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Plant, Cell and Environment (2015) 38, 1312–1320

doi: 10.1111/pce.12478

Original Article

The calcium-dependent protein kinase CPK7 acts on root hydraulic conductivity Guowei Li1*, Marie Boudsocq2†, Sonia Hem3, Jérôme Vialaret3‡, Michel Rossignol3, Christophe Maurel1 & Véronique Santoni1 1

Biochimie et Physiologie Moléculaire des Plantes, INRA/CNRS/SupAgro/UM2, UMR 5004, 2 Place Viala, Montpellier Cedex 1 34060, France 2Saclay Plant Sciences, Institut des Sciences du Végétal, UPR2355, 1 Avenue de la Terrasse, Gif-sur-Yvette Cedex 91198, France 3Laboratoire de Protéomique Fonctionnelle, UR1199, 1 Place Viala, Montpellier Cedex 1 34060, France

ABSTRACT

INTRODUCTION

The hydraulic conductivity of plant roots (Lpr) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post-translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lpr of knockout Arabidopsis plants for four Ca2+-dependent protein kinases. cpk7 plants showed a 30% increase in Lpr because of a higher aquaporin activity. A quantitative proteomic analysis of wild-type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lpr of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7dependent stability of specific membrane proteins.

The plant water status tightly depends on soil water availability and is constantly challenged by environmental variations. Short-term responses of plants to their environment thereby include rapid changes in their root water transport capacity (root hydraulic conductivity; Lpr). Lpr is determined in large part by the function of channel proteins called aquaporins, which mediate the transport across cell membranes of water and small neutral solutes (Boursiac et al. 2008; Kaldenhoff et al. 2008). Aquaporins form a multigenic family, with 35 members in Arabidopsis, subdivided in four subfamilies (Johanson et al. 2001; Quigley et al. 2002). One major subfamily is composed of plasma membrane intrinsic proteins (PIPs) and is subdivided in two homology groups, PIP1 and PIP2, with five and eight members, respectively, in Arabidopsis. Over the last decade, the application of welldeveloped proteomics and mass spectrometry techniques has led to a comprehensive description of co-translational and post-translational modifications of plant aquaporins (Santoni et al. 2006; Maurel 2007; Prak et al. 2008; Casado-Vela et al. 2010; Di Pietro et al. 2013). Phosphorylation of PIPs was specifically investigated in several species growing under various environmental conditions (Niittylä et al. 2007; Prak et al. 2008; Van Wilder et al. 2008; Kline et al. 2010; Di Pietro et al. 2013; Prado et al. 2013; Wu et al. 2013). In particular, PIPs of Arabidopsis showed multiple and interdependent phosphorylations at adjacent sites of their C-terminal tail (Prak et al. 2008), which were modulated by numerous stimuli including salt, oxidative stress or light (Di Pietro et al. 2013; Prado et al. 2013). Functional studies in plants or heterologous expression systems have shown that phosphorylation stimulates aquaporin water transport activity (Maurel et al. 1995; Johansson et al. 1998; Guenther et al. 2003; Prado et al. 2013). The molecular bases of PIP aquaporin gating, as elucidated from the atomic structure of spinach SoPIP2;1 (Tornroth-Horsefield et al. 2006), suggested a role for phosphorylation of cytosolic loop B and C-terminal residue Ser280 in water channel gating. Furthermore, phosphorylation of Arabidopsis AtPIP2;1 at Ser283 was shown to interfere with its subcellular localization in roots under resting and salt stress conditions (Prak et al. 2008). However, knowledge on the protein kinases and protein phosphatases determining reversible aquaporin

Key-words: aquaporin; CDPK; phosphorylation; PIP; quantitative proteomics; root water transport. Abbreviations: AQP, aquaporin; CDPK, Ca2+-dependent protein kinase; DWr, root dry weight; Jv, sap flow; Lpr, root hydraulic conductivity; PIP, plasma membrane intrinsic protein; TIP, tonoplast intrinsic protein.

Correspondence: V. Santoni. Fax: 33 (0)4 67 52 57 37; e-mail: [email protected] Current addresses: *Bio-Tech Research Center, Shandong Academy of Agricultural Sciences, 250100 Jinan, China. † Unité de Recherche en Génomique Végétale, INRA-UEVE UMR1165, CNRS ERL8196, Saclay Plant Sciences, 2 rue Gaston Crémieux, 91057 Evry cedex, France. ‡ CHU Montpellier, Institut de Recherches en Biothérapie, 80 Avenue A. Fliche, F-34295 Montpellier cedex 5, France 1312

© 2014 John Wiley & Sons Ltd

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rene raft floating in a basin filled with 8 L hydroponic culture medium and grown in a growth chamber at 70% relative humidity with cycles of 16 h of light (250 μmol photons m−2 s−1) and 8 h of dark at 20 °C. Twenty-one-dayold plants were used for Lpr measurements, qRT-PCR, ELISA experiments and proteomic analyses. Hydroponic culture medium was replaced weekly.

phosphorylation is still scarce (Sjovall-Larsen et al. 2006; Wu et al. 2013). Ca2+-dependent protein kinases (CDPKs), which form a large family of 34 members in Arabidopsis, are enzymes that directly bind Ca2+ ions and phosphorylate downstream targets acting in plant responses to various stresses and developmental cues (Cheng et al. 2002; Hrabak et al. 2003; Ludwig et al. 2004; Boudsocq & Sheen 2013; Schulz et al. 2013). Several Arabidopsis CDPKs have been reported to be involved in abiotic and abscisic acid (ABA) signalling pathways. In particular AtCPK3 and AtCPK6 act as positive regulators of plant tolerance to salt stress (Mehlmer et al. 2010; Xu et al. 2010). AtCPK10 is involved in drought tolerance and plays important roles in ABA- and Ca2+-triggered stomatal movements (Zou et al. 2010). CDPKs substrates include proteins involved in multiple cellular processes such as metabolism, stress response, transcription and signalling (Patharkar & Cushman 2000; Rodriguez Milla et al. 2006; Uno et al. 2009; Curran et al. 2011). Furthermore, CDPKs were proposed to play a role in in vitro and in vivo phosphorylation of PIP1s and/or PIP2s in maize and spinach (Aasamaa & Sober 2005; Sjovall-Larsen et al. 2006; Van Wilder et al. 2008) but no functional role in planta was demonstrated. In the present work, we investigated a putative role of CDPKs in regulating root water transport. We therefore investigated the Lpr of knockout Arabidopsis plants for four CDPKs. Our work reveals a link between AtCPK7 and aquaporin function in the Arabidopsis root.

Measurement of root hydraulic conductivity (Lpr, in mL g-1 h−1 MPa−1) was essentially performed as described (Javot et al. 2003). Briefly, the root system of a freshly detopped plant was inserted into a pressure chamber. The rate of pressure (P)-induced sap flow (Jv) exuded from the hypocotyl section was determined by a flowmeter. For that, excised roots were subjected to a pretreatment at 350 kPa for 10 min to reach equilibrium, and Jv was measured successively at 320, 160 and 240 kPa. After flow measurements, root dry weight (DWr) was measured.The Lpr was calculated from the following equation: Lpr = Jv/P/DWr. NaN3 experiments were performed essentially as described (Tournaire-Roux et al. 2003). Briefly, root systems were equilibrated in a standard hydroponic solution at 320 kPa for 20 min. NaN3 was then applied by complementing the root bathing solution with 1 mm NaN3. Jv was measured at 320 kPa during the 30 min following the treatment. In all cases, the maximal percentage inhibition of Jv, and therefore of Lpr, was deduced by fitting the kinetic curves with a negative exponential function.

MATERIAL AND METHODS

RT-PCR and qRT-PCR analysis

Screening of loss-of-function cpk mutants

Total RNA was extracted from roots using a Qiagen RNeasy plant mini kit with on-column DNase treatment (RNase-free DNase set, Qiagen). Poly(dT) cDNA was prepared from 2 μg total RNA using the transcription first-strand cDNA synthesis kit (Promega, Madison, WI, USA). To check CPK expression in the cpk mutants, RT-PCR was conducted with gene-specific primers and Actin2 (At3g18780) was used as a control (Supporting Information Table S1).The expression of all 13 PIP genes was quantified by qRT-PCR using the genespecific primers described in (Postaire et al. 2010). The geNORM method (Vandesompele et al. 2002) was used to identify within several genes (UBQ10 [At4g05320], TIP41like [At4g34270], F-box family protein [At5g15710], ACT7 [At5g09810], EF-1-α [At5g60390], PP2A3 [At1g13320], SAND family protein [SFP; At2g28390]) the most stable reference genes in our conditions. In practice, SPF, ACT7 and PP2A3 were used for subsequent data normalization.

T-DNA insertion lines for the CPK7 (At5g12480), CPK8 (At5g19450) and CPK9 (At3g20410) genes in the Arabidopsis thaliana Col-0 accession were obtained from the Nottingham Arabidopsis Stock Center (NASC), and corresponded to SALK_127223 (cpk7-1), SALK_035601 (cpk7-2), SALK_036581 (cpk8-1) and GABI_386D12 (cpk9-1). Homozygous knockout mutants were isolated by genotyping, using pairs of primers specific for CPK genes (forward and reverse primer) or the CPK genes and the T-DNA left border (LBb1) (Supporting Information Table S1). Furthermore, the expression of CPK7, CPK8 and CPK9 in cpk7-1, cpk7-2, cpk8-1, cpk9-1 and WT plants was checked by RT-PCR as described below. The cpk11-2 mutant line was previously described (Zhu et al. 2007; Boudsocq et al. 2010).

Root hydraulic conductivity measurements

Plant materials and growth conditions All experiments were performed using Arabidopsis thaliana accession Col-0 or Col-0-derived mutants. Surface-sterilized seeds were grown on vertical 1/2 Murashige and Skoog (MS) plates according to (Sutka et al. 2011). The plates were kept for 2 d at 4 °C, incubated vertically for 10 d at 20 °C in the light, and seedlings were transplanted into hydroponic culture. Plants were mounted on a 35 × 35 × 0.6 cm polysty-

Microsomal protein purification The whole procedure was performed at 4 °C. Roots from 21-day-old plants were grinded with a roll mill (C. Fauvel, INRA Avignon, France) with 0.25 g mL−1 of grinding buffer (500 mm sucrose, 10% glycerol, 20 mm EDTA, 20 mm EGTA, 50 mm NaF, 5 mm β–glycerophosphate, 1 mm phenantroline, 0.6% PVP, 10 mm ascorbic acid, 50 mm Tris, pH 8 with Mes,

© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 38, 1312–1320

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1 μm leupeptine, 5 mm DTT, 1 mm stabilized vanadate, 1 mm PMSF) and then centrifuged at 800 g for 2 min. The supernatant was then centrifuged at 9000 g for 12 min, and the resulting supernatant was centrifuged at 50 000 g for 12 min. The resulting pellet was resuspended in a minimal volume of storage buffer (10 mm Tris, 10 mm borate, 300 mm sucrose, 9 mm KCl, 5 mm EDTA, 5 mm EGTA, 50 mm NaF, 4.2 μm leupeptine, 1 mm PMSF, 5 mm DTT, pH 8.3) and stored at −80 °C. Protein concentration was estimated using a modified Bradford procedure (Stoscheck 1990). Microsomes were stripped by incubation in 4 m urea, pH 11, and 20 mm NaOH according to a procedure previously described (Santoni et al. 2003). Proteins were then solubilized in 2 × Laemmli buffer (2% SDS, 10% glycerol, 100 mm DTT, 0.001% bromophenol blue, 50 mm Tris, pH 6.8) during 3 h under shaking. After centrifugation (16 000 g for 2 min), the supernatant that contained solubilized proteins was stored at −20 °C.

and statistical analyses of data. Three independent biological experiments and two technical repeats by experiment were used to calculate quantitative ratios and corresponding P-values (Di Pietro et al. 2013).

Immunodetections The ELISA assay was performed as previously described (Santoni et al. 2003). Serial two-fold dilutions in a carbonate buffer (30 mm Na2CO3, 60 mm NaHCO3, pH 9.5) of 0.5 μg of microsomal extracts were loaded in triplicate onto immunoplates (Maxisorp Thermo Scientific, Roskilde, Denmark). The anti-PIP1 antibody, raised against 4 out of 5 PIP1s (PIP1;1, PIP1;2, PIP1;3 and PIP1;4) and anti-PIP2 antibody raised against 3 out of 8 PIP2s (PIP2;1, PIP2;2 and PIP2;3) were used at a 1:2000 dilution.

RESULTS

Proteomic analyses Protein digestion, phospho-peptide purification, peptide identification and label-free peptide quantification were performed as previously described (Di Pietro et al. 2013; Vialaret et al. 2014). Briefly, in solution, reductions/alkylations were performed simultaneously with detergent removing by the filter-aided sample preparation (FASP) protocol (Wis´niewski et al. 2009). These steps were followed by a digestion using Lys-C (Roche Applied Science (Indianapolis, IN, USA)) for 4 h at 37 °C and trypsin (Sequencing Grade Modified, Promega) overnight at 37 °C. After desalting, dried peptides were fractionated with a strong cation exchange chromatography and were submitted to an additional enrichment step in phosphopeptides with a TiO2 chromatographic column (Di Pietro et al. 2013). Peptides were separated with a reversedphase capillary column (Dionex, C18 PepMap100, 75 μm × 250 mm, 3 μm, 100 A) over a 140 min gradient and fragmented with a QTOF tandem MS system equipped with a nano-ESI source (Maxis, Bruker, Bremen, Germany) performed in the positive ion mode (Vialaret et al. 2014). The corresponding data were interrogated with Mascot 2.2.07 (Matrix Science, http://www.matrixscience.com) against the Arabidopsis proteome (Tair10, 31960 entries, http:// www.arabidopsis.org/). Parameters of interrogation accepted one putative missed cleavage, a 15 ppm and 0.05 Da mass range for the parent peptide and the MS/MS fragment, respectively. The selected enzyme was trypsin. Carbamidomethylation was taken as a fixed modification. Variable modifications were N-terminal acetylation, deamidation of asparagines and glutamines, methionine oxidation, phosphorylation of serines, threonines and tyrosines, methylation of aspartic and glutamic acids and of serines. Using the above criteria for protein identification, the rate of false peptide sequence assignment (FDR) as determined by the ‘decoy database’ function implemented in Mascot version 2.2.07 was 1%. Bruker softwares (DataAnalysis, ProfileAnalysis) provided a workflow including peak detection, peak matching/ alignment, normalization, detection of differential peptides

Identification of T-DNA insertion mutants of cpk7, cpk8 and cpk9 We isolated two independent cpk7 mutant lines, cpk7-1 (SALK_127223) and cpk7-2 (SALK_035601), a cpk8 mutant (SALK_036581) and a cpk9 mutant (GABI-386D12), all in a Columbia-0 (Col-0) background (Fig. 1a, Supporting Information Fig. S1). The T-DNA insertions were characterized by PCR analysis of the Arabidopsis genome (Fig. 1b) with specific sets of primers (Supporting Information Table S1). To confirm that cpk7-1, cpk7-2, cpk8-1 or cpk9-1 are transcriptnull mutants, RT-PCR was performed with RNA isolated from wild-type (WT) and mutant plants. The results showed that whereas WT plants produced native CPK7, CPK8 and CPK9 mRNAs (Fig. 1c), the four mutants did not display the RT-PCR products corresponding to the mutated genes. Thus, expression of a specific CDPK was fully knocked out in each of the mutants. A cpk11-2 mutant (SALK_054495) previously described by (Zhu et al. 2007; Boudsocq et al. 2010) was also used in this study.

Root hydraulic conductivity (Lpr) in cpk plants We used a previously described device (Boursiac et al. 2005) to characterize the Lpr of WT and cpk plants. For that, the entire root system of hydroponically grown plants was excised, inserted into a pressure chamber and submitted to various pressures (Boursiac et al. 2005) to induce sap flow (Jv). Lpr was deduced from the pressure-to-sap flow relationship.The Lpr of cpk8-1, cpk9-1 and cpk11-2 plants was similar to that of WT. By contrast, the Lpr of cpk7-1 and cpk7-2 plants was found to be increased by 27.1% and 34.2%, respectively, when compared to that of WT plants (Fig. 2). Changes in root hydraulics may be related to adjustments to distinct shoot and root relative sizes. However, no difference could be noticed in the ratio between shoot and root dry weight in WT and cpk7 plants (Supporting Information Fig. S2). Overall, the data indicate that CPK7 may be involved in Lpr down-regulation.


CPK7-dependent regulation of root water transport 1315 cpk7-1

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Figure 2. Lpr of WT, cpk7-1, cpk7-2, cpk8-1, cpk9-1 and cpk11-2 plants. Lpr (mean ± SE) was expressed as percentage of mean Lpr of WT plants (Lpr = 122.4 mL g-1 h-1 MPa-1). Data were pooled from three independent plant cultures and number of plants measured are indicated. Asterisks (**) indicate a statistically significant difference from WT (P < 0.05).

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Figure 1. Molecular characterization of cpk T-DNA insertion mutants. (a) Schematic representation of genomic DNA structure of CPK7, CPK8 and CPK9 with the T-DNA insertion sites in cpk7-1 (SALK_127223), cpk7-2 (SALK_035601), cpk8-1 (SALK_036581) and cpk9-1 (Gabi_585B10). Exons are indicated as black rectangles and introns are displayed as black bars. For more details, see Supporting Information Fig. S1. (b) PCR analysis of genomic DNA of WT, cpk7-1, cpk7-2, cpk8-1, cpk9-1 plants, using a pair of primers, either both specific of the CPK gene (lane 1), or with one primer recognizing the T-DNA insertion (lane 2). The sequences of primers are provided in Supporting Information Table S1. (c) RT-PCR analysis of CPK mRNA expression in WT and T-DNA insertion mutants, cpk7-1, cpk7-2, cpk8-1, cpk9-1 (lane 1). Actin2 amplification was used as a control for cDNA integrity (lane 2).

attributed to an increased aquaporin-dependent pathway. Thus, CPK7 appears to down-regulate aquaporin function in roots.

PIP gene expression in cpk7 plants The transcript abundance of each of the 13 PIP genes was investigated using quantitative real-time RT-PCR (qRTPCR) in roots of cpk7 and WT plants. For each gene, expression in cpk7 was normalized to expression in WT.

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Aquaporin activity in cpk7 plants According to the composite root water transport model, Lpr can be contributed by an apoplastic pathway, which is aquaporin-independent, and a cell-to-cell pathway, which is aquaporin-dependent (Steudle & Peterson 1998). Respiratory blockers such as azide (NaN3) have been identified as potent inhibitors of aquaporin-mediated water transport in roots (Tournaire-Roux et al. 2003; Sutka et al. 2011) and can be used to distinguish between the two pathways. Water flow from a root system was measured in a standard solution and after treatment with 1 mm NaN3 for 30 min. These measurements were used to calculate the inhibitable and residual root water flow. The latter was similar in cpk7 plants and in WT plants (Fig. 3). However, the higher inhibitable flow in cpk7 plants (Fig. 3, Supporting Information Fig. S3) when compared with that of WT plants, suggested that the increased Lpr value in cpk7 plants could be

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Figure 3. Contribution of aquaporins to water transport in WT and cpk7 roots. Jv of excised roots was measured by the pressure chamber technique before and after addition of 1 mM azide for 30 min. These measurements allow to evaluate the total root hydraulic conductivity and the aquaporin-independent conductivity, respectively (see Supporting Information Fig. S3). They were used to split the relative Lpr of WT, cpk7-1 and cpk7-2 into an inhibitable (□) and residual fraction (■), respectively. Data were pooled from two independent plant cultures and the number of plants measured is indicated. The values are means ± SE.


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Figure 5. Relative abundance of PIP1 and PIP2 isoforms in WT and cpk7 plants. ELISA assays were performed with microsomal protein extracts purified from roots of WT and cpk7 plants, using anti-PIP1 (a) and anti-PIP2 (b) antibodies. Values were expressed in percentage of corresponding value in WT. Data were from three individual ELISA assays with samples from three independent cultures. Asterisks (**) indicate a statistically significant difference from WT (P < 0.01). It was previously shown that the two antibodies do not cross-react with other proteins than PIPs in Arabidopsis root extracts (Santoni et al. 2003).

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Figure 4. Relative PIP expression in WT and cpk7 plants. The transcript abundance of all 13 PIP genes was measured in roots of WT and cpk7 plants grown in standard conditions. The PIP transcript abundance in cpk7-1 (□) and cpk7-2 (■) roots was plotted as relative expression (fold) to WT (■). Expression of PIP1 and PIP2 genes is shown in a and b, respectively. The values are means ± SE and asterisks (*) indicate a statistically significant difference from WT (P < 0.05 ).

Interestingly, the expression profile of PIP genes was not altered in the cpk7 plants, except for PIP2;6, with a transcript abundance increased by two-fold in cpk7 plants (Fig. 4). However, PIP2;6 is known to be lowly expressed in roots (Boursiac et al. 2005; Monneuse et al. 2011). Thus, additional post-transcriptional mechanisms affecting abundant PIP isoforms should be considered to account for the higher aquaporin activity in cpk7 plants.

Aquaporin abundance in cpk7 plants We used anti-PIP1 and anti-PIP2 antibodies that recognize the PIP1;1 to PIP1;4 isoforms and the PIP2;1 to PIP2;3 isoforms (Santoni et al. 2003), respectively, to quantify these aquaporins in microsomal extracts purified from WT and cpk7 plants. The abundance of PIP2 isoforms was unchanged in cpk7 plants when compared to WT plants, while the PIP1 amount was significantly increased (Fig. 5). This effect could contribute to the increase in Lpr displayed by cpk7 plants (Fig. 2). This result suggests that CPK7 contributes to the control of PIP1 cellular abundance.

Proteomic analysis of cpk7 plants A quantitative proteomic approach was then used to refine the changes in the abundance of individual aquaporins and

investigate their phosphorylation state in cpk7 plants. For that, we performed a quantitative label-free proteomic analysis of hydrophobic microsomal proteins of WT, cpk7-1 and cpk7-2 plants. Fractions were profiled by LC-MS and then identified by tandem MS. This procedure led to identification of about 1000 proteins (Supporting Information Table S2; Supporting Information Appendix S1) across the three plant genotypes. More than one half of the proteins was predicted to display at least one transmembrane domain (http:// aramemnon.botanik.uni-koeln.de/; data not shown) suggesting an overall enrichment in membrane proteins.A total of 37 aquaporin peptides could be identified and revealed the presence of all 13 PIP isoforms except PIP2;6, and of 2 out of 9 tonoplast intrinsic protein (TIP) isoforms (TIP2;2 and TIP2;3; Supporting Information Table S3, Supporting information Appendix S1). MS analyses also revealed that PIPs can be modified by phosphorylation, deamidation, methylation and oxidation (Supporting Information Table S3) as recently described (Di Pietro et al. 2013). A quantitative analysis of peptide and phosphopeptide abundance was then performed to highlight quantitative protein variations between WT and cpk7 plants. We considered a quantitative variation as significant when a similar type of variation (increase or decrease) was obtained for both allelic mutants compared with WT, with P < 0.05. Otherwise, the peptide was considered as quantitatively invariant between WT and cpk7 plants.Among the 37 aquaporin peptides quantified in the two cpk7 lines (Supporting Information Table S3), five peptides belonging to PIP1;1 and PIP1;2 in their non-modified, deamidated and phosphorylated forms showed a significant increase in abundance in cpk7-2 that was even more pronounced in cpk7-1 (Fig. 6, Supporting Information Table S3). Consistent with our previous ELISA measurements, this result suggests that the entire cellular pool of PIP1;1 and PIP1;2 (i.e. their non-modified and modified forms) increased in cpk7 plants when compared with WT plants. We also noticed that the abundance of TIP2;3,


CPK7-dependent regulation of root water transport 1317 permease PUP18) and proteins involved in endomembrane trafficking such as SEC12, SECY and SYP122 (Supporting Information Table S4).

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DISCUSSION

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The central aim of this work was to search for a role of CDPKs in regulation of root aquaporin function. This question was addressed by studying the Lpr of cpk plants. CDPKs belong to a multigene family, with 34 members in Arabidopsis distributed in four subfamilies. CDPKs display highly variable Ca2+ dependencies for their kinase activities, which make them suitable sensors of the various Ca2+ signatures induced by environmental stimuli (Boudsocq et al. 2012). An elegant genetic screen recently identified OSCA1 as a Ca2+-permeable channel involved in generating Ca2+ influx in response to hyperosmolarity (Yuan et al. 2014). The particular combination of stimulus-dependent Ca2+ channels with downstream Ca2+ decoders like CDPKs likely contributes to the specificity of Ca2+ signaling. Here, we studied the influence on Arabidopsis Lpr of four CDPKs (CPK7, CPK8, CPK9 and CPK11) belonging to three subfamilies. CPK7 was the only one to interfere with Lpr in standard conditions. Intriguingly, unlike CPK9 and CPK11, CPK7 was found to be insensitive to Ca2+ on generic substrates while it was still able to bind Ca2+ in vitro (Boudsocq et al. 2012). Although the molecular mechanism remains unclear, the data suggest that CPK7 may be involved in Ca2+ signalling. Surprisingly, CPK7 appeared as a negative regulator of Lpr. Pharmacological evidence showed that 80–90% of Arabidopsis Lpr can be attributed to aquaporin activities (Tournaire-Roux et al. 2003). In the present work, we used azide as an aquaporin inhibitor to show that aquaporin activity is higher in cpk7 plants. Thus, CPK7 negatively interferes with aquaporin function. Biochemical and physiological studies have demonstrated that the phosphorylation of PIP2s acts on both their gating and subcellular localization (Tornroth-Horsefield et al. 2006; Prak et al. 2008) and that changes in phosphorylation of PIPs are positively correlated to changes in Lpr induced by abiotic constraints (Di Pietro et al. 2013). In addition, genetic studies revealed that aquaporin abundance is a critical determinant of Lpr. For instance, the study of pip1;2 plants revealed that PIP1;2 can account for 20% of Lpr and a double pip2;1 × pip2;2 knockout mutant showed a 40% decrease in Lpr (Peret et al. 2012). Finally, we recently quantified PIPs in roots of Col-0 plants using a multiple reaction monitoring approach and showed that PIP1;1, PIP1;2, PIP2;1, PIP2;2, PIP2;4 and PIP2;7 are the most abundant isoforms, ranging from 70 to 280 pmol mg−1 of membrane proteins (Monneuse et al. 2011). Thus, the regulation of either PIP phosphorylation or PIP abundance could significantly contribute to the observed changes in Lpr. Using a quantitative proteomic approach with peptide fractionation, we quantified the abundance of individual PIP isoforms in their native and modified forms. We, however, did not observe any alteration of the phosphorylation pattern of PIP2s in cpk7 plants. This suggests that CPK7 does not

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Figure 6. Relative abundance of PIP1 peptides in WT (white), cpk7-1 (black) and cpk7-2 (grey) plants. All peptide abundance ratio in cpk7 plants are significantly different from unity (P < 0.05). The peptide sequences and modifications are as follows: PIP1;1 157–174: 157QYQALGGGANTVAHGYTK174; PIP1;1 157–174 Dea: 157QYQALGGGANTVAHGYTK174 with a deamidated residue (Q159 or N166 or Q157); PIP1;2 157–174: 157QYQALGGGANTIAHGYTK174; PIP1;2 157–174 Dea: 157QYQALGGGANTIAHGYTK174 with a deamidated residue (Q159 or N166); PIP1;1 1;2 P: 19QPIGTSAQSDK29 with a phosphorylated residue (Ser27); additional information on peptides is given in Supporting Information Table S2.

increased in cpk7 plants (Supporting Information Table S3). By contrast, we did not detect any quantitative variation of the phosphorylation of the C-terminal tail of PIP2;1/2;2/2;3 (Supporting Information Table S3). These data suggest that CPK7 plays a specific role in controlling PIP1 and TIP abundance. This study also revealed significant increases and decreases in abundance for 12 and 7 additional proteins, respectively. In each case, both the native and modified forms showed similar variations (Supporting Information Table S4). In addition, the phosphorylation of H+-ATPase AHA11 and mechanosensitive channel MSL9 was specifically increased in cpk7 plants (Supporting Information Table S4). Additional proteins were also quantified but with a unique phosphopeptide (Supporting Information Table S4). Without any quantification of the corresponding unmodified peptide, it remains unclear whether the variation in phosphopeptide abundance corresponds to a genuine quantitative variation of phosphorylation or an overall change in protein abundance. Overall, CPK7 interferes with expression and/or phosphorylation of four main classes of proteins including protein kinases (CPK3, CIPK8), proteins involved in glycosylation (ribophorin and DGL1), membrane transporters and channels (with, in addition to AHA11 and MSL9, nitrate transporter NRT2;1, ABC transporter PGP19, Ca2+ATPase ECA3, ammonium transporter AMT1;3, potassium transporter KUP4, chloride channel CLCc, and purine


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phosphorylate the C-terminal tail of PIP2 aquaporins.Yet, we cannot exclude that moderate changes in phosphorylation of PIP2 aquaporins that occurred in specific root cell layers, may have escaped our analysis. By contrast, proteomic analyses together with ELISA measurements revealed in cpk7 plants, an increased cellular abundance of PIP1s but not of PIP2. More precisely, quantitative proteomics revealed that cpk7 plants showed a specific accumulation of PIP1;1 and PIP1;2 isoforms, in their non-modified and modified forms including deamidated and phosphorylated forms, suggesting a global increase of the PIP1;1 and PIP1;2 cellular pool (Supporting Information Table S3). In addition, we observed an overall lack of correlation between PIP1 gene and protein expression indicating that the down-regulation of PIP1 abundance driven by CPK7 in the WT plant is determined at the posttranscriptional level. The overall results suggest that CPK7 negatively interferes with the cellular accumulation of PIP1;1 and PIP1;2, contributing to lower the Lpr in WT plants compared with cpk7 plants. Interestingly, while PIP2s were proposed to play a major role in water transport (Otto et al. 2010), the present work supports previous studies (Postaire et al. 2010) showing that PIP1s also significantly contribute to Lpr. The mechanisms of PIP degradation are as yet poorly known. PIP ubiquitination seems to contribute to the decrease in PIP abundance at the plasma membrane in response to drought (Lee et al. 2009). Recent results in animals indicate that the hormone arginine vasopressin enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and MAPKdependent pathway while PKC mediates angiotensin regulation of AQP2 abundance (Nedvetsky et al. 2010; Bagnasco 2012). Thus, the present work similarly links plant aquaporin stability to protein kinase activities. However, the signals acting upstream of CPK7 and acting on PIP1 stability remain as yet unknown. Proteomic analyses also revealed that beyond aquaporins, CPK7 interferes with the expression of a few other membrane proteins. Interestingly, connections between water and nutrient transport could be anticipated from the proteomic data, in particular because of similar variation profiles of PIP1 aquaporins and several transporters and channels (i.e. nitrate transporter NRT2;1, potassium transporter KUP4, ammonium transporter AMT1;3 and anion channel CLC-c) (Supporting Information Table S4). In particular, the abundance of NRT2;1 in its non-modified and phosphorylated forms increased in cpk7 plants suggesting that CPK7 negatively interferes with the abundance and phosphorylation of NRT2;1 and thus, with nitrate uptake. Interestingly, the present work also shows that CPK7 negatively interferes with CIPK8 phosphorylation (Supporting Information Table S4). CIPK8 was shown to be involved in the induction of NRT2;1 expression by nitrate and thus to be a positive regulator of the primary nitrate response (Hu et al. 2009). One hypothesis is that CPK7 would contribute (1) to prevent CIPK8 phosphorylation and (2) to decrease the cellular abundance of NRT2;1, thereby reducing nitrate absorption. The involvement of Ca2+ and protein phosphorylation in the

primary nitrate response has been already suggested (Sakakibara et al. 1997; Ludwig et al. 2004; Hu et al. 2009; Luan 2009). Our results reinforce the idea that a protein kinase cascade and potentially Ca2+ signalling are involved in nitrate sensing and help identify the targets involved. The potassium transporter KUP4 was shown to be phosphorylated at residue Ser647 by CPK1, CPK10 and CPK34 (Curran et al. 2011). The present work showed that the phosphorylation of this and/or of a close residue, Ser645, increased in cpk7 mutants (Supporting Information Table S4). This suggests that CPK7 might negatively interfere with other CPKs involved in phosphorylation of KUP4. However, the present work does not provide evidence for modulation of CPK1, CPK10 and CPK34 abundance by CPK7 but rather shows that the abundance of CPK3 is increased in cpk7 mutants (Supporting Information Table S4). Interestingly, CPK3 was recently shown to phosphorylate and regulate the activity of the potassium channel TPK1 (Latz et al. 2013). These observations and our work together indicate that complex connections between potassium transport proteins and CDPKs exist that would explain how CPK7 activity leads to a decreased phosphorylation of KUP4. CLC-c and AMT1;3 represent two additional classes of transporters the phosphorylation of which is antagonized by CPK7, suggesting that CPK7 also interferes with anion and ammonium homeostasis. Altogether, and beyond the study of the regulation of root water transport, the present proteomic study reveals a coordinated regulation of membrane transport proteins, including channels and transporters, to support an efficient uptake of nutrients and water from the soil. It also points to a critical role of CPK7 in this regulation.

ACKNOWLEDGMENTS This research work was supported by a grant from the Agence Nationale de la Recherche (PhosphoStim, ANR-08GENM-013). The authors are grateful to Christiane Laurière and Laurence Lejay for fruitful discussions and Marie-Jo Droillard for technical assistance.

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Received 21 August 2014; received in revised form 24 October 2014; accepted for publication 27 October 2014

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. T-DNA insertion sites in cpk7-1, cpk7-2, cpk8-1 and cpk9-1 plants. Figure S2. Ratio between shoot and root dry weight in WT and cpk7 plants. Figure S3. Sap flow (Jv) inhibition (A) and residual Jv (B) after NaN3 treatment. Table S1. Sequence of primers used for CDPK mutants identification. Table S2. Proteins identified in the membrane proteome. Table S3. Aquaporins peptides. Table S4. List of peptides with significant quantitative variations between cpk7 mutants and WT plants. Appendix S1. Protein report.
