Research ArticleResearch Article

MAPKs Influence Pollen Tube Growth by Controlling the Formation of Phosphatidylinositol 4,5-Bisphosphate in an Apical Plasma Membrane Domain

Franziska Hempel, Irene Stenzel, Mareike Heilmann, Praveen Krishnamoorthy, Wilhelm Menzel, Ralph Golbik, Stefan Helm, Dirk Dobritzsch, Sacha Baginsky, Justin Lee, Wolfgang Hoehenwarter, Ingo Heilmann

Published December 2017. DOI: https://doi.org/10.1105/tpc.17.00543

Abstract

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 phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen 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 on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth.

INTRODUCTION

Pollen tubes serve as models for the study of polar tip growth and cellular polarization (Kost, 2008; Ischebeck et al., 2010a; Rounds and Bezanilla, 2013; Franciosini et al., 2017). Tip-growing cells can attain length/width ratios exceeding 1000 (Kost, 2008; Riquelme, 2013; Rounds and Bezanilla, 2013) and share structural features and regulatory mechanisms that have been conserved in evolution (Kost, 2008; Ischebeck et al., 2010a; Rounds and Bezanilla, 2013). The apical expansion of a cell requires the transport of membrane and cell wall material by directional vesicle trafficking to the growing apex and the retrieval of unloaded vesicles (Thole and Nielsen, 2008; Moscatelli and Idilli, 2009; Ischebeck et al., 2010a; Hepler and Winship, 2015; Franciosini et al., 2017). The polarized expansion of pollen tubes and some other cell types is furthermore responsive to exogenous cues (Duan et al., 2010; Kessler and Grossniklaus, 2011; Lindner et al., 2012; Dresselhaus and Franklin-Tong, 2013; Higashiyama and Takeuchi, 2015; Dresselhaus et al., 2016). For instance, pollen tubes grow toward the ovules within flowers of a compatible genotype to achieve fertilization, guided by cues emitted by the female organs (Dresselhaus and Franklin-Tong, 2013; Higashiyama and Takeuchi, 2015; Dresselhaus et al., 2016). So far, it is unclear how the perception of exogenous cues controls the 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 plasma membrane domain in pollen tubes (Kost et al., 1999; Ischebeck et 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 root hairs (Vincent et al., 2005; Preuss et al., 2006; Kusano et al., 2008; Stenzel et al., 2008; Ghosh et al., 2015) and fungal hyphae (Mähs et al., 2012). PtdIns(4,5)P2 acts as a ligand to target proteins, which are regulated in their biochemical activity or subcellular localization by the protein-lipid interaction (Hammond and Balla, 2015; Heilmann, 2016; Gerth et al., 2017). Arabidopsis thaliana plants displaying T-DNA- or RNAi-mediated underexpression of the pollen-specific PI4P 5-kinase isoforms PIP5K4 and PIP5K5 (Ischebeck et al., 2008; Sousa et al., 2008), PIP5K6 (Zhao et al., 2010), or PIP5K10 and PIP5K11 (Ischebeck et al., 2011) display substantially reduced rates of pollen germination and pollen tube expansion. Therefore, the PtdIns(4,5)P2 domain in the apical plasma membrane of pollen tubes is thought to be essential for polar cell expansion (Kost et al., 1999; Vincent et al., 2005; Ischebeck et al., 2008, 2010b, 2011; Sousa et al., 2008; Zhao et al., 2010). A critical role of PtdIns(4,5)P2 in the control of apical secretion of cell wall material and directional cell expansion of pollen tubes was previously described mainly based on the effects of overexpressing PI4P 5-kinases (Ischebeck et al., 2008, 2010b; Sousa et al., 2008; Zhao et al., 2010; Stenzel et al., 2012). In these studies, the overproduction of PtdIns(4,5)P2 resulted in increased apical deposition of pectin and characteristic morphological defects, including pollen tube tip branching and protoplast trapping, which have previously been summarized as “secretion phenotypes” (Ischebeck et al., 2010b). It is evident that correct amounts of PtdIns(4,5)P2 are important for the control of apical cell expansion of pollen tubes and that PtdIns(4,5)P2 production must be tightly controlled. However, it is unknown how the biosynthesis of PtdIns(4,5)P2 or other phosphoinositides is regulated by upstream signaling pathways.

As PI4P 5-kinases in the apical plasma membrane of pollen tubes generate the PtdIns(4,5)P2 membrane domain important for cell expansion, we examined whether these enzymes are candidates for regulation. Our working hypothesis was that PI4P 5-kinases in the pollen tube are regulated by phosphorylation, as has previously been found for the PI4P 5-kinase Mss4p from Saccharomyces cerevisiae (Audhya and Emr, 2003) and for PI4P 5-kinases from Schizosaccharomyces pombe and human, which display decreased catalytic activity in vitro when phosphorylated (Vancurova et al., 1999; Park et al., 2001). Information of posttranslational control of the plant phosphoinositide system is scarce. The Arabidopsis PI4P 5-kinase PIP5K1 can be phosphorylated in vitro by mammalian protein kinase A (PKA) (Westergren et al., 2001). However, the relevant phosphosites or regulatory consequences of this phosphorylation have not been determined, and endogenous protein kinases acting upstream of PI4P 5-kinases remain unknown in plants. While a role for protein phosphorylation in the control of phosphoinositide biosynthesis in pollen tubes has not been reported, protein kinases are required for the regulation of pollen tube growth (Higashiyama and Takeuchi, 2015). For instance, Arabidopsis plants carrying lesions in the genes encoding the mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 display pollen tube guidance defects (Guan et al., 2014), suggesting that a MAPK cascade is involved in the transduction of exogenous guidance cues in pollen tubes (Higashiyama and Takeuchi, 2015). While these findings indicate a role for MAPKs in the control of pollen tube growth, it is currently unclear how MAPK-mediated protein phosphorylation might be linked to the machinery for apical cell expansion.

Here, we demonstrate that the pollen-expressed PI4P 5-kinases AtPIP5K6 from Arabidopsis and NtPIP5K6 from tobacco (Nicotiana tabacum) are phosphorylated by protein kinase activities in pollen tube extracts. We identify MPK6 and its tobacco homolog, SALICYLIC ACID INDUCED PROTEIN KINASE (SIPK), as protein kinases that bind and phosphorylate PIP5K6 homologs from Arabidopsis and tobacco, respectively. Phosphorylation by the MAPKs inhibits PI4P 5-kinase activity in vitro. In vivo, expression of AtMPK6 reduces the plasma membrane association of a fluorescent reporter for PtdIns(4,5)P2, inhibits endocytosis, and modulates pollen tube growth. The data demonstrate an unexpected regulatory link between MAPKs and the apical production of PtdIns(4,5)P2 required for pollen tube expansion.

RESULTS

AtPIP5K6 and NtPIP5K6 Are Phosphorylated by MAPKs from Tobacco Pollen Tube Extracts

The PI4P 5-kinase AtPIP5K6 and its tobacco homolog NtPIP5K6 have reported roles in the control of membrane trafficking in pollen tubes (Zhao et al., 2010; Stenzel et al., 2012). To test for phosphorylation of these enzymes, purified recombinant AtPIP5K6 or NtPIP5K6 was incubated in the presence of [γ-33P]ATP with extracts obtained from germinated tobacco pollen tubes (Figure 1A). Both AtPIP5K6 and NtPIP5K6 were radiolabeled by the incubation (Figure 1A). For AtPIP5K6, a time course of increasing incorporation of the radiolabel is shown (Figure 1A, left panels). Controls without added recombinant enzyme or without added pollen tube extract did not result in a radiolabeled band (Figure 1A, right panels). The data indicate that the pollen tube extract contained protein kinase activities capable of phosphorylating recombinant AtPIP5K6 and NtPIP5K6 in vitro. Relevant protein kinases from pollen tube extracts were identified by a nontargeted in-gel kinase assay. Protein extracts of pollen tubes were electrophoresed on SDS-PAGE gels containing purified recombinant AtPIP5K6 protein embedded in the gel matrix. Negative controls were performed without added recombinant AtPIP5K6 in the gels. After renaturing and washing, the gels were incubated with [γ-33P]ATP to assess the in-gel protein kinase activity against the supplied AtPIP5K6 substrate (Figure 1B). In the absence of AtPIP5K6 in the gel, there was no phosphorylation signal with the PKA control, and only weak phosphorylation signals resulted from the application of the pollen tube extract (Figure 1B, left panel), which presumably represent kinase autophosphorylation. By contrast, the presence of recombinant AtPIP5K6 protein in the gels yielded radiolabeled signals with the PKA positive control as well as enhanced signals with the pollen tube extract (Figure 1B, right panel), indicating phosphorylation of the substrate protein, AtPIP5K6, or possibly enhanced autophosphorylation. In-gel kinase assays performed in parallel with added AtPIP5K6 protein but without radiolabel were excised in the range of observed bands, and the proteins were reextracted, subjected to tryptic digestion, and analyzed by liquid chromatography high-definition multiparallel collision-induced dissociation mass spectrometry (nano-LC-HD-MSE) to identify protein kinase candidates. The underlying mass spectrometry data have been deposited into the ProteomeXchange Consortium with the data set identifier PXD006067. Tryptic peptides identified by the analysis were annotated according to the Arabidopsis genome database. Among the 260 detected proteins, four candidates for relevant protein kinases represented MAPK sequences (Figure 1C). The candidate MPK6 was selected for further analysis because the encoding gene displays an expression pattern similar to AtPIP5K6 and is highly expressed in pollen according to expression patterns available in publicly accessible databases (Winter et al., 2007).

Figure 1.
Figure 1.

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

(A) Purified recombinant MBP-AtPIP5K6 or MBP-NtPIP5K6 was incubated with pollen tube extracts in the presence of [γ-33P]ATP, proteins were separated by SDS-PAGE, and the incorporation of the radiolabel was analyzed by phosphor imaging. MBP-ATPIP5K6 was analyzed in a time-course experiment (left panels). NtPIP5K6 was analyzed together with control experiments (right panels). The presence of the recombinant proteins (arrowheads) is indicated by Coomassie Brilliant Blue-stained gels (top panels). Radiolabeled bands detected by phosphor imaging (mid panels) and a quantification of the radiolabeling signals (lower panels) are also shown. Controls included assays with no added recombinant protein or without added pollen extract, as indicated. Data are from a representative experiment. The experiments were performed three times with similar results. Plus and 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 containing purified recombinant AtPIP5K6 protein as part of the gel matrix. After washing and renaturing, nontargeted in-gel protein kinase assays were performed by incubating the gels with [γ-33P]ATP and visualizing radiolabeled bands by phosphor imaging. SDS-PAGE gels without added recombinant protein were used as a negative control (left panel); mammalian PKA was used as a positive control, as indicated. Bands observed in the 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 genome yielded 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.

Recombinant AtPIP5K6 Is Phosphorylated by Recombinant MPK6 in Two Bona Fide MAPK Recognition Motifs

To verify phosphorylation of the PIP5K6 homologs from Arabidopsis and tobacco by MPK6 in a targeted analysis, purified activated recombinant MPK6 was incubated with AtPIP5K6 or NtPIP5K6 in the presence of [γ-33P]ATP (Figure 2A). The data indicate that MPK6 is capable of phosphorylating AtPIP5K6 and NtPIP5K6 in vitro. To test the specificity of this reaction, other recombinant PI4P 5-kinases from Arabidopsis were also tested (Supplemental Figure 1), among which AtPIP5K6 displayed the strongest phosphorylation signal, followed by the pollen-specific AtPIP5K5 and a weaker signal for AtPIP5K1 (Supplemental Figure 1). Recombinant AtPIP5K6 pretreated with activated recombinant MPK6 was subjected to tryptic digestion and the phosphorylation sites were determined as T590 and T597 by liquid chromatography online with high-resolution accurate-mass mass spectrometry (HR/AM LC-MS) at a sequence coverage of ∼70% (Supplemental Figure 2A). Both phosphorylation sites represent TP (a phosphorylated threonine followed by a proline) phosphorylation motifs characteristic of MAPKs and are located in the variable insert region of the catalytic domain of AtPIP5K6 (Figure 2B; Supplemental Figure 2B). Phosphorylation of AtPIP5K6 was further verified by treating AtPIP5K6 with MPK6 and [γ-33P]ATP, followed by incubation with serine/threonine protein phosphatase 1 (PP1), resulting in a reduction of the incorporated [33P] label to below 5% of the control (Figure 2D). Furthermore, T590A and T597A alanine substitution variants of AtPIP5K6 were generated, in which the phosphorylation sites were eliminated. Using the variant recombinant proteins, MPK6-mediated phosphorylation was gradually reduced in the T590A and T597A substitution variants, respectively, and weakest in the T590A T597A double substitution variant (AtPIP5K6 AA) (Figure 2E). Differences were statistically significant, despite a high sd of the control measurements. Residual phosphorylation of AtPIP5K6 AA by MPK6 (Figure 2E) was accompanied by phosphorylation of residues which were not identified in other experiments and are not part of characteristic MAPK phosphorylation motifs (Supplemental Figure 2C). These phosphorylation events likely represent nonspecific modifications of the recombinant AtPIP5K6 AA protein by MPK6 in vitro. Together, the data indicate that purified recombinant AtPIP5K6 was phosphorylated in vitro by purified recombinant MPK6 in positions T590 and T597.

Figure 2.
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 [γ-33P]ATP, proteins were separated by SDS-PAGE, and the incorporation of the radiolabel was analyzed by phosphor imaging. All proteins were expressed in E. coli and added at 5 µg of PIP5K6 and 0.2 µg of MPK6 protein, respectively, when required. The presence of the recombinant proteins was tested by Coomassie Brilliant Blue (top panels). Radiolabeled bands were detected by phosphor imaging (lower panels). Controls included assays using MBP instead of MBP-PIP5K6, omitting MPK6 protein, or using α-labeled instead of γ-labeled ATP, as indicated. The experiments were performed three times with similar results. Plus and minus symbols indicate added and omitted MPK6, respectively.

(B) and (C) HR/AM LC-MS analysis of tryptic peptides of MPK6-phosphorylated AtPIP5K6 revealed the presence of two phosphorylated residues, T590 and T597, 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, dimerization 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 PI4P 5-kinases, as indicated. The positions of T590 and T597 are indicated by arrowheads. Black, identical residues in the same position in three or more sequences; 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 [γ-33P]ATP.

(D) Reduced radiolabel upon treatment of prephosphorylated AtPIP5K6 protein with serine/threonine PP1 phosphatase. Plus and minus symbols indicate added and omitted components, as indicated.

(E) Reduced phosphorylation of alanine substitution variants T590A, T597A, and T590A T597A (AA) by MPK6. The experiments were performed four times. Data represent mean ± sd. Letters a to c in (E) indicate categories of values that display significant differences from each other, according to Student’s t tests (for different categories all P < 0.05).

Phosphorylation of AtPIP5K6 at T590 and T597 Inhibits Catalytic Activity

To investigate possible effects of the MPK6-mediated phosphorylation on AtPIP5K6 activity, the enzyme was preincubated with recombinant MPK6 and ATP for 1 h, followed by the determination of specific PI4P 5-kinase activity. Enzyme activity was assessed 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 activity of AtPIP5K6 AA (Figure 3A), where both T590 and T597 were substituted with alanine and can no longer be phosphorylated in vitro (compared with Figure 2E). The data indicate that the phosphorylation of AtPIP5K6 in positions T590 and T597 reduced the catalytic activity of AtPIP5K6 in vitro. To further characterize the contribution of these positions to the catalytic activity of AtPIP5K6, we tested substitution variants of AtPIP5K6 carrying either an alanine or an aspartate in the respective positions for PI4P 5-kinase activity in vitro (Figure 3B). The substitution variants T590A, T590D, and T597A did not display altered catalytic activity. By contrast, the activity of the substitution variant T597D was reduced by ∼75% (Figure 3B), suggesting that T597 might be a relevant residue exerting an effect on the catalytic activity of the enzyme when targeted by the posttranslational modification or when carrying a negative charge. This notion is supported by the conservation of this residue within a TP motif of other pollen expressed PI4P 5-kinases, such as NtPIP5K6 or AtPIP5K5 (compared with Figure 2C).

Figure 3.
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 activity against the lipid substrate, PtdIns4P. The reduction in catalytic activity was calculated relative to the activity of non-treated controls. The data represent the mean ± sd from four experiments. The asterisks indicate a significant difference from the non-treated control, according to a Student’s t test (**P < 0.01). Plus and 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 lipid substrate, PtdIns4P. The data represent the mean ± sd from three experiments. Letters a to c indicate categories of values that display significant differences from each other, according to Student’s t tests (for different categories all P < 0.01).

(C) The CD of purified recombinant PIP5K6 variants was analyzed in buffer containing 20 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 1 mM EDTA, and 10 mM maltose, 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.

As the double substitution variant T590A T597A (AA) displayed enhanced catalytic activity in vitro while the phosphomimetic T590 D T597D (DD) variant showed an intermediate effect, we hypothesized that these phosphorylation sites may lie in a region of 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 UV range (Figure 3C). The far UV data from the CD spectroscopy (Figure 3D) indicate that the substitution variants all retained a similar secondary structure, with no gross differences in the content of alpha-helices or beta-sheets. By contrast, we observed changed patterns in the near UV CD spectra (Figure 3E), indicative of differences in the tertiary structures of some of the substitution variants. Interestingly, T597D displayed the most deviant tertiary structure, which is striking because only this variant displayed reduced catalytic activity consistent with the effects of phosphorylation of T597 (compared with Figure 3B). The CD spectroscopy data do not indicate substantial differences in secondary or tertiary structure between PIP5K6 AA and PIP5K6 DD (Figures 3D and 3E). Overall, the CD spectroscopy data indicate that the introduction of a negative charge especially at position 597 results in a change in the tertiary structure of the PIP5K6 protein, with little or no effect on its secondary structure.

MPK6 Interacts Physically with AtPIP5K6

To further test the interplay between MPK6 and PIP5K6, physical interaction of the proteins was tested by split-ubiquitin-based yeast two-hybrid analysis (Figure 4A) using the DualMembrane system (Johnsson and Varshavsky, 1994; Stagljar et al., 1998; Möckli et al., 2007). In these experiments, AtPIP5K6 was immobilized as a bait protein at the endoplasmic reticulum by a C-terminally fused OST4 anchor, and the MPK6 prey protein was freely diffusible. Only upon recruitment of MPK6 to the endoplasmic reticulum by the interaction with the bait protein will the yeast grow on the restrictive selection media. On selective SD-LWH media, yeast cells expressing both tested proteins displayed growth comparable to that of positive controls and substantially stronger than that of the negative controls (Figure 4A). Similar results were obtained for NtPIP5K6 and SIPK (Supplemental Figure 4). The interaction was verified by immuno-pull-down experiments using purified recombinant GST-MPK6 and MPB-PIP5K6 proteins (Figure 4B). In these experiments, immobilized GST-MPK6, but not GST alone, could bind to MBP-PIP5K6. Furthermore, bimolecular fluorescence complementation (BiFC) was used to verify the interaction of MPK6 and AtPIP5K6 (Figure 4C). The coexpression of AtPIP5K6-YFPN with MPK6-YFPC in tobacco pollen tubes resulted in the reconstitution of fluorescence at the apical plasma membrane. No fluorescence was observed when AtPIP5K6-YFPN or MPK6-YFPC was expressed together with YFPC or YFPN, respectively, suggesting that the reconstituted fluorescence of the protein fusions was not a consequence of reassembling YFP halves. However, the BiFC experiment has to be interpreted with caution, as the transient pollen tube expression system does not permit the recommended analysis for protein integrity by immunodetection (Kudla and Bock, 2016). In sum, the data indicate a (possibly weak) physical interaction of AtPIP5K6 with MPK6, which presumably takes place at the apical plasma membrane of pollen tubes.

Figure 4.
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-based yeast two-hybrid system. The AtPIP5K6-bait protein was expressed as a fusion to an OST4 anchor, which attaches the protein to the cytosolic face of the endoplasmic reticulum. The MPK6 fusion was expressed as a soluble cytoplasmic protein. Interaction is indicated by yeast growth under selective (−LWH) conditions. The experiment was performed three times with similar results.

(B) Immuno-pull-down experiments. Recombinant MPK6 expressed as a GST fusion or a GST control were immobilized and incubated with purified recombinant MBP-PIP5K6 protein. Upon washing of the resin, interacting MBP-PIP5K6 protein was analyzed by immunodetection using an anti-MBP antibody. Left panel, protein detected by anti-GST antibody; right panel, pull-down, detected by the anti-MBP-antibody. The experiment was performed three times with similar results.

(C) Analysis by BiFC. Fusions of AtPIP5K6 and the N-terminal half of YFP (AtPIP5K6-YFPN), and of MPK6 and the C-terminal half of YFP (MPK6-YFPC) at their respective C termini were transiently expressed in tobacco pollen tubes. Negative controls included the coexpression of AtPIP5K6-YFPN with YFPC, and of YFPN with MPK6-YFPC. An mCherry marker was always coexpressed as a reporter for positive transformation events. Reconstitution of YFP fluorescence indicates close physical proximity of AtPIP5K6-YFPN and MPK6-YFPC. Bars = 10 µm. The experiments were performed four times with similar results. −LW, media lacking leucine and tryptophan; −LWH, media lacking leucine, tryptophan, and histidine; MBP, maltose binding protein; OST4, yeast oligosaccharyl transferase 4-kD subunit; pAI-Alg5, positive control; pDL2-Alg5, negative control.

Expression of MPK6 Reduces Plasma Membrane Association 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 with a fluorescent probe for PtdIns(4,5)P2, Red StarPLC-PH (König et al., 2008), and the fluorescence distribution of the probe was analyzed by confocal microscopy (Figure 5). In control pollen tubes expressing EYFP together with Red StarPLC-PH, the PtdIns(4,5)P2-probe decorated the apical plasma membrane in the previously reported pattern (Figure 5A, upper panels). By contrast, membrane association of the PtdIns(4,5)P2 probe was reduced when MPK6-EYFP was coexpressed with Red StarPLC-PH, (Figure 5A, lower panels). The effect of MPK6-EYFP expression on the membrane association of Red StarPLC-PH was numerically assessed (Figures 5B and 5C) based on fluorescence intensity profiles recorded as indicated in Figure 5B. A decreasing plasma membrane-associated (PM) versus cytoplasmic (cyt) intensity ratio of Red StarPLC-PH fluorescence indicates reduced plasma membrane association of the reporter. The PM versus cyt intensity ratio of Red StarPLC-PH fluorescence dropped with increasing expression of MPK6-EYFP (Figure 5C, closed circles), whereas the ratio remained roughly constant with increasing expression of the EYFP control (Figure 5C, open circles). Reduced membrane association of the PtdIns(4,5)P2-specific probe upon expression of MPK6-EYFP is consistent with MPK6-mediated inhibition of PIP5K6 (compared with Figure 3A) and suggests reduced PtdIns(4,5)P2 formation in the apical plasma membrane of the pollen tubes in vivo. While this observation cannot be directly verified by biochemical analysis of PtdIns(4,5)P2 levels in pollen tubes transiently overexpressing PIP5K6-EYFP due to the low transformation frequency, pollen tubes of tobacco plants, in which the expression of the endogenous MAPKs, SIPK and WIPK, is RNAi suppressed (Seo et al., 2007), displayed elevated levels of PtdIns(4,5)P2 (Figure 5D; see Supplemental Figure 5 for transcript reduction in pollen tubes from the RNAi lines). These data support the notion that the formation of PtdIns(4,5)P2 in pollen tubes is controlled by the MAPKs.

Figure 5.
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, Red StarPLC-PH.

(A) Red StarPLC-PH was coexpressed with either an EYFP control (upper panels) or with MPK6-EYFP (lower panels), and fluorescence distribution was assessed by confocal microscopy. Dotted lines, position for collecting fluorescence intensity profiles for quantitative analysis ([B] and [C]). Bars = 10 µm. For each condition (EYFP versus MPK6-EYFP), four independent transformations were set up to coexpress these markers with Red StarPLC-PH. Only data from morphologically unaltered pollen tubes were included. Images are representative for 14 individual transformations from two independent experiments 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 Red StarPLC-PH profiles were interpreted as PM-associated fluorescence and the central regions as cytoplasmic fluorescence (cyt), as indicated. The intensity of the coexpressed EYFP or MPK6-EYFP was determined as indicated. The values were used to calculate the ratios in (C).

(C) Decreasing plasma membrane association of the Red StarPLC-PH probe with increasing expression of MPK6-EYFP. Mean intensities for PM and cyt fluorescence of the Red StarPLC-PH probe were calculated for each transformed cell, and the PM versus cyt ratios of these means were plotted against the mean 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 independent experiments 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 independent transgenic lines (WS2 and WS3), in which SIPK and WIPK are RNAi suppressed. Data indicate mean ± sd from four experiments. Asterisks indicate a 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.

Expression of MPK6 Does Not Interfere with Membrane Association of PIP5K6 in Pollen Tubes

One mode to regulate PI4P 5-kinase activity in animal cells is by an electrostatic switch mechanism, where the introduction of negative charges, e.g., upon protein phosphorylation, interferes with membrane association of the enzymes (Rao et al., 1998; Burden et al., 1999; Fairn et al., 2009). Therefore, we analyzed next whether the coexpression of MPK6-mCherry would influence the apical membrane association of PIP5K6-EYFP in pollen tubes (Figure 6). When PIP5K6-EYFP was coexpressed with either an mCherry control or with MPK6-mCherry, membrane 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 on PtdIns(4,5)P2 production, which were observed in the same cell types at identical conditions (compared with Figure 5), were not mediated by displacement of PIP5K6 from the apical plasma membrane.

Figure 6.
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 an mCherry control (upper panels) or with MPK6-mCherry (lower panels), and the fluorescence distribution was assessed by confocal microscopy. Dotted lines, position for collecting fluorescence intensity profiles for quantitative analysis ([B] and [C]). Bars = 10 µm. For each condition (mCherry versus MPK6-mCherry), four independent transformations were set up to coexpress these markers with PIP5K6-EYFP. Only data from morphologically unaltered pollen tubes were included. Images are representative for 16 individual transformations from two independent experiments 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 cytoplasmic fluorescence (cyt), as indicated. The intensity of the coexpressed mCherry or MPK6-mCherry was determined as indicated. The values were used to 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 or MPK6-mCherry. Open circles, mCherry control; closed circles, MPK6-mCherry. The data represent 16 individual transformation events for each condition, obtained in four independent experiments.

Membrane Trafficking Is Influenced by the Expression of MPK6 in Pollen Tubes

Next, we examined whether PtdIns(4,5)P2-dependent processes were influenced by MPK6 in vivo. As PIP5K6 is required for clathrin-mediated endocytosis in pollen tubes (Zhao et al., 2010), we analyzed the endocytosis of the membrane dye, FM 4-64, over time (Figure 7). The distribution of FM 4-64 was imaged after 15 to 25 min, 35 to 50 min, and again after 65 to 85 min upon dye application in control pollen tubes expressing EYFP (Figure 7A, upper panels) and in pollen tubes expressing MPK6-EYFP (Figure 7A, lower panels). In the control pollen tubes, substantial incorporation of the dye into endomembranes was observed after 35 to 50 min. By contrast, only limited incorporation was apparent after this time in pollen tubes expressing MPK6-EYFP (Figure 7A). Numerically, a decreasing PM versus cyt intensity ratio of FM 4-64 fluorescence indicates internalization of the dye into the cytoplasm. After 15 to 25 min of dye application, the PM versus cyt intensity ratios were comparable between EYFP controls and MPK6-EYFP expressers (P < 0.045), whereas after 35 to 50 min and 65 to 85 min the internalization of the dye was significantly faster (P < 0.01 in both cases) in the control pollen tubes compared with the cells expressing MPK6-EYFP (Figure 7B). This pattern indicates reduced endocytosis of FM 4-64 in pollen tubes expressing MPK6-EYFP. We also tested whether the expression of MPK6 would influence pollen tube growth, which requires tip-directed membrane trafficking and apical pectin secretion. After 10 h of incubation, pollen tubes expressing MPK6 were slightly shorter than control pollen tubes. Furthermore, pectin secretion according to staining of pollen tubes with ruthenium red was reduced when MPK6-EYFP was expressed (Supplemental Figure 6). Together, these data indicate that different aspects of apical membrane trafficking are influenced by the overexpression of MPK6-EYFP, consistent with MPK6-mediated inhibition of PIP5K6 and PtdIns(4,5)P2 production in vivo.

Figure 7.
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 each condition (EYFP versus MPK6-EYFP), five independent transformations were set up to analyze the effect on FM 4-64 uptake. Only data from morphologically unaltered 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 to 85 min, as indicated. Intensity profiles for FM 4-64 were recorded as indicated by the dotted lines, and the mean PM-associated and mean cyt fluorescence was 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 µm.

(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).

Coexpression with MAPKs Attenuates the Effects of PIP5K6 Homologs from Arabidopsis or Tobacco on Pollen Tube Growth

To further delineate the functional relevance of the modification of PI4P 5-kinases by MPK6 in vivo, we scored the incidence of morphological alterations resulting from overexpressing AtPIP5K6 or NtPIP5K6 as a quantitative readout for physiological functionality of the enzymes, as was previously described (Ischebeck et al., 2008, 2010b, 2011; Stenzel et al., 2012). Pollen tubes used in our experiments displayed the morphological alterations previously found to result from overexpression of AtPIP5K6 and NtPIP5K6 (Figure 8A, left panel). As in previous studies (Ischebeck et al., 2008, 2011), the distribution of phenotypic categories was associated with the degree of expression, as assessed by fluorescence intensity of the expressed proteins (Figure 8A, right panel). When pollen tubes coexpressing AtPIP5K6-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 with these controls, coexpression of AtPIP5K6-EYFP with MPK6-mCherry resulted in a significant shift toward weaker morphological defects, with now 22% normal, 16% branched, and only 56% stunted morphologies (P < 0.05 for the increase in normal pollen tubes). When AtPIP5K6 AA-EYFP or AtPIP5K6 DD-EYFP were each coexpressed with either an mCherry control or with MPK6-mCherry (Figure 8B), the resulting patterns did not differ from one another, nor from that observed upon coexpression of AtPIP5K6-EYFP with the mCherry control, indicating that MPK6 did not exert an inhibitory influence on the function of AtPIP5K6 AA or AtPIP5K DD in vivo. These data indicate that the phosphosites T590 and/or T597 are critical for the MPK6-mediated regulation of PIP5K6 effects on cell morphology. When the respective experiments were performed using the homologous tobacco enzymes NtPIP5K6 and SIPK, equivalent observations were made (Figure 8C). Pollen tubes coexpressing NtPIP5K6-EYFP and an mCherry control displayed normal, branched, and stunted morphologies for ∼10%, 39%, and 45% of fluorescing cells, respectively. In comparison, coexpression of NtPIP5K6-EYFP with SIPK-mCherry again resulted in a significant shift toward weaker morphological defects, with now 39% normal, 29% branched, and only 31% stunted morphologies (P < 0.01 for the increase in normal pollen tubes). In the NtPIP5K6 sequence, only one of the two residues phosphorylated in AtPIP5K6 is conserved (T651, which corresponds to T597 of AtPIP5K6; compared with Figure 2B). To test for functional relevance of this residue, an NtPIP5K6 variant was generated in which T651 was substituted with alanine (NtPIP5K6 A). When NtPIP5K6 A-EYFP was coexpressed with mCherry or SIPK-mCherry, there was no difference in the resulting patterns, indicating that SIPK did not exert an inhibitory influence on the function of the variant NtPIP5K6 A protein in vivo.

Figure 8.
Figure 8.

Coexpression with MAPKs Attenuates the Effects of PIP5K6 Homologs from Arabidopsis or Tobacco on Pollen Tube Growth and Cell Morphology.

The effects of MAPKs on the functionality of AtPIP5K6 or NtPIP5K6 were tested in vivo by coexpressing the enzymes with either mCherry controls or with the MAPKs, MPK6 or SIPK, respectively, and scoring the incidence of morphological alterations during pollen tube growth, as previously described (Ischebeck et al., 2010b).

(A) The overexpression of type B PI4P 5-kinases, including AtPIP5K6, in tobacco pollen tubes results in morphological changes related to an increased apical secretion of pectin (Ischebeck et al., 2008, 2010b). Left: Phenotypic categories observed upon expression of AtPIP5K6, as indicated. Bars = 10 µm. Right: Correlation of the categories with fluorescence intensities of the expressed proteins. Letters a to c indicate categories of values that display significant differences 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 ± sd from seven (B) or six (C) experiments, each representing > 100 pollen tubes analyzed. Asterisks indicate significant changes compared with the mCherry control experiments according to a Student’s t test (*P < 0.05; **P < 0.01).

Effects of PIP5K6 Homologs from Arabidopsis or Tobacco Are Delimited in Pollen Tubes upon RNAi Suppression of Intrinsic MAPK Expression

The in vivo effects of MAPKs on PI4P 5-kinases were further characterized in a reciprocal experiment. For this purpose, AtPIP5K6 or NtPIP5K6 was expressed in pollen tubes of tobacco plants, in which expression of the endogenous MAPKs, SIPK and WOUNDING-INDUCED PROTEIN KINASE (WIPK), was suppressed by RNAi (Seo et al., 2007) (Figure 9). Pollen tubes of tobacco plants expressing an empty control construct were used as a reference. When AtPIP5K6-EYFP or NtPIP5K6-EYFP was expressed in pollen tubes of the control plants, for AtPIP5K6, 11% of transgenic pollen tubes were normal, 22% branched, and 50% stunted (Figure 9A), whereas for NtPIP5K6, 11% of transgenic pollen tubes were normal, 38% branched, and 35% stunted. By contrast, the expression of AtPIP5K6-EYFP or NtPIP5K6-EYFP in either of the two independent RNAi lines resulted in significant shifts of the phenotypic categories toward more severe phenotypes (P < 0.01 for the increases in stunted pollen tubes in all four cases). The data indicate that RNAi suppression of endogenous MAPKs enhanced the effects of co-overexpressing PIP5K6 in vivo, consistent with a regulatory effect of the MAPKs on PtdIns(4,5)P2 production. Importantly, the distribution of phenotypic categories resulting from the expression of AtPIP5K6 AA-EYFP or NtPIP5K6 A-EYFP in pollen tubes of the control plants or the two RNAi lines did not differ (Figures 9C and 9D), indicating that the effects of MAPK RNAi suppression on PI4P 5-kinase function depended on the threonine residues found to be phosphorylated by the MAPKs. Overall, the assessment of the in vivo influence of MAPKs from Arabidopsis or tobacco on the function of AtPIP5K6 and NtPIP5K6 is consistent with the reduced PtdIns(4,5)P2 formation upon phosphorylation of PI4P 5-kinases determined in vitro.

Figure 9.
Figure 9.

Effects of PIP5K6 Homologs from Arabidopsis or Tobacco Are Delimited in Pollen Tubes upon RNAi Suppression of Intrinsic MAPK Expression.

The effects of MAPKs on the functionality of AtPIP5K6 or NtPIP5K6 were further tested in vivo by expressing the enzymes in pollen tubes of tobacco plants RNAi-suppressed for the endogenous MAPKs, SIPK and WIPK (Seo et al., 2007), and scoring the incidence of morphological alterations during pollen tube growth, as previously described (Ischebeck et al., 2010b). Three transgenic tobacco lines (Seo et al., 2007) were used, a control carrying an empty expression construct (white bars) and two independent RNAi-suppressed lines (WS1, light gray bars; WS2, dark-gray bars). The data represent the mean ± sd from five experiments. 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.

DISCUSSION

This study addressed the regulation of PI4P 5-kinases, which are key enzymes of plant phosphoinositide biosynthesis, by protein phosphorylation. Our results demonstrate that the MAPK MPK6 (1) mediates phosphorylation of PIP5K6 (Figures 1 and 2), (2) mediates a concomitant inhibition of PtdIns(4,5)P2 formation in vitro (Figure 3), (3) interacts with AtPIP5K6 (Figure 4), (4) causes reduced formation of PtdIns(4,5)P2 and reduced endocytosis at the apical plasma membrane in vivo (Figures 5 and 7), and (5) attenuates PtdIns(4,5)P2-dependent effects on pollen tube morphologies (Figures 8 and 9).

The phosphorylation sites T590 and T597 determined in the PIP5K6 protein by MS-based approaches and subsequent substitution experiments represent bona fide MAPK-targeted S/TP motifs. In the course of this study, a global analysis of the Arabidopsis pollen phosphoproteome was published (Mayank et al., 2012), and both AtPIP5K6 phosphorylation sites determined in our in vitro approach were also identified in planta. In vivo phosphorylation of AtPIP5K6 in pollen has thus been independently demonstrated. Based on our data, phosphorylation of the sites T590 and T597 can now be recognized as MPK6-mediated signal transduction events limiting PtdIns(4,5)P2 production in the apical plasma membrane of pollen tubes.

At first approximation, the inhibitory effects of phosphorylation on activity and physiological function of PIP5K6 appear to be consistent with data on mammalian phosphoinositide kinases, which are thought to be regulated by phosphorylation via an electrostatic switch model (Rao et al., 1998; Burden et al., 1999; Fairn et al., 2009). In this model, the introduction of negative charges by phosphorylation of residues at the protein-membrane interface mediates the dissociation of the protein from the membrane and its anionic substrate lipids. However, as the membrane association of PIP5K6-EYFP did not change in pollen tubes upon coexpression of MPK6-mCherry (Figure 6), we conclude that the regulation of PIP5K6 by MPK6 might not involve an electrostatic switch mechanism. To delineate the mechanistic details of PIP5K6 regulation by MPK6, further analyses will be necessary. In the absence of structural data on plant PI4P 5-kinases, we can currently only speculate whether phosphorylation of T590 and/or T597 in the variable insert region of the catalytic domain might exert a regulatory effect on PIP5K6 by mediating conformational changes that may directly influence the conformation of the catalytic site. The CD spectroscopy data obtained for the substitution variants of PIP5K6 (Figures 3C to 3E) indicate that the introduction of a negative charge in position(s) 590 and/or 597 results in a change in the protein’s tertiary structure. As the near UV CD spectra reflect aromatic residues, which are concentrated in the N-terminal region of PIP5K6, we speculate that phosphorylation of PIP5K6 by MPK6 mediates a conformational change involving the NT and MORN domains. This notion is consistent with the previous report that N-terminal domains of Arabidopsis PI4P 5-kinases of subfamily B have a role in regulating catalytic activity (Im et al., 2007; Stenzel et al., 2012), possibly controlled by reversible phosphorylation of PIP5K6. Based on the analysis of single-substitution variants (Figure 3B), a phosphorylation site relevant for such regulation might be T597. However, our studies do not reveal the in vivo stoichiometry of singly or doubly phosphorylated AtPIP5K6. It is possible that the two detected phosphosites are sequentially phosphorylated by MPK6 under physiological conditions. Such a scenario may explain the “compensatory” effect of the dual phospho-mimetic substitution of PIP5K6 DD on the reduced kinase activity (Figure 3B) or the tertiary structure (Figure 3E) of the single T597D substitution variant. With regard to the activity of the double substitution variants, it should also be noted that the substitution of phosphorylation sites by charged or uncharged residues will not always faithfully reflect the behavior of the protein when phosphorylated or dephosphorylated (Dissmeyer and Schnittger, 2011). While we are providing evidence for regulation of PIP5K6 by phosphorylation, the effects of this modification might thus be more complex. The survey by Mayank et al. (2012) reports further phosphorylation sites in PIP5K6, and it appears likely that the enzyme is targeted also by other protein kinases, which might have interplay with the MPK6-mediated phosphorylation or include examples for regulation by an electrostatic switch mechanism.

PI4P 5-kinases and their product, PtdIns(4,5)P2, control membrane trafficking in pollen tubes (Kost et al., 1999; Ischebeck et al., 2008, 2010b, 2011; Sousa et al., 2008; Zhao et al., 2010; Stenzel et al., 2012). A main trafficking route in these cells involves the directional delivery of secretory vesicles to their target membrane and the endocytotic retrieval of vesicles upon cargo release (Thole and Nielsen, 2008). The reduced plasma membrane association of the Red StarPLC-PH reporter (Figure 5) and the impaired endocytosis of FM 4-64 with expression of MPK6-EYFP (Figure 7) are consistent with reported roles of PtdIns(4,5)P2 (Ischebeck et al., 2008, 2013; König et al., 2008; Sousa et al., 2008) and PIP5K6 (Zhao et al., 2010) in the control of membrane trafficking and clathrin-mediated endocytosis. The discovery of the pollen-expressed PI4P 5-kinases AtPIP5K6 and NtPIP5K6 as targets for regulation by MPK6 and the related tobacco SIPK and/or WIPK, respectively, suggests a 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 observation that the interaction of the cytoplasmic MPK6 with PIP5K6 may occur at the apical plasma membrane (Figure 4C) is consistent with the report that in Arabidopsis a subset of MPK6 protein colocalizes with FM 4-64, indicating membrane association (Müller et al., 2010). A regulatory function of MPK6 in apical PtdIns(4,5)P2 formation is also consistent with reduced rates of pollen tube expansion (Supplemental Figure 6) and the manifestation of morphological alterations observed in pollen tubes resulting from altered apical pectin secretion (Figures 8 and 9).

The reported pollen tube guidance defect of Arabidopsis mpk6 mutants furthermore suggests relevance for MPK6-dependent PIP5K6 regulation in signal transduction events linking extracellular cues emitted by the ovules with the control of the secretory machinery of the pollen tubes (Dresselhaus and Franklin-Tong, 2013; Higashiyama and Takeuchi, 2015; Dresselhaus et al., 2016). A number of guidance signals have been reported, which are perceived at the cell surface of pollen tubes (Dresselhaus and Franklin-Tong, 2013; Higashiyama and Takeuchi, 2015; Dresselhaus et al., 2016). Signal transduction events mediating pollen tube guidance have been proposed to involve MAPKs, including MPK6 (Guan et al., 2014; Higashiyama and Takeuchi, 2015), in analogy to other receptor-dependent signaling cascades known in plants (Pitzschke et al., 2009). Our data suggest that pollen tube guidance cues transduced via MPK6 may result in an inhibition of PtdIns(4,5)P2 formation and reduced apical pollen tube expansion, as illustrated in the model shown in Figure 10. A topical inhibition of secretion by extracellular guidance cues might contribute to asymmetric pollen tube growth toward the ovules for fertilization. However, as this notion is currently not supported by experimental evidence, it is also possible that MPK6 influences overall pollen tube growth with no links to guidance. In either case, directional cell expansion and its responsiveness to exogenous cues are a biological phenomenon shared by polar growing cells from various models, including plants, fungi, and possibly even mammalian cells, and these models share all the regulatory elements investigated in our study (Ischebeck et al., 2010a). Therefore, the proposed mode of regulation may have relevance across eukaryotic kingdoms. Future research will aim to establish whether these proposed regulatory circuits contribute to pollen tube growth and/or guidance in response to exogenous signals, and whether such regulation has been conserved in evolution.

Figure 10.
Figure 10.

Model for the Effects of MAPK-Mediated Limitation of Apical PtdIns(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 symmetric expansion, PIP5K6 and other PI4P 5-kinase isoforms mediate the expansion of pollen tubes at the apex. Bottom half: External guidance cues are perceived by cell surface receptors, likely initiating a MAPK cascade involving MPK6. Activated MPK6 might locally transduce this signal to the machinery for apical membrane trafficking by targeting PIP5K6. The MPK6-mediated phosphorylation of PIP5K6 might result in reduced apical expansion of the cell, possibly resulting in an asymmetric expansion and curvature toward the guidance cues. Other explanations are possible. Blue arrows, promoting influences; blue T-bars; inhibiting influences; orange arrows, simplified representation of vesicle movement for secretion and endocytotic recycling in the expanding pollen tube tip; ellipses, enzymes as indicated; red circles, phosphorylation events. CME, clathrin-mediated endocytosis.

METHODS

Plant Material

Experiments were performed using source material from Arabidopsis thaliana Col-0 grown under a short-day regime (8 h light at ∼140 μmol photons m−2 s−1, 16 h dark) or pollen from tobacco (Nicotiana tabacum; ecotype Samsun N) grown in a greenhouse. The transgenic tobacco plants carrying RNAi constructs against SIPK and WIPK (Seo et al., 2007) were a gift from Shigemi Seo and Shinpei Katou (National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan).

Preparation of Pollen Extracts

Ripe pollen from tobacco flowers was harvested, solubilized, and germinated 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, 1 mM KCl, 0.8 mM H3BO3, 3 μM CuSO4, and 10 μg/mL rifampicin). After the germinated pollen was separated from the growth media, the pollen tube material was frozen in liquid nitrogen and stored at −80°C until use. For the protein extraction, ice-cold protein extraction buffer containing 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 2.5 mM NaF, 1 mM Na3VO4, 0.1 mM EDTA, 0.1 mM DTT, PhosSTOP (Roche), and protease inhibitor cocktail (Sigma-Aldrich) was added to the material, and the cells were broken with a mini pestle. The suspension was cleared by centrifuging at 20,000g and 4°C for 20 min. The extract was kept on ice to prevent protein degradation and protein kinase inactivation.

cDNA Cloning

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

Constructs for Bacterial Expression

For Escherichia coli expression, the cDNAs for AtPIP5K6 and NtPIP5K6 were cloned into the expression plasmid pMALc5g (NEB). To obtain an amplicon that is in frame with the sequence for the N-terminal MBP-tag, AtPIP5K6 and the cDNAs encoding the T590A/D,T597A/D and T590A/D_T597A/D variants of AtPIP5K6 were amplified with the primer combination AtPIP5K6 NdeI for, 5′-GCCATGCCATATGATGTCGGTAGCACACGCAGATGA-3′/AtPIP5K6 His6 SalI rev, 5′-GCCATGCGTCGACTCAGTGGTGGTGGTGGTGGTGAGCGTCTTCAACGAAGACCC-3′, and moved as NdeI/SalI fragments into pMALc5G. The tobacco homolog NtPIP5K6 and the cDNAs for the respective A and D variants were amplified with primer combinations previously described (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 cDNA encoding the endoplasmic reticulum transmembrane oligosaccharyl transferase4 (Ost4p) from yeast via the XbaI restriction site upstream of the multiple cloning site of pBT3-C (DualSystems Biotech), as previously described (Möckli et al., 2007). To clone AtPIP5K6 and NtPIP5K6 into pBT3-C-OST4, the open reading frames were amplified with forward and reverse primers adding SfiI restrictions sites to the 5′- and 3′-ends of the cDNAs, respectively. Additionally, the 5′-amplification primers introduced an additional cytidine base between the SfiI restriction site and the ATG codon. The amplicons of AtPIP5K6 or NtPIP5K6 were then cloned via the SfiI sites into pBT3-C-OST4, yielding pBT3-C-Ost4p-AtPIP5K6 and pBT3-C-Ost4p-NtPIP5K6. To clone pPR3-N-MPK6 and pPR3-N-NtSIPK, the open reading frames of MPK6 and NtSIPK were amplified with primers introducing 5′and 3′ SfiI restriction sites and moved as SfiI fragments into pPR3-N.

Constructs for BiFC Analysis

Constructs for BiFC studies (Kerppola, 2008) were based on the plasmids pEntryA and pEntryD, which are pUC18 based and contain separate multiple cloning sites for the insertion of promoter sequences and a gene of choice and differ by the nature of the att sites for homologous recombination. For the BiFC analyses, expression from a CaMV 35S promoter was chosen to enable weak expression in pollen tubes to observe interactions at close to physiological conditions (Sun et al., 2015). The coding sequences for AtPIP5K6 and NtPIP5K6 were amplified using the primer combinations AtPIP5K6 AscI for, 5′-ATGCGGCGCGCCATGTCGGTAGCACACGCAGA-3′/AtPIP5K6 XhoI rev, 5′-ATGCCTCGAGAGCGTCTTCAACGAAGACCC-3′ and NtPIP5K6 AscI for, 5′-ATGCGGCGCGCCATGAGCAAAGAATTTAGTGG-3′/NtPIP5K6 XhoI rev, 5′-ATGCCTCGAGAGTGTCTTCTGCAAAAACTT-3′, respectively, and moved as AscI/XhoI fragments in reading frame with a downstream coding sequence for the N-terminal half of YFP, YFPN, yielding pEntryA-CaMV35S:AtPIP5K6-YFPN and pEntryA-ProCaMV35S:NtPIP5K6-YFPN. The coding sequences of MPK6 and SIPK were amplified using the primer combinations MPK6 SalI for, 5′-ATGCGTCGACATGGACGGTGGTTCAGGTCA-3′/ MPK6 XbaI rev, 5′-ATGCTCTAGATTGCTGATATTCTGGATTGA-3′ or NtSIPK AscI for, 5′-ATGCGGCGCGCCATGGATGGTTCTGGTCAGCA-3′/NtSIPK XhoI rev, 5′-ATGCCTCGAGCATATGCTGGTATTCAGGAT-3′, respectively, and moved as SalI/XbaI or AscI/XhoI fragments in frame with the downstream coding sequence for the C-terminal half of YFP (YFPc) present in the pEntryD plasmid, yielding pEntryD-ProLat52:MPK6-YFPc and pEntryD-ProLat52:NtSIPK-YFPc. For both BiFC partners, the YFP halves were thus fused at the C termini of the fusion proteins.

Constructs for Transient Expression in Pollen Tubes

mCherry was amplified using the primer combination mCherry-AscI-for, 5′-ATGCGGCGCGCCAATGGTGAGCAAGGGCGAGGA-3′/mCherry-BamHI-rev, 5′-ATGCGGATCCCTACTTGTACAGCTCGTCCAT-3′, adding an AscI restriction site at the 5′-end of mCherry cDNA and a BamHI restriction site at the 3′-end of the mCherry sequence. Between the AscI restriction site and the ATG of the mCherry sequence, the primer introduced an additional adenine base to ensure cloning in frame. After restriction, the mCherry fragment was moved into pEntryA-pLat52 as an AscI-BamHI fragment. cDNAs for MPK6 or SIPK were amplified using primers introducing a 5′-end SalI and a 3′-end AscI restriction site to their cDNA sequences. After digestion, the MPK6 or SIPK fragments were moved into the pEntryA-ProLat52:mCherry vector, 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 conducted by QuickChange technology (Stratagene). In this PCR-based approach, primers containing the desired base exchange were used to amplify the DNA template. The PCR was performed with Phusion High Fidelity Polymerase (NEB) in a 50-μL reaction according to the manufacturer’s instructions. The following thermal cycling steps were used for amplification: 98°C for 30 s as initial denaturation step, 18 cycles at 98°C for 20 s, and annealing between 55 and 65°C for 30 s and 72°C for 1 min/kb for the elongation of the amplicon. The following primers were used: PIPK6 T590A for, 5′-CTGCTATCAAGGACTCTGCCGCTCCTACTTCCGGCGCTCGAAC-3′/PIPK6 T590A rev, 5′-GTTCGAGCGCCGGAAGTAGGAGCGGCAGAGTCCTTGATAGCAG-3′; PIPK6 T590D for, 5′-CTGCTATCAAGGACTCTGCCGATCCTACTTCCGGCGCTCGAAC-3′/PIPK6 T590D rev, 5′-GTTCGAGCGCCGGAAGTAGGATCGGCAGAGTCCTTGATAGCAG-3′; PIPK6 T597A for, 5′-CTACTTCCGGCGCTCGAGCCCCTACCGGAAATTCAGA-3′/PIPK6 T597A rev, 5′-TCTGAATTTCCGGTAGGGGCTCGAGCGCCGGAAGTAG-3′; PIPK6 T597D for, 5′-CTACTTCCGGCGCTCGAGACCCTACCGGAAATTCAGA-3′/PIPK6 T597D rev, 5′-TCTGAATTTCCGGTAGGGTCTCGAGCGCCGGAAGTAG-3′. The mixture of template and amplicon was digested with 10 units of the methylation-dependent restriction enzyme DpnI to degrade all DNA of bacterial origin. After digestion, the nonmethylated amplicon DNA was transformed into chemically-competent E. coli. From the respective pEntry plasmids, amplicons were moved to the vector pLatGW (Ischebeck et al., 2008) using Gateway technology (Invitrogen). To confirm successful cloning and to verify site-directed mutations in plasmids, DNA was sequenced using a commercial service (GATC).

Expression and Purification of Recombinant Proteins in E. coli

Recombinant PIP5K6 was expressed as a fusion to an N-terminal MBP-tag in E. coli Rosetta 2 cells (Merck). Starter cultures were incubated overnight with 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 selection and used to inoculate main cultures in baffled flasks. Cells were grown until an OD600 of 0.6 to 0.8, and expression was induced with 0.1 mM IPTG, unless stated otherwise. The fusion proteins MBP-AtPIP5K1 and pMAL-AtPIP5K6 were best expressed in 1-liter cultures induced with 0.1 mM IPTG at 22°C for 4 h. MBP-NtPIP5K6 was expressed in 3 liters of cultures at 37°C with 30 min induction. Cells were harvested by centrifugation for 20 min at 4000g, and the bacterial pellet was immediately frozen in liquid nitrogen and stored until use at −20°C. Cell disruption was initiated with the addition of lysozyme (Serva) to digest the bacterial cell wall. Ultrasound was used to disrupt small volumes, while larger volumes were homogenized by a high-pressure cell disruption French Press system (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 proteins was performed by affinity chromatography using an MBPTrap column (GE Life Sciences).

GST-MPK6 and GST were recombinantly expressed from pGEX4T1 plasmids (GE Healthcare Europe) in E. coli BL21(DE) cells. Starter cultures with 50 mL of LB media were inoculated with single colonies and grown at 30°C overnight with shaking at 180 rpm. Expression cultures were inoculated at an OD600 of 0.1 and grown in 200 mL of LB media in Erlenmeyer flasks at 37°C with shaking at 180 rpm. Expression was induced with 0.1 mM IPTG at an OD600 of 0.6. After induction, the cultures were shaken at 22°C and 180 rpm for 20 h. Then, 50-mL culture aliquots were harvested by centrifugation for 10 min at 3220g. Bacterial pellets from 50 mL of expression culture were resuspended in 2 to 4 mL of 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl, containing protease inhibitor cocktail (Sigma-Aldrich) and 1 mg mL−1 lysozyme (Serva Electrophoresis). After incubation on ice for 30 min, cells were further disrupted by sonication. Cell debris was removed 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 kinase assay was performed as previously described (Zhang and Klessig, 1997). In brief, pollen tube extracts (80 µg) were loaded on a 10% SDS gel. As a substrate for the protein kinases from the extract, 0.25 mg/mL of MBP-AtPIP5K6 was copolymerized into the resolving gel. Higher concentrations were not used due to limited amounts of material. A control with no protein embedded in the gel was used as an autophosphorylation control. The catalytic subunit of PKA from bovine heart (1 unit; Sigma-Aldrich) was used as a positive control. After electrophoresis, the gel was washed three times for 30 min with washing buffer (25 mM Tris, pH 7.5, 0.5 mM DTT, 0.1 mM Na3V04, 5 mM NaF, 0.5 mg/mL [w/v] BSA, and 0.1% [v/v] Triton X-100) at room temperature with gentle agitation to remove SDS. To renature protein kinases, the gel was incubated in renaturing buffer (25 mM Tris, pH 7.5, 0.5 mM DTT, 0.1 mM Na3V04, and 5 mM NaF) overnight at 4°C with three changes of buffer. The gel was equilibrated in 25 mM Tris, pH 7.5, 2 mM EGTA,12 mM MgCl2, 10 mM CaCl2, 1 mM DTT, 0.1 mM Na3VO4, and the kinase reaction was started in a volume of 30 mL by the addition of 200 nM ATP containing 50 µCi [γ-33P]ATP (10 mCi/mL; Hartmann). The gel was incubated for 60 min with gentle agitation, and the reaction was stopped by the addition of 5% (w/v) trichloroacetic acid and 1% (w/v) sodium pyrophosphate to fix proteins in the gel and remove unbound [γ-33P]ATP for 6 h with at least five changes of buffer. The control gel was stained with Coomassie Brilliant Blue. A prestained protein ladder was used to estimate sizes of phosphorylated proteins. After washing, the gels were dried overnight and radiolabeled bands were visualized using a radiosensitive imager screen (BAS MP 2040s; 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 of radiolabeled γ-phosphate of [γ-33P]ATP into proteins. Purified recombinant PI4P 5-kinases (5–10 µg) were incubated with 15 µg of freshly prepared pollen extract in the presence of 50 µm ATP containing 10 µCi [γ-33P]ATP (10 mCi/mL; Hartmann) in 1× kinase buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, and PhosSTOP) in a volume of 50 μL for 30 min. Variations to the experiments performed with recombinant protein kinases are described below. For kinase assays performed with recombinant, activated MPK6, the sample volume was 20 µL. For each reaction, 0.2 µg of MPK6 was used. MPK6 was purified as described previously (Pecher et al., 2014) and obtained from Pascal Pecher (IPB Halle, Germany). During incubation, samples were gently agitated. The reaction was stopped with SDS sample buffer and the sample was applied to SDS-PAGE. The gel was stained with Coomassie Brilliant Blue and dried overnight. Radiolabeled bands were visualized using a radiosensitive imager screen (BAS MP 2040s; Fujifilm) and the extent of 33P-incorporation was quantified by phosphor imaging (BAS 1500; Fujifilm).

Circular Dichroism

Measurements of dichroic properties of the proteins were performed on a Jasco J-810 spectropolarimeter with the following instrumental setup: 1-nm pitch, 40 accumulations, 50-nm per min scan speed, 1-nm slit widths, and 1-s response time. All experiments were performed in buffer composed of 20 mM Tris/HCl and 200 mM NaCl, pH 7.5, supplemented with 1 mM EDTA and 10 mM maltose at a temperature of 20°C (Peltier element). CD spectra were recorded at a protein concentration of 260 to 530 µg mL−1 (1.21–4.28 µM) using cuvettes with optical path lengths of 1 mm for both far and near UV. Acquired protein spectra were corrected for buffer contribution using Spectra Manager I software (Jasco). The data were converted to mean residue ellipticity (Kelly et al., 2005).

Tryptic Protein Digest

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

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 preselection of precursor ions (Helm et al., 2014). Glu-Fib (Glu-1-Fibrinopeptide B) was used as lock mass (m/z = 785.8426, z = 2), and mass correction was applied 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 processing parameters were set as described (Helm et al., 2014). The intensity of precursor ions was ≥180 counts and for fragment ions ≥15 counts to be distinguished from noise. The designation of fragment ions to precursor ions was achieved by PLGS 3.0 based on peak form, retention time, isotope cluster, and m/z value as well as ion mobility. Further data analysis was performed by PLGS 3.0. Then, MSE data were searched against the modified Arabidopsis database (TAIR10; www.arabidopsis.org) containing common contaminants such as keratin (ftp.thegpm.org/fasta/cRAP/crap.fasta). Protein identification required the detection of two fragment ions per peptide and a minimum of five fragment ions and two peptide matches. Primary digest reagent was trypsin with one missed cleavage allowed, as previously described (Helm et al., 2014).

Identification of Phosphopeptides

AtPIP5K6 residues phosphorylated by MPK6 were identified by liquid chromatography online with HR/AM LC-MS using an Orbitrap Velos Pro System (Thermo Scientific). Proteins separated by SDS-PAGE were subjected to in-gel tryptic digestion, and peptides were analyzed by a data-dependent acquisition scan strategy with inclusion list to specifically select and isolate AtPIP5K6 phosphorylated peptides for MS/MS peptide sequencing. An inclusion list was used to identify low abundant species in the survey scan (targeted data-dependent acquisition). Multi-stage activation (MSA) was applied to further fragment ion peaks resulting from neutral loss of the phosphate moiety by dissociation of the high energy phosphate bond to generate 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 (AtPIP5K6 T590A, AtPIP5K6 T597A, and AtPIP5K6 T590A T597A) with the Mascot software v.2.5 integrated in Proteome Discoverer v.1.4. Phosphopeptides with an ion score surpassing the Mascot significance threshold (P < 0.05) were accepted. The phosphoRS module was used to localize phosphorylation sites within the primary structure of the peptide.

Data Availability

All mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Vizcaíno et al., 2016) partner repository with the data set identifier PXD006067.

Lipid Kinase Assays

The catalytic activity of recombinant PI4P 5-kinases was determined based on their ability to phosphorylate PtdIns4P in the presence of [γ-33P]ATP as previously described (Perera et al., 2005). The extent of 33P-incorporation was 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 (Dualsystems Biotech) as previously described (Johnsson and Varshavsky, 1994). Bait and prey constructs coding for AtPIP5K6 versus MPK6 or for NtPIP5K6 versus NtSIPK, respectively, were cotransformed in the yeast strain NMY51 (Dualsystems Biotech). The bait protein was cotransformed with a positive and a negative control. The positive control consisted of a 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, and histidine (SD-LWH). For higher stringency, selection was performed on SD-LWH plates supplemented with 10 mM 3-amino-1,2,4-triazole (SD-LWHA).

Immuno-Pull-Down

Recombinant GST or GST-MPK6 proteins were immobilized on glutathione-agarose (Thermo Fisher Scientific) and incubated with purified recombinant MBP-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 was detected using a polyclonal anti-GST antibody (GE Healthcare; product number 27-4577-01).

Transient Expression of cDNA Constructs in Tobacco Pollen Tubes

Transient expression of cDNA constructs in tobacco pollen tubes was performed by particle bombardment as previously described (Ischebeck et al., 2008).

Fluorescence Microscopy

Pollen phenotypes were analyzed using an Axio ImagerM1 fluorescence microscope (Carl Zeiss) and an AxioCam MRm gray-scale camera. The observation of phenotypes was performed at 20× magnification using filter set 38 high efficiency (HE) for EYFP detection and filter set 43 HE for mCherry detection (all filters from Carl Zeiss). EYFP was excited at 514 nm and imaged using a FT 495-nm beam splitter and a 470/40-nm band-pass filter; mCherry was excited at 561 nm and imaged using a FT 570-nm beam splitter and a 550/25-nm band-pass filter. Images were taken with the corresponding software program (Axio Vision; Carl Zeiss). Localization studies were performed using LSM780 or LSM880 laser scanning confocal microscopes (Carl Zeiss) with a 40× magnifying objective, unless specified otherwise. EYFP was excited with an argon laser at 488 nm and detected at 493 to 598 nm; mCherry was excited with a DPSS laser at 561 nm and detected at 578 to 696 nm. Images were taken using Zen (Carl Zeiss).

Staining of Tobacco Pollen Tubes

To test for defects in endocytosis, FM 4-64 staining was performed on pollen tubes grown on glass slides for 3 to 4 h after bombardment. FM 4-64 dye (from a stock solution of 50 µm diluted in pollen tube growth medium) was added to a final concentration of 2.5 µM. To test for defects in pectin secretion, a stock solution of 0.01% Ruthenium red (Sigma-Aldrich) was added drop wise to the glass slides containing pollen tubes 3 to 4 h after bombardment.

BiFC

BiFC experiments were performed in tobacco pollen tubes as a physiologically relevant cell type. The constructs were transiently transformed into tobacco pollen and the cells were grown for 7 to 10 h until microscopy evaluation. An mCherry fluorophore under the control of a LAT52 promoter was cotransformed as a marker for transformed cells.

Image Analysis

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

Statistical Evaluation

All quantitative data were tested for statistical significance using two-tailed Student’s t tests. Confidence intervals are given in the figure legends for each data set.

Accession Numbers

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

Supplemental Data

Acknowledgments

We thank the following individuals: Shigemi Seo and Shinpei Katou (both National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan) for the tobacco RNAi plants; Pascal Pecher, Petra Majovsky, and Lennart Eschen-Lippold (all Leibniz Institute of Plant Biochemistry, Halle, Germany) for activated recombinant MPK6 protein, technical assistance, and helpful discussion, respectively; Jennifer Lerche (Institute for Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg) for helpful discussion; and Sven-Erik Behrens (Institute for Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg) for access to spectroscopy equipment. We acknowledge funding from the German Research Foundation (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 the experiments. 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.

Footnotes

  • Received July 12, 2017.
  • Revised October 12, 2017.
  • Accepted November 18, 2017.
  • Published November 22, 2017.

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MAPKs Influence Pollen Tube Growth by Controlling the Formation of Phosphatidylinositol 4,5-Bisphosphate in an Apical Plasma Membrane Domain
Franziska Hempel, Irene Stenzel, Mareike Heilmann, Praveen Krishnamoorthy, Wilhelm Menzel, Ralph Golbik, Stefan Helm, Dirk Dobritzsch, Sacha Baginsky, Justin Lee, Wolfgang Hoehenwarter, Ingo Heilmann
The Plant Cell Dec 2017, 29 (12) 3030-3050; DOI: 10.1105/tpc.17.00543
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