Pollen tubes require a tightly regulated pectin secretion machinery to sustain the cell wall plasticity required for polar tip growth. Involved in this regulation at the apical plasma membrane are proteins and signaling molecules, including phosphoinositides and phosphatidic acid (PA). However, the contribution of diacylglycerol kinases (DGKs) is not clear. We transiently expressed tobacco DGKs in pollen tubes to identify a plasma membrane (PM)-localized isoform, and then to study its effect on pollen tube growth, pectin secretion and lipid signaling. In order to potentially downregulate DGK5 function, we overexpressed an inactive variant. Only one of eight DGKs displayed a confined localization at the apical PM. We could demonstrate its enzymatic activity and that a kinase-dead variant was inactive. Overexpression of either variant led to differential perturbations including misregulation of pectin secretion. One mode of regulation could be that DGK5-formed PA regulates phosphatidylinositol 4-phosphate 5-kinases, as overexpression of the inactive DGK5 variant not only led to a reduction of PA but also of phosphatidylinositol 4,5-bisphosphate levels and suppressed related growth phenotypes. We conclude that DGK5 is an additional player of polar tip growth that regulates pectin secretion probably in a common pathway with PI4P 5-kinases.

Department of Experimental Plant Biology Charles University Prague 12844 Czech Republic

Department of Plant Biochemistry Albrecht von Haller Institute for Plant Sciences and Göttingen Center for Molecular Biosciences University of Göttingen Göttingen 37077 Germany

Green Biotechnology Institute of Plant Biology and Biotechnology University of Münster Münster 48143 Germany

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

Plant Cell Biology Swammerdam Institute for Life Sciences University of Amsterdam Amsterdam 1000 BE the Netherlands

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Angkawijaya AE, Nguyen VC, Gunawan F, Nakamura Y. 2020. A pair of Arabidopsis diacylglycerol kinases essential for gametogenesis and endoplasmic reticulum phospholipid metabolism in leaves and flowers. Plant Cell 32: 2602-2620.

Arisz SA, Testerink C, Munnik T. 2009. Plant PA signaling via diacylglycerol kinase. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1791: 869-875.

Arisz SA, van Wijk R, Roels W, Zhu J-K, Haring MA, Munnik T. 2013. Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase. Frontiers in Plant Science 4: 1.

Bloch D, Pleskot R, Pejchar P, Potocký M, Trpkošová P, Cwiklik L, Vukašinović N, Sternberg H, Yalovsky S, Žárský V. 2016. Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth. Plant Physiology 172: 980-1002.

Bosch M, Cheung AY, Hepler PK. 2005. Pectin methylesterase, a regulator of pollen tube growth. Plant Physiology 138: 1334-1346.

Cacas J-L, Gerbeau-Pissot P, Fromentin J, Cantrel C, Thomas D, Jeannette E, Kalachova T, Mongrand S, Simon-Plas F, Ruelland E. 2017. Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein: control of RBOHD activity by DGK. Plant, Cell & Environment 40: 585-598.

Chang L-C, Guo C-L, Lin Y-S, Fu H, Wang C-S, Jauh G-Y. 2009. Pollen-specific SKP1-like proteins are components of functional scf complexes and essential for lily pollen tube elongation. Plant & Cell Physiology 50: 1558-1572.

Chebli Y, Kaneda M, Zerzour R, Geitmann A. 2012. The cell wall of the Arabidopsis pollen tube-spatial distribution, recycling, and network formation of polysaccharides. Plant Physiology 160: 1940-1955.

Cheung AY, Chen CY-h, Glaven RH, de Graaf BHJ, Vidali L, Hepler PK, Wu H. 2002. Rab2 GTPase regulates vesicle trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth. Plant Cell 14: 945-962.

Colongonzalez F, Kazanietz M. 2006. C1 domains exposed: from diacylglycerol binding to protein-protein interactions. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1761: 827-837.

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

Corbalan-Garcia S, Gómez-Fernández JC. 2014. Signaling through C2 domains: more than one lipid target. Biochimica et Biophysica Acta (BBA) - Biomembranes 1838: 1536-1547.

Dowd PE, Coursol S, Skirpan AL, Kao T, Gilroy S. 2006. Petunia phospholipase c1 is involved in pollen tube growth. Plant Cell 18: 1438-1453.

Elias M, Drdova E, Ziak D, Bavlnka B, Hala M, Cvrckova F, Soukupova H, Zarsky V. 2003. The exocyst complex in plants. Cell Biology International 27: 199-201.

Fayant P, Girlanda O, Chebli Y, Aubin C-E, Villemure I, Geitmann A. 2010. Finite element model of polar growth in pollen tubes. Plant Cell 22: 2579-2593.

Feijó JA, Sainhas J, Holdaway-Clarke T, Cordeiro MS, Kunkel JG, Hepler PK. 2001. Cellular oscillations and the regulation of growth: the pollen tube paradigm. BioEssays 23: 86-94.

Franks CE, Campbell ST, Purow BW, Harris TE, Hsu K-L. 2017. The ligand binding landscape of diacylglycerol kinases. Cell Chemical Biology 24: 870-880.e5.

Furt F, König S, Bessoule J-J, Sargueil F, Zallot R, Stanislas T, Noirot E, Lherminier J, Simon-Plas F, Heilmann I et al. 2010. Polyphosphoinositides are enriched in plant membrane rafts and form microdomains in the plasma membrane. Plant Physiology 152: 2173-2187.

Furt F, Simon-Plas F, Mongrand S. 2011. Lipids of the plant plasma membrane. In: Murphy AS, Schulz B, Peer W, eds. The plant plasma membrane. Berlin, Heidelberg, Germany: Springer Berlin Heidelberg, 3-30.

Gómez-Merino FC, Arana-Ceballos FA, Trejo-Téllez LI, Skirycz A, Brearley CA, Dörmann P, Mueller-Roeber B. 2005. Arabidopsis AtDGK7, the smallest member of plant diacylglycerol kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration: the DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alters plant growth and development. Journal of Biological Chemistry 280: 34888-34899.

Gómez-Merino FC, Brearley CA, Ornatowska M, Abdel-Haliem MEF, Zanor M-I, Mueller-Roeber B. 2004. AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phosphorylates 1-stearoyl-2-arachidonoyl-sn -glycerol and 1,2-dioleoyl-sn-glycerol and exhibits cold-inducible gene expression. Journal of Biological Chemistry 279: 8230-8241.

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

Haduch-Sendecka A, Pietruszka M, Zajdel P. 2014. Power spectrum, growth velocities and cross-correlations of longitudinal and transverse oscillations of individual Nicotiana tabacum pollen tube. Planta 240: 263-276.

Hanahan DJ, Chaikoff IL. 1947. A new phospholipide-splitting enzyme specific for the ester linkage between the nitrogenous base and the phosphoric acid grouping. Journal of Biological Chemistry 169: 699-705.

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

Helling D, Possart A, Cottier S, Klahre U, 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.

Hille B, Dickson EJ, Kruse M, Vivas O, Suh B-C. 2015. Phosphoinositides regulate ion channels. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1851: 844-856.

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

Honys D, Twell D. 2004. Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biology 5: R85.

Im YJ, Davis AJ, Perera IY, Johannes E, Allen NS, Boss WF. 2007. The N-terminal membrane occupation and recognition nexus domain of Arabidopsis phosphatidylinositol phosphate kinase 1 regulates enzyme activity. Journal of Biological Chemistry 282: 5443-5452.

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

Ivakov A, Persson S. 2013. Plant cell shape: modulators and measurements. Frontiers in Plant Science 4: 439.

Johnson MA, Harper JF, Palanivelu R. 2019. A fruitful journey: pollen tube navigation from germination to fertilization. Annual Review of Plant Biology 70: 809-837.

de Jong F, Munnik T. 2021. Attracted to membranes: lipid-binding domains in plants. Plant Physiology 185: 707-723.

Julkowska MM, Rankenberg JM, Testerink C. 2013. Liposome-binding assays to assess specificity and affinity of phospholipid-protein interactions. In: Munnik T, Heilmann I, eds. Methods in molecular biology. Plant lipid signaling protocols. Totowa, NJ, USA: Humana Press, 261-271.

Kim S-C, Wang X. 2020. Phosphatidic acid: an emerging versatile class of cellular mediators. Essays in Biochemistry 64: 533-546.

Klahre U, Becker C, Schmitt AC, Kost B. 2006. Nt-RhoGDI2 regulates Rac/Rop signaling and polar cell growth in tobacco pollen tubes. The Plant Journal 46: 1018-1031.

König S, Hoffmann M, Mosblech A, Heilmann I. 2008. Determination of content and fatty acid composition of unlabeled phosphoinositide species by thin-layer chromatography and gas chromatography. Analytical Biochemistry 378: 197-201.

Kost B, Lemichez E, Spielhofer P, Hong Y, Tolias K, Carpenter C, Chua N-H. 1999. Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth. The Journal of Cell Biology 145: 317-330.

Kost B, Spielhofer P, 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. The Plant Journal 16: 393-401.

Kroeger JH, Zerzour R, Geitmann A. 2011. Regulator or driving force? The role of turgor pressure in oscillatory plant cell growth. PLoS ONE 6: e18549.

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

Martin TFJ. 2012. Role of PI(4,5)P2 in vesicle exocytosis and membrane fusion. Subcellular Biochemistry 59: 111-130.

Mori T, Kuroiwa H, Higashiyama T, Kuroiwa T. 2006. GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization. Nature Cell Biology 8: 64-71.

Müller AO, Blersch KF, Gippert AL, Ischebeck T. 2017. Tobacco pollen tubes - a fast and easy tool for studying lipid droplet association of plant proteins. The Plant Journal 89: 1055-1064.

Müller AO, Ischebeck T. 2018. Characterization of the enzymatic activity and physiological function of the lipid droplet-associated triacylglycerol lipase AtOBL1. New Phytologist 217: 1062-1076.

Munnik T. 2001. Phosphatidic acid: an emerging plant lipid second messenger. Trends in Plant Science 6: 227-233.

Munnik T, Laxalt AM. 2013. Measuring PLD activity in vivo. In: Munnik T, Heilmann I, eds. Methods in molecular biology. Plant lipid signaling protocols. Totowa, NJ, USA: Humana Press, 219-231.

Munnik T, Testerink C. 2009. Plant phospholipid signaling: ‘in a nutshell’. Journal of Lipid Research 50: S260-S265.

Munnik T, Zarza X. 2013. Analyzing plant signaling phospholipids through 32Pi-labeling and TLC. Methods in Molecular Biology 1009: 3-15.

Nishizuka Y. 1988. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334: 661-665.

Noack LC, Bayle V, Armengot L, Rozier F, Mamode-Cassim A, Stevens FD, Caillaud MC, Munnik T, Mongrand S, Pleskot R et al. 2021. A nanodomain-anchored scaffolding complex is required for the function and localization of phosphatidylinositol 4-kinase alpha in plants. Plant Cell koab135. doi: 10.1093/plcell/koab135.

Noack LC, Jaillais Y. 2020. Functions of anionic lipids in plants. Annual Review of Plant Biology 71: 71-102.

Pejchar P, Sekereš J, Novotný O, Žárský V, Potocký M. 2020. Functional analysis of phospholipase Dδ family in tobacco pollen tubes. The Plant Journal 103: 212-226.

Platre MP, Jaillais Y. 2017. Anionic lipids and the maintenance of membrane electrostatics in eukaryotes. Plant Signaling & Behavior 12: e1282022.

Platre MP, Noack LC, Doumane M, Bayle V, Simon MLA, Maneta-Peyret L, Fouillen L, Stanislas T, Armengot L, Pejchar P et al. 2018. A combinatorial lipid code shapes the electrostatic landscape of plant endomembranes. Developmental Cell 45: 465-480.

Pleskot R, Li J, Žárský V, Potocký M, Staiger CJ. 2013. Regulation of cytoskeletal dynamics by phospholipase D and phosphatidic acid. Trends in Plant Science 18: 496-504.

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

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

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

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

Read SM, Clarke AE, Bacic A. 1993. Stimulation of growth of cultured Nicotiana tabacum W 38 pollen tubes by poly(ethylene glycol) and Cu(II) salts. Protoplasma 177: 1-14.

Röckel N, Wolf S, Kost B, Rausch T, Greiner S. 2008. Elaborate spatial patterning of cell-wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the distribution of esterified and de-esterified pectins. The Plant Journal 53: 133-143.

Rotsch AH, Kopka J, Feussner I, Ischebeck T. 2017. Central metabolite and sterol profiling divides tobacco male gametophyte development and pollen tube growth into eight metabolic phases. The Plant Journal 92: 129-146.

Ruelland E, Cantrel C, Gawer M, Kader J-C, Zachowski A. 2002. Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis suspension cells. Plant Physiology 130: 999-1007.

Sanjuán MA, Jones DR, Izquierdo M, Mérida I. 2001. Role of diacylglycerol kinase α in the attenuation of receptor signaling. The Journal of Cell Biology 153: 207-220.

Scheible N, McCubbin A. 2019. Signaling in pollen tube growth: beyond the tip of the polarity iceberg. Plants 8: 156.

Scholz P, Anstatt J, Krawczyk HE, Ischebeck T. 2020. Signalling pinpointed to the tip: the complex regulatory network that allows pollen tube growth. Plants 9: 1098.

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

Sessions A, Burke E, Presting G, Aux G, McElver J, Patton D, Dietrich B, Ho P, Bacwaden J, Ko C et al. 2002. A high-throughput Arabidopsis reverse genetics system. Plant Cell 14: 2985-2994.

Simon MLA, Platre MP, Marquès-Bueno MM, Armengot L, Stanislas T, Bayle V, Caillaud M-C, Jaillais Y. 2016. A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants. Nature Plants 2: 16089.

Sousa E, Kost B, 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, Heilmann I. 2012. Variable regions of PI4P 5-kinases direct PtdIns(4,5)P2 toward alternative regulatory functions in tobacco pollen tubes. Frontiers in Plant Science 2: 114.

Stephan O, Cottier S, Fahlén S, Montes-Rodriguez A, Sun J, Eklund DM, Klahre U, Kost B. 2014. RISAP is a TGN-associated RAC5 effector regulating membrane traffic during polar cell growth in tobacco. Plant Cell 26: 4426-4447.

Synek L, Pleskot R, Sekereš J, Serrano N, Vukašinović N, Ortmannová J, Klejchová M, Pejchar P, Batystová K, Gutkowska M et al. 2021. Plasma membrane phospholipid signature recruits the plant exocyst complex via the EXO70A1 subunit. Proceedings of the National Academy of Sciences, USA 118: e2105287118.

Tan W-J, Yang Y-C, Zhou Y, Huang L-P, Xu L, Chen Q-F, Yu L-J, Xiao S. 2018. DIACYLGLYCEROL ACYLTRANSFERASE and DIACYLGLYCEROL KINASE modulate triacylglycerol and phosphatidic acid production in the plant response to freezing stress. Plant Physiology 177: 1303-1318.

TerBush DR, Maurice T, Roth D, Novick P. 1996. The exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO Journal 15: 6483-6494.

Testerink C, Munnik T. 2005. Phosphatidic acid: a multifunctional stress signaling lipid in plants. Trends in Plant Science 10: 368-375.

Testerink C, Munnik T. 2011. Molecular, cellular, and physiological responses to phosphatidic acid formation in plants. Journal of Experimental Botany 62: 2349-2361.

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

Vermeer JEM, van Wijk R, Goedhart J, Geldner N, Chory J, Gadella TWJ, Munnik T. 2017. In vivo imaging of diacylglycerol at the cytoplasmic leaflet of plant membranes. Plant & Cell Physiology 58: 1196-1207.

Wilson ZA, Morroll SM, Dawson J, Swarup R, Tighe PJ. 2001. The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors. The Plant Journal 28: 27-39.

Wissing J, Heim S, Wagner KG. 1989. Diacylglycerol kinase from suspension cultured plant cells: purification and properties. Plant Physiology 90: 1546-1551.

Wong A, Donaldson L, Portes MT, Eppinger J, Feijó J, Gehring C. 2020. The Arabidopsis diacylglycerol kinase 4 is involved in nitric oxide-dependent pollen tube guidance and fertilization. Development 147: dev183715.

Yamamoto E, Domański J, Naughton FB, Best RB, Kalli AC, Stansfeld PJ, Sansom MSP. 2020. Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes. Science Advances 6: eaay5736.

Yao H-Y, Xue H-W. 2018. Phosphatidic acid plays key roles regulating plant development and stress responses. Journal of Integrative Plant Biology 60: 851-863.

Zerzour R, Kroeger J, Geitmann A. 2009. Polar growth in pollen tubes is associated with spatially confined dynamic changes in cell mechanical properties. Developmental Biology 334: 437-446.

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

Zhukovsky MA, Filograna A, Luini A, Corda D, Valente C. 2019. Phosphatidic acid in membrane rearrangements. FEBS Letters 593: 2428-2451.

Zonia L, Müller M, Munnik T. 2006. Hydrodynamics and cell volume oscillations in the pollen tube apical region are integral components of the biomechanics of Nicotiana tabacum pollen tube growth. Cell Biochemistry and Biophysics 46: 209-232.

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