Dataset Information

A mitogen-activated protein kinase limits the apical formation of phosphatidylinositol 4,5-bisphosphate during polarized cell expansion of pollen tubes

ABSTRACT: Mitogen activated protein kinases (MAPKs) and phosphoinositides contribute to establishment and maintenance of polarity in eukaryotic cells. However, interactions between these important signaling pathways are largely unclear. Pollen tubes are an important model for the study of polarized cell expansion, in which PI4P 5-kinases control apical secretion and MAPKs contribute to pollen tube guidance. Here we report on the regulation of the Arabidopsis PI4P 5-kinase, AtPIP5K6, by the MAPK, MPK6. Purified recombinant AtPIP5K6 was phosphorylated in vitro upon incubation with pollen tube extracts. Non-targeted in-gel-kinase assays of pollen tube extracts followed by mass-spectrometry identified MPK6 as a candidate protein kinase upstream of AtPIP5K6. MPK6 interacted with AtPIP5K6 in yeast-two-hybrid tests, and bimolecular fluorescence complementation verified the interaction at the apical plasma membrane of pollen tubes. Recombinant AtPIP5K6 was phosphorylated in vitro by recombinant MPK6, resulting in reduced catalytic activity of AtPIP5K6. Alanine-substitution of phosphorylation sites, T590A and T597A, abrogated phosphorylation of AtPIP5K6 by MPK6 and prevented the in vitro inhibition. A T597D substitution resulted in reduced catalytic activity of the modified AtPIP5K6 protein in vitro. Coexpression of MPK6 with AtPIP5K6 in pollen tubes resulted in attenuation of PI4P 5-kinase-mediated polarity defects in vivo. Reciprocally, AtPIP5K6 effects were enhanced upon concomitant RNAi-inhibition of endogenous tobacco MAPKs. Both effects were absent when a AtPIP5K6 T590A T597A-variant was expressed, which could no longer be phosphorylated. We conclude that MPK6 limits PI4P 5-kinase-dependent polarized secretion to effect pollen tube guidance, revealing a new link between conserved signaling pathways with potential ramifications across eukaryotic kingdoms.

INSTRUMENT(S): LTQ Orbitrap Velos, Synapt MS

ORGANISM(S): Nicotiana Tabacum (common Tobacco) Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Pollen

SUBMITTER: Sacha Baginsky

LAB HEAD: Ingo Heilmann

PROVIDER: PXD006067 | Pride | 2022-02-22

REPOSITORIES: Pride

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Publications

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

Hempel Franziska F Stenzel Irene I Heilmann Mareike M Krishnamoorthy Praveen P Menzel Wilhelm W Golbik Ralph R Helm Stefan S Dobritzsch Dirk D Baginsky Sacha S Lee Justin J Hoehenwarter Wolfgang W Heilmann Ingo I

The Plant cell 20171122 12

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P<sub>2</sub>] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P<sub>2</sub> 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 identif ...[more]

PMID: 29167320

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Project description:As in animals, cell-cell communication plays pivotal role in male-female recognition during plant sexual reproduction. Prelaid peptides secreted from the female reproductive tissues guide pollen tubes towards ovules for fertilization. However, the elaborate mechanisms for this dialogue have remained elusive, particularly from the male perspective. We perform genome-wide quantitative liquid chromatography coupled tandem mass spectrometry of a pistil-stimulated pollen tube secretome and identify 801 pollen tube-secreted proteins. Interestingly, in silico analysis reveals that the pollen tube-secretome is dominated by unconventional-type secreted proteins representing 57% of the total secretome. In support, we show that unconventional-type protein, translationally controlled tumor protein, is secreted to the apoplast. Remarkably, we discover that this protein could be secreted by infiltrating through the initial phases of the conventional secretory pathway and could reach the apoplast via exosomes as demonstrated by co-localization with Oleisin1 exosome marker. We demonstrate that Arabidopsis thaliana translationally controlled tumor protein-knockdown plants have pollen tubes that poorly navigate to the target ovule, and theÂ knocked down allele is poorly transmitted through the male. We show that regulators of the endoplasmic reticulum-trans-golgi network protein secretory pathway control secretion of pollen tube-secreted cysteine-rich proteins, including pollen tube attractants, and are essential for pollen tube growth and guidance, as well as ovule-targeting competence. This work, the first pollen tube secretome study, identifies novel genome-wide pollen tube-secreted proteins with potential function in pollen tube-ovule guidance for sexual reproduction. Functional analysis highlights a potential mechanism for pollen tube unconventional protein secretion and reveals likely regulators of pollen tube protein secretion. The association of pollen tube-secreted proteins with marker proteins shown to be secreted via exosomes in other species suggest secretion via exosomes as a possible mechanism for cell-cell communication between the pollen tube and female reproductive cells. For processed dataset with quantitative information, see Hafidh S, Potesil D, Fila J et al. Genome Bilogy 2016.

Dataset Information

A mitogen-activated protein kinase limits the apical formation of phosphatidylinositol 4,5-bisphosphate during polarized cell expansion of pollen tubes

Publications

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

Similar Datasets

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