Phosphatidic acid binds to and regulates guanine nucleotide exchange factor 8 (GEF8) activity in Arabidopsis
Chunyan Cao A * , Peipei Wang A * , Hongdi Song A , Wen Jing A , Like Shen A , Qun Zhang A B and Wenhua Zhang A B
+ Author Affiliations
- Author Affiliations
A College of Life Sciences, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, PR China.
B Corresponding authors. Email: zhangqun@njau.edu.cn; whzhang@njau.edu.cn
Functional Plant Biology 44(10) 1029-1038 https://doi.org/10.1071/FP17113
Submitted: 11 January 2017 Accepted: 15 June 2017 Published: 14 July 2017
Abstract
Phosphatidic acid (PA) forms part of plant lipid metabolism and is a signalling molecule used in response to various external stresses. Guanine nucleotide exchange factors (GEFs) activate small GTPase ROPs, serving as molecular switches in a wide range of signalling pathways. However, the interaction between PA and GEFs in plants has not yet been reported. Here we show that PA bound specifically to GEF8 by using fat-Western blot and isothermal titration calorimetry assays. A C-terminal truncation of GEF8 exhibited strong PA binding, and mutation of lysines 13 and 18 in GEF8 PRONE domain caused a total loss of binding to PA. Two ROPs, ROP7 and ROP10, were identified as preferred substrates of GEF8 by pull-down and bimolecular fluorescence complementation assays. GEF8 activity towards ROP7, but not ROP10, was stimulated by PA both in vitro and in cells. Moreover, the PA- or ABA-induced activation of GEF8 was completely lost in the mutant GEF8, which did not bind to PA. Together, these findings identify a direct interconnection between PA-mediated GEFs activity and small GTPase signalling in plants and provide evidence for a synergistic activation of GEF8 by direct PA-binding to its PRONE domain.
Additional keywords: abscisic acid, guard cells, ROP, signaling, signalling.
References
Berken A, Thomas C, Wittinghofer A (2005) A new family of RhoGEFs activates the Rop molecular switch in plants. Nature 436, 1176–1180.| A new family of RhoGEFs activates the Rop molecular switch in plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXovVOgsbY%3D&md5=1365f67656fa343c437fea709a513547CAS |
Brembu T, Winge P, Bones AM (2005) The small GTPase AtRAC2/ROP7 is specifically expressed during late stages of xylem differentiation in Arabidopsis. Journal of Experimental Botany 56, 2465–2476.
| The small GTPase AtRAC2/ROP7 is specifically expressed during late stages of xylem differentiation in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtVahtLjI&md5=de95ef477a6f58156445c3ed621c801aCAS |
Brugnera E, Haney L, Grimsley C, Lu M, Walk SF, Tosello-Trampont AC, Macara IG, Madhani H, Fink GR, Ravichandran KS (2002) Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex. Nature Cell Biology 4, 574–582.
Eklund DM, Svensson EM, Kost B (2010) Physcomitrella patens: a model to investigate the role of RAC/ROP GTPase signalling in tip growth. Journal of Experimental Botany 61, 1917–1937.
| Physcomitrella patens: a model to investigate the role of RAC/ROP GTPase signalling in tip growth.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXlsFGjsLg%3D&md5=df68c853d57caf50808d2ce894f03408CAS |
Gu Y, Li S, Lord EM, Yang Z (2006) Members of a novel class of Arabidopsis Rho guanine nucleotide exchange factors control Rho GTPase-dependent polar growth. The Plant Cell 18, 366–381.
| Members of a novel class of Arabidopsis Rho guanine nucleotide exchange factors control Rho GTPase-dependent polar growth.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xhs1egu7g%3D&md5=8d279c79d41aefea7eb5d504735f1ab7CAS |
Guo L, Devaiah SP, Narasimhan R, Pan X, Zhang Y, Zhang W, Wang X (2012) Cytosolic glyceraldehyde-3-phosphate dehydrogenases interact with phospholipase Dδ to transduce hydrogen peroxide signals in the Arabidopsis response to stress. The Plant Cell 24, 2200–2212.
| Cytosolic glyceraldehyde-3-phosphate dehydrogenases interact with phospholipase Dδ to transduce hydrogen peroxide signals in the Arabidopsis response to stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhtVOktb3M&md5=cf40cef0d7a2654999c0ca07400c48aaCAS |
Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Molecular Cell 9, 789–798.
| Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XjsFWgsLY%3D&md5=0faed41e3c57fb5bc1215646ad4cd38eCAS |
Lemichez E, Wu Y, Sanchez JP, Mettouchi A, Mathur J, Chua N (2001) Inactivation of AtRac1 by abscisic acid is essential for stomatal closure. Genes & Development 15, 1808–1816.
| Inactivation of AtRac1 by abscisic acid is essential for stomatal closure.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXlsFanu7o%3D&md5=73cf66f105525466937eebd9c1af02cdCAS |
Leonard DA, Evans T, Hart M, Cerione RA, Manor D (1994) Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy. Biochemistry 33, 12323–12328.
| Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXlvFyru7Y%3D&md5=729e01c37c89c07c8b7541e500324cc0CAS |
Li ZX, Liu D (2012) ROPGEF1 and ROPGEF4 are functional regulators of ROP11 GTPase in ABA-mediated stomatal closure in Arabidopsis. FEBS Letters 586, 1253–1258.
| ROPGEF1 and ROPGEF4 are functional regulators of ROP11 GTPase in ABA-mediated stomatal closure in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38Xlt1ylt7o%3D&md5=53f3f7f638cefeb49c8d69bfd4b83a58CAS |
Liu Y, Su Y, Wang X (2013) Phosphatidic acid-mediated signaling. Advances in Experimental Medicine and Biology 991, 159–176.
| Phosphatidic acid-mediated signaling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhvVWmsL%2FN&md5=e0dac4f3b93fef3887b34a4884c68bdaCAS |
Manser E, Loo TH, Koh CG, Zhao ZS, Chen XQ, Tan L, Tan I, Leung T, Lim L (1998) PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Molecular Cell 1, 183–192.
| PAK kinases are directly coupled to the PIX family of nucleotide exchange factors.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXhs1yjurw%3D&md5=3cb86ace548144bb7a3fc52722dfd815CAS |
Meller N, Irani-Tehrani M, Kiosses WB, Del Pozo MA, Schwartz MA (2002) Zizimin1, a novel Cdc42 activator, reveals a new GEF domain for Rho proteins. Nature Cell Biology 4, 639–647.
| Zizimin1, a novel Cdc42 activator, reveals a new GEF domain for Rho proteins.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xms1aht78%3D&md5=ba80b4ae1132187fa13c8159253bc651CAS |
Mishra G, Zhang W, Deng F, Zhao J, Wang X (2006) A bifurcating pathway directs abscisic acid effects on stomatal closure and opening in Arabidopsis. Science 312, 264–266.
| A bifurcating pathway directs abscisic acid effects on stomatal closure and opening in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XjtlOjsr0%3D&md5=70116b45ac86a62e97a7d601110e9e37CAS |
Nishikimi A, Fukuhara H, Su WJ, Hongu T, Takasuga S, Mihara H, Cao QH, Fumiyuki S, Kanai M, Hasegawa H, Tanaka Y, Shibasaki M, Kanaho Y, Sasaki T, Frohman MA, Fukui Y (2009) Sequential regulation of DOCK2 dynamics by two phospholipids during neutrophil chemotaxis. Science 324, 384–387.
| Sequential regulation of DOCK2 dynamics by two phospholipids during neutrophil chemotaxis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXksVOlsbc%3D&md5=fc2e5e8fbf79a16e288f9895ceb9c9a2CAS |
Nomanbhoy TK, Leonard DA, Manor D, Cerione RA (1996) Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using extrinsic reporter group fluorescence. Biochemistry 35, 4602–4608.
| Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using extrinsic reporter group fluorescence.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28Xhs12nu7s%3D&md5=e394e97abbc5b585114e8c7de4923f2cCAS |
Rossman KL, Der CJ, Sondek J (2005) GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nature Reviews. Molecular Cell Biology 6, 167–180.
| GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXoslOmtA%3D%3D&md5=7fec70ac0f70e18f07d1b5a2ea16df10CAS |
Sanematsu F, Nishikimi A, Watanabe M, Hongu T, Tanaka Y, Kanaho Y, Côté JF, Fukui Y (2013) Phosphatidic acid-dependent recruitment and function of the Rac activator DOCK1 during dorsal ruffle formation. Journal of Biological Chemistry 288, 8092–8100.
| Phosphatidic acid-dependent recruitment and function of the Rac activator DOCK1 during dorsal ruffle formation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXksVyqtbk%3D&md5=e13803b20931331520867ab292afc863CAS |
Waadt R, Schmidt LK, Lohse M, Hashimoto K, Bock R, Kudla J (2008) Multicolor bimolecular fluorescence complement reveals simultaneous formation of alternative C complexes in planta. The Plant Journal 56, 505–516.
| Multicolor bimolecular fluorescence complement reveals simultaneous formation of alternative C complexes in planta.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsVGmtbvK&md5=fd75483862cc5dfc623aeb49062363e5CAS |
Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. The Plant Journal 40, 428–438.
| Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhtVShurnE&md5=a9e1981453f98bea82619b69609b5ac0CAS |
Wang X, Devaiah SP, Zhang W, Welti R (2006) Signaling functions of phosphatidic acid. Progress in Lipid Research 45, 250–278.
| Signaling functions of phosphatidic acid.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XjvVWqtrY%3D&md5=2c486fd4dcca9359267d0cc0d8f48315CAS |
Wang X, Su Y, Liu Y, Kim SC, Fanella B (2014) Phosphatidic acid as lipid messenger and growth regulators in plants. In ‘Signaling and communication in plants. Vol. 20’. pp. 69–92. (Springer: Berlin)
Wu G, Li H, Yang Z (2000) Arabidopsis RopGAPs are a novel family of Rho GTPase-activating proteins that require the Cdc42/Rac-interactive binding motif for Rop-specific GTPase stimulation. Plant Physiology 124, 1625–1636.
| Arabidopsis RopGAPs are a novel family of Rho GTPase-activating proteins that require the Cdc42/Rac-interactive binding motif for Rop-specific GTPase stimulation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXitVWksA%3D%3D&md5=02526ffdbef2055c16f5d0c6e1cd6217CAS |
Yalovsky S (2015) Protein lipid modifications and the regulation of ROP GTPase function. Journal of Experimental Botany 66, 1617–1624.
| Protein lipid modifications and the regulation of ROP GTPase function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXitVOks73F&md5=b28c3170037cc289dadf3cc9c44e3309CAS |
Yan M, Jing W, Xu N, Shen L, Zhang Q, Zhang W (2016) Arabidopsis thaliana constitutively active ROP11 interacts with the NADPH oxidase respiratory burst oxidase homologue F to regulate reactive oxygen species production in root hairs. Functional Plant Biology 43, 221–231.
| Arabidopsis thaliana constitutively active ROP11 interacts with the NADPH oxidase respiratory burst oxidase homologue F to regulate reactive oxygen species production in root hairs.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC28XivVekurk%3D&md5=fe9b50d23cba073fabbad5f46a9fb4ccCAS |
Yang Z (2002) Small GTPases: versatile signaling switches in plants. The Plant Cell 14, S375–S388.
Yang Z, Fu Y (2007) ROP/RAC GTPase signaling. Current Opinion in Plant Biology 10, 490–494.
| ROP/RAC GTPase signaling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtFekurjI&md5=fa34bc6b5e2a95199166e815dc23af13CAS |
Zhang Y, Du G (2009) Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases. Biochimica et Biophysica Acta 1791, 850–855.
| Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtVensbbE&md5=e5f1ca7155f81c9b9e26524157788296CAS |
Zhang Y, McCormick S (2007) A distinct mechanism regulating a pollen-specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 104, 18830–18835.
| A distinct mechanism regulating a pollen-specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtl2ksrfE&md5=d3d0f4873e12fc64ae716702c2271466CAS |
Zhang WH, Qin C, Zhao J, Wang XM (2004) Phospholipase Dα1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and regulates abscisic acid signaling. Proceedings of the National Academy of Sciences of the United States of America 101, 9508–9513.
| Phospholipase Dα1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and regulates abscisic acid signaling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXltlKjtr4%3D&md5=e09582dc1682ba1f065c9e3d70131480CAS |
Zhang Y, Zhu H, Zhang Q, Li M, Yan M, Wang R, Wang L, Welti R, Zhang W, Wang X (2009) Phospholipase D α1 and phosphatidic acid regulate NADPH oxidase activity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis. The Plant Cell 21, 2357–2377.
| Phospholipase D α1 and phosphatidic acid regulate NADPH oxidase activity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXht12qsLrI&md5=0b21cff0c751e1fbf61cbdd18c3083b5CAS |
Zhao J (2015) Phospholipase D and phosphatidic acid in plant defence response: from protein–protein and lipid–protein interactions to hormone signaling. Journal of Experimental Botany 66, 1721–1736.
| Phospholipase D and phosphatidic acid in plant defence response: from protein–protein and lipid–protein interactions to hormone signaling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXitVGjs7zL&md5=2e164d7cbc0b41a481b0629678c4dcdcCAS |
Zheng Z, Nafisi M, Tam A, Li H, Crowell D, Chary N, Schroeder J, Shen J, Yang Z (2002) Plasma membrane–associated ROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis. The Plant Cell 14, 2787–2797.
| Plasma membrane–associated ROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XovF2qur0%3D&md5=39bb7b2f0ac8b5c8d945e45a63474821CAS |
