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• W2516044865 abstract "PIP1;2 and PIP2;1 are aquaporins that are highly expressed in roots and bring a major contribution to root water transport and its regulation by hormonal and abiotic factors. Interactions between cellular proteins or with other macromolecules contribute to forming molecular machines. Proteins that molecularly interact with PIP1;2 and PIP2;1 were searched to get new insights into regulatory mechanisms of root water transport. For that, a immuno-purification strategy coupled to protein identification and quantification by mass spectrometry (IP-MS) of PIPs was combined with data from the literature, to build thorough PIP1;2 and PIP2;1 interactomes, sharing about 400 interacting proteins. Such interactome revealed PIPs to behave as a platform for recruitment of a wide range of transport activities and provided novel insights into regulation of PIP cellular trafficking by osmotic and oxidative treatments. This work also pointed a role of lipid signaling in PIP function and enhanced our knowledge of protein kinases involved in PIP regulation. In particular we show that 2 members of the receptor-like kinase (RLK) family (RKL1 (At1g48480) and Feronia (At3g51550)) differentially modulate PIP activity through distinct molecular mechanisms. The overall work opens novel perspectives in understanding PIP regulatory mechanisms and their role in adjustment of plant water status. PIP1;2 and PIP2;1 are aquaporins that are highly expressed in roots and bring a major contribution to root water transport and its regulation by hormonal and abiotic factors. Interactions between cellular proteins or with other macromolecules contribute to forming molecular machines. Proteins that molecularly interact with PIP1;2 and PIP2;1 were searched to get new insights into regulatory mechanisms of root water transport. For that, a immuno-purification strategy coupled to protein identification and quantification by mass spectrometry (IP-MS) of PIPs was combined with data from the literature, to build thorough PIP1;2 and PIP2;1 interactomes, sharing about 400 interacting proteins. Such interactome revealed PIPs to behave as a platform for recruitment of a wide range of transport activities and provided novel insights into regulation of PIP cellular trafficking by osmotic and oxidative treatments. This work also pointed a role of lipid signaling in PIP function and enhanced our knowledge of protein kinases involved in PIP regulation. In particular we show that 2 members of the receptor-like kinase (RLK) family (RKL1 (At1g48480) and Feronia (At3g51550)) differentially modulate PIP activity through distinct molecular mechanisms. The overall work opens novel perspectives in understanding PIP regulatory mechanisms and their role in adjustment of plant water status. The absorption of soil water by roots is crucial for plants to maintain their water status. Studies in various plant species have shown that the root water permeability (root hydraulic conductivity; Lpr 1The abbreviations used are:Lprroot hydraulic conductivityABAabscisic acidBRbrassinosteroidFLIMFluorescence Lifetime Imaging MicroscopyFRETFörster resonance energy transferGOGene OntologyIPimmuno-purificationLRRLeucine-rich-repeatNIPnodulin-26-like proteinPAphosphatidic acidPfosmotic water permeabilityPIPplasma membrane intrinsic proteinPKprotein kinaseRLKreceptor-like-kinaseSIPsmall basic intrinsic proteinTIPtonoplast intrinsic proteinTSPOtryptophan-rich sensory protein/translocator.) is constantly adjusted depending on the developmental stage of the plant, its nutritional or hormonal status, or multiple environmental stimuli (1.Di Pietro M. Vialaret J. Li G-W Hem S. Prado K. Rossignol M. Maurel C. Santoni V. Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots.Mol. Cell. Proteomics. 2013; 12: 3886-3897Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 2.Maurel C. Verdoucq L. Luu D-T Santoni V. Plant aquaporins: membrane channels with multiple integrated functions.Annu. Rev. Plant Biol. 2008; 59: 595-624Crossref PubMed Scopus (928) Google Scholar). These adjustments depend in large part on the function and regulation of aquaporins, a large class of channel proteins that facilitate the diffusion of water and small neutral solutes across cell membranes (2.Maurel C. Verdoucq L. Luu D-T Santoni V. Plant aquaporins: membrane channels with multiple integrated functions.Annu. Rev. Plant Biol. 2008; 59: 595-624Crossref PubMed Scopus (928) Google Scholar, 3.Kaldenhoff R. Ribas-Carbo M. Flexas J. Lovisolo C. Heckwolf M. Uehlein N. Aquaporins and plant water balance.Plant Cell Environ. 2008; 31: 658-666Crossref PubMed Scopus (223) Google Scholar). Aquaporins are 25–30 kDa proteins with 6 membrane-spanning domains and five connecting loops (A-E), the N- and C-terminal tails being exposed to the cytosol (4.Tornroth-Horsefield S. Wang Y. Hedfalk K. Johanson U. Karlsson M. Tajkhorshid E. Neutze R. Kjellbom P. Structural mechanism of plant aquaporin gating.Nature. 2006; 439: 688-694Crossref PubMed Scopus (655) Google Scholar). Plant aquaporins show a high multiplicity of isoforms. Thirty-five homologs comprised in four homology subclasses have been identified in Arabidopsis. The plasma membrane intrinsic proteins (PIPs) (with 13 isoforms further subdivided in the PIP1 and PIP2 subgroups), and the tonoplast intrinsic proteins (TIPs) (with 10 homologs) are the most abundant aquaporins in the plasma membrane and the tonoplast, respectively (5.Johanson U. Gustavsson S. A new subfamily of major intrinsic proteins in plants.Mol. Biol. Evol. 2002; 19: 456-461Crossref PubMed Scopus (114) Google Scholar, 6.Quigley F. Rosenberg J.M. Shachar-Hill Y. Bohnert H.J. From genome to function: the Arabidopsis aquaporins.Genome Biol. 2002; 3: 1-17Google Scholar). Two other subclasses include nodulin-26-like proteins (NIPs) and small basic intrinsic proteins (SIPs), with nine and three homologs, respectively (5.Johanson U. Gustavsson S. A new subfamily of major intrinsic proteins in plants.Mol. Biol. Evol. 2002; 19: 456-461Crossref PubMed Scopus (114) Google Scholar, 6.Quigley F. Rosenberg J.M. Shachar-Hill Y. Bohnert H.J. From genome to function: the Arabidopsis aquaporins.Genome Biol. 2002; 3: 1-17Google Scholar, 7.Ishikawa F. Suga S. Uemura T. Sato M.H. Maeshima M. Novel type aquaporins SIPs are mainly localized to the ER membrane and show cell-specific expression in Arabidopsis thaliana.Febbs Let. 2005; 579: 5814-5820Crossref PubMed Scopus (168) Google Scholar). root hydraulic conductivity abscisic acid brassinosteroid Fluorescence Lifetime Imaging Microscopy Förster resonance energy transfer Gene Ontology immuno-purification Leucine-rich-repeat nodulin-26-like protein phosphatidic acid osmotic water permeability plasma membrane intrinsic protein protein kinase receptor-like-kinase small basic intrinsic protein tonoplast intrinsic protein tryptophan-rich sensory protein/translocator. The response of plant roots to environmental and hormonal stimuli is mediated through long-term transcriptional control of aquaporin functions, together with multiple post-translational mechanisms, such as phosphorylation, that affect the activity of aquaporins, their targeting to their destination compartment, or their stability. PIP aquaporins show a conserved phosphorylation site in their first cytosolic loop (loop B) and, in the case of PIP2 isoforms, multiple phosphorylations in adjacent sites of their C-terminal tail (1.Di Pietro M. Vialaret J. Li G-W Hem S. Prado K. Rossignol M. Maurel C. Santoni V. Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots.Mol. Cell. Proteomics. 2013; 12: 3886-3897Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), PhosPhAt database (http://phosphat.mpimp-golm.mpg.de/). Aquaporin phosphorylation is a significant component of plant responses to stresses. For instance, following exposure of Arabidopsis roots to salt (NaCl) or hydrogen peroxide (H2O2), AtPIP2;1 phosphorylation was decreased and increased, respectively (8.Prak S. Hem S. Boudet J. Viennois J. Sommerer N. Rossignol R. Maurel C. Santoni V. Multiple phosphorylations in the C-terminal tail of plant plasma membrane aquaporins. Role in sub-cellular trafficking of AtPIP2;1 in response to salt stress.Mol. Cell. Proteomics. 2008; 7: 1019-1030Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). A recent quantitative phosphoproteomic work showed a strong correlation between the level of PIP phosphorylation and Lpr, under a wide range of environmental conditions (1.Di Pietro M. Vialaret J. Li G-W Hem S. Prado K. Rossignol M. Maurel C. Santoni V. Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots.Mol. Cell. Proteomics. 2013; 12: 3886-3897Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). However, knowledge of the protein kinases (PKs) that phosphorylate aquaporins is still scarce. Two PKs that phosphorylate SoPIP2;1 at Ser115 and Ser274 have been purified from spinach (9.Sjovall-Larsen S. Alexandersson E. Johansson I. Karlsson M. Johanson U. Kjellbom P. Purification and characerization of two protein kinases acting on the aquaporin SoPIP2;1.Biochim. Biophys. Acta. 2006; 1758: 1157-1164Crossref PubMed Scopus (27) Google Scholar). A very recent work revealed that Open stomata 1 (OST1)/Snf1-related PK 2.6 (SnRK2.6), a central PK in guard cell abscisic acid (ABA) signaling, was able to phosphorylate a cytosolic AtPIP2;1 peptide at Ser121, this modification being necessary during ABA-induced stomatal closure (10.Grondin A. Rodrigues O. Verdoucq L. Merlot S. Leonhardt N. Maurel C. Aquaporins contribute to ABA-triggered stomatal closure through OST1-mediated phosphorylation.Plant Cell. 2015; 27: 1-11Crossref PubMed Scopus (205) Google Scholar). Receptor-like-kinases (RLKs) constitute a class of serine/threonine kinases that perceive environmental and extracellular developmental signals and transduce them via their intracellular kinase domain (11.Morillo S.A. Tax F.E. Functional analysis of receptor-like kinases in monocots and dicots.Curr. Opin. Plant Biol. 2006; 9: 460-469Crossref PubMed Scopus (168) Google Scholar). Two RLKs, SIRK1 (At5g10020) and BSK8 (At5g41260), a leucine-rich-repeat (LRR)-RLK and a receptor-like cytoplasmic kinase (RLCK), respectively, were shown to act on PIPs (12.Niittylä T. Fuglsang A.T. Palmgren M.G. Frommer W.B. Schulze W.X. Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis.Mol. Cell. Proteomics. 2007; 6: 1711-1726Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 13.Wu X.N. Sanchez-Rodriguez C. Pertl-Obermeyer H. Obermeyer G. Schulze W.X. Sucrose-induced receptor kinase SIRK1 regulates plasma membrane aquaporins in Arabidopsis.Mol. Cell. Proteomics. 2013; 12: 2856-2873Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In particular, phosphorylation of five PIPs (AtPIP2;1–2;4, AtPIP2;7) upon stimulation by sucrose of carbon-starved seedlings was reduced in the sirk1 mutant, and SIRK1 was confirmed to phosphorylate AtPIP2;4 at Ser283 and Ser286. A rice RLK (LP2, Os02g40240), was recently shown to interact with three PIPs in vivo (14.Wu F.Q. Sheng P.K. Tan J.J. Chen X.L. Lu G.W. Ma W.W. Heng Y.Q. Lin Q.B. Zhu S.S. Wang J.L. Wang J. Guo X.P. Zhang X. Lei C.L. Wan J.M. Plasma membrane receptor-like kinase leaf panicle 2 acts downstream of the drought and salt tolerance transcription factor to regulate drought sensitivity in rice.J. Exp. Bot. 2015; 66: 271-281Crossref PubMed Scopus (70) Google Scholar) but the functional role of this interactions remains unknown. The identification of cellular interaction partners is fundamental for understanding cellular and physiological processes. In recent years, crucial experimental approaches for protein interaction mapping such as yeast two hybrid or split ubiquitin, have begun to unravel the complex interacting networks of plant proteins (15.Chen J. Lalonde S. Obrdlik P. Noorani Vatani A. Parsa S.A. Vilarino C. Revuelta J.L. Frommer W.B. Rhee S.Y. Uncovering Arabidopsis membrane protein interactome enriched in transporters using mating-based split ubiquitin assays and classification models.Front. Plant Sci. 2012; 3: 124Crossref PubMed Scopus (36) Google Scholar, 16.Jones A.M. Xuan Y.H. Xu M. Wang R.S. Ho C.H. Lalonde S. You C.H. Sardi M.I. Parsa S.A. Smith-Valle E. Su T.Y. Frazer K.A. Pilot G. Pratelli R. Grossmann G. Acharya B.R. Hu H.C. Engineer C. Villiers F. Ju C.L. Takeda K. Su Z. Dong Q.F. Assmann S.M. Chen J. Kwak J.M. Schroeder J.I. Albert R. Rhee S.Y. Frommer W.B. Border control - A membrane-linked interactome of Arabidopsis.Science. 2014; 344: 711-716Crossref PubMed Scopus (159) Google Scholar, 17.Lalonde S. Sero A. Pratelli R. Pilot G. Chen J. Sardi M.I. Parsa S.A. Kim D.Y. Acharya B.R. Stein E.V. Hu H.C. Villiers F. Takeda K. Yang Y. Han Y.S. et al.A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2.Front. Physiol. 2010; 1: 24Crossref PubMed Scopus (109) Google Scholar, 18.Braun P. Carvunis A.R. Charloteaux B. Dreze M. Ecker J.R. Hill D.E. Roth F.P. Vidal M. Galli M. Balumuri P. Bautista V. Chesnut J.D. Kim R.C. de los Reyes C. Gilles P. Kim C.J. Matrubutham U. Mirchandani J. Olivares E. Patnaik S. Quan R. Ramaswamy G. Shinn P. Swamilingiah G.M. Wu S. Byrdsong D. Dricot A. Duarte M. Gebreab F. Gutierrez B.J. MacWilliams A. Monachello D. Mukhtar M.S. Poulin M.M. Reichert P. Romero V. Tam S. Waaijers S. Weiner E.M. Cusick M.E. Tasan M. Yazaki J. Ahn Y.Y. Barabasi A.L. Chen H.M. Dangl J.L. Fan C.Y. Gai L.T. Ghoshal G. Hao T. Lurin C. Milenkovic T. Moore J. Pevzner S.J. Przulj N. Rabello S. Rietman E.A. Rolland T. Santhanam B. Schmitz R.J. Spooner W. Stein J. Vandenhaute J. Ware D. Arabidopsis Interactome Mapping, C Evidence for network evolution in an Arabidopsis Interactome map.Science. 2011; 333: 601-607Crossref PubMed Google Scholar). Analysis of protein complexes through immuno-purification (IP) followed by mass spectrometry (MS) (19.Dedecker M. Van Leene J. De Jaeger G. Unravelling plant molecular machineries through affinity purification coupled to mass spectrometry.Cur. Op. Plant Biol. 2015; 24: 1-9Crossref PubMed Scopus (22) Google Scholar) is also a widely employed technique because of its high throughput and sensitivity. Most importantly, this technique addresses the properties of protein-protein interactions occurring in the plant. However, suitable controls and quantitative proteomics are required to distinguish between bona fide binders and background contaminants (20.Marcilla M. Albar J.P. Quantitative proteomics: A strategic ally to map protein interaction networks.IUBMB Life. 2013; 65: 9-16Crossref PubMed Scopus (17) Google Scholar). Data on plant aquaporin interactomes are starting to emerge. Yeast-two hybrid (18.Braun P. Carvunis A.R. Charloteaux B. Dreze M. Ecker J.R. Hill D.E. Roth F.P. Vidal M. Galli M. Balumuri P. Bautista V. Chesnut J.D. Kim R.C. de los Reyes C. Gilles P. Kim C.J. Matrubutham U. Mirchandani J. Olivares E. Patnaik S. Quan R. Ramaswamy G. Shinn P. Swamilingiah G.M. Wu S. Byrdsong D. Dricot A. Duarte M. Gebreab F. Gutierrez B.J. MacWilliams A. Monachello D. Mukhtar M.S. Poulin M.M. Reichert P. Romero V. Tam S. Waaijers S. Weiner E.M. Cusick M.E. Tasan M. Yazaki J. Ahn Y.Y. Barabasi A.L. Chen H.M. Dangl J.L. Fan C.Y. Gai L.T. Ghoshal G. Hao T. Lurin C. Milenkovic T. Moore J. Pevzner S.J. Przulj N. Rabello S. Rietman E.A. Rolland T. Santhanam B. Schmitz R.J. Spooner W. Stein J. Vandenhaute J. Ware D. Arabidopsis Interactome Mapping, C Evidence for network evolution in an Arabidopsis Interactome map.Science. 2011; 333: 601-607Crossref PubMed Google Scholar) and split-ubiquitin (15.Chen J. Lalonde S. Obrdlik P. Noorani Vatani A. Parsa S.A. Vilarino C. Revuelta J.L. Frommer W.B. Rhee S.Y. Uncovering Arabidopsis membrane protein interactome enriched in transporters using mating-based split ubiquitin assays and classification models.Front. Plant Sci. 2012; 3: 124Crossref PubMed Scopus (36) Google Scholar, 16.Jones A.M. Xuan Y.H. Xu M. Wang R.S. Ho C.H. Lalonde S. You C.H. Sardi M.I. Parsa S.A. Smith-Valle E. Su T.Y. Frazer K.A. Pilot G. Pratelli R. Grossmann G. Acharya B.R. Hu H.C. Engineer C. Villiers F. Ju C.L. Takeda K. Su Z. Dong Q.F. Assmann S.M. Chen J. Kwak J.M. Schroeder J.I. Albert R. Rhee S.Y. Frommer W.B. Border control - A membrane-linked interactome of Arabidopsis.Science. 2014; 344: 711-716Crossref PubMed Scopus (159) Google Scholar) studies have identified, about 200 proteins that seem to interact, with a high confidence, with PIP aquaporins ((21.Maurel C. Boursiac Y. Luu D-T Santoni V. Shahzad Z. Verdoucq L. Aquaporins in Plants.Physiol. Rev. 2015; 95: 1321-1358Crossref PubMed Scopus (481) Google Scholar) for review). In addition, more focused recent studies have revealed that PIPs can functionally interact with several classes of proteins. For instance, PIP1-PIP2 interactions were shown to be required for in planta trafficking of PIP1s to the plasma membrane (22.Chen W. Yin X. Wang L. Tian J. Yang R.Y. Liu D.F. Yu Z.H. Ma N. Gao J.P. Involvement of rose aquaporin RhPIP1;1 in ethylene-regulated petal expansion through interaction with RhPIP2;1.Plant Mol. Biol. 2013; 83: 219-233Crossref PubMed Scopus (63) Google Scholar, 23.Li D.D. Ruan X.M. Zhang J. Wu Y.J. Wang X.L. Li X.B. Cotton plasma membrane intrinsic protein 2s (PIP2s) selectively interact to regulate their water channel activities and are required for fibre development.New Phytol. 2013; 199: 695-707Crossref PubMed Scopus (71) Google Scholar, 24.Zelazny E. Borst J.W. Muylaert M. Batoko H. Hemminga M.A. Chaumont F. FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12359-12364Crossref PubMed Scopus (265) Google Scholar). PIP2s were also shown to functionally interact with syntaxins, a family of proteins involved in vesicle trafficking (25.Besserer A. Burnotte E. Bienert G.P. Chevalier A.S. Errachid A. Grefen C. Blatt M.R. Chaumont F. Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121.Plant Cell. 2012; 24: 3463-3481Crossref PubMed Scopus (98) Google Scholar, 26.Hachez C. Laloux T. Reinhardt H. Cavez D. Degand H. Grefen C. De Rycke R. Inze D. Blatt M.R. Russinova E. Chaumont F. Arabidopsis SNAREs SYP61 and SYP121 coordinate the trafficking of plasma membrane aquaporin PIP2;7 to modulate the cell membrane water permeability.Plant Cell. 2014; 26: 3132-3147Crossref PubMed Scopus (127) Google Scholar). In addition, the tryptophan-rich sensory protein/translocator (TSPO), a multistress regulator that is transiently induced by osmotic stress, and that is degraded through a selective autophagic pathway, physically interact with AtPIP2;7 (27.Hachez C. Veljanovski V. Reinhardt H. Guillaumot D. Vanhee C. Chaumont F. Batoko H. The Arabidopsis abiotic stress-induced TSPO-related protein reduces cell-surface expression of the aquaporin PIP2;7 through protein-protein interactions and autophagic degradation.Plant Cell. 2014; 26: 4974-4990Crossref PubMed Scopus (99) Google Scholar). PIPs also functionally interact with Rma1H1, a membrane-anchor E3 ubiquitin ligase homolog, to regulate aquaporin levels via ubiquitination (28.Lee H.K. Cho S.K. Son O. Xu Z. Hwang I. Kim W.T. Drought stress-induced Rma1H1, a RING membrane-anchor E3 ubiquitin ligase homolog, regulates aquaporin levels via ubiquitination in transgenic Arabidopsis plants.Plant Cell. 2009; 21: 622-641Crossref PubMed Scopus (241) Google Scholar). One major objective of the present work was to investigate as a whole the plant PIP1;2 and PIP2;1 interactome. A quantitative IP-MS strategy, together with data from available databases, allowed to build an interconnected PIP network of about 900 proteins. Next, we focused on those protein interaction partners (next called interactants) that show a physical interaction with PIPs. We hypothesized that these interactants may provide novel insights into the molecular regulation of PIP aquaporins. Here, we explore novel functional roles of phospholipases D and RLKs. The latter can have opposite effects on aquaporin activity through specific molecular mechanisms. Arabidopsis thaliana (Col-0 ecotype) transgenic plants expressing GFP, GFP-PIP2;1, GFP-PIP1;2 under the control of a constitutive 35S promotor were used (29.Cutler S.R. Ehrhardt D.W. Griffitts J.S. Sommerville C.R. Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 3718-3723Crossref PubMed Scopus (770) Google Scholar) for proteomic analysis (see below). Arabidopsis seeds were sown in vitro on a MS/2 medium (30.Murashige T. Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol. Plant. 1962; 15: 473-497Crossref Scopus (53704) Google Scholar) complemented with 1% sucrose, 0.05% MES and 7 g/l agar. Seeds were kept at 4 °C for 48 h and cultivated in vitro during 9 days (16 h light (250 μmol photons/m2/s), 20 °C, 70% relative humidity). The effect of NaCl and H2O2 were studied by bathing plantlets with 100 mm NaCl during 2 h, and 500 μm H2O2 for 20 min, respectively. Additional transgenic plants were used: promAMT1;3::AMT1;3-GFP (31.Lima J.E. Kojima S. Takahashi H. von Wiren N. Ammonium triggers lateral root branching in Arabidopsis in an ammonium transporter1;3-dependent manner.Plant Cell. 2010; 22: 3621-3633Crossref PubMed Scopus (248) Google Scholar), promPGP4::PGP4-GFP in a pgp4 background (32.Cho M. Lee S.H. Cho H.T. P-glycoprotein4 displays auxin efflux transporter-like action in Arabidopsis root hair cells and tobacco cells.Plant Cell. 2007; 19: 3930-3943Crossref PubMed Scopus (172) Google Scholar), promPGP19::GFP-PGP19 (33.Lewis D.R. Wu G.S. Ljung K. Spalding E.P. Auxin transport into cotyledons and cotyledon growth depend similarly on the ABCB19 multidrug resistance-like transporter.Plant J. 2009; 60: 91-101Crossref PubMed Scopus (41) Google Scholar). Nicotiana tabacum plants were cultivated in soil for 4–6 weeks (8 h light (120 μmol photons/m2/s, 20 °C, 65% relative humidity). All constructs were obtained using Gateway cloning technology (Invitrogen) according to the manufacturer's instructions. The cDNAs of RKL1 (At1g48480), RLK902 (At3g17840), Feronia (At3g51550), PLDδ (At4g35790), PLDγ1 (At4g11850), and NHL3 (At5g06320) were amplified by PCR using the primers described in supplemental Table S1 followed by a second PCR with the primers AttB1 or AttB1′ and AttB2 or AttB2′ (supplemental Table S1) allowing the addition of attB recombination sites and cloned into a pDONOR 207 vector using a Gateway® BP Clonase enzyme mix (Invitrogen). Annexin4 clone (At2g38750) was obtained from ARBC (U15576 clone). For FLIM experiments, cDNAs were transferred into binary destination vectors pGWB5 and pGWB6 (Dr. Nakagawa, Shimane University, Matsue, Japan) to allow fusion of eGFP at the C- and N terminus of the proteins of interest, respectively, by using a Gateway® LR Clonase enzyme mix (Invitrogen). To fuse mRFP at the C terminus of the proteins, cDNAs were transferred into a pB7WGR2 vector. GFP- and mCherry- tagged PIP2;1 are described in (34.Boursiac Y. Chen S. Luu D-T Sorieul M. van den Dries N. Maurel C. Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression.Plant Physiol. 2005; 139: 790-805Crossref PubMed Scopus (436) Google Scholar, 35.Wudick M.M. Li X. Valentini V. Geldner N. Chory J. Lin J. Maurel C. Doan-Trung L. Subcellular redistribution of root aquaporins induced by hydrogen peroxide.Mol. Plant. 2015; 8: 1103-1114Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). A. tumefaciens strain GV3101 was transformed with the constructs of interest, selected for resistance toward rifampicin (50 mg/l), gentamycin (25 mg/l), and kanamycin (50 mg/l) in the case of pGWB vectors, and for resistance toward spectinomycin (100 mg/l) in the case of pB7WGR2 vector. For oocytes experiments, cDNAs were transferred into a xenopus oocyte expression vector pGEM-GWC using a Gateway® LR Clonase enzyme mix (Invitrogen) and E. Coli strain DH5α was transformed and selected for ampicillin (50 mg/l) resistance. IP of GFP-tagged proteins was performed at 4 °C from 9 day-old plantlets. Plantlets were treated with 1% formaldehyde for 15 min under gentle shaking. Formaldehyde was then quenched with the addition of 300 mm glycine under continuous shaking. After for 30 min, plantlets were rinsed twice with phosphate-buffered saline (4 mm KH2PO4, 16 mm Na2HPO4, 115 mm NaCl, pH 7.4). One to two grams of roots were collected, chopped with a scalpel in the presence of 2.1 ml/g fresh weight (FW) of a buffer made of NaCl 300 mm, Triton X-100 1% (w/v), Na deoxycholate 0.5% (w/v), SDS 0.1% (w/v), Tris-HCl 100 mm pH 8, leupeptin 2 mm, DTT 5 mm, AEBSF 1 mm, and then potterised. After centrifugation at 10,000 × g for 15 min, the supernatant was again centrifuged in the same conditions. IP was performed from the supernatant with an antibody against GFP using a μMACSTM anti-GFP Microbeads kit (Miltenyi Biotec, Paris, France) according to manufacturer's conditions. Briefly, sample was incubated at 4 °C for 1h with a volume equivalent to 35 μl/g FW of an anti-GFP Microbeads solution. The sample was then loaded onto μMACS columns that were previously conditioned with 200 μl of a lysis buffer (NaCl 150 mm, Triton X-100 1% (w/v), Tris-HCl 50 mm pH 8). After 4 washings with 200 μl of a buffer made of NaCl 150 mm and Igepal CA-630 1% (v/v) and an additional washing step with 100 μl of Tris-HCl 20 mm pH 7,5, proteins were eluted with 50 μl of elution buffer (Tris-HCl 50 mm pH 6.8, DTT 50 mm, SDS 1% (w/v), EDTA 1 mm, bromophenol blue 0.005% (v/v), glycerol 10% (v/v)). Formaldehyde fixation was reversed by heating eluted proteins at 100 °C during 20 min. In-solution reductions/alkylations were performed simultaneously with detergent removing by the filter-aided sample preparation protocol (1.Di Pietro M. Vialaret J. Li G-W Hem S. Prado K. Rossignol M. Maurel C. Santoni V. Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots.Mol. Cell. Proteomics. 2013; 12: 3886-3897Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 36.Wiœniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5043) Google Scholar). These steps were followed by a endolysin-C (Sequencing Grade Modified, Promega, Madison, WI) digestion 3 h at 37 °C followed by a trypsin (Sequencing Grade Modified, Promega) digestion overnight at 37 °C. Peptides were eluted by step elutions with 50 mm ammonium bicarbonate, followed by 50% acetonitrile and then 0.5 m NaCl. Peptides were desalted on C18 columns (Sep-Pak® VactC18 cartridge 3cc, Waters, Guyancourt, France). After solvent evaporation, peptides were resuspended in 2% formic acid. The protein digests were analyzed using a Q-TOF mass spectrometer (Maxis Impact; Bruker Daltonik GmbH, Bremen, Germany), interfaced with a nano-HPLC U3000 system (Thermo Scientific, Waltham, MA). Samples were concentrated with a pre-column (Thermo Scientific, C18 PepMap100, 300 μm × 5 mm, 5 μm, 100 A) at a flow rate of 20 μl/min using 0.1% formic acid. After preconcentration, peptides were separated with a reversed-phase capillary column (Thermo Scientific, C18 PepMap100, 75 μm × 250 mm, 3 μm, 100 A) at a flow rate of 0.3 μl/min using a two steps gradient (8% to 28% acetonitrile in 40 min, then 28% to 42% in 10 min), and eluted directly into the mass spectrometer. Proteins were identified by MS/MS by information-dependent acquisition of fragmentation spectra of multiple charged peptides. Up to twenty data-dependent MS/MS spectra were acquired in positive ion mode. MS/MS raw data were analyzed using Bruker Compass Data Analysis software (Automatic Engine Version 4.1 (Build 359)) to generate the peak lists. The non-redundant Arabidopsis protein database (TAIR10, version 20110823, 35386 entries, http://www.arabidopsis.org) was locally queried using X!Tandem search engine (version 2013.09.01; http://www.thegpm.org/tandem/) with the following parameters: trypsin as enzyme, 1 allowed missed cleavage, carbamidomethylation of cystein as fixed modification and N-terminal acetylation of protein, deamidation of asparagine and glutamine, N-terminal-pyroglutamylation of glutamine and glutamate, oxidation of methionine, phosphorylation of serine, threonine and tyrosine, and methylation of glutamate and aspartate as variable modifications. Mass tolerance was set to 10 ppm on full scans and 0.05 Da for fragment ions. Identified proteins were filtered and grouped using the X!Tandem pipeline v3.3.1 (http://pappso.inra.fr/bioinfo/xtandempipeline/). Identified proteins were filtered according to the following criteria: at least two different trypsin peptides with an E value below 0.05 and a protein E value smaller than 0.025 were required. Using the above criteria, the rate of false peptide sequence assignment and false protein identification as determined by the “decoy database” function implemented in X!tandem pipeline was lower than 1.5 and 5%, respectively. The X!tandem grouping function allows to take into account redundancy and proteins with at least one common peptide were grouped. Within each group, proteins with at least one specific peptide relative to other members of the group were reported as subgroups. Relative label-free quantification was carried out with the MassChroQ software (version 2.1) based on extracted ion chroma" @default.
• W2516044865 created "2016-09-16" @default.
• W2516044865 creator A5012893037 @default.
• W2516044865 creator A5022733184 @default.
• W2516044865 creator A5030079555 @default.
• W2516044865 creator A5054868166 @default.
• W2516044865 creator A5057108463 @default.
• W2516044865 creator A5067931598 @default.
• W2516044865 creator A5088916575 @default.
• W2516044865 date "2016-11-01" @default.
• W2516044865 modified "2023-10-18" @default.
• W2516044865 title "Novel Aquaporin Regulatory Mechanisms Revealed by Interactomics" @default.
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