited apical localization in some cell ﬁles and apolar localization in others in both wild-type and P35S:PID backgrounds (Figures 7E and 7F), further indicating that the polar localization of the phosphorylation mutant PIN1:GFP proteins occurred independent of PID overexpression. Consistent with the PIN1 function of mediating auxin transport to root tips, PIN1: GFP S1,3A induced an enhancement of auxin reporter PDR5: GFP signal compared with the control PIN1:GFP roots (Figures 7G and 7I; see Supplemental Figure 7 online; Student’s t test, P < 0.05), consistent with the basal localization of PIN1:GFP S1,3A protein. By contrast, the PDR5:GFP signal was signiﬁcantly reduced in PIN1:GFP S1,2,3E root tips (Figure 7K; see Supple- mental Figure 7 online; Student’s t test, P < 0.05), in line with its preferably apical localization. In the P35S:PID background, basally accumulated PIN1:GFP S1,3A resulted in higher auxin accumulation compared with PIN1:GFP (Figures 7J and 7H; see Supplemental Figure 7 online; Student’s t test, P < 0.05) and as a result signiﬁcantly reduced the P35S:PID-induced root collapse frequency (Figure 7N). By con- trast, PIN1:GFP S1,2,3E had no signiﬁcant effect on P35S:PID- induced root collapse (Figure 7N). This might be due to the already maximal effect of PID overexpression on PIN apicaliza- tion and auxin depletion, as no signiﬁcant reduction of auxin accumulation was detected (Figures 7B and 7F; see Supple- mental Figure 7 online). The genetic interactions between phos- phomutants and PID conﬁrmed that our identiﬁed Ser residues are phosphotargets of PID. Together, these results linked phosphorylation of the con- served Ser residues to polar PIN1 driven differential auxin dis- tribution in roots and further demonstrated that the conserved phosphoserines are key determinants in modulating plant archi- tecture by instructing PIN1 polarity. DISCUSSION Previously, it has been shown that the PID kinase and PP2A phosphatases antagonistically regulate PIN polarity by reversible phosphorylation of the PINHL (Michniewicz et al., 2007). How- Figure 6. Embryo Defects Induced by PIN1:GFP S1,2,3A Mislocalization Are Due to Disturbed Auxin Distribution. ever, no functional evidence was provided to support the im- portance of this phosphorylation in planta. In this study, we (A) and (B) Differential interference contrast microscopy images of identiﬁed Ser residues centrally located in three conserved +/ +/ embryos from wild-type (A) and pin1 PIN1:GFP S1,2,3A#23 (B) TPRXS(N/S) motifs within the PIN1HL that are phosphorylated plants. Text at the bottom of each image indicates the developmental by PID in vitro. Subsequent in planta analyses of loss-of- stage of the embryo. For the defective embryos in (B), the developmental stage was based on a rough estimate of the cell number. Bars = 10 mm. phosphorylation and phosphomimic PIN1:GFP mutants proved (C) to (E) Confocal laser scanning microscopy images of pin1 PIN1:GFP that reversible phosphorylation of all three residues is required heart-stage embryos (C) and wild-type looking (D) and defective looking and sufﬁcient for proper PIN1 polar localization and auxin- +/ +/ (E) embryos from pin1 PIN1:GFP S1,2,3A#23 plants from the same regulated plant development. developmental stage. Insets in (C) and (D) represent confocal scans through the epidermal cell layer of cotyledon primordia. White arrows in (C) and (D) indicate the PIN1:GFP polarity, and a star in (D) indicates the Ser Resides in the Conserved TPRXS(N/S) Motifs Are Crucial absence of basally localized PIN1:GFP S1,2,3A protein. Phosphorylation Targets in the PIN1HL (F) to (H) Confocal laser scanning microscopy images of PDR5:GFP +/ auxin distribution in embryos from pin1 PIN1:GFP (F) and pin1 PIN1: Several Ser and Thr residues within the PIN1HL have been +/ GFP S1,2,3A#23 ([G] and [H]) plants, showing a reduced (G) or identiﬁed as phosphorylation substrates in vivo, and not surpris- mislocalized (H) auxin maximum. ingly, S2 and S3 are among them (Nu¨ hse et al., 2004; Benschop et al., 2007). Two other phosphorylation targets in the PIN1HL identiﬁed by mass spectrometry analysis are Ser-337 and Conserved Serines Direct PIN1 Polarity 1137 Figure 7. PIN1:GFP Polarity Changes Induced by Manipulation of Phosphoserines Correlate with Changes in the Auxin Maximum in the Root Tip. (A) to (F) Confocal laser scanning microscopy of primary roots of 5-d-old seedlings expressing PIN1:GFP ([A] and [B]), PIN1:GFP S1,3A#10 ([C] and [D]), and PIN1:GFP S1,2,3E#8 ([E] and [F]) in the wild-type ([A], [C],and [E])or P35S:PID ([B], [D],and [F]) background. The white dashed boxes in the overview images (top) indicate the position of the zoomed-in images (bottom), in which the PIN1:GFP polarity is indicated by arrows. The seedlings are homozygous for the indicated T-DNA constructs. Bars = 5 mm. (G) to (L) Confocal laser scanning microscopy of PDR5:GFP signals in 3-d-old seedling root tips expressing PIN1:GFP ([G] and [H]), PIN1:GFP S1,3A#10 ([I] and [J]), and PIN1:GFP S1,2,3E#8 ([K] and [L]) in the wild-type ([G], [I],and [K])or P35S:PID ([H], [J],and [L]) background. The seedlings are heterozygous for the PDR5:GFP reporter. Bar = 50 mm. (M) PIN2 and PIN4 immunolocalization in 3-d-old P35S:PID PIN1:GFP S1,3A#10 seedling roots. The arrows indicate the apical PIN2 and PIN4 localization induced by PID overexpression. Bars = 5 mm. (N) Quantiﬁcation of the effects of wild-type, loss-of-phosphorylation, or phosphomimic PIN1:GFP expression on the PID overexpression-induced root meristem collapse phenotype. Percentages are based on scoring 153, 144, 179, and 98 seedlings at 4, 6, and 8 d after germination. 1138 The Plant Cell Thr-340 in the MFSPNTG sequence (Benschop et al., 2007; In the epidermis of the shoot apex, loss-of-phosphorylation Michniewicz et al., 2007). Recent functional analysis of these res- PIN1:GFP S1,3A and PIN1:GFP S1,2,3A proteins were targeted idues in planta has shown that their phosphorylation states are to the basal side (Figures 5C and 5D), whereas the phospho- important for PIN1 polarity (Zhang et al., 2010). However, these mimic PIN1:GFP S1,2,3E protein was apicalized (Figure 5B). This residues are not directly phosphorylated by PID (Zhang et al., pattern is consistent with the binary switch mode, which pro- 2010), suggesting that other protein kinases could coordinately poses that no or low kinase activity (in the pid mutant) results in regulate PIN polarity with PID by phosphorylating Ser-337 and PIN1 localization at the basal membrane and that above thresh- Thr-340. Ser-337 could be a target of mitogen-activated protein old kinase activity (in the wild type) directs PIN1 apical localiza- kinases (Benschop et al., 2007), as mitogen-activated protein tion (Friml et al., 2004). Despite its apical localization, PIN1:GFP kinases preferably phosphorylate Ser or Thr residues followed by S1,2,3E could not complement pin1 inﬂorescence defects (Fig- a Pro in both plant and animal systems (Pearson et al., 2001; Liu ure 4E), a result that is seemingly contradictory to the observa- and Zhang, 2004). tion that in the shoot apex of wild-type plants, PIN1 is apically The TPRXS(N/S) motifs in the HL are highly conserved among localized. However, PIN1 polarity and the resulting auxin maxima six Arabidopsis PIN proteins (Figure 1) and among PIN1 homo- in the shoot apex have been reported to be highly dynamic logs from other land plant species (see Supplemental Figure 4 (Heisler et al., 2005), and a constitutively apical-localized PIN1: online), suggesting functional conservation of the motifs. Inter- GFP S1,2,3E would obviously interfere with auxin-mediated estingly, PID orthologs have been identiﬁed in maize (Zea mays) organ initiation. In addition, the shoot defects could also be and rice (Oryza sativa; McSteen et al., 2007; Morita and Kyozuka, attributable to PIN1:GFP S1,2,3E apical localization in the vas- 2007), and it has been shown that the maize ortholog BARREN cular tissues where PIN1 polarity is normally basal. INFLORESCENCE2 phosphorylates Zm PIN1a, the maize ortho- In seedling roots, loss-of-phosphorylation PIN1:GFP S1,3A log of Arabidopsis PIN1 in vitro, and that it regulates the subcel- was localized on the basal membrane of root stele cells, in both lular localization of Zm PIN1a in vivo (Skirpan et al., 2009). Further the wild type and P35S:PID background (Figures 7C and 7D), research is needed, however, to establish whether this functional indicating that the protein is unresponsive to PID activity. Even conservation extends to the conserved TPRXS(N/S) motifs in all though endogenous PIN2 and PIN4 in the same roots underwent PIN1-type proteins in Arabidopsis and in other plant species. the basal-to-apical polarity shift induced by PID overexpression For the Arabidopsis PIN1-type proteins (PIN1, 2, 3, 4, and 7), (Figure 7M), the root collapse was delayed (Figure 7N). Previ- the predicted protein structure consists of two sets of ﬁve ously, we have shown that pin2 and pin4 loss-of-function mu- transmembrane domains that are linked by a HL (Ga¨ lweiler tants delay P35S:PID-mediated root meristem collapse (Friml et al., 1998; Mu¨ ller et al., 1998). On the other hand, for the PIN5- et al., 2004). It is therefore not surprising that the basally localized type proteins (PIN5, 6, and 8), two sets of ﬁve and four trans- phosphorylation-deﬁcient PIN1 is able to reduce the frequency membrane domains, respectively, are predicted (Mravec et al., of root meristem collapse. 2009). PIN5 and PIN8 clearly lack a large central HL (Mravec By contrast, PIN1:GFP S1,2,3E was preferably targeted to the et al., 2009), but our alignment suggests that a shorter HL is apical membrane of root stele cells (Figure 7E). It has been shown present in PIN6 and that it contains two TPRXS(N/S) motifs that apical localized PIN proteins are more resistant to BFA- (Figure 1). Immunohistochemical analyses and studies using induced internalization (Kleine-Vehn et al., 2008). Consistently, reporter fusion proteins have shown that PIN1, 2, 3, 4, and 7 PIN1:GFP S1,2,3E was more resistant to BFA treatment than are localized at the PM (Ga¨ lweiler et al., 1998; Mu¨ ller et al., 1998; wild-type PIN1:GFP (Kleine-Vehn et al., 2009). This, together with Friml et al., 2002a, 2002b, 2003), whereas PIN5, 6, and 8 are the reduction of the PDR5:GFP signal in root tips compared with ER localized (Mravec et al., 2009). This suggests that for ER- that in PIN1:GFP roots (Figures 7K and 7G; see Supplemental localized PIN proteins, there has been no selective advantage Figure 7 online; Student’s t test, P < 0.05), strongly support that to maintain PINHL-located polarity determinants. Alternatively, PIN1:GFP S1,2,3E predominantly localizes to the apical mem- the loss of phosphorylation motifs may have been crucial for brane. The apical localization of phosphomimic PIN1:GFP did allowing the diversiﬁcation of PIN proteins function from the PM not induce root meristem collapse on its own. This can be to the ER. explained by the fact that the PID overexpression-induced root meristem collapse is caused by the basal-to-apical polarity change of three PIN proteins (PIN1, PIN2, and PIN4), of which Phosphorylation of the Conserved Ser Residues Directs PIN2 and PIN4 are crucial players (Friml et al., 2004). PIN1 Polar Localization and Auxin-Regulated Moreover, PIN1:GFP S1,2,3E apicalization was not complete, Plant Development as in certain cell ﬁles, apolar PIN1:GFP S1,2,3E localization was In wild-type plants, PIN1 proteins are basally localized in (pro) also detected (Figures 7E and 7F). Possibly, Glu is not a perfect vascular tissues in embryos, leaves, and roots and are apically phosphomimic in these cell ﬁles. Alternatively, there could still be localized in the epidermis of shoot apices and embryos additional PID phosphorylation targets in the PIN1HL, as the (Ga¨ lweiler et al., 1998; Benkova et al., 2003; Friml et al., 2003, S1,2,3A mutations did not completely abolish PID phosphory- 2004; Reinhardt et al., 2003). Our analyses of the subcellular lation of the PIN1HL in vitro. Nonetheless, the basal localization localization of mutant PIN1:GFP proteins in different tissues of loss-of-phosphorylation PIN1 and the apical localization showed that manipulation of the phosphoserines leads to of phosphomimic PIN1, together with their opposite effects on changes in PIN1 polarity, most (but not all) of which are consis- PID overexpression-induced root collapse, indicated that our tent with the PID binary switch function (Friml et al., 2004). identiﬁed phosphoserines are functional targets of PID. Conserved Serines Direct PIN1 Polarity 1139 During embryogenesis, phosphorylation of the conserved Ser complete loss of phosphorylation leads to not only PID-related residues seems to play a role in the maintenance of PIN1 PM morphological and cellular defects, but also defects never ob- localization rather than polarity alteration, as a complete loss of served in PID-regulated processes. This leads us to hypothesize phosphorylation induces PIN1:GFP intracellular accumulation that the three phosphoserines identiﬁed here are not only targets (Figures 6D and 6E). This mislocalized PIN1:GFP S1,2,3A inter- of PID, but also of other AGC3 kinases, and might explain why pid fered with auxin accumulation (Figures 6G and 6H), resulting in loss of function does not lead to apical-to-basal PIN2 polarity strong embryo defects (Figure 6B), similar to embryo defects changes in the root (Sukumar et al., 2009) or why the strong of gnom and pp2aa1,3 loss-of-function (Mayer et al., 1993; embryo defects induced by PIN1:GFP S1,2,3A are not observed Michniewicz et al., 2007) or PID gain-of-function (RPS5A>>PID) in pid mutants. Further research is needed to validate this (Friml et al., 2004) mutants. The common reason for the embryo hypothesis. defects in these different mutants is that the basal localization of Our data, together with the conclusion that PIN proteins and PIN1 in the provascular cells is lost, disturbing auxin accumula- PID-like kinases coevolved during the transition of plants from tion in the basal tier of the embryo (Steinmann et al., 1999; Friml water to land (Galva´ n-Ampudia and Offringa, 2007), as well as the et al., 2004; Michniewicz et al., 2007; Figures 6G and 6H), which demonstrated functional relationship between PID and PINs leads to auxin-regulated embryo defects. The enhanced intra- (Friml et al., 2004), lead us to propose that phosphorylation of the cellular accumulation of loss-of-phosphorylation PIN1 (PIN1: conserved Ser residues plays an important role in PIN-dependent GFP S1,2,3A) is in line with the observation of PIN2 accumulation auxin transport throughout the evolution of plants. However, in endomembrane structures in pid-9 loss-of-function mutant many questions still need to be answered. Functional analysis roots (Sukumar et al., 2009), which suggests that low levels of concerning phosphorylation of the three Ser residues in other PID activity result in intracellular accumulation of PIN proteins. PIN proteins and the regulation of PIN proteins by other AGC Loss-of-phosphorylation-induced intracellular PIN1 accumula- kinases will be the next challenges for the coming years. tion might be a result of reduced recycling to the PM, increased endocytosis, or reduced sorting from endosomes to the vacu- METHODS oles. Further research is needed to distinguish between these possibilities. Plant Material, Growth Conditions, and Phenotypic Analysis For all experiments, Arabidopsis thaliana of ecotype Col-0 was used. AGC Kinases and PIN Proteins: A Stable Marriage in Construction of PIN1:GFP (to produce PIN1:GFP fusion proteins) and Plant Evolution P35S:PID (to overexpress PID) and the corresponding Arabidopsis lines were described previously (Benjamins et al., 2001; Benkova et al., 2003). PID belongs to the plant-speciﬁc AGCVIII family of protein The loss-of-function alleles pid-14 (SALK_049736) and pin1 (SALK_047613) kinases, which are plant orthologs of the mammalian cAMP- were obtained from the Nottingham Arabidopsis Stock Centre. dependent PKA, cGMP-dependent protein kinase G, and protein Seedlings were grown on MA medium (Masson and Paszkowski, 1992) kinase C (Bo¨ gre et al., 2003). Among the three identiﬁed Ser at 218C and a 16-h-light/8-h-dark photoperiod. One-week-old seedlings residues, two (S2 and S3) are recognized by NetPhos as PKA were transferred to MA medium supplemented with 50 mM naphthylph- phosphorylation targets (Blom et al., 1999), corroborating previ- thalamic acid (Pfaltz and Bauer), an auxin transport inhibitor, to induce pin ous suggestions that the plant AGCVIII kinases might have been inﬂorescences. The number of seedlings with root meristem collapse was derived from the same ancestral kinase as animal PKAs (Bo¨ gre counted 4, 6, and 8 d after germination. Plants were grown on a mixture of et al., 2003; Galva´ n-Ampudia and Offringa, 2007). 9:1 substrate soil and sand (Holland Potgrond) at 218C, a 16-h photope- riod, and 70% relative humidity. In Arabidopsis, PID groups together with 22 other AGCVIII protein kinases (Bo¨ gre et al., 2003; Galva´ n-Ampudia and Offringa, 2007), of which the blue light receptors PHOT1 and PHOT2, the DNA Constructs, Sequence Alignment, and Plant Transformation root growth regulators WAG1 and WAG2, and the D6 protein Molecular cloning, DNA sequence analysis, and DNA and protein se- kinases (D6PKs) have also been shown to be involved in auxin quence alignments were performed using the Vector NTI 10 software transport-regulated plant development (Sakai et al., 2001; (Invitrogen). For the in silico prediction of putative phosphorylation sites, Santner and Watson, 2006; Zourelidou et al., 2009). Previously, we used the NetPhos software (Blom et al., 1999). The pET-PIN1HLsv it was shown that the PIN1HL is also phosphorylated by D6PKs (Galweiler et al., 1998) and pGEX-PID (Benjamins et al., 2003) constructs (Zourelidou et al., 2009). Although the D6PK genes showed a have been described before. The pET-PID fusion construct was gener- genetic interaction with PIN1, the D6PK protein had no effect on ated by cloning the PID cDNA into the pET16B (Novagen) derivative pET16H, which was kindly provided by Johan Memelink. The pGEX- PIN1 polarity regulation (Friml et al., 2004; Michniewicz et al., PIN1HL fusion was generated by cloning the PIN1HL SmaI/SalI fragment 2007; Zourelidou et al., 2009). Further investigation into the from pACT2-PIN1HL into the corresponding restriction sites of plasmid possible D6PK phosphorylation targets in PINs should provide pGEX-KG (Guan and Dixon, 1991). For construct pGreen0229 PPIN1: insight into the differential action of the distinct regulatory path- PIN1:GFP,the PIN1 gene was ampliﬁed from Col-0 DNA using prim- ways by PID and D6PKs. PID, together with WAG1, WAG2, and ers PIN1F 59-CGAATTCATTATTCCATTGGCGTTGTC-39 and PIN1R AGC3-4, groups to the AGC3 clade within the AGCVIII family 59-CAGGTACCCACTTCTTATTTTGGTGAGA-39, and the fragment was (Galva´ n-Ampudia and Offringa, 2007). The localization of all four digested with EcoRI and KpnI, and cloned into the corresponding sites of kinases at the plasma membrane (Galva´ n-Ampudia and Offringa, pGreen0229. Subsequently, the BstAPI fragment in this genomic clone 2007), and their genetic interactions (Cheng et al., 2008), suggest was exchanged for the BstAPI fragment containing the PIN1:GFP trans- functional redundancy among the AGC3 kinases. In our studies, lational fusion from pBIN-PPIN1:PIN1:GFP (Friml et al., 2003). The 1140 The Plant Cell Quickchange XL site-directed mutagenesis kit (Stratagene) was used to Membrane Protein Extraction and Protein Gel Blot Analysis generate mutant constructs. Oligonucleotides used for mutagenesis are For the protein gel blot analysis, shoots from 7-d-old seedlings (grown on listed in Supplemental Table 1 online. Arabidopsis plants were transformed 1% agar medium containing 0.53 Gambourg-B5 and 1% sucrose, with a by the ﬂoral dip method as described (Clough and Bent, 1998). 16-h-light/8-h-dark photoperiod) were used to extract the membrane protein fraction. For the PIN1:GFP S1,2,3A mutant, shoots from 3-week- Protein Puriﬁcation and in Vitro Phosphorylation Assays old heterozygote seedlings selected on antibiotic plates were used. Membrane protein extraction and protein gel blot analyses were de- Protein puriﬁcation and in vitro phosphorylation assays were performed scribed recently (Abas and Luschnig, 2010). as described before (Benjamins et al., 2003; Michniewicz et al., 2007), with the following speciﬁcations. GST/HIS-tagged full-length PID and different mutant versions of GST/HIS-tagged PIN1HL proteins were used Immunolocalization, Microscopy, and Signal Analysis in in vitro phosphorylation assays. Cultures of Escherichia coli strain Whole-mount immunolocalization was performed as described (Friml Rosetta (Novagen) containing the constructs were grown at 378Cto et al., 2004), using rabbit anti-PIN2 (dilution 1:200; Abas et al., 2006) and OD = 0.6 in 50 mL LC supplemented with 100 mg/mL carbenicillin, 30 rabbit anti-PIN4 (dilution 1:200; Friml et al., 2004) as the primary antibody mg/mL chloramphenicol, and 25 mg/mL kanamycin. The cultures were and an anti-rabbit Alexa 488 conjugate as the secondary antibody then induced for 4 h with 1 mM isopropyl b-D-1-thiogalactopyranoside at (dilution 1:200; Molecular Probes). 308C, after which cells were harvested by centrifugation (20 min at 4000 PIN1:GFP signal in shoots and embryos and DR5:GFP signal in roots rpm, 48C in tabletop centrifuge) and frozen in liquid nitrogen. Precipitated (used in Figures 7G to 7L) were visualized in water without ﬁxation. PIN1: cells were resuspended in 2 mL extraction buffer (EB; 13 PBS, 2 mM GFP signal in 3-d-old seedling roots (used in Figures 7A to 7F) was EDTA, and 2 mM DTT, pH 8.0) supplemented with 0.1% Tween 20 and 0.1 visualized with ﬁxation and permeation steps as described (Friml et al., mM of the protease inhibitors phenylmethanesulfonyl ﬂuoride, leupeptin, 2004). Signals were detected with confocal laser scanning microscopy and aprotinin (Sigma-Aldrich) and sonicated for 2 min on ice. From this (Zeiss LSM 5 confocal microscope). The images were processed by point on, all steps were performed at 48C. Eppendorf tubes containing the ImageJ software (http://rsb.info.nih.gov/ij/) and assembled in Adobe sonicated cells were centrifugated at full speed (14,000 rpm) for 20 min, Photoshop CS2. PDR5:GFP signal intensity was measured by ImageJ, and the supernatants were transferred to 15-mL tubes containing 100 mL and error bars were obtained based on the measurement of three to six preequilibrated glutathione sepharose resin from GE Healthcare (pre- seedling roots per line. The Y value is the average DR5 signal intensity of equilibration performed with three washes of EB). Resin-containing each line relative to that in wild-type PIN1:GFP line. mixtures were incubated with gentle agitation for 1 h, subsequently Embryo development was analyzed by differential interference con- centrifuged at 500 relative centrifugal force for 3 min, and the precipitated trast microscopy (Zeiss Axioplan2) on cleared ovules (1-h treatment in the resin was washed three times with 20 resin volumes of EB. Then, three clearing solution of chloral hydrate:H O:glycerol = 8:3:1). resin volumes of glutathione elution buffer (10 mM reduced glutathione and 50 mM Tris-HCl, pH 8.0) were added to the glutathione sepharose Accession Numbers resin, and the mixture was agitated for 10 min at room temperature with gentle agitation. The resin was subsequently centrifuged for 3 min at 500 Sequence data from this article can be found in The Arabidopsis In- RCF, and the supernatant containing the desired protein was transferred formation Resource (http://www.Arabidopsis.org/) or GenBank/EMBL to a new tube; this process was repeated twice more. The solutions con- databases under the following accession numbers: Arabidopsis taining the proteins were diluted 1000-fold in Tris buffer (25 mM Tris-HCl, PIN1 (gi:15219501), Arabidopsis PIN2 (gi:42558886), Arabidopsis PIN3 pH 7.5, and 1 mM DTT) and concentrated to a workable volume (usually (gi:42558887), Arabidopsis PIN4 (gi:42558871), Arabidopsis PIN6 50 mL) using Vivaspin microconcentrators with a 10-kD cutoff and a (gi:42558888), Arabidopsis PIN7 (gi:42558877), barrel medic (Medicago maximum capacity of 600 mL (Vivascience). Glycerol was added as preser- truncatula) PIN1 (gi:25986771), rice (Oryza sativa) PIN1 (gi:75251559), vative to a ﬁnal concentration of 10%, and samples were stored at 2808C. moss (Physcomitrella patens) PIN1 (gi:55859521), and maize (Zea mays) Approximately 1 mg of each puriﬁed GST/HIS-tagged protein (PID and PIN1c (gi:171850415). Arabidopsis T-DNA insertion mutants representing substrates) was added to a 20 mL kinase reaction mix, containing 13 the loss-of-function alleles are SALK_049736 (pid-14) and SALK_047613 kinase buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, and 5 mM MgCl )and (pin1). Nottingham Arabidopsis Stock Centre identiﬁcation numbers for 13 labeled ATP solution (100 mM MgCl /ATP and 1 mCi [g- P]ATP). the transgenic Arabidopsis lines PIN1:GFP and P35S:PID are N9362 and Reactions were incubated at 308C for 30 min and stopped by addition N9867, respectively. of 5 mLof 53 protein loading buffer (310 mM Tris-HCl, pH 6.8, 10% SDS, 50% glycerol, 750 mM b-mercaptoethanol, and 0.125% bromophenol Supplemental Data blue) and boiling for 5 min. Reactions were subsequently separated over 10% acrylamide gels, which were washed three times for 30 min with The following materials are available in the online version of this article. Kinase Gel wash buffer (5% trichoroacetic acid and 1% Na H P O ), 2 2 2 7 Supplemental Figure 1. Amino Acid Sequence of the Arabidopsis stained with Coomassie Brilliant Blue, and dried. Autoradiography was PIN1 Protein. performed for 24 to 48 h at 2808C using Fuji Super RX x-ray ﬁlms and Supplemental Figure 2. Relative in Vitro Phosphorylation Intensities intensiﬁer screens. The relative intensity of phosphorylation bands was of Peptides Containing Putative Phosphorylation Sites in the PIN1HL analyzed using ImageJ software (http://rsbweb.nih.gov/ij/). (Boxed) by PID. For the peptide assays, 1 mg of puriﬁed PID was incubated with 4 nmol mer of 9 biotinilated peptides (Pepscan) in a phosphorylation reaction as Supplemental Figure 3. In Vitro Phosphorylation Assays of Predicted described above. Reaction processing, spotting, and washing of the Phosphorylation Targets in the PIN1HL by PID. SAM Biotin Capture Membrane (Promega) were performed according to Supplemental Figure 4. Alignment of the Amino Acid Sequences of the protocol of the manufacturer. Following washing, the membranes the PIN1 Proteins from Five Plant Species Showing the Evolutionary were sealed in plastic wrap and exposed to x-ray ﬁlms for 24 to 48 h at Conservation of the Three TPRXS(N/S) Motifs. 2808C using intensiﬁer screens. The phosphorylation intensities of the peptides were determined by densitometry analysis of the autoradio- Supplemental Figure 5. Quantiﬁcation of Protein Expression by graphs using the ImageQuant software (Molecular Dynamics). Western Blot Analysis Using PIN1-Speciﬁc Antibody. Conserved Serines Direct PIN1 Polarity 1141 Supplemental Figure 6. Confocal Laser Scanning Microscopy Im- Blom, N., Gammeltoft, S., and Brunak, S. (1999). Sequence and ages of Mutated PIN1:GFP Localization. structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 294: 1351–1362. Supplemental Figure 7. Quantiﬁcation of Auxin Reporter PDR5:GFP Bo¨ gre, L., Okre´ sz, L., Henriques, R., and Anthony, R.G. (2003). Expression in Root Tips. Growth signalling pathways in Arabidopsis and the AGC protein Supplemental Table 1. Oligonucleotides Used in Site-Directed Mu- kinases. Trends Plant Sci. 8: 424–431. tagenesis. Carraro, N., Forestan, C., Canova, S., Traas, J., and Varotto, S. (2006). 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Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport

Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport

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Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport

Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport

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