Phosphatidic acid (PA), an important signalling and metabolic phospholipid, is predominantly localized in the subapical plasma membrane (PM) of growing pollen tubes. PA can be produced from structural phospholipids by phospholipase D (PLD), but the isoforms responsible for production of PM PA were not identified yet and their functional roles remain unknown. Following genome-wide bioinformatic analysis of the PLD family in tobacco, we focused on the pollen-overrepresented PLDδ class. Combining live-cell imaging, gene overexpression, lipid-binding and structural bioinformatics, we characterized five NtPLDδ isoforms. Distinct PLDδ isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane-bound PLDδs are tightly bound to PM, primarily via the central catalytic domain. Overexpression analyses suggested isoform PLDδ3 as the most important member of the PLDδ subfamily active in pollen tubes. Moreover, only PLDδ3 shows significant constitutive PLD activity in vivo and, in turn, PA promotes binding of PLDδ3 to the PM. This forms a positive feedback loop leading to PA accumulation and the formation of massive PM invaginations. Tightly controlled production of PA generated by PLDδ3 at the PM is important for maintaining the balance between various membrane trafficking processes that are crucial for plant cell tip growth.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague 16628 Prague 6 Czech Republic

Department of Experimental Plant Biology Charles University 128 44 Prague 2 Czech Republic

Institute of Experimental Botany of the Czech Academy of Sciences 16502 Prague 6 Czech Republic

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Abrams, Z.B., Johnson, T.S., Huang, K., Payne, P.R.O. and Coombes, K. (2019) A protocol to evaluate RNA sequencing normalization methods. BMC Bioinform. 20, 679.

Andreeva, Z., Ho, A.Y.Y., Barthet, M.M., Potocký, M., Bezvoda, R., Žárský, V. and Marc, J. (2009) Phospholipase D family interactions with the cytoskeleton: isoform delta promotes plasma membrane anchoring of cortical microtubules. Funct. Plant Biol. 36, 600.

Arisz, S.A., Testerink, C. and Munnik, T. (2009) Plant PA signalling via diacylglycerol kinase. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1791, 869-875.

Baker, N.A., Sept, D., Joseph, S., Holst, M.J. and McCammon, J.A. (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10 037-10 041.

Barrett, T., Wilhite, S.E., Ledoux, P. et al. (2013) NCBI GEO: archive for functional genomics data sets-update. Nucleic Acids Res. 41, D991-D995.

Boisson-Dernier, A., Lituiev, D.S., Nestorova, A., Franck, C.M., Thirugnanarajah, S. and Grossniklaus, U. (2013) ANXUR Receptor-like kinases coordinate cell wall integrity with growth at the pollen tube tip via NADPH oxidases. PLoS Biol. 11, e1001719.

Bosch, M., Cheung, A.Y. and Hepler, P.K. (2005) Pectin methylesterase, a regulator of pollen tube growth. Plant Physiol. 138, 1334-1346.

Chen, J., Wang, P., de Graaf, B.H.J., Zhang, H., Jiao, H., Tang, C., Zhang, S. and Wu, J. (2018) Phosphatidic acid counteracts S-RNase signaling in pollen by stabilizing the actin cytoskeleton. Plant Cell 30, 1023-1039.

Cho, W. and Stahelin, R.V. (2005) Membrane-protein interactions in cell signaling and membrane trafficking. Annu. Rev. Biophys. Biomol. Struct. 34, 119-151.

Cole, R.A. and Fowler, J.E. (2006) Polarized growth: maintaining focus on the tip. Curr. Opin. Plant Biol. 9, 579-588.

Conze, L.L., Berlin, S., Le Bail, A. and Kost, B. (2017) Transcriptome profiling of tobacco (Nicotiana tabacum) pollen and pollen tubes. BMC Genom. 18, 581.

Dhonukshe, P., Laxalt, A.M., Goedhart, J., Gadella, T.W.J. and Munnik, T. (2003) Phospholipase D activation correlates with microtubule reorganization in living plant cells. Plant Cell 15, 2666-2679.

Edwards, K.D., Fernandez-Pozo, N., Drake-Stowe, K. et al. (2017) A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genom. 18, 448.

Eliáš, M., Potocký, M., Cvrčková, F. and Žárský, V. (2002) Molecular diversity of phospholipase D in angiosperms. BMC Genom. 3, 2.

Foreman, J., Demidchik, V., Bothwell, J.H.F. et al. (2003) Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422, 442-446.

Furt, F., Simon-Plas, F. and Mongrand, S. (2011) Lipids of the plant plasma membrane. In The Plant Plasma Membrane (Murphy, A.S., Schulz, B. and Peer, W., eds). Berlin: Springer, pp. 3-30.

Gerth, K., Lin, F., Daamen, F., Menzel, W., Heinrich, F. and Heilmann, M. (2017) Arabidopsis phosphatidylinositol 4-phosphate 5-kinase 2 contains a functional nuclear localization sequence and interacts with alpha-importins. Plant J. 92, 862-878.

Ghosh, S. and Chan, C.-K.K. (2016) Analysis of RNA-seq data using TopHat and Cufflinks. In Methods in Molecular Biology. Plant Bioinformatics: Methods and Protocols (Edwards, D., ed.). New York, NY: Springer, pp. 339-361.

Goodstein, D.M., Shu, S., Howson, R. et al. (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 40, D1178-D1186.

Grebnev, G., Ntefidou, M. and Kost, B. (2017) Secretion and endocytosis in pollen tubes: models of tip growth in the spot light. Front. Plant Sci. 8, 154.

Gronnier, J., Gerbeau-Pissot, P., Germain, V., Mongrand, S. and Simon-Plas, F. (2018) Divide and rule: plant plasma membrane organization. Trends Plant Sci. 23, 899-917.

Guindon, S., Dufayard, J.-F., Lefort, V., Anisimova, M., Hordijk, W. and Gascuel, O. (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307-321.

Heilmann, I. and Ischebeck, T. (2016) Male functions and malfunctions: the impact of phosphoinositides on pollen development and pollen tube growth. Plant Reprod. 29, 3-20.

Helling, D., Possart, A., Cottier, S., Klahre, U. and Kost, B. (2006) Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling. Plant Cell 18, 3519-3534.

Herberich, E., Sikorski, J. and Hothorn, T. (2010) A robust procedure for comparing multiple means under heteroscedasticity in unbalanced designs. PLoS ONE 5, e9788.

Hong, Y., Devaiah, S.P., Bahn, S.C., Thamasandra, B.N., Li, M., Welti, R. and Wang, X. (2009) Phospholipase Dε and phosphatidic acid enhance Arabidopsis nitrogen signaling and growth. Plant J. 58, 376-387.

Hong, Y., Zhao, J., Guo, L., Kim, S.-C., Deng, X., Wang, G., Zhang, G., Li, M. and Wang, X. (2016) Plant phospholipases D and C and their diverse functions in stress responses. Prog. Lipid Res. 62, 55-74.

Ischebeck, T., Stenzel, I. and Heilmann, I. (2008) Type B Phosphatidylinositol-4-Phosphate 5-Kinases mediate Arabidopsis and Nicotiana tabacum pollen tube growth by regulating apical pectin secretion. Plant Cell 20, 3312-3330.

Ischebeck, T., Seiler, S. and Heilmann, I. (2010) At the poles across kingdoms: phosphoinositides and polar tip growth. Protoplasma 240, 13-31.

Ischebeck, T., Stenzel, I., Hempel, F., Jin, X., Mosblech, A. and Heilmann, I. (2011) Phosphatidylinositol-4,5-bisphosphate influences Nt-Rac5-mediated cell expansion in pollen tubes of Nicotiana tabacum. Plant J. 65, 453-468.

Katoh, K. and Standley, D.M. (2013) MAFFT Multiple Sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772-780.

Kaya, H., Nakajima, R., Iwano, M. et al. (2014) Ca2+-activated Reactive oxygen species production by Arabidopsis RbohH and RbohJ Is essential for proper pollen tube tip growth. Plant Cell 26, 1069-1080.

Klahre, U., Becker, C., Schmitt, A.C. and Kost, B. (2006) Nt-RhoGDI2 regulates Rac/Rop signaling and polar cell growth in tobacco pollen tubes. Plant J. 46, 1018-1031.

Ko, J., Park, H., Heo, L. and Seok, C. (2012) GalaxyWEB server for protein structure prediction and refinement. Nucleic Acids Res. 40, W294-W297.

Korver, R.A., van den Berg, T., Meyer, A.J., Galvan-Ampudia, C.S., ten Tusscher, K.H.W.J. and Testerink, C. (2019) Halotropism requires phospholipase Dζ1-mediated modulation of cellular polarity of auxin transport carriers. Plant Cell Environ. 43(1), 143-158.

Kost, B., Spielhofer, P. and Chua, N.-H. (1998) A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J. 16, 393-401.

Kubátová, Z., Pejchar, P., Potocký, M., Sekereš, J., Žárský, V. and Kulich, I. (2019) Arabidopsis trichome contains two plasma membrane domains with different lipid compositions which attract distinct EXO70 subunits. Int. J. Mol. Sci. 20, 3803.

Lassig, R., Gutermuth, T., Bey, T.D., Konrad, K.R. and Romeis, T. (2014) Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth. Plant J. 78, 94-106.

Lee, Y.J., Szumlanski, A., Nielsen, E. and Yang, Z. (2008) Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth. J. Cell. Biol. 181, 1155-1168.

Li, J. and Wang, X. (2019) Phospholipase D and phosphatidic acid in plant immunity. Plant Sci. 279, 45-50.

Li, G. and Xue, H.-W. (2007) Arabidopsis PLDζ2 regulates vesicle trafficking and is required for auxin response. Plant Cell 19, 281-295.

Li, M., Hong, Y. and Wang, X. (2009) Phospholipase D- and phosphatidic acid-mediated signaling in plants. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1791, 927-935.

Li, J., Yu, F., Guo, H., Xiong, R., Zhang, W., He, F., Zhang, M. and Zhang, P. (2020) Crystal structure of plant PLDα1 reveals catalytic and regulatory mechanisms of eukaryotic phospholipase D. Cell Res. 30, 61-69.

Lu, S., Fadlalla, T., Tang, S., Li, L., Ali, U., Li, Q. and Guo, L. (2019) Genome-wide analysis of phospholipase D gene family and profiling of phospholipids under abiotic stresses in Brassica napus. Plant Cell Physiol. 60, 1556-1566.

Mancuso, S., Marras, A.M., Mugnai, S., Schlicht, M., Žárský, V., Li, G., Song, L., Xue, H.-W. and Baluška, F. (2007) Phospholipase Dζ2 drives vesicular secretion of auxin for its polar cell-cell transport in the transition zone of the root apex. Plant Signal. Behav. 2, 240-244.

Monteiro, D., Liu, Q., Lisboa, S., Scherer, G.F.E., Quader, H. and Malhó, R. (2005) Phosphoinositides and phosphatidic acid regulate pollen tube growth and reorientation through modulation of [Ca2+]c and membrane secretion. J. Exp. Bot. 56, 1665-1674.

Mueller, L.A., Solow, T.H., Taylor, N. et al. (2005) The SOL genomics network. A comparative resource for solanaceae biology and beyond. Plant Physiol. 138, 1310-1317.

Nakanishi, H., de los Santos, P. and Neiman, A.M. (2004) Positive and negative regulation of a SNARE protein by control of intracellular localization. Mol. Biol. Cell 15, 1802-1815.

Noack, L.C. and Jaillais, Y. (2017) Precision targeting by phosphoinositides: how PIs direct endomembrane trafficking in plants. Curr. Opin. Plant Biol. 40, 22-33.

Orlando, K. and Guo, W. (2009) Membrane organization and dynamics in cell polarity. Cold Spring Harbor Perspect. Biol. 1, a001321.

Pinosa, F., Buhot, N., Kwaaitaal, M., Fahlberg, P., Thordal-Christensen, H., Ellerstrom, M. and Andersson, M.X. (2013) Arabidopsis phospholipase Dδ is involved in basal defense and nonhost resistance to powdery mildew fungi. Plant Physiol. 163, 896-906.

Platre, M.P., Noack, L.C., Doumane, M. et al. (2018) A combinatorial Lipid code shapes the electrostatic landscape of plant endomembranes. Dev. Cell 45, 465-480.

Pleskot, R., Potocký, M., Pejchar, P., Linek, J., Bezvoda, R., Martinec, J., Valentová, O., Novotná, Z. and Žárský, V. (2010) Mutual regulation of plant phospholipase D and the actin cytoskeleton. Plant J. 62, 494-507.

Pleskot, R., Pejchar, P., Bezvoda, R., Lichtscheidl, I.K., Wolters-Arts, M., Marc, J., Žárský, V. and Potocký, M. (2012) Turnover of phosphatidic acid through distinct signaling pathways affects multiple aspects of pollen tube growth in tobacco. Front. Plant Sci. 3, 54.

Pleskot, R., Pejchar, P., Staiger, C.J. and Potocký, M. (2014) When fat is not bad: the regulation of actin dynamics by phospholipid signaling molecules. Front. Plant Sci. 5, 5.

Potocký, M., Eliáš, M., Profotová, B., Novotná, Z., Valentová, O. and Žárský, V. (2003) Phosphatidic acid produced by phospholipase D is required for tobacco pollen tube growth. Planta 217, 122-130.

Potocký, M., Jones, M.A., Bezvoda, R., Smirnoff, N. and Žárský, V. (2007) Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytol. 174, 742-751.

Potocký, M., Pejchar, P., Gutkowska, M., Jiménez-Quesada, M.J., Potocká, A., de Alché, J., Kost, B. and Žárský, V. (2012) NADPH oxidase activity in pollen tubes is affected by calcium ions, signaling phospholipids and Rac/Rop GTPases. J. Plant Physiol. 169, 1654-1663.

Potocký, M., Pleskot, R., Pejchar, P., Vitale, N., Kost, B. and Žárský, V. (2014) Live-cell imaging of phosphatidic acid dynamics in pollen tubes visualized by Spo20p-derived biosensor. New Phytol. 203, 483-494.

Proost, S. and Mutwil, M. (2018) CoNekT: an open-source framework for comparative genomic and transcriptomic network analyses. Nucleic Acids Res. 46, W133-W140.

Qin, C. and Wang, X. (2002) The Arabidopsis phospholipase D family. Characterization of a calcium-independent and phosphatidylcholine-selective PLDζ1 with distinct regulatory domains. Plant Physiol. 128, 1057-1068.

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A. and Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539-542.

Sekereš, J., Pleskot, R., Pejchar, P., Žárský, V. and Potocký, M. (2015) The song of lipids and proteins: dynamic lipid-protein interfaces in the regulation of plant cell polarity at different scales. J. Exp. Bot. 66, 1587-1598.

Sekereš, J., Pejchar, P., Šantrůček, J., Vukašinović, N., Žárský, V. and Potocký, M. (2017) Analysis of exocyst subunit EXO70 family reveals distinct membrane polar domains in tobacco pollen tubes. Plant Physiol. 173, 1659-1675.

Sierro, N., Battey, J.N., Ouadi, S., Bovet, L., Goepfert, S., Bakaher, N., Peitsch, M.C. and Ivanov, N.V. (2013) Reference genomes and transcriptomes of Nicotiana sylvestris and Nicotiana tomentosiformis. Genome Biol. 14, R60.

Sousa, E., Kost, B. and Malhó, R. (2008) Arabidopsis Phosphatidylinositol-4-Monophosphate 5-Kinase 4 regulates pollen tube growth and polarity by modulating membrane recycling. Plant Cell 20, 3050-3064.

Stenzel, I., Ischebeck, T., Quint, M. and Heilmann, I. (2012) Variable regions of PI4P 5-kinases direct PtdIns(4,5)P2 toward alternative regulatory functions in tobacco pollen tubes. Front. Plant Sci. 2, 114.

Sung, T.-C., Roper, R.L., Zhang, Y., Rudge, S.A., Temel, R., Hammond, S.M., Morris, A.J., Moss, B., Engebrecht, J. and Frohman, M.A. (1997) Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity. EMBO J. 16, 4519-4530.

Takáč, T., Novák, D. and Šamaj, J. (2019) Recent advances in the cellular and developmental biology of phospholipases in plants. Front. Plant Sci. 10, 362.

Testerink, C. and Munnik, T. (2011) Molecular, cellular, and physiological responses to phosphatidic acid formation in plants. J. Exp. Bot. 62, 2349-2361.

Vaškovičová, K., Žárský, V., Rösel, D., Nikolič, M., Buccione, R., Cvrčková, F. and Brábek, J. (2013) Invasive cells in animals and plants: searching for LECA machineries in later eukaryotic life. Biol. Direct 8, 8.

Vaz Dias, F., Serrazina, S., Vitorino, M., Marchese, D., Heilmann, I., Godinho, M., Rodrigues, M. and Malhó, R. (2019) A role for diacylglycerol kinase 4 in signalling crosstalk during Arabidopsis pollen tube growth. New Phytol. 222, 1434-1446.

Waterhouse, A.M., Procter, J.B., Martin, D.M.A., Clamp, M. and Barton, G.J. (2009) Jalview Version 2-a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189-1191.

Webb, B. and Sali, A. (2016) Comparative protein structure modeling using MODELLER. Curr. Protoc. Protein Sci. 86, 2.9.1-2.9.37.

Žárský, V. and Potocký, M. (2009). Signaling in vesicle traffic: protein-lipid interface in regulation of plant endomembrane dynamics. In Signaling in Plants (Mancuso, S. and Baluška, F., eds). Berlin: Springer, pp. 107-133.

Žárský, V. and Potocký, M. (2010) Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst. Biochem. Soc. Trans. 38, 723-728.

Zeniou-Meyer, M., Zabari, N., Ashery, U. et al. (2007) Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage. J. Biol. Chem. 282, 21746-21757.

Zhang, W., Wang, C., Qin, C., Wood, T., Olafsdottir, G., Welti, R. and Wang, X. (2003) The oleate-stimulated phospholipase D, PLDδ and phosphatidic acid decrease H2O2-induced cell death in Arabidopsis. Plant Cell, 15, 2285-2295.

Zhao, Y., Yan, A., Feijó, J.A., Furutani, M., Takenawa, T., Hwang, I., Fu, Y. and Yang, Z. (2010) Phosphoinositides regulate clathrin-dependent endocytosis at the tip of pollen tubes in Arabidopsis and tobacco. Plant Cell 22, 4031-4044.

Zhao, J., Wang, C., Bedair, M., Welti, R., W. Sumner, L., Baxter, I. and Wang, X. (2011) Suppression of phospholipase Dγs confers increased aluminum resistance in Arabidopsis thaliana. PLoS ONE 6, e28086.

Zhao, L.-J., Yuan, H.-M., Guo, W.-D. and Yang, C.-P. (2016) Digital gene expression analysis of Populus simonii × P. nigra pollen germination and tube growth. Front. Plant Sci. 7, 825.

Zonia, L. and Munnik, T. (2004) Osmotically induced cell swelling versus cell shrinking elicits specific changes in phospholipid signals in tobacco pollen tubes. Plant Physiol. 134, 813-823.

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