The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P 2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)-green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A-induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxindependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P 2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P 2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.
In plants, vacuolar H + -ATPase (V-ATPase) activity acidifies both the trans-Golgi network/early endosome (TGN/EE) and the vacuole. This dual V-ATPase function has impeded our understanding in how the pH homeostasis within the plant TGN/EE controls exo-and endocytosis.⋆ Staffan. Persson@unimelb.au.edu, karin.schumacher@cos.uni-heidelberg.de, and eugenia.russinova@psb.vib-ugent. Additional informationSupplementary information is available on line. Competing interestsThe authors declare no competing financial interests Europe PMC Funders GroupAuthor Manuscript Nat Plants. Author manuscript; available in PMC 2016 June 13. Here, we show that the weak V-ATPase mutant deetiolated3 (det3) displayed a pH increase in the TGN/EE, but not in the vacuole, strongly impairing secretion and recycling of the brassinosteroid receptor and the cellulose synthase complexes to the plasma membrane, in contrast to mutants lacking tonoplast-localized V-ATPase activity only. The brassinosteroid insensitivity and the cellulose deficiency defects in det3 were tightly correlated with reduced Golgi and TGN/EE motility. Thus, our results provide strong evidence that acidification of the TGN/EE, but not of the vacuole, is indispensable for functional secretion and recycling in plants.Plant exo-and endocytic pathways converge at the trans-Golgi network/early endosome (TGN/EE) compartment where different cargos are sorted to further destinations1,2. In animal and yeast cells, acidification of intracellular organelles is crucial for the function of the secretory and endocytic pathways and requires proton pumping activity of the vacuolar H + -ATPases (V-ATPase)3-5. The V-ATPase is conserved across species and consists of multiple subunits that are organized in a cytosolic V1 domain, which is important for the ATP hydrolysis (including A, B, C, D, E, F, G, and H subunits), and an integral membrane V0 domain, which forms the proton pore (including a, d, c, c" and e subunits)3. In Arabidopsis thaliana, the V-ATPase activity is associated with both the TGN/EEs and the tonoplast that are marked by the differential localization of the membraneVHA-a1, VHA-a2 and VHA-a3 isoforms1,6,7. The vha-a3 mutant and the vha-a2 vha-a3 double mutant that lack the tonoplast V-ATPase activity do not display severe defects in cell expansion, whereas the inducible inhibition of the TGN/EE-localized VHA-a1 isoform constrains it7,8. Treatment with the V-ATPase inhibitor concanamycinA (ConcA) resulted in loss of the TGN/EE identity and interfered with the trafficking of endocytic and secretory cargos1,2. Given the differential localization of the V-ATPases, the reduced cell expansion has been concluded to be caused by defects in TGN/EE compartments rather than in the vacuole8, but the nature of these defects has not been clarified. In contrast, the cytosolic V-ATPase subunit C (VHA-C), encoded by the single-copy VHA-C/DEETIOLATED3 (DET3) gene, is required for V-ATPase activity at the TGN/EEs and at the vacuole9. A knockdown allele of DET3 displayed pleiotropic phe...
Coccoliths are calcitic particles produced inside the cells of unicellular marine algae known as coccolithophores. They are abundant components of sea-floor carbonates, and the stoichiometry of calcium to other elements in fossil coccoliths is widely used to infer past environmental conditions. Here we study cryo-preserved cells of the dominant coccolithophore Emiliania huxleyi using state-of-the-art nanoscale imaging and spectroscopy. We identify a compartment, distinct from the coccolith-producing compartment, filled with high concentrations of a disordered form of calcium. Co-localized with calcium are high concentrations of phosphorus and minor concentrations of other cations. The amounts of calcium stored in this reservoir seem to be dynamic and at a certain stage the compartment is in direct contact with the coccolith-producing vesicle, suggesting an active role in coccolith formation. Our findings provide insights into calcium accumulation in this important calcifying organism.
Eukaryotic membranes contain small amounts of phospholipids that have regulatory effects on the physiological functions of cells, tissues, and organs. Phosphoinositides (PIs)-the phosphorylated derivatives of phosphatidylinositol-are one example of such regulatory lipids. Although PIs were described in plants decades ago, their contribution to the regulation of physiological processes in plants is not well understood. In the past few years, evidence has emerged that PIs are essential for plant function and development. Recently reported phenotypes associated with the perturbation of different PIs suggest that some subgroups of PIs influence specific processes. Although the molecular targets of PI-dependent regulation in plants are largely unknown, the effects of perturbed PI metabolism can be used to propose regulatory modules that involve particular downstream targets of PI regulation. This review summarizes phenotypes associated with the perturbation of the plant PI network to categorize functions and suggest possible downstream targets of plant PI regulation.
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