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

Abstract

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P 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)P 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)P production and membrane trafficking in pollen tubes, possibly contributing to directional growth.

A dual role for cell plate‐associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis

“…We could confirm this phosphorylation event in vitro using recombinant proteins (Appendix Fig S10A), and the modification of purified PI4Kb1 by purified MPK4 supports a direct physical interaction of the proteins in vitro (cf. In analogy to the in vitro inhibition of the phosphoinositide kinase PIP5K6 by the MAPK, MPK6 (Hempel et al, 2017), we then tested the catalytic activity of PI4Kb1 protein which had been prephosphorylated in vitro by MPK4, but found no change (Appendix Fig S10B). In analogy to the in vitro inhibition of the phosphoinositide kinase PIP5K6 by the MAPK, MPK6 (Hempel et al, 2017), we then tested the catalytic activity of PI4Kb1 protein which had been prephosphorylated in vitro by MPK4, but found no change (Appendix Fig S10B).…”

“…MPK4 acts in the Arabidopsis equivalent of the tobacco NACK-PQR pathway and is thus required for maintaining microtubule organization (Beck et al, , 2011. As we have previously described a regulatory link between MAPKs and phosphoinositide kinases in plants (Hempel et al, 2017), we next asked whether similarly MPK4 is functionally linked to the role of PI4Kbs in phragmoplast progression during cytokinesis. As we have previously described a regulatory link between MAPKs and phosphoinositide kinases in plants (Hempel et al, 2017), we next asked whether similarly MPK4 is functionally linked to the role of PI4Kbs in phragmoplast progression during cytokinesis.…”

A PAMP‐triggered MAPK cascade inhibits phosphatidylinositol 4,5‐bisphosphate production by PIP5K6 in Arabidopsis thaliana

“…The gel shift was more pronounced for myc‐MVQ1, in line with polyphosphorylation of this protein (Pecher et al ., ), whereas only two phosphorylation sites were reported for MPK6‐mediated phosphorylation of PIP5K6 (Hempel et al ., ). To characterize flg22‐dependent phosphorylation of PIP5K6 in more detail, we next analyzed PIP5K6‐HA in comparison to a PIP5K6‐HA AA variant, which can no longer be phosphorylated by MPK6, because residues T590 and T597 were substituted for alanines (Hempel et al ., ) (Fig. b).…”

“…So far, information on cytoplasmic effects of MAPKs is limited. We have previously reported that MPK6 influences pollen tube growth by targeting enzymes with key roles in the biosynthesis of phosphoinositides (PIs) at the plasma membrane (Hempel et al ., ). This finding suggests a regulatory link between MAPKs and PIs, which is probably not limited to pollen tubes and which we aimed to explore further.…”

“…The best‐studied role of PtdIns(4,5)P 2 in plants is its requirement for clathrin‐mediated endocytosis (CME) (König et al ., ; Zhao et al ., ; Ischebeck et al ., ). As in pollen tubes MAPK‐mediated phosphorylation of PIP5K6 in positions T590 and T597 inhibited the catalytic activity of PIP5K6 (Hempel et al ., ), phosphorylation of PIP5K6 by MPK6 resulted in reduced rates of endocytosis at the plasma membrane (Hempel et al ., ), an effect that might also be relevant in other cell types and tissues. A prominent group of CME cargo proteins influenced by altered concentrations of PtdIns(4,5)P 2 is the family of PIN‐FORMED (PIN) auxin efflux transporters (Mei et al ., ; Ischebeck et al ., ; Tejos et al ., ).…”