MAPKs Influence Pollen Tube Growth by Controlling theFormation of Phosphatidylinositol 4,5-Bisphosphate in anApical Plasma Membrane Domain

FranziskaHempel,a Irene Stenzel,aMareikeHeilmann,a PraveenKrishnamoorthy,aWilhelmMenzel,a RalphGolbik,b

Stefan Helm,c Dirk Dobritzsch,c Sacha Baginsky,c Justin Lee,d Wolfgang Hoehenwarter,e and Ingo Heilmanna,1

a Department of Cellular Biochemistry, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg,06120 Halle (Saale), GermanybDepartment of Microbial Biotechnology, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg,06120 Halle (Saale), GermanycDepartment of Plant Biochemistry, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg,06120 Halle (Saale), GermanydDepartment of Stress and Developmental Biology, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germanye Proteome Analytics, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany

ORCID IDs: 0000-0002-5440-3898 (S.B.); 0000-0001-8269-7494 (J.L.); 0000-0002-7669-7524 (W.H.); 0000-0002-2324-1849 (I.H.)

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate[PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based onan untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts couldbe attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalyticdomain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybridtests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane ofpollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2

reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression onpollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Ourdata indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing todirectional growth.

INTRODUCTION

Pollen tubes serve asmodels for the study of polar tip growth andcellular polarization (Kost, 2008; Ischebeck et al., 2010a; RoundsandBezanilla, 2013;Franciosini et al., 2017). Tip-growingcells canattain length/width ratios exceeding 1000 (Kost, 2008; Riquelme,2013; Rounds and Bezanilla, 2013) and share structural featuresand regulatorymechanisms thathavebeenconserved inevolution(Kost, 2008; Ischebeck et al., 2010a; RoundsandBezanilla, 2013).The apical expansion of a cell requires the transport of membraneand cell wall material by directional vesicle trafficking to thegrowing apex and the retrieval of unloaded vesicles (Thole andNielsen, 2008; Moscatelli and Idilli, 2009; Ischebeck et al., 2010a;Hepler and Winship, 2015; Franciosini et al., 2017). The polarizedexpansionofpollen tubesandsomeother cell types is furthermoreresponsive to exogenous cues (Duan et al., 2010; Kessler and

Grossniklaus, 2011; Lindner et al., 2012;DresselhausandFranklin-Tong, 2013; Higashiyama and Takeuchi, 2015; Dresselhaus et al.,2016). For instance, pollen tubes grow toward the ovules withinflowers of a compatible genotype to achieve fertilization, guided bycuesemittedby the femaleorgans (DresselhausandFranklin-Tong,2013; Higashiyama and Takeuchi, 2015; Dresselhaus et al., 2016).So far, it is unclear how the perception of exogenous cues controlsthe machinery for apical cell expansion of pollen tubes.The apical cell expansion of tip-growing cells is regulated in

part by phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which occupies an apical plasmamembrane domain in pollen tubes (Kost et al., 1999; Ischebecket al., 2008; Sousa et al., 2008; Ischebeck et al., 2010b; Zhao et al.,2010; Ischebeck et al., 2011; Stenzel et al., 2012) as well as roothairs (Vincent et al., 2005; Preuss et al., 2006; Kusano et al., 2008;Stenzel et al., 2008; Ghosh et al., 2015) and fungal hyphae (Mähset al., 2012).PtdIns(4,5)P2 actsasa ligand to targetproteins,whichare regulated in their biochemical activity or subcellular locali-zation by the protein-lipid interaction (Hammond and Balla, 2015;Heilmann, 2016; Gerth et al., 2017). Arabidopsis thaliana plantsdisplaying T-DNA- or RNAi-mediated underexpression of thepollen-specific PI4P 5-kinase isoforms PIP5K4 and PIP5K5

1Address correspondence to ingo.heilmann@biochemtech.uni-halle.de.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Ingo Heilmann (ingo.heilmann@biochemtech.uni-halle.de).www.plantcell.org/cgi/doi/10.1105/tpc.17.00543

The Plant Cell, Vol. 29: 3030–3050, December 2017, www.plantcell.org ã 2017 ASPB.

(Ischebeck et al., 2008; Sousa et al., 2008), PIP5K6 (Zhao et al.,2010), or PIP5K10 and PIP5K11 (Ischebeck et al., 2011) displaysubstantially reduced rates of pollen germination and pollen tubeexpansion. Therefore, the PtdIns(4,5)P2 domain in the apicalplasma membrane of pollen tubes is thought to be essential forpolar cell expansion (Kost et al., 1999; Vincent et al., 2005;Ischebecket al., 2008, 2010b, 2011;Sousaet al., 2008;Zhaoet al.,2010). A critical role of PtdIns(4,5)P2 in the control of apical se-cretion of cell wall material and directional cell expansion of pollentubes was previously described mainly based on the effects ofoverexpressing PI4P 5-kinases (Ischebeck et al., 2008, 2010b;Sousa et al., 2008; Zhao et al., 2010; Stenzel et al., 2012). In thesestudies, the overproduction of PtdIns(4,5)P2 resulted in increasedapical deposition of pectin and characteristic morphologicaldefects, including pollen tube tip branching and protoplasttrapping, which have previously been summarized as “secretionphenotypes” (Ischebeck et al., 2010b). It is evident that correctamountsofPtdIns(4,5)P2 are important for thecontrol of apical cellexpansion of pollen tubes and that PtdIns(4,5)P2 productionmustbe tightly controlled. However, it is unknownhow thebiosynthesisof PtdIns(4,5)P2 or other phosphoinositides is regulated by up-stream signaling pathways.

As PI4P 5-kinases in the apical plasma membrane of pollentubes generate the PtdIns(4,5)P2membrane domain important forcell expansion, we examined whether these enzymes are can-didates for regulation. Our working hypothesis was that PI4P5-kinases in the pollen tube are regulated by phosphorylation, ashas previously been found for the PI4P 5-kinase Mss4p fromSaccharomyces cerevisiae (Audhya and Emr, 2003) and for PI4P5-kinases from Schizosaccharomyces pombe and human, whichdisplaydecreasedcatalytic activity in vitrowhenphosphorylated(Vancurova et al., 1999; Park et al., 2001). Information of post-translational control of the plant phosphoinositide system isscarce. The Arabidopsis PI4P 5-kinase PIP5K1 can be phos-phorylated in vitro by mammalian protein kinase A (PKA)(Westergren et al., 2001). However, the relevant phosphosites orregulatory consequences of this phosphorylation have not beendetermined, andendogenousprotein kinasesactingupstreamofPI4P5-kinases remain unknown in plants.While a role for proteinphosphorylation in the control of phosphoinositide biosynthesisin pollen tubes has not been reported, protein kinases are re-quired for the regulation of pollen tube growth (Higashiyama andTakeuchi, 2015). For instance, Arabidopsis plants carrying le-sions in the genes encoding the mitogen-activated proteinkinases (MAPKs) MPK3 andMPK6 display pollen tube guidancedefects (Guan et al., 2014), suggesting that a MAPK cascade isinvolved in the transduction of exogenous guidance cues inpollen tubes (Higashiyama and Takeuchi, 2015). While thesefindings indicate a role for MAPKs in the control of pollen tubegrowth, it is currently unclear how MAPK-mediated proteinphosphorylation might be linked to the machinery for apical cellexpansion.

Here, we demonstrate that the pollen-expressed PI4P 5-kinasesAtPIP5K6 from Arabidopsis and NtPIP5K6 from tobacco (Nicotianatabacum)arephosphorylatedbyproteinkinaseactivities inpollentubeextracts. We identify MPK6 and its tobacco homolog, SALICYLICACID INDUCED PROTEIN KINASE (SIPK), as protein kinases thatbindandphosphorylatePIP5K6homologs fromArabidopsis and

tobacco, respectively. Phosphorylation by the MAPKs inhibitsPI4P 5-kinase activity in vitro. In vivo, expression of AtMPK6reduces the plasma membrane association of a fluorescentreporter for PtdIns(4,5)P2, inhibits endocytosis, and modulatespollen tube growth. The data demonstrate an unexpected reg-ulatory link between MAPKs and the apical production ofPtdIns(4,5)P2 required for pollen tube expansion.

RESULTS

AtPIP5K6 and NtPIP5K6 Are Phosphorylated by MAPKsfrom Tobacco Pollen Tube Extracts

The PI4P 5-kinase AtPIP5K6 and its tobacco homolog NtPIP5K6have reported roles in the control ofmembrane trafficking in pollentubes (Zhao et al., 2010; Stenzel et al., 2012). To test for phos-phorylation of these enzymes, purified recombinant AtPIP5K6 orNtPIP5K6 was incubated in the presence of [g-33P]ATP with ex-tracts obtained fromgerminated tobaccopollen tubes (Figure 1A).Both AtPIP5K6 and NtPIP5K6 were radiolabeled by the incuba-tion (Figure 1A). For AtPIP5K6, a time course of increasing in-corporation of the radiolabel is shown (Figure 1A, left panels).Controls without added recombinant enzyme or without addedpollen tube extract did not result in a radiolabeled band (Figure 1A,right panels). The data indicate that the pollen tube extract con-tained protein kinase activities capable of phosphorylating re-combinant AtPIP5K6 and NtPIP5K6 in vitro. Relevant proteinkinases frompollen tube extractswere identifiedby a nontargetedin-gel kinase assay. Protein extracts of pollen tubes were elec-trophoresed on SDS-PAGE gels containing purified recombinantAtPIP5K6 protein embedded in the gel matrix. Negative controlswere performed without added recombinant AtPIP5K6 in thegels. After renaturing and washing, the gels were incubated with[g-33P]ATP to assess the in-gel protein kinase activity againstthe supplied AtPIP5K6 substrate (Figure 1B). In the absence ofAtPIP5K6 in the gel, there was no phosphorylation signal with thePKA control, and only weak phosphorylation signals resultedfrom the application of the pollen tube extract (Figure 1B, leftpanel), which presumably represent kinase autophosphor-ylation. By contrast, the presence of recombinant AtPIP5K6protein in the gels yielded radiolabeled signals with the PKApositive control as well as enhanced signals with the pollen tubeextract (Figure 1B, right panel), indicating phosphorylation of thesubstrate protein, AtPIP5K6, or possibly enhanced autophos-phorylation. In-gel kinase assays performed in parallel withadded AtPIP5K6 protein but without radiolabel were excised inthe range of observed bands, and the proteins were reextracted,subjected to tryptic digestion, and analyzed by liquid chro-matography high-definition multiparallel collision-induceddissociation mass spectrometry (nano-LC-HD-MSE) to iden-tify protein kinase candidates. The underlying mass spec-trometry data have been deposited into the ProteomeXchangeConsortium with the data set identifier PXD006067. Trypticpeptides identified by the analysis were annotated accordingto theArabidopsis genomedatabase. Among the 260 detectedproteins, four candidates for relevant protein kinases repre-sented MAPK sequences (Figure 1C). The candidate MPK6

MPK6-Mediated Phosphorylation of PIP5K6 3031

Figure 1. In Vitro Phosphorylation of AtPIP5K6 and NtPIP5K6 by Protein Kinases from Tobacco Pollen Tube Extracts.

(A)Purified recombinantMBP-AtPIP5K6orMBP-NtPIP5K6was incubatedwith pollen tubeextracts in thepresenceof [g-33P]ATP, proteinswere separatedby SDS-PAGE, and the incorporation of the radiolabel was analyzed by phosphor imaging. MBP-ATPIP5K6 was analyzed in a time-course ex-periment (left panels). NtPIP5K6 was analyzed together with control experiments (right panels). The presence of the recombinant proteins (ar-rowheads) is indicated by Coomassie Brilliant Blue-stained gels (top panels). Radiolabeled bands detected by phosphor imaging (mid panels) anda quantification of the radiolabeling signals (lower panels) are also shown. Controls included assays with no added recombinant protein or withoutadded pollen extract, as indicated. Data are from a representative experiment. The experiments were performed three timeswith similar results. Plusand minus symbols indicate added and omitted components, as indicated.(B) and (C) Identification of candidate protein kinases mediating the phosphorylation of AtPIP5K6.(B) To identify protein kinase candidates, protein extracts of germinated pollen tubes were electrophoresed on SDS-PAGE gels containingpurified recombinant AtPIP5K6 protein as part of the gel matrix. After washing and renaturing, nontargeted in-gel protein kinase assays wereperformed by incubating the gels with [g-33P]ATP and visualizing radiolabeled bands by phosphor imaging. SDS-PAGE gels without addedrecombinant protein were used as a negative control (left panel); mammalian PKAwas used as a positive control, as indicated. Bands observed inthe range of 50 kD were excised from gels in assays performed in the absence of radiolabel, subjected to tryptic digestion and analyzed by nano-LC-HD-MSE.(C) Mass spectrometric analysis and comparison of the identified peptides to amino acid sequences deduced from the annotated Arabidopsis genomeyielded hits for MAPKs. Detailed mass spectrometric data on the identified peptides are available online via ProteomeXchange with identifier PXD006067.The in-gel protein kinase assays and candidate identification was performed twice. PSL, photostimulated luminescence.

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was selected for further analysis because the encoding genedisplays an expression pattern similar to AtPIP5K6 and ishighly expressed in pollen according to expression patternsavailable in publicly accessible databases (Winter et al., 2007).

Recombinant AtPIP5K6 Is Phosphorylated by RecombinantMPK6 in Two Bona Fide MAPK Recognition Motifs

To verify phosphorylation of the PIP5K6 homologs from Arabi-dopsis and tobacco by MPK6 in a targeted analysis, purifiedactivated recombinant MPK6 was incubated with AtPIP5K6 orNtPIP5K6 in the presence of [g-33P]ATP (Figure 2A). The dataindicate that MPK6 is capable of phosphorylating AtPIP5K6 andNtPIP5K6 in vitro. To test the specificity of this reaction, otherrecombinant PI4P 5-kinases from Arabidopsis were also tested(Supplemental Figure 1), among which AtPIP5K6 displayed thestrongest phosphorylation signal, followed by the pollen-specificAtPIP5K5 and a weaker signal for AtPIP5K1 (SupplementalFigure 1). Recombinant AtPIP5K6 pretreated with activatedrecombinant MPK6 was subjected to tryptic digestion andthe phosphorylation sites were determined as T590 and T597by liquid chromatography online with high-resolution accurate-mass mass spectrometry (HR/AM LC-MS) at a sequencecoverage of ;70% (Supplemental Figure 2A). Both phos-phorylation sites represent TP (a phosphorylated threoninefollowed by a proline) phosphorylation motifs characteristic ofMAPKs and are located in the variable insert region of thecatalytic domain of AtPIP5K6 (Figure 2B; Supplemental Figure2B). Phosphorylation of AtPIP5K6 was further verified bytreating AtPIP5K6 with MPK6 and [g-33P]ATP, followed by in-cubation with serine/threonine protein phosphatase 1 (PP1),resulting in a reduction of the incorporated [33P] label to below5% of the control (Figure 2D). Furthermore, T590A and T597Aalanine substitution variants of AtPIP5K6 were generated, inwhich the phosphorylation sites were eliminated. Using thevariant recombinant proteins, MPK6-mediated phosphorylationwasgradually reduced in theT590AandT597Asubstitutionvariants,respectively, and weakest in the T590A T597A double substitutionvariant (AtPIP5K6 AA) (Figure 2E). Differences were statisticallysignificant, despite a high SD of the control measurements. Re-sidual phosphorylation of AtPIP5K6 AA by MPK6 (Figure 2E) wasaccompanied by phosphorylation of residues which were notidentified in other experiments and are not part of characteristicMAPK phosphorylation motifs (Supplemental Figure 2C). Thesephosphorylation events likely represent nonspecificmodificationsof the recombinant AtPIP5K6 AA protein by MPK6 in vitro. To-gether, the data indicate that purified recombinant AtPIP5K6 wasphosphorylated in vitrobypurified recombinantMPK6 inpositionsT590 and T597.

Phosphorylation of AtPIP5K6 at T590 and T597 InhibitsCatalytic Activity

To investigate possible effects of the MPK6-mediated phos-phorylation on AtPIP5K6 activity, the enzyme was preincubatedwith recombinant MPK6 and ATP for 1 h, followed by the de-termination of specificPI4P5-kinase activity. Enzymeactivitywasassessed according to PtdIns(4,5)P2 formation after 30 min of

incubation, thus within the linear range of product increase(Supplemental Figure 3). Preincubation with MPK6 resulted in an;60% decrease in catalytic activity of AtPIP5K6 (Figure 3A).Importantly, incubation with MPK6 did not decrease the ac-tivity of AtPIP5K6 AA (Figure 3A), where both T590 and T597were substituted with alanine and can no longer be phos-phorylated in vitro (comparedwith Figure 2E). Thedata indicatethat the phosphorylation of AtPIP5K6 in positions T590 andT597 reduced the catalytic activity of AtPIP5K6 in vitro. Tofurther characterize the contribution of these positions to thecatalytic activity of AtPIP5K6, we tested substitution variantsof AtPIP5K6 carrying either an alanine or an aspartate in therespective positions for PI4P 5-kinase activity in vitro (Figure3B). The substitution variants T590A, T590D, and T597A didnot display altered catalytic activity. By contrast, the activity ofthe substitution variant T597D was reduced by ;75% (Figure3B), suggesting that T597 might be a relevant residue exertingan effect on the catalytic activity of the enzyme when targetedby the posttranslational modification or when carrying a neg-ative charge. This notion is supported by the conservation ofthis residue within a TP motif of other pollen expressed PI4P5-kinases, such as NtPIP5K6 or AtPIP5K5 (compared withFigure 2C).As thedouble substitutionvariantT590AT597A (AA) displayed

enhanced catalytic activity in vitro while the phosphomimeticT590 D T597D (DD) variant showed an intermediate effect, wehypothesized that these phosphorylation sitesmay lie in a regionof the PIP5K6 protein that mediates conformational changes.This hypothesis was tested by analyzing the circular dichroism(CD) of purified recombinant PIP5K6 variants (Figures 3C to 3E).CD spectroscopy data were obtained for the far and near UVrange (Figure 3C). The far UV data from the CD spectroscopy(Figure 3D) indicate that the substitution variants all retaineda similar secondary structure, with no gross differences in thecontent of alpha-helices or beta-sheets. By contrast, we ob-served changed patterns in the near UV CD spectra (Figure 3E),indicative of differences in the tertiary structures of some of thesubstitution variants. Interestingly, T597D displayed the mostdeviant tertiary structure, which is striking because only thisvariant displayed reduced catalytic activity consistent with theeffects of phosphorylation of T597 (compared with Figure 3B).The CD spectroscopy data do not indicate substantial differ-ences in secondary or tertiary structure between PIP5K6 AA andPIP5K6 DD (Figures 3D and 3E). Overall, the CD spectroscopydata indicate that the introduction of a negative charge espe-cially at position 597 results in a change in the tertiary structure ofthe PIP5K6 protein, with little or no effect on its secondarystructure.

MPK6 Interacts Physically with AtPIP5K6

To further test the interplay between MPK6 and PIP5K6, physicalinteraction of the proteins was tested by split-ubiquitin-basedyeast two-hybrid analysis (Figure 4A) using the DualMembranesystem (Johnsson and Varshavsky, 1994; Stagljar et al., 1998;Möckli et al., 2007). In these experiments, AtPIP5K6 was im-mobilized as a bait protein at the endoplasmic reticulum bya C-terminally fused OST4 anchor, and the MPK6 prey protein

MPK6-Mediated Phosphorylation of PIP5K6 3033

Figure 2. In Vitro Phosphorylation of TP Motifs in the Catalytic Domain of AtPIP5K6 by Recombinant MPK6.

(A) Purified recombinant MBP-fusions of Arabidopsis PI4P 5-kinase isoforms were incubated with activated recombinant MPK6 in the presence of[g-33P]ATP, proteinswere separatedbySDS-PAGE, and the incorporation of the radiolabelwasanalyzedbyphosphor imaging.All proteinswere expressed inE. coli and added at 5 mg of PIP5K6 and 0.2 mg of MPK6 protein, respectively, when required. The presence of the recombinant proteins was tested byCoomassieBrilliant Blue (toppanels). Radiolabeledbandswere detectedbyphosphor imaging (lower panels). Controls includedassaysusingMBP instead ofMBP-PIP5K6, omitting MPK6 protein, or using a-labeled instead of g-labeled ATP, as indicated. The experiments were performed three times with similarresults. Plus and minus symbols indicate added and omitted MPK6, respectively.(B)and (C)HR/AMLC-MSanalysis of tryptic peptidesofMPK6-phosphorylatedAtPIP5K6 revealed thepresenceof twophosphorylated residues, T590andT597, which are located in the catalytic domain of AtPIP5K6.(B) Schematic representation of AtPIP5K6 with the positions of T590 and T597, which are located in the variable insert of the catalytic domain of AtPIP5K6(indicated by arrowheads). NT, N-terminal domain; MORN, membrane occupation and recognition nexus repeat-domain; Lin, linker domain; Dim, di-merization domain; Cat, catalytic domain; Var, variable insert.(C) Local alignment of the sequence region of AtPIP5K6 around T590 and T597 with the corresponding sequences of other pollen-expressed PI4P5-kinases, as indicated. The positions of T590 and T597 are indicated by arrowheads. Black, identical residues in the same position in three or moresequences; gray, residues with similar properties in the same position in three or more sequences.(D) and (E) Verification of phosphorylation events upon preincubation of AtPIP5K6 with activated MPK6 and [g-33P]ATP.

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was freely diffusible. Only upon recruitment of MPK6 to theendoplasmic reticulum by the interaction with the bait proteinwill the yeast grow on the restrictive selection media. On se-lective SD-LWH media, yeast cells expressing both testedproteins displayed growth comparable to that of positivecontrols and substantially stronger than that of the negativecontrols (Figure 4A). Similar resultswereobtained forNtPIP5K6and SIPK (Supplemental Figure 4). The interaction was verifiedby immuno-pull-down experiments using purified recombinantGST-MPK6 and MPB-PIP5K6 proteins (Figure 4B). In theseexperiments, immobilized GST-MPK6, but not GST alone, couldbind to MBP-PIP5K6. Furthermore, bimolecular fluorescencecomplementation (BiFC) was used to verify the interaction ofMPK6andAtPIP5K6 (Figure4C). ThecoexpressionofAtPIP5K6-YFPN with MPK6-YFPC in tobacco pollen tubes resulted in thereconstitution of fluorescence at the apical plasma membrane.No fluorescencewas observedwhenAtPIP5K6-YFPNorMPK6-YFPCwas expressed togetherwithYFPCorYFPN, respectively,suggesting that the reconstituted fluorescence of the proteinfusions was not a consequence of reassembling YFP halves.However, the BiFC experiment has to be interpreted with cau-tion, as the transient pollen tube expression system does notpermit the recommended analysis for protein integrity by im-munodetection (Kudla andBock, 2016). In sum, the data indicatea (possibly weak) physical interaction of AtPIP5K6 with MPK6,which presumably takes place at the apical plasmamembrane ofpollen tubes.

Expression of MPK6 Reduces Plasma MembraneAssociation of a Fluorescent Reporter for PtdIns(4,5)P2

To test for in vivo effects of MPK6 expression on PtdIns(4,5)P2

formation in pollen tubes, MPK6-EYFP was coexpressed witha fluorescent probe for PtdIns(4,5)P2, Red StarPLC-PH (König et al.,2008), and thefluorescencedistributionof theprobewasanalyzedby confocal microscopy (Figure 5). In control pollen tubes ex-pressing EYFP together with Red StarPLC-PH, the PtdIns(4,5)P2-probe decorated the apical plasma membrane in the previouslyreported pattern (Figure 5A, upper panels). By contrast,membrane association of the PtdIns(4,5)P2 probe was reducedwhen MPK6-EYFP was coexpressed with Red StarPLC-PH,(Figure 5A, lower panels). The effect of MPK6-EYFP expressionon themembrane association of RedStarPLC-PHwas numericallyassessed (Figures 5B and 5C) based on fluorescence intensityprofiles recordedas indicated inFigure5B.Adecreasingplasmamembrane-associated (PM) versus cytoplasmic (cyt) intensityratio of Red StarPLC-PH fluorescence indicates reduced plasmamembrane association of the reporter. The PM versus cyt in-tensity ratio of Red StarPLC-PH fluorescence dropped with

increasing expression of MPK6-EYFP (Figure 5C, closed cir-cles), whereas the ratio remained roughly constant with in-creasing expression of the EYFP control (Figure 5C, opencircles). Reduced membrane association of the PtdIns(4,5)P2-specific probe upon expression of MPK6-EYFP is consistentwith MPK6-mediated inhibition of PIP5K6 (compared withFigure 3A) and suggests reduced PtdIns(4,5)P2 formation inthe apical plasma membrane of the pollen tubes in vivo.While this observation cannot be directly verified by biochem-ical analysis of PtdIns(4,5)P2 levels in pollen tubes transientlyoverexpressing PIP5K6-EYFP due to the low transformationfrequency, pollen tubes of tobacco plants, in which the ex-pression of the endogenous MAPKs, SIPK and WIPK, is RNAisuppressed (Seo et al., 2007), displayed elevated levels ofPtdIns(4,5)P2 (Figure 5D; see Supplemental Figure 5 for tran-script reduction in pollen tubes from the RNAi lines). These datasupport the notion that the formation of PtdIns(4,5)P2 in pollentubes is controlled by the MAPKs.

Expression of MPK6 Does Not Interfere with MembraneAssociation of PIP5K6 in Pollen Tubes

One mode to regulate PI4P 5-kinase activity in animal cells isby an electrostatic switch mechanism, where the introduction ofnegative charges, e.g., upon protein phosphorylation, interfereswith membrane association of the enzymes (Rao et al., 1998;Burden et al., 1999; Fairn et al., 2009). Therefore, we analyzednext whether the coexpression of MPK6-mCherry would in-fluence the apical membrane association of PIP5K6-EYFP inpollen tubes (Figure 6). When PIP5K6-EYFP was coexpressedwith either an mCherry control or with MPK6-mCherry, mem-brane association of PIP5K6-EYFP was not abated (Figure 6)even with higher expression levels of the coexpressed markers(Figure 6C). The data suggest that the effects of MPK6 onPtdIns(4,5)P2 production, which were observed in the same celltypes at identical conditions (compared with Figure 5), were notmediated by displacement of PIP5K6 from the apical plasmamembrane.

Membrane Trafficking Is Influenced by the Expression ofMPK6 in Pollen Tubes

Next, we examined whether PtdIns(4,5)P2-dependent processeswere influenced by MPK6 in vivo. As PIP5K6 is required for cla-thrin-mediated endocytosis in pollen tubes (Zhao et al., 2010), weanalyzed the endocytosis of the membrane dye, FM 4-64, overtime (Figure 7). The distribution of FM4-64was imaged after 15 to25 min, 35 to 50 min, and again after 65 to 85 min upon dyeapplication in control pollen tubes expressing EYFP (Figure 7A,

Figure 2. (continued).

(D) Reduced radiolabel upon treatment of prephosphorylated AtPIP5K6 protein with serine/threonine PP1 phosphatase. Plus andminus symbols indicateadded and omitted components, as indicated.(E)Reduced phosphorylation of alanine substitution variants T590A, T597A, and T590A T597A (AA) byMPK6. The experimentswere performed four times.Data representmean6 SD. Letters a toc in (E) indicate categoriesof values that displaysignificantdifferences fromeachother, according toStudent’s t tests(for different categories all P < 0.05).

MPK6-Mediated Phosphorylation of PIP5K6 3035

upper panels) and in pollen tubes expressingMPK6-EYFP (Figure7A, lower panels). In the control pollen tubes, substantial in-corporation of the dye into endomembranes was observed after35 to 50min. By contrast, only limited incorporation was apparentafter this time in pollen tubes expressingMPK6-EYFP (Figure 7A).

Numerically, a decreasingPMversus cyt intensity ratio of FM4-64fluorescence indicates internalization of the dye into the cyto-plasm. After 15 to 25 min of dye application, the PM versus cytintensity ratios were comparable between EYFP controls andMPK6-EYFP expressers (P < 0.045), whereas after 35 to 50 min

Figure 3. Inhibition of AtPIP5K6 Catalytic Activity by MPK6-Mediated Phosphorylation in Positions T590 and T597.

(A) Recombinant AtPIP5K6 or AtPIP5K6 AA protein was preincubated with recombinant activated MPK6 and subsequently analyzed for catalytic activityagainst the lipid substrate, PtdIns4P. The reduction in catalytic activitywas calculated relative to the activity of non-treated controls. The data represent themean6 SD from four experiments.Theasterisks indicate asignificantdifference from thenon-treatedcontrol, according toaStudent’s t test (**P<0.01). Plusand minus symbols indicate added and omitted MPK6, respectively.(B) The intrinsic catalytic activity of AtPIP5K6 variants, in which T590 and/or T597 were substituted with either A or D, was analyzed against the lipidsubstrate,PtdIns4P.Thedata represent themean6 SD fromthreeexperiments. Lettersa toc indicatecategoriesof values thatdisplaysignificantdifferencesfrom each other, according to Student’s t tests (for different categories all P < 0.01).(C)TheCDofpurified recombinantPIP5K6variantswasanalyzed inbuffer containing20mMTris-HCl, pH7.5, 0.2MNaCl, 1mMEDTA, and10mMmaltose,using an optical path length of 1 mm. CD spectroscopy data were obtained for the far and near-UV range.(D) Far-UV CD spectra indicative of protein secondary structure.(E) Near-UV CD spectra indicative of protein tertiary structure. Protein variants as indicated. Recombinant MBP was used as a control protein.

3036 The Plant Cell

Figure 4. Physical Interaction of MPK6 with AtPIP5K6.

An interaction between MPK6 and AtPIP5K6 was tested by split-ubiquitin-based yeast two-hybrid analysis, immuno-pull-down experiments, and BiFC.(A)Analysis by the split-ubiquitin-basedyeast two-hybrid system. TheAtPIP5K6-bait proteinwasexpressed asa fusion to anOST4anchor, which attachesthe protein to the cytosolic face of the endoplasmic reticulum. TheMPK6 fusionwas expressed as a soluble cytoplasmic protein. Interaction is indicated byyeast growth under selective (2LWH) conditions. The experiment was performed three times with similar results.(B) Immuno-pull-down experiments. RecombinantMPK6expressed as aGST fusion or aGST control were immobilized and incubatedwith purified recombinantMBP-PIP5K6 protein. Upon washing of the resin, interacting MBP-PIP5K6 protein was analyzed by immunodetection using an anti-MBP antibody. Left panel,protein detectedby anti-GSTantibody; right panel, pull-down, detectedby the anti-MBP-antibody. The experimentwasperformed three timeswith similar results.(C)AnalysisbyBiFC.FusionsofAtPIP5K6and theN-terminal half ofYFP (AtPIP5K6-YFPN), andofMPK6andtheC-terminalhalf ofYFP (MPK6-YFPC)at theirrespective C termini were transiently expressed in tobacco pollen tubes. Negative controls included the coexpression of AtPIP5K6-YFPNwith YFPC, and ofYFPNwithMPK6-YFPC. AnmCherry marker was always coexpressed as a reporter for positive transformation events. Reconstitution of YFP fluorescenceindicates closephysical proximityofAtPIP5K6-YFPNandMPK6-YFPC.Bars=10mm.Theexperimentswereperformed four timeswith similar results.2LW,media lacking leucine and tryptophan;2LWH,media lacking leucine, tryptophan, and histidine;MBP,maltose binding protein; OST4, yeast oligosaccharyltransferase 4-kD subunit; pAI-Alg5, positive control; pDL2-Alg5, negative control.

MPK6-Mediated Phosphorylation of PIP5K6 3037

Figure 5. Reduced Plasma Membrane Association of the Red StarPLC-PH Reporter upon Coexpression with MPK6-EYFP.

The effect of MPK6-EYFP expression on the apical formation of PtdIns(4,5)P2 was assessed using the PtdIns(4,5)P2-specific fluorescent reporter, RedStarPLC-PH.(A) Red StarPLC-PH was coexpressed with either an EYFP control (upper panels) or with MPK6-EYFP (lower panels), and fluorescence distribution wasassessedbyconfocalmicroscopy.Dotted lines,position for collectingfluorescence intensityprofiles forquantitativeanalysis ([B]and [C]).Bars=10mm.Foreach condition (EYFP versusMPK6-EYFP), four independent transformations were set up to coexpress thesemarkers with Red StarPLC-PH. Only data frommorphologically unaltered pollen tubes were included. Images are representative for 14 individual transformations from two independent experiments foreach condition.(B) Fluorescence intensity profiles (as indicated in [A]) were analyzed using the Fiji software package. Profiles shown are from the representative images in(A). Bottom diagram: Peripheral regions of the Red StarPLC-PH profiles were interpreted as PM-associated fluorescence and the central regions as cy-toplasmic fluorescence (cyt), as indicated. The intensity of the coexpressed EYFP or MPK6-EYFP was determined as indicated. The values were used tocalculate the ratios in (C).

3038 The Plant Cell

and 65 to 85 min the internalization of the dye was significantlyfaster (P < 0.01 in both cases) in the control pollen tubescompared with the cells expressing MPK6-EYFP (Figure 7B).This pattern indicates reduced endocytosis of FM 4-64 in pollentubes expressing MPK6-EYFP. We also tested whether theexpression of MPK6 would influence pollen tube growth, whichrequires tip-directed membrane trafficking and apical pectinsecretion. After 10 h of incubation, pollen tubes expressingMPK6 were slightly shorter than control pollen tubes. Further-more, pectin secretion according to staining of pollen tubes withruthenium red was reduced when MPK6-EYFP was expressed(Supplemental Figure 6). Together, these data indicate thatdifferent aspects of apical membrane trafficking are influencedby the overexpression of MPK6-EYFP, consistent with MPK6-mediated inhibition of PIP5K6 and PtdIns(4,5)P2 productionin vivo.

Coexpression with MAPKs Attenuates the Effects of PIP5K6Homologs from Arabidopsis or Tobacco on PollenTube Growth

To further delineate the functional relevance of the modificationof PI4P 5-kinases by MPK6 in vivo, we scored the incidenceof morphological alterations resulting from overexpressingAtPIP5K6 or NtPIP5K6 as a quantitative readout for physiologi-cal functionality of the enzymes, as was previously described(Ischebeck et al., 2008, 2010b, 2011; Stenzel et al., 2012). Pollentubes used in our experiments displayed the morphologicalalterations previously found to result from overexpression ofAtPIP5K6 and NtPIP5K6 (Figure 8A, left panel). As in previousstudies (Ischebeck et al., 2008, 2011), the distribution of pheno-typic categorieswas associatedwith the degree of expression, asassessed by fluorescence intensity of the expressed proteins(Figure8A, rightpanel).WhenpollentubescoexpressingAtPIP5K6-EYFP with an mCherry control were scored (Figure 8B), normal,branched, and stunted morphologies were observed for;12%,10%, and 66%of fluorescing cells, respectively. Compared withthese controls, coexpression of AtPIP5K6-EYFP with MPK6-mCherry resulted in a significant shift toward weaker morpho-logical defects, with now 22% normal, 16% branched, and only56% stunted morphologies (P < 0.05 for the increase in normalpollen tubes). When AtPIP5K6 AA-EYFP or AtPIP5K6 DD-EYFPwere each coexpressed with either an mCherry control or withMPK6-mCherry (Figure 8B), the resulting patterns did not differfrom one another, nor from that observed upon coexpression ofAtPIP5K6-EYFP with the mCherry control, indicating that MPK6

did not exert an inhibitory influence on the function of AtPIP5K6AA or AtPIP5K DD in vivo. These data indicate that the phos-phosites T590 and/or T597 are critical for the MPK6-mediatedregulation of PIP5K6 effects on cell morphology. When therespective experiments were performed using the homologoustobacco enzymesNtPIP5K6 and SIPK, equivalent observationswere made (Figure 8C). Pollen tubes coexpressing NtPIP5K6-EYFP and an mCherry control displayed normal, branched, andstunted morphologies for;10%, 39%, and 45% of fluorescingcells, respectively. In comparison, coexpression of NtPIP5K6-EYFP with SIPK-mCherry again resulted in a significant shifttoward weaker morphological defects, with now 39% normal,29% branched, and only 31% stunted morphologies (P < 0.01for the increase in normal pollen tubes). In the NtPIP5K6sequence, only one of the two residues phosphorylated inAtPIP5K6 is conserved (T651, which corresponds to T597 ofAtPIP5K6; compared with Figure 2B). To test for functionalrelevance of this residue, an NtPIP5K6 variant was generated inwhich T651 was substituted with alanine (NtPIP5K6 A). WhenNtPIP5K6 A-EYFP was coexpressed with mCherry or SIPK-mCherry, there was no difference in the resulting patterns, in-dicating that SIPK did not exert an inhibitory influence on thefunction of the variant NtPIP5K6 A protein in vivo.

Effects of PIP5K6 Homologs from Arabidopsis or TobaccoAre Delimited in Pollen Tubes upon RNAi Suppression ofIntrinsic MAPK Expression

The in vivo effects of MAPKs on PI4P 5-kinases were furthercharacterized in a reciprocal experiment. For this purpose,AtPIP5K6 or NtPIP5K6 was expressed in pollen tubes of tobaccoplants, in which expression of the endogenousMAPKs, SIPK andWOUNDING-INDUCED PROTEIN KINASE (WIPK), was sup-pressed by RNAi (Seo et al., 2007) (Figure 9). Pollen tubes oftobacco plants expressing an empty control construct were usedas a reference. When AtPIP5K6-EYFP or NtPIP5K6-EYFP wasexpressed inpollen tubesof the control plants, forAtPIP5K6, 11%of transgenic pollen tubes were normal, 22%branched, and 50%stunted (Figure 9A), whereas for NtPIP5K6, 11% of transgenicpollen tubes were normal, 38% branched, and 35% stunted. Bycontrast, the expression of AtPIP5K6-EYFP or NtPIP5K6-EYFP ineither of the two independent RNAi lines resulted in significantshifts of the phenotypic categories toward more severe pheno-types (P < 0.01 for the increases in stunted pollen tubes in all fourcases). The data indicate that RNAi suppression of endogenousMAPKs enhanced the effects of co-overexpressing PIP5K6 in vivo,

Figure 5. (continued).

(C) Decreasing plasma membrane association of the Red StarPLC-PH probe with increasing expression of MPK6-EYFP. Mean intensities for PM and cytfluorescence of the Red StarPLC-PH probe were calculated for each transformed cell, and the PM versus cyt ratios of these means were plotted against themean intensity of either EYFP or MPK6-EYFP. A decreasing PM versus cyt ratio indicates reduced plasma membrane association of the PtdIns(4,5)P2-reporter. Open circles, EYFP control; closed circles, MPK6-EYFP. The data represent 14 individual transformation events obtained in two independentexperiments for each condition.(D) The levels of PtdIns(4,5)P2 were analyzed in pollen tubes from nontransformed tobacco plants, from empty vector controls, or from two independenttransgenic lines (WS2 and WS3), in which SIPK and WIPK are RNAi suppressed. Data indicate mean 6 SD from four experiments. Asterisks indicatea significant change compared with the empty vector control according to a Student’s t test (*P < 0.05). AU, arbitrary units; cyt, cytoplasmic fluorescence;PM, PM-associated fluorescence.

MPK6-Mediated Phosphorylation of PIP5K6 3039

Figure 6. Unaltered Plasma Membrane Association of PIP5K6-EYFP upon Coexpression with MPK6-mCherry.

The effect of MPK6-EYFP expression on the apical plasma membrane localization of PIP5K6-EYFP was assessed in coexpression experiments.(A) PIP5K6-EYFP was coexpressed with either anmCherry control (upper panels) or with MPK6-mCherry (lower panels), and the fluorescence distributionwas assessed by confocal microscopy. Dotted lines, position for collecting fluorescence intensity profiles for quantitative analysis ([B] and [C]). Bars =10mm.For each condition (mCherry versusMPK6-mCherry), four independent transformationswere set up to coexpress thesemarkerswithPIP5K6-EYFP.Only data from morphologically unaltered pollen tubes were included. Images are representative for 16 individual transformations from two independentexperiments for each condition.(B) Fluorescence intensity profiles (as indicated in [A]) were analyzed using the Fiji software package. Profiles shown are from the representative images in(A). Bottom diagram: Peripheral regions of the PIP5K6-EYFP profiles were interpreted as PM-associated fluorescence, and the central regions as cy-toplasmic fluorescence (cyt), as indicated. The intensity of the coexpressedmCherry orMPK6-mCherrywasdeterminedas indicated. Thevalueswere usedto calculate the ratios in (C).(C) PM association of PIP5K6-EYFP with increasing expression of either EYFP or MPK6-EYFP. Mean intensities for PM and cyt fluorescence of PIP5K6-EYFP were calculated for each transformed cell, and the PM versus cyt ratios of these means were plotted against the mean intensity of either mCherry orMPK6-mCherry. Open circles, mCherry control; closed circles, MPK6-mCherry. The data represent 16 individual transformation events for each condition,obtained in four independent experiments.

3040 The Plant Cell

consistent with a regulatory effect of the MAPKs on PtdIns(4,5)P2

production. Importantly, the distribution of phenotypic catego-ries resulting from the expression of AtPIP5K6 AA-EYFP orNtPIP5K6 A-EYFP in pollen tubes of the control plants or the twoRNAi lines did not differ (Figures 9C and 9D), indicating that theeffects of MAPK RNAi suppression on PI4P 5-kinase functiondepended on the threonine residues found to be phosphorylatedby theMAPKs. Overall, the assessment of the in vivo influence ofMAPKs fromArabidopsis or tobaccoon the functionofAtPIP5K6and NtPIP5K6 is consistent with the reduced PtdIns(4,5)P2

formation upon phosphorylation of PI4P 5-kinases determinedin vitro.

DISCUSSION

This study addressed the regulation of PI4P 5-kinases, which arekey enzymes of plant phosphoinositide biosynthesis, by proteinphosphorylation. Our results demonstrate that the MAPKMPK6(1) mediates phosphorylation of PIP5K6 (Figures 1 and 2), (2)mediates a concomitant inhibition of PtdIns(4,5)P2 formationin vitro (Figure3), (3) interactswithAtPIP5K6 (Figure4), (4) causesreduced formation of PtdIns(4,5)P2 and reduced endocytosisat the apical plasma membrane in vivo (Figures 5 and 7), and(5) attenuates PtdIns(4,5)P2-dependent effects on pollen tubemorphologies (Figures 8 and 9).

Figure 7. Reduced Endocytosis of FM 4-64 upon Coexpression with MPK6-EYFP.

The effects of MPK6-EYFP on endocytosis were assessed by monitoring the uptake of the membrane dye, FM 4-64, over time by confocal microscopy.(A) Fluorescence distribution of FM 4-64 (red) during coexpression with an EYFP control (upper panels) or with MPK6-EYFP (lower panels). For eachcondition (EYFPversusMPK6-EYFP),five independent transformationsweresetup toanalyze theeffectonFM4-64uptake.Onlydata frommorphologicallyunaltered pollen tubes were included. Representative images are shown for three time points after dye application, 15 to 25 min, 35 to 50 min, and 65 to85min, as indicated. Intensity profiles for FM4-64were recorded as indicated by the dotted lines, and themeanPM-associated andmean cyt fluorescencewas calculated (as described in the diagrams in Figures 5 and 6). From these values, the PM versus cyt intensity ratios were calculated and plotted in (B).Bars = 10 mm.(B) A decreasing PM versus cyt ratio indicates progressing endocytosis and internalization of the dye. Open circles, EYFP control; closed circles, MPK6-EYFP. Data represent five independent transformation experiments for each condition and each time point, as follows: 15 to 25 min (EYFP, n = 38; MPK6-EYFP, n = 46), 35 to 50 min (EYFP, n = 54; MPK6-EYFP, n = 57), and 65 to 85 min (EYFP, n = 24; MPK6-EYFP, n = 26).

MPK6-Mediated Phosphorylation of PIP5K6 3041

The phosphorylation sites T590 and T597 determined in thePIP5K6 protein by MS-based approaches and subsequent sub-stitution experiments represent bona fide MAPK-targeted S/TPmotifs. In the course of this study, a global analysis of the Ara-bidopsis pollen phosphoproteome was published (Mayank et al.,2012), andbothAtPIP5K6phosphorylationsitesdetermined inour

in vitro approach were also identified in planta. In vivo phos-phorylation of AtPIP5K6 in pollen has thus been independentlydemonstrated. Based on our data, phosphorylation of the sitesT590 and T597 can now be recognized asMPK6-mediated signaltransduction events limiting PtdIns(4,5)P2 production in the apicalplasma membrane of pollen tubes.

Figure 8. Coexpression with MAPKs Attenuates the Effects of PIP5K6 Homologs from Arabidopsis or Tobacco on Pollen Tube Growth and CellMorphology.

TheeffectsofMAPKson the functionalityofAtPIP5K6orNtPIP5K6were tested in vivobycoexpressing theenzymeswitheithermCherry controls orwith theMAPKs,MPK6or SIPK, respectively, and scoring the incidence ofmorphological alterations during pollen tube growth, as previously described (Ischebecket al., 2010b).(A) The overexpression of type B PI4P 5-kinases, including AtPIP5K6, in tobacco pollen tubes results in morphological changes related to an increasedapical secretion of pectin (Ischebeck et al., 2008, 2010b). Left: Phenotypic categories observed upon expression of AtPIP5K6, as indicated. Bars = 10 mm.Right:Correlationof thecategorieswithfluorescence intensities of theexpressedproteins. Letters a toc indicate categoriesof values that displaysignificantdifferences from each other, according to pairwise Student’s t tests (for different categories all P < 0.01).(B)Distribution of phenotypes upon coexpression of AtPIP5K6-EYFP, AtPIP5K6 AA-EYFP, or AtPIP5K6DD-EYFP with either mCherry or MPK6-mCherry.(C) Distribution of phenotypes upon coexpression of NtPIP5K6-EYFP, NtPIP5K6 T651A-EYFP (NtPIP5K6 A-EYFP), or NtPIP5K6 T651D-EYFP(NtPIP5K6 D-EYFP) with either mCherry or SIPK-mCherry. The data reflect the mean 6 SD from seven (B) or six (C) experiments, each representing>100pollen tubes analyzed. Asterisks indicate significant changes comparedwith themCherry control experiments according to aStudent’s t test (*P<0.05; **P < 0.01).

3042 The Plant Cell

At first approximation, the inhibitory effects of phosphorylationon activity and physiological function of PIP5K6 appear to beconsistent with data on mammalian phosphoinositide kinases,which are thought to be regulated by phosphorylation via anelectrostatic switch model (Rao et al., 1998; Burden et al., 1999;Fairn et al., 2009). In this model, the introduction of negativecharges by phosphorylation of residues at the protein-membraneinterface mediates the dissociation of the protein from themembrane and its anionic substrate lipids. However, as themembrane association of PIP5K6-EYFP did not change inpollen tubes upon coexpression of MPK6-mCherry (Figure 6),we conclude that the regulation of PIP5K6 by MPK6 might notinvolve an electrostatic switch mechanism. To delineate themechanistic details of PIP5K6 regulation by MPK6, furtheranalyses will be necessary. In the absence of structural data onplant PI4P 5-kinases, we can currently only speculate whetherphosphorylation of T590 and/or T597 in the variable insert

region of the catalytic domainmight exert a regulatory effect onPIP5K6 by mediating conformational changes that may di-rectly influence the conformation of the catalytic site. The CDspectroscopy data obtained for the substitution variants ofPIP5K6 (Figures 3C to 3E) indicate that the introduction ofa negative charge in position(s) 590 and/or 597 results ina change in the protein’s tertiary structure. As the near UV CDspectra reflect aromatic residues,which are concentrated in theN-terminal region of PIP5K6, we speculate that phosphoryla-tion of PIP5K6 by MPK6 mediates a conformational changeinvolving the NT and MORN domains. This notion is consistentwith the previous report thatN-terminal domains of ArabidopsisPI4P 5-kinases of subfamily B have a role in regulating catalyticactivity (Im et al., 2007; Stenzel et al., 2012), possibly controlledby reversible phosphorylation of PIP5K6. Based on the analysisof single-substitution variants (Figure 3B), a phosphorylationsite relevant for such regulation might be T597. However, our

Figure 9. Effects of PIP5K6 Homologs fromArabidopsis or Tobacco Are Delimited in Pollen Tubes upon RNAi Suppression of IntrinsicMAPK Expression.

The effects ofMAPKs on the functionality of AtPIP5K6 or NtPIP5K6were further tested in vivo by expressing the enzymes in pollen tubes of tobacco plantsRNAi-suppressed for the endogenousMAPKs, SIPK andWIPK (Seo et al., 2007), and scoring the incidence ofmorphological alterations during pollen tubegrowth, aspreviouslydescribed (Ischebecketal., 2010b).Three transgenic tobacco lines (Seoetal., 2007)wereused,acontrol carryinganemptyexpressionconstruct (white bars) and two independent RNAi-suppressed lines (WS1, light gray bars;WS2, dark-gray bars). The data represent themean6 SD from fiveexperiments. Asterisks indicate significant changes compared with the vector control experiments according to a Student’s t test (*P < 0.05; **P < 0.01).(A) Distribution of phenotypes upon expression of AtPIP5K6-EYFP.(B) Distribution of phenotypes upon expression of NtPIP5K6-EYFP.(C) Distribution of phenotypes upon expression of AtPIP5K6 T590A T597A-EYFP.(D) Distribution of phenotypes upon expression of NtPIP5K6 T651A-EYFP.

MPK6-Mediated Phosphorylation of PIP5K6 3043

studies do not reveal the in vivo stoichiometry of singly ordoubly phosphorylated AtPIP5K6. It is possible that the twodetected phosphosites are sequentially phosphorylated byMPK6 under physiological conditions. Such a scenario mayexplain the “compensatory” effect of thedual phospho-mimeticsubstitutionofPIP5K6DDon the reducedkinaseactivity (Figure3B) or the tertiary structure (Figure 3E) of the single T597Dsubstitution variant. With regard to the activity of the doublesubstitution variants, it should also be noted that the sub-stitution of phosphorylation sites by charged or unchargedresidues will not always faithfully reflect the behavior of theprotein when phosphorylated or dephosphorylated (Dissmeyerand Schnittger, 2011). While we are providing evidence forregulation of PIP5K6 by phosphorylation, the effects of thismodification might thus be more complex. The survey byMayank et al. (2012) reports further phosphorylation sites inPIP5K6, and it appears likely that the enzyme is targeted alsoby other protein kinases, which might have interplay with theMPK6-mediated phosphorylation or include examples forregulation by an electrostatic switch mechanism.

PI4P 5-kinases and their product, PtdIns(4,5)P2, control mem-brane trafficking in pollen tubes (Kost et al., 1999; Ischebecket al., 2008, 2010b, 2011; Sousa et al., 2008; Zhao et al., 2010;Stenzel et al., 2012). A main trafficking route in these cellsinvolves the directional delivery of secretory vesicles to theirtargetmembrane and the endocytotic retrieval of vesicles uponcargo release (Thole and Nielsen, 2008). The reduced plasmamembrane association of the Red StarPLC-PH reporter (Figure 5)and the impaired endocytosis of FM 4-64 with expression ofMPK6-EYFP (Figure 7) are consistent with reported roles ofPtdIns(4,5)P2 (Ischebeck et al., 2008, 2013; König et al., 2008;Sousa et al., 2008) and PIP5K6 (Zhao et al., 2010) in the controlof membrane trafficking and clathrin-mediated endocyto-sis. The discovery of the pollen-expressed PI4P 5-kinasesAtPIP5K6 and NtPIP5K6 as targets for regulation byMPK6 andthe related tobacco SIPK and/or WIPK, respectively, suggestsa MAPK-dependent regulatory circuit controlling PtdIns(4,5)P2

production and apical membrane trafficking (Ischebeck et al.,2008, 2010b; Sousa et al., 2008; Zhao et al., 2010). The ob-servation that the interaction of the cytoplasmic MPK6 withPIP5K6 may occur at the apical plasma membrane (Figure 4C)is consistent with the report that in Arabidopsis a subset ofMPK6 protein colocalizes with FM 4-64, indicating membraneassociation (Müller et al., 2010). A regulatory function of MPK6in apical PtdIns(4,5)P2 formation is also consistent with re-duced rates of pollen tube expansion (Supplemental Figure 6)and themanifestation of morphological alterations observed inpollen tubes resulting from altered apical pectin secretion(Figures 8 and 9).

The reported pollen tube guidance defect of Arabidopsismpk6mutants furthermore suggests relevance for MPK6-dependentPIP5K6 regulation in signal transduction events linking ex-tracellular cues emitted by the ovules with the control of thesecretory machinery of the pollen tubes (Dresselhaus andFranklin-Tong, 2013; Higashiyama and Takeuchi, 2015;Dresselhaus et al., 2016). A number of guidance signals havebeen reported, which are perceived at the cell surface of pollentubes (Dresselhaus and Franklin-Tong, 2013; Higashiyama and

Takeuchi, 2015; Dresselhaus et al., 2016). Signal transductionevents mediating pollen tube guidance have been proposed toinvolveMAPKs, includingMPK6 (Guan et al., 2014; Higashiyamaand Takeuchi, 2015), in analogy to other receptor-dependentsignaling cascades known in plants (Pitzschke et al., 2009). Ourdata suggest that pollen tube guidance cues transduced viaMPK6 may result in an inhibition of PtdIns(4,5)P2 formation andreduced apical pollen tube expansion, as illustrated in themodelshown in Figure 10. A topical inhibition of secretion by extra-cellular guidance cues might contribute to asymmetric pollentube growth toward the ovules for fertilization. However, as thisnotion is currently not supported by experimental evidence, itis also possible that MPK6 influences overall pollen tube growthwith no links to guidance. In either case, directional cell ex-pansion and its responsiveness to exogenous cues are a bi-ological phenomenon sharedbypolar growingcells fromvariousmodels, including plants, fungi, and possibly even mammaliancells, and these models share all the regulatory elements in-vestigated in our study (Ischebeck et al., 2010a). Therefore, theproposed mode of regulation may have relevance across eu-karyotic kingdoms. Future researchwill aim to establish whetherthese proposed regulatory circuits contribute to pollen tubegrowth and/or guidance in response to exogenous signals, andwhether such regulation has been conserved in evolution.

Figure 10. Model for the Effects of MAPK-Mediated Limitation of ApicalPtdIns(4,5)P2 Formation in Pollen Tubes.

PtdIns(4,5)P2 is involved in the apical control of membrane trafficking,influencing apical pectin secretion and CME. Top half: During symmetricexpansion, PIP5K6 and other PI4P 5-kinase isoforms mediate the ex-pansion of pollen tubes at the apex. Bottom half: External guidance cuesare perceived by cell surface receptors, likely initiating a MAPK cascadeinvolving MPK6. Activated MPK6 might locally transduce this signal tothe machinery for apical membrane trafficking by targeting PIP5K6. TheMPK6-mediated phosphorylation of PIP5K6might result in reduced apicalexpansion of the cell, possibly resulting in an asymmetric expansion andcurvature toward theguidance cues.Other explanations are possible. Bluearrows, promoting influences; blue T-bars; inhibiting influences; orangearrows, simplified representation of vesicle movement for secretion andendocytotic recycling in theexpandingpollen tube tip; ellipses, enzymesasindicated; red circles, phosphorylation events. CME, clathrin-mediatedendocytosis.

3044 The Plant Cell

METHODS

Plant Material

Experiments were performed using source material from Arabidopsisthaliana Col-0 grown under a short-day regime (8 h light at ;140 mmolphotons m22 s21, 16 h dark) or pollen from tobacco (Nicotiana tabacum;ecotypeSamsunN) grown in a greenhouse. The transgenic tobaccoplantscarrying RNAi constructs against SIPK and WIPK (Seo et al., 2007) werea gift from Shigemi Seo and Shinpei Katou (National Institute of Agro-biological Sciences, Tsukuba, Ibaraki, Japan).

Preparation of Pollen Extracts

Ripe pollen from tobacco flowers was harvested, solubilized, and germi-nated in pollen growth media (5% [w/v] sucrose, 12.5% [w/v] PEG-6000,0.03% [w/v] casein hydrolysate, 15 mM MES-KOH, pH 5.8, 1 mM CaCl2,1mMKCl, 0.8mMH3BO3, 3mMCuSO4, and10mg/mL rifampicin). After thegerminated pollen was separated from the growth media, the pollen tubematerial was frozen in liquid nitrogen and stored at280°C until use. For theprotein extraction, ice-cold protein extraction buffer containing 10 mMTris-HCl, pH 7.5, 10 mMMgCl2, 50 mMNaCl, 2.5 mMNaF, 1 mMNa3VO4,0.1 mM EDTA, 0.1 mM DTT, PhosSTOP (Roche), and protease inhibitorcocktail (Sigma-Aldrich) was added to the material, and the cells werebroken with a mini pestle. The suspension was cleared by centrifuging at20,000g and 4°C for 20min. The extract was kept on ice to prevent proteindegradation and protein kinase inactivation.

cDNA Cloning

Total RNA was isolated from Arabidopsis or tobacco flowers using theTRIzol method (Chomczynski and Mackey, 1995) and used as a templatefor cDNA synthesis using RevertAid H Minus Reverse Transcriptase(Fermentas) and oligo(dT)-primers according to the manufacturer’srecommendations.

Constructs for Bacterial Expression

For Escherichia coli expression, the cDNAs for AtPIP5K6 and NtPIP5K6 wereclonedintotheexpressionplasmidpMALc5g(NEB).Toobtainanampliconthatis in frame with the sequence for the N-terminal MBP-tag, AtPIP5K6 and thecDNAs encoding the T590A/D,T597A/D and T590A/D_T597A/D variants ofAtPIP5K6 were amplified with the primer combination AtPIP5K6 NdeI for, 59-GCCATGCCATATGATGTCGGTAGCACACGCAGATGA-39/AtPIP5K6 His6SalI rev, 59-GCCATGCGTCGACTCAGTGGTGGTGGTGGTGGTGAG-CGTCTTCAACGAAGACCC-39, and moved as NdeI/SalI fragments intopMALc5G. The tobacco homologNtPIP5K6 and the cDNAs for the respectiveAandDvariantswereamplifiedwithprimercombinationspreviouslydescribed(Stenzel et al., 2012) and moved as NotI/SalI fragments into pMALc5G.

Constructs for Yeast Two-Hybrid Analysis

The bait vector pBT3-C-OST4 was obtained by introducing the cDNAencoding the endoplasmic reticulum transmembrane oligosaccharyltransferase4 (Ost4p) from yeast via the XbaI restriction site upstreamof the multiple cloning site of pBT3-C (DualSystems Biotech), aspreviously described (Möckli et al., 2007). To clone AtPIP5K6 andNtPIP5K6 into pBT3-C-OST4, the open reading frames were amplifiedwith forward and reverse primers adding SfiI restrictions sites to the 59-and 39-ends of the cDNAs, respectively. Additionally, the 59-amplificationprimers introduced an additional cytidine base between the SfiI restriction

site and theATGcodon. The amplicons of AtPIP5K6orNtPIP5K6were thencloned via the SfiI sites into pBT3-C-OST4, yielding pBT3-C-Ost4p-AtPIP5K6 and pBT3-C-Ost4p-NtPIP5K6. To clone pPR3-N-MPK6 andpPR3-N-NtSIPK, the open reading frames of MPK6 and NtSIPK wereamplified with primers introducing 59and 39 SfiI restriction sites andmoved as SfiI fragments into pPR3-N.

Constructs for BiFC Analysis

Constructs for BiFC studies (Kerppola, 2008) were based on the plasmidspEntryA and pEntryD, which are pUC18 based and contain separatemultiple cloning sites for the insertion of promoter sequences and a geneof choice and differ by the nature of the att sites for homologous re-combination.For theBiFCanalyses,expression fromaCaMV35Spromoterwas chosen to enable weak expression in pollen tubes to observe interactions atclose to physiological conditions (Sun et al., 2015). The coding sequences forAtPIP5K6andNtPIP5K6were amplifiedusing theprimer combinationsAtPIP5K6AscI for, 59-ATGCGGCGCGCCATGTCGGTAGCACACGCAGA-39/AtPIP5K6XhoI rev, 59-ATGCCTCGAGAGCGTCTTCAACGAAGACCC-39 andNtPIP5K6AscI for,59-ATGCGGCGCGCCATGAGCAAAGAATTTAGTGG-39/NtPIP5K6XhoIrev, 59-ATGCCTCGAGAGTGTCTTCTGCAAAAACTT-39, respectively, andmoved as AscI/XhoI fragments in reading frame with a downstream codingsequence for the N-terminal half of YFP, YFPN, yielding pEntryA-CaMV35S:AtPIP5K6-YFPNandpEntryA-ProCaMV35S:NtPIP5K6-YFPN. The codingsequences of MPK6 and SIPK were amplified using the primer combi-nations MPK6SalI for, 59-ATGCGTCGACATGGACGGTGGTTCAGGTCA-39/MPK6XbaI rev, 59-ATGCTCTAGATTGCTGATATTCTGGATTGA-39orNtSIPKAscI for, 59-ATGCGGCGCGCCATGGATGGTTCTGGTCAGCA-39/NtSIPKXhoI rev, 59-ATGCCTCGAGCATATGCTGGTATTCAGGAT-39, respectively, andmoved as SalI/XbaI or AscI/XhoI fragments in framewith the downstream codingsequence for the C-terminal half of YFP (YFPc) present in the pEntryD plasmid,yielding pEntryD-ProLat52:MPK6-YFPc and pEntryD-ProLat52:NtSIPK-YFPc.ForbothBiFCpartners, theYFPhalveswerethus fusedat theCterminiof the fusion proteins.

Constructs for Transient Expression in Pollen Tubes

mCherry was amplified using the primer combination mCherry-AscI-for, 59-ATGCGGCGCGCCAATGGTGAGCAAGGGCGAGGA-39/mCherry-BamHI-rev,59-ATGCGGATCCCTACTTGTACAGCTCGTCCAT-39, adding an AscI restric-tionsiteat the59-endofmCherrycDNAandaBamHI restrictionsiteat the39-end of themCherry sequence. Between theAscI restriction site and theATGof the mCherry sequence, the primer introduced an additional adeninebase to ensure cloning in frame. After restriction, the mCherry fragmentwas moved into pEntryA-pLat52 as an AscI-BamHI fragment. cDNAs forMPK6or SIPKwere amplified using primers introducing a 59-endSalI anda 39-end AscI restriction site to their cDNA sequences. After digestion, theMPK6 or SIPK fragments were moved into the pEntryA-ProLat52:mCherryvector, yielding pEntryA-ProLat52:MPK6-mCherry or pEntryA-ProLat52:SIPK-mCherry, respectively.

Site-Directed Mutagenesis

The site-directed exchange of bases within a DNA sequence was con-ducted by QuickChange technology (Stratagene). In this PCR-based ap-proach,primerscontainingthedesiredbaseexchangewereusedtoamplify theDNAtemplate.ThePCRwasperformedwithPhusionHighFidelityPolymerase(NEB) in a 50-mL reaction according to the manufacturer’s instructions. Thefollowing thermal cycling steps were used for amplification: 98°C for 30 s asinitial denaturation step, 18 cycles at 98°C for 20 s, and annealing between

MPK6-Mediated Phosphorylation of PIP5K6 3045

55 and 65°C for 30 s and 72°C for 1min/kb for the elongation of the amplicon.The following primers were used: PIPK6 T590A for, 59-CTGCTATCAAG-GACTCTGCCGCTCCTACTTCCGGCGCTCGAAC-39/PIPK6 T590A rev,59-GTTCGAGCGCCGGAAGTAGGAGCGGCAGAGTCCTTGATAGCAG-39;PIPK6 T590D for, 59-CTGCTATCAAGGACTCTGCCGATCCTACTT-CCGGCGCTCGAAC-39/PIPK6 T590D rev, 59-GTTCGAGCGCCG-GAAGTAGGATCGGCAGAGTCCTTGATAGCAG-39; PIPK6 T597A for,59-CTACTTCCGGCGCTCGAGCCCCTACCGGAAATTCAGA-39/PIPK6T597A rev, 59-TCTGAATTTCCGGTAGGGGCTCGAGCGCCGGAAGTAG-39;PIPK6 T597D for, 59-CTACTTCCGGCGCTCGAGACCCTACCGGAAATTCA-GA-39/PIPK6 T597D rev, 59-TCTGAATTTCCGGTAGGGTCTCGAGCGCCG-GAAGTAG-39. The mixture of template and amplicon was digested with10 units of the methylation-dependent restriction enzyme DpnI to degrade allDNAof bacterial origin. After digestion, the nonmethylated ampliconDNAwastransformed into chemically-competent E. coli. From the respective pEntryplasmids,ampliconsweremovedto thevectorpLatGW(Ischebecketal.,2008)using Gateway technology (Invitrogen). To confirm successful cloning and toverify site-directed mutations in plasmids, DNA was sequenced usinga commercial service (GATC).

Expression and Purification of Recombinant Proteins in E. coli

RecombinantPIP5K6wasexpressedas a fusion to anN-terminalMBP-tagin E. coliRosetta 2 cells (Merck). Starter cultures were incubated overnightwith continuous shaking at 30°C in 2YT medium (1.6% [w/v] peptone, 1%[w/v] yeast extract, and 0.5% [w/v] NaCl) with appropriate antibiotic se-lection and used to inoculate main cultures in baffled flasks. Cells weregrownuntil anOD600 of 0.6 to 0.8, and expressionwas inducedwith 0.1mMIPTG, unless stated otherwise. The fusion proteins MBP-AtPIP5K1 andpMAL-AtPIP5K6 were best expressed in 1-liter cultures induced with0.1 mM IPTG at 22°C for 4 h. MBP-NtPIP5K6 was expressed in 3 liters ofcultures at 37°C with 30 min induction. Cells were harvested by cen-trifugation for 20 min at 4000g, and the bacterial pellet was immediatelyfrozen in liquidnitrogenandstoreduntil useat220°C.Cell disruptionwasinitiated with the addition of lysozyme (Serva) to digest the bacterial cellwall. Ultrasoundwas used to disrupt small volumes, while larger volumeswere homogenized by a high-pressure cell disruption French Presssystem (Gaulin, APV Homogenizer) at 1200 bar. After both treatments,crude lysate was cleared by centrifugation at 20,000g for 20 min at 4°C,kept on ice until further use. Purification of the full-length fusion proteinswas performed by affinity chromatography using an MBPTrap column(GE Life Sciences).

GST-MPK6 and GST were recombinantly expressed from pGEX4T1plasmids (GE Healthcare Europe) in E. coli BL21(DE) cells. Starter cultureswith 50 mL of LBmedia were inoculated with single colonies and grown at30°C overnight with shaking at 180 rpm. Expression cultures were in-oculated at anOD600 of 0.1 and grown in 200mLof LBmedia in Erlenmeyerflasks at 37°C with shaking at 180 rpm. Expression was induced with0.1mM IPTGat anOD600 of 0.6. After induction, thecultureswere shakenat22°Cand 180 rpm for 20 h. Then, 50-mLculture aliquotswere harvested bycentrifugation for 10 min at 3220g. Bacterial pellets from 50 mL of ex-pression culture were resuspended in 2 to 4mL of 50mMTris-HCl, pH 8.0,and 150 mM NaCl, containing protease inhibitor cocktail (Sigma-Aldrich)and 1 mg mL21 lysozyme (Serva Electrophoresis). After incubation on icefor 30 min, cells were further disrupted by sonication. Cell debris wasremoved by centrifugation for 15 min at 20,000g and 4°C.

Protein amounts were estimated with the Bradford assay (Bradford,1976) calibrated against BSA.

In-Gel Protein Phosphorylation

The in-gel kinaseassaywasperformedaspreviouslydescribed (ZhangandKlessig, 1997). In brief, pollen tube extracts (80 mg) were loaded on a 10%SDSgel.Asasubstrate for theproteinkinases fromtheextract, 0.25mg/mL

of MBP-AtPIP5K6 was copolymerized into the resolving gel. Higherconcentrationswere not used due to limited amounts ofmaterial. A controlwith no protein embedded in the gel was used as an autophosphorylationcontrol. The catalytic subunit of PKA from bovine heart (1 unit; Sigma-Aldrich) was used as a positive control. After electrophoresis, the gel waswashed three times for 30 min with washing buffer (25 mM Tris, pH 7.5,0.5mMDTT, 0.1mMNa3V04, 5mMNaF, 0.5mg/mL [w/v] BSA, and 0.1%[v/v] Triton X-100) at room temperature with gentle agitation to removeSDS. To renature protein kinases, the gel was incubated in renaturingbuffer (25 mM Tris, pH 7.5, 0.5 mM DTT, 0.1 mMNa3V04, and 5 mMNaF)overnight at 4°C with three changes of buffer. The gel was equilibrated in25mMTris, pH 7.5, 2 mMEGTA,12mMMgCl2, 10mMCaCl2, 1 mMDTT,0.1mMNa3VO4, and the kinase reactionwas started in a volumeof 30mLby the addition of 200 nM ATP containing 50 mCi [g-33P]ATP (10 mCi/mL;Hartmann). The gel was incubated for 60 min with gentle agitation, andthe reaction was stopped by the addition of 5% (w/v) trichloroacetic acidand 1% (w/v) sodiumpyrophosphate to fix proteins in the gel and removeunbound [g-33P]ATP for 6 h with at least five changes of buffer. Thecontrol gel was stained with Coomassie Brilliant Blue. A prestainedprotein ladder was used to estimate sizes of phosphorylated proteins.Afterwashing, thegelsweredriedovernight and radiolabeledbandswerevisualized using a radiosensitive imager screen (BASMP2040s; Fujifilm).The extent of 33P-incorporation was quantified by phosphor imaging(BAS 1500; Fujifilm).

In Vitro Protein Phosphorylation

Transphosphorylation was detected by monitoring the incorporation ofradiolabeled g-phosphate of [g-33P]ATP into proteins. Purified re-combinant PI4P 5-kinases (5–10 mg) were incubated with 15 mg offreshly prepared pollen extract in the presence of 50 mmATP containing10 mCi [g-33P]ATP (10 mCi/mL; Hartmann) in 13 kinase buffer (10 mMTris-HCl, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 0.1 mM EDTA, 0.1 mMDTT, and PhosSTOP) in a volume of 50 mL for 30 min. Variations to theexperiments performedwith recombinant protein kinasesare describedbelow. For kinase assays performed with recombinant, activatedMPK6, the sample volumewas 20 mL. For each reaction, 0.2 mg ofMPK6was used. MPK6 was purified as described previously (Pecher et al.,2014) and obtained from Pascal Pecher (IPB Halle, Germany). Duringincubation, samples were gently agitated. The reaction was stoppedwith SDS sample buffer and the sample was applied to SDS-PAGE. Thegel was stained with Coomassie Brilliant Blue and dried overnight.Radiolabeled bands were visualized using a radiosensitive imagerscreen (BASMP2040s; Fujifilm) and the extent of 33P-incorporationwasquantified by phosphor imaging (BAS 1500; Fujifilm).

Circular Dichroism

Measurements of dichroic properties of the proteins were performed ona Jasco J-810 spectropolarimeter with the following instrumental setup:1-nmpitch, 40accumulations, 50-nmperminscanspeed,1-nmslitwidths,and 1-s response time. All experiments were performed in buffer com-posed of 20 mM Tris/HCl and 200 mM NaCl, pH 7.5, supplemented with1mMEDTA and 10mMmaltose at a temperature of 20°C (Peltier element).CDspectrawere recordedat aprotein concentrationof 260 to530mgmL21

(1.21–4.28mM)using cuvetteswith optical path lengths of 1mm for both farand near UV. Acquired protein spectra were corrected for buffer contri-bution using SpectraManager I software (Jasco). The datawere convertedto mean residue ellipticity (Kelly et al., 2005).

Tryptic Protein Digest

Protein bands of interest were excised and digested with trypsin as pre-viously described (Shevchenko et al., 1996).

3046 The Plant Cell

Mass Spectrometry

Identification of Candidate Protein Kinases from Pollen Tube Extracts

Protein kinase candidates in pollen tube extracts were identified by nano-LC-HD-MSE using an Acquity UPLC system and a coupled Synapt G2-S(Waters) in resolution mode with positive ionization (Helm et al., 2014).Peptides were analyzed in data independent acquisition mode without pre-selectionofprecursor ions (Helmetal., 2014).Glu-Fib (Glu-1-FibrinopeptideB)was used as lock mass (m/z = 785.8426, z = 2), and mass correction wasapplied to the spectra during data processing in a ProteinLynx Global Server(PLGS 3.0, Apex3D algorithm v. 2.128.5.0, 64 bit; Waters). The processingparametersweresetasdescribed (Helmetal.,2014).The intensityofprecursorions was$180 counts and for fragment ions$15 counts to be distinguishedfromnoise.Thedesignationof fragment ionstoprecursor ionswasachievedbyPLGS3.0basedonpeak form, retention time, isotopecluster,andm/zvalueaswell as ion mobility. Further data analysis was performed by PLGS 3.0. Then,MSEdatawere searched against themodifiedArabidopsis database (TAIR10;www.arabidopsis.org) containing common contaminants such as keratin (ftp.thegpm.org/fasta/cRAP/crap.fasta). Protein identification required the de-tection of two fragment ions per peptide and aminimum of five fragment ionsand twopeptidematches.Primarydigest reagentwas trypsinwithonemissedcleavage allowed, as previously described (Helm et al., 2014).

Identification of Phosphopeptides

AtPIP5K6 residues phosphorylated by MPK6 were identified by liquidchromatography online with HR/AM LC-MS using an Orbitrap Velos ProSystem (Thermo Scientific). Proteins separated by SDS-PAGE were sub-jected to in-gel tryptic digestion, and peptides were analyzed by a data-dependent acquisition scan strategy with inclusion list to specifically selectand isolate AtPIP5K6 phosphorylated peptides for MS/MS peptide se-quencing. An inclusion list was used to identify low abundant species in thesurvey scan (targeted data-dependent acquisition). Multi-stage activation(MSA)wasapplied to further fragment ionpeaks resulting fromneutral lossofthe phosphatemoiety by dissociation of the high energy phosphate bond togenerate b- and y- fragment ion series rich in peptide sequence information.MS/MS spectra were used to search the TAIR10 database (ftp://ftp.arabidopsis.org) amended with mutant AtPIP5K6 sequences (AtPIP5K6T590A, AtPIP5K6 T597A, and AtPIP5K6 T590A T597A) with the Mascotsoftware v.2.5 integrated in Proteome Discoverer v.1.4. Phosphopeptideswith an ion score surpassing the Mascot significance threshold (P < 0.05)were accepted. The phosphoRS module was used to localize phosphory-lation sites within the primary structure of the peptide.

Data Availability

All mass spectrometry proteomics data have been deposited to theProteomeXchangeConsortiumvia thePRIDE (Vizcaíno et al., 2016) partnerrepository with the data set identifier PXD006067.

Lipid Kinase Assays

Thecatalyticactivityof recombinantPI4P5-kinaseswasdeterminedbasedon their ability to phosphorylate PtdIns4P in the presence of [g-33P]ATP aspreviously described (Perera et al., 2005). The extent of 33P-incorporationwas quantified by phosphor imaging (BAS-MP 2040s; Fujifilm) using BAS-1500 imager screens (Fujifilm).

Yeast Two-Hybrid Analysis

Protein-protein interactions were tested using the split-ubiquitin (Ub)membrane-based yeast two-hybrid system (SUS) Dualmembrane Kit

3 (DualsystemsBiotech) as previously described (Johnsson and Varshavsky,1994). Bait and prey constructs coding for AtPIP5K6 versus MPK6 or forNtPIP5K6 versus NtSIPK, respectively, were cotransformed in the yeaststrain NMY51 (Dualsystems Biotech). The bait protein was cotransformedwith a positive and a negative control. The positive control consisted ofa native Ub-half (NubI) fused to endoplasmic reticulum-localized Alg5.NubG fused to Alg5 served as a negative control. To test for interactions,single positive yeast clones were grown on SD-media lacking leucine,tryptophan, andhistidine (SD-LWH). For higher stringency, selectionwasperformed onSD-LWHplates supplementedwith 10mM3-amino-1,2,4-triazole (SD-LWHA).

Immuno-Pull-Down

RecombinantGSTorGST-MPK6proteinswere immobilizedonglutathione-agarose (Thermo Fisher Scientific) and incubatedwith purified recombinantMBP-PIP5K6 protein for 60 min at 4°C. Upon washing of the resin, GST-bound proteins were eluted with 50 mM glutathione. Interacting MBP-PIP5K6 protein was detected using a monoclonal anti-MBP antibody(New England Biolabs; product number E8032S). Protein input wasdetected using a polyclonal anti-GST antibody (GEHealthcare; productnumber 27-4577-01).

Transient Expression of cDNA Constructs in Tobacco Pollen Tubes

Transient expression of cDNA constructs in tobacco pollen tubes wasperformed by particle bombardment as previously described (Ischebecket al., 2008).

Fluorescence Microscopy

Pollen phenotypes were analyzed using an Axio ImagerM1 fluorescencemicroscope (Carl Zeiss) and an AxioCam MRm gray-scale camera. Theobservationofphenotypeswasperformedat203magnificationusing filterset 38 high efficiency (HE) for EYFP detection and filter set 43 HE formCherry detection (all filters from Carl Zeiss). EYFP was excited at514 nm and imaged using a FT 495-nm beam splitter and a 470/40-nmband-pass filter; mCherry was excited at 561 nm and imaged using a FT570-nm beam splitter and a 550/25-nm band-pass filter. Images weretaken with the corresponding software program (Axio Vision; Carl Zeiss).Localization studies were performed using LSM780 or LSM880 laserscanning confocal microscopes (Carl Zeiss) with a 403 magnifyingobjective, unless specified otherwise. EYFP was excited with an argonlaser at 488 nmand detected at 493 to 598 nm;mCherry was excitedwithaDPSS laser at 561nmanddetectedat578 to696nm. Imageswere takenusing Zen (Carl Zeiss).

Staining of Tobacco Pollen Tubes

To test for defects in endocytosis, FM 4-64 staining was performed onpollen tubes grownonglass slides for 3 to 4 h after bombardment. FM4-64dye (from a stock solution of 50 mm diluted in pollen tube growth medium)was added to a final concentration of 2.5 mM. To test for defects in pectinsecretion, a stock solution of 0.01% Ruthenium red (Sigma-Aldrich) wasadded drop wise to the glass slides containing pollen tubes 3 to 4 h afterbombardment.

BiFC

BiFC experiments were performed in tobacco pollen tubes as a physio-logically relevant cell type. The constructs were transiently transformedinto tobacco pollen and the cells were grown for 7 to 10 h until microscopyevaluation. AnmCherry fluorophore under the control of a LAT52 promoterwas cotransformed as a marker for transformed cells.

MPK6-Mediated Phosphorylation of PIP5K6 3047

Image Analysis

Images were analyzed using the open source Fiji image analysis software(Schindelin et al., 2012).

Statistical Evaluation

All quantitative datawere tested for statistical significanceusing two-tailedStudent’s t tests.Confidence intervalsaregiven in thefigure legends foreachdata set.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under the following accession numbers: AtPIP5K6, At3g07960;NtPIP5K6, JQ219669; AtMPK6, At2g43790; SIPK, NP_001312060; andWIPK, NP_001313013.

Supplemental Data

Supplemental Figure 1. In vitro phosphorylation of pollen-expressedPI4P 5-kinase isoforms by MPK6.

Supplemental Figure 2. Sequence coverage and mass spectra ofphosphopeptide identification by HR/AM LC-MS.

Supplemental Figure 3. Reaction kinetics for purified recombinantMBP-AtPIP5K6 and MBP-NtPIP5K6.

Supplemental Figure 4. Interaction of NtPIP5K6 and SIPK.

Supplemental Figure 5. Reduced transcript levels of SIPK and WIPKin pollen tubes of tobacco plants expressing RNAi constructs.

Supplemental Figure 6. Reduced pollen tube growth and apicalpectin secretion in pollen tubes overexpressing MPK6-EYFP.

ACKNOWLEDGMENTS

We thank the following individuals: Shigemi Seo and Shinpei Katou (bothNational Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan) forthe tobacco RNAi plants; Pascal Pecher, Petra Majovsky, and LennartEschen-Lippold (all Leibniz Institute of Plant Biochemistry, Halle, Germany)for activated recombinant MPK6 protein, technical assistance, and helpfuldiscussion, respectively; Jennifer Lerche (Institute for Biochemistry andBiotechnology, Martin-Luther-University Halle-Wittenberg) for helpful dis-cussion; and Sven-Erik Behrens (Institute for Biochemistry and Biotechnol-ogy,Martin-Luther-University Halle-Wittenberg) for access to spectroscopyequipment. We acknowledge funding from the German Research Founda-tion (Grants He3424/3-1, He3424/6-1, and CRC648 TP10 to I.H.).

AUTHOR CONTRIBUTIONS

F.H., I.S., M.H., P.K., W.M., R.G., S.H., D.D., and W.H. performed theexperiments. F.H., I.S., M.H., P.K., R.G., S.H., D.D., S.B., W.H., and I.H.analyzed the data. F.H., M.H., J.L., and I.H. designed the research. I.H.wrote the manuscript.

ReceivedJuly12, 2017; revisedOctober12, 2017; acceptedNovember18,2017; published November 22, 2017.

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3050 The Plant Cell

DOI 10.1105/tpc.17.00543; originally published online November 22, 2017; 2017;29;3030-3050Plant Cell

HeilmannGolbik, Stefan Helm, Dirk Dobritzsch, Sacha Baginsky, Justin Lee, Wolfgang Hoehenwarter and Ingo

Franziska Hempel, Irene Stenzel, Mareike Heilmann, Praveen Krishnamoorthy, Wilhelm Menzel, Ralph4,5-Bisphosphate in an Apical Plasma Membrane Domain

MAPKs Influence Pollen Tube Growth by Controlling the Formation of Phosphatidylinositol

 This information is current as of March 20, 2020

 

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References /content/29/12/3030.full.html#ref-list-1

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