selective regulation of maize plasma membrane aquaporin

Selective Regulation of Maize Plasma Membrane AquaporinTrafficking and Activity by the SNARE SYP121W

Arnaud Besserer,a Emeline Burnotte,a Gerd Patrick Bienert,a Adrien S. Chevalier,a Abdelmounaim Errachid,a

Christopher Grefen,b Michael R. Blatt,b and François Chaumonta,1

a Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgiumb Laboratory of Plant Physiology and Biophysics, Institute of Molecular, Cell, and Systems Biology, University of Glasgow,Glasgow G12 8QQ, United Kingdom

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. Wepreviously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmicreticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulatedby the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasmamembrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K+ channels. We demonstrate that theexpression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads toa decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo(bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approachesallowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role ofSYP121 in regulating K+ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to theregulation of the cell osmotic homeostasis.

INTRODUCTION

Water uptake and maintenance of the cell osmotic homeostasisare major constraints encountered by living organisms. One ofthe protein families involved in the regulation of these processesis the aquaporin family. Aquaporins are small (28 to 34 kD)membrane proteins that form tetrameric channels facilitating themovement of water and/or small neutral solutes through cellularmembranes (Gomes et al., 2009). Compared with mammals,plants express a high number of aquaporin isoforms, underliningtheir fundamental roles in growth, development, and abioticstress tolerance (Maurel et al., 2008; Gomes et al., 2009; Heinenet al., 2009). Among the plant aquaporins, the plasma mem-brane intrinsic proteins (PIPs) are subdivided into two sequence-related groups, PIP1s and PIP2s, the members of which exhibitdifferent water channel activities when expressed in Xenopuslaevis oocytes (Chaumont et al., 2000; Temmei et al., 2005;Vandeleur et al., 2009; Bellati et al., 2010; Otto et al., 2010).When expressed alone, maize (Zea mays) PIP1s are inactive,whereas Zm-PIP2s cause a marked increase in the osmoticwater permeability coefficient, Pf. However, when coexpressed,Zm-PIP1s and Zm-PIP2s physically interact to increase the Pf

compared with oocytes expressing Zm-PIP2s alone (Fetteret al., 2004).

As integral membrane proteins, PIPs are synthesized in theendoplasmic reticulum (ER) and then transported along the se-cretory pathway to reach the plasma membrane. We previouslyshowed that PIP trafficking is regulated by a heterooligomeriza-tion process (Fetter et al., 2004; Zelazny et al., 2007). Whentransiently expressed alone in maize cells, Zm-PIP1s and Zm-PIP2s differ in their subcellular localization. Zm-PIP1s are re-tained in the ER, whereas Zm-PIP2s are targeted to the plasmamembrane (Zelazny et al., 2007). However, upon coexpression,Zm-PIP1s are relocalized from the ER to the plasma membraneas a result of their physical interaction with Zm-PIP2s, demon-strated by Förster resonance energy transfer (FRET) and fluo-rescence lifetime imaging microscopy experiments. A diacidicmotif, DIE (Asp-Ile-Glu) at position 4 to 6 in the N terminus ofZm-PIP2;5 is essential for ER export (Zelazny et al., 2009). Thismotif is conserved and functional in Zm-PIP2;4 but is absent inZm-PIP2;1. However, the replacement of the N terminus of Zm-PIP1;2 by the N terminus of Zm-PIP2;5 containing the diacidicmotif is not sufficient to cause the export of Zm-PIP1;2 from theER. A functional diacidic motif has also been identified in the Nterminus of Arabidopsis thaliana PIP2;1 (Sorieul et al., 2011). Inaddition, the traffic and plasma membrane abundance of the At-PIP2;1 is regulated by different posttranslational modifications,including phosphorylation and ubiquitylation (Prak et al., 2008;Lee et al., 2009). Once located in the plasma membrane, At-PIP2;1 can move into or out of membrane microdomains andundergoes constitutive cycling, involving clathrin-dependent andraft-associated endocytosis (Paciorek et al., 2005; Dhonuksheet al., 2007; Li et al., 2011). The presence of PIPs in endosomesreflects a dynamic regulation of their density at the plasmamembrane and their sorting between storage or degradation

1 Address correspondence to [email protected] author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: François Chaumont([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.112.101758

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been

edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online

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paths (Wudick et al., 2009; Luu et al., 2012). In this process, thetrans-Golgi network (TGN) could act as a late sorting platform inthe secretory pathway (Robinson et al., 2008; Richter et al.,2009), but the molecular actors regulating the post-Golgi traf-ficking of PIPs and their insertion in the plasma membrane re-main elusive.

In mammals, the proper trafficking and anchoring of aqua-porin2 (AQP2) in the apical membrane of collecting duct epi-thelial cells are specifically regulated by SNARE (for solubleN-ethylmaleimide-sensitive factor protein attachment proteinreceptor) complexes (Mistry et al., 2009; Wang et al., 2010).SNAREs are membrane proteins that facilitate vesiculartrafficking in eukaryotes. They are subdivided into Q- and R-SNAREs, according to their conserved residues within theSNARE motif (Fasshauer et al., 1998). SNAREs are present in themembranes of all subcellular compartments and play a majorrole in vesicular fusion and membrane biosynthesis. Genomeanalysis showed that the SNARE family is larger in plants than inother eukaryotes (Sanderfoot, 2007). Indeed, more than 60SNARE isoforms have been identified in the Arabidopsis ge-nome, and orthologs are found in rice (Oryza sativa) for most ofthem, supporting the idea that they are conserved betweenmonocots and dicots (Sutter et al., 2006a). Syntaxins belong tothe Q-SNARE subfamily and, in Arabidopsis, seven of them lo-calize to the plasma membrane and are involved in traffic at thismembrane (Grefen et al., 2011). Among these plasma mem-brane resident syntaxins, SYP121 has been the most intensivelystudied and is known to mediate vesicle traffic between theGolgi complex and plasma membrane (Geelen et al., 2002).Disrupting its function by overexpressing a dominant-negativecytosolic (so-called Sp2) fragment specifically affected thetrafficking to the plasma membrane (Tyrrell et al., 2007). In to-bacco (Nicotiana tabacum), SYP121-Sp2 suppressed the mo-bility and delivery of the potassium channel KAT1 to the plasmamembrane, but not of the H+-ATPase (Sutter et al., 2006b). In-terestingly, SYP121 was recently shown to regulate the gating ofthe AKT1/KC1 K+ channel complex through a direct interactioninvolving an FxRF motif located within the first 12 residues of theprotein (Honsbein et al., 2009; Grefen et al., 2010). Altogether,these data raise the hypothesis that SYP121 acts as a moleculargovernor, coordinating the gating of ion channels in parallel withmembrane traffic and, therefore, ion uptake with increasedmembrane surface area through vesicle fusion during cell ex-pansion (Grefen and Blatt, 2008; Honsbein et al., 2011).

As water movement is a key element of the mechanism thatregulates cell osmotic homeostasis, we wondered if SYP121could also regulate the traffic and activity of PIP aquaporins. Inbarley (Hordeum vulgare), PIP2 colocalized in ROR2/SYP121vesicle-like compartments, supporting this hypothesis (Kwaaitaalet al., 2010). We report here that the SYP121-Sp2 from Arab-idopsis and its putative ortholog from maize affect the delivery ofZm-PIP2;5 to the plasma membrane in maize mesophyll proto-plasts and epidermal cells. Furthermore, the traffic inhibition iscorrelated with a decrease in the protoplasts Pf. As Zm-PIP2;5water channel activity is also reduced in the presence of theZmSYP121-Sp2 fragment in Xenopus oocytes, we tested thepossibility that both proteins directly interact. Physical interactionbetween both proteins was demonstrated in vitro and in vivo by

different technical approaches. These data indicate that SNAREmight play an important role in the regulation and maintenance ofosmolarity in the cytosol through a coordinated regulation ofaquaporins and K+ channels.

RESULTS

The AtSYP121-Sp2 Fragment Reduces Plasma MembraneDelivery of Zm-PIP2;5

When expressed in maize mesophyll protoplasts, monomericyellow fluorescent protein (mYFP):ZmPIP2;5 accumulates in theplasma membrane (Zelazny et al., 2007). To determine whetherthis localization is dependent on the activity of the syntaxinSYP121 or not, we coexpressed mYFP:ZmPIP2;5 and the Arab-idopsis dominant-negative AtSYP121-Sp2 fragment fused tothe C terminus of monomeric cyan fluorescent protein (mCFP)and analyzed the cellular localization and intensity of the fluo-rescent proteins using confocal laser scanning microscopy(CLSM). Fusions of the fluorescent proteins to the N terminus ofAt-SYP121 and Zm-PIP2;5 have been previously shown not toaffect the subcellular localization of both proteins in protoplasts(Uemura et al., 2004; Zelazny et al., 2007). When expressedalone, mYFP:ZmPIP2;5 mainly accumulated in the cell periphery(Figure 1A). The fluorescent signal colocalized with the styryl dyeFM4-64 (Figures 1Aa to 1Ac) and mCFP:tagged Np-PMA2H+-ATPase (Lefebvre et al., 2004) (Figures 1Ad to 1Af), dem-onstrating that ZmPIP2;5 was mainly present in the plasmamembrane. When coexpressed with mCFP:AtSYP121-Sp2,mYFP:ZmPIP2;5 was still present in the plasma membrane, butthe fluorescent signal intensity was much weaker than in cellsexpressing mYFP:PIP2;5 alone (Figures 1Ag to 1Ai). mYFP:ZmPIP2;5 also labeled internal structures. To quantify the plasmamembrane fluorescence intensity, an image analysis procedurewas developed. Briefly, the average thickness of the plasmamembrane was determined using the colocalized mYFP:ZmPIP2;5 and FM4-64 fluorescent signals, and the differencebetween the overall and intracellular fluorescent signals was usedto calculate the plasma membrane fluorescence (for more details,see Methods). The mYFP plasmamembrane fluorescence intensitywas significantly lower (P < 0.05) in protoplasts coexpressing themYFP:ZmPIP2;5 and mCFP:AtSYP121-Sp2 fragment than in pro-toplasts expressing mYFP:ZmPIP2;5 alone (Figure 1B), indicatingthat AtSYP121-Sp2 interferes with Zm-PIP2;5 delivery to theplasma membrane.

Zm-SYP121 Is the Functional Ortholog of At-SYP121

Maize database analysis allowed the identification of a maizeSYP121 protein that shared 54% amino acid identity with At-SYP121. Both proteins cluster in the same phylogenetic groupas shown by the neighbor-joining tree (see Supplemental Figure1 and Supplemental Data Set 1 online). To test if this sequencesimilarity could reflect a conservation in the regulation of Zm-PIP2;5 delivery to the plasma membrane, the SYP121 cDNA wascloned from total RNA extracted from maize roots and fusedto mCFP and mYFP sequences in plant expression vectors. A

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Figure 1. Both AtSYP121-Sp2 and ZmSYP121-Sp2 Fragments Selectively Reduce the Accumulation of Zm-PIP2;5 in the Plasma Membrane of MaizeMesophyll Protoplasts.

PIP Aquaporin Regulation by SNARE 3 of 19

truncated version of the cDNA was also prepared to produce theZmSYP121-Sp2 fragment. These constructs were then trans-fected in maize mesophyll protoplasts. Figures 1C and 1D showthe subcellular localization of mYFP:ZmPIP2;5 and the plasmamembrane fluorescence signal intensity, respectively, in proto-plasts transiently expressing mYFP:ZmPIP2;5 alone (Figures1Ca and 1Cb) and coexpressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121 (Figures 1Ce and 1Cf) or mCFP:ZmSYP121-Sp2(Figures 1Cg and 1Ch). The soluble mCFP was coexpressedwith mYFP:ZmPIP2;5 as a control (Figures 1Cc and 1Cd). Whenexpressed alone or coexpressed with mCFP or mCFP:ZmSYP121, mYFP:ZmPIP2;5 accumulated in the cell plasmamembrane (Figures 1Cb, 1Cd, and 1Cf). Quantification of theplasma membrane fluorescence intensities in the mYFP channelshowed that the mYFP:ZmPIP2;5 and mCFP:ZmSYP121 coex-pressing protoplasts had a slightly weaker plasma membranefluorescence than protoplasts expressing mYFP:ZmPIP2;5 alone,but the intensities were not significantly different (Figure 1D). Bycontrast, upon coexpression with the mCFP:ZmSYP121-Sp2fragment, the mYFP:ZmPIP2;5 plasma membrane signal intensitywas strongly decreased (Figures 1Ch and 2Af), indicating thatZm-SYP121 is a functional ortholog of AtSYP121. In protoplastscotransfected with equal amounts of plasmids encoding mCFP:-ZmSYP121-Sp2 or mYFP:ZmPIP2;5, mYFP:ZmPIP2;5 only poorlyaccumulated in internal structures. However, when protoplastswere transfected with a 3:1 ratio of mCFP:ZmSYP121-Sp2 andmYFP:ZmPIP2;5 plasmids, a significant accumulation of mYFP:ZmPIP2;5 in internal structures was observed (see SupplementalFigure 2 online).

For comparison, we examined the fluorescence distribution ofmCFP:NpPMA2 expressed in maize protoplasts. It was pre-viously shown that the delivery of Np-PMA2 to the plasmamembrane was not affected by AtSYP121-Sp2 in tobacco epi-dermal cells (Sutter et al., 2006a). When coexpressed withmYFP:ZmSYP121-Sp2 in maize protoplasts, no modification in

mCFP:NpPMA2 plasma membrane localization and intensitywas detected (Figures 1E and 1F).To analyze the dynamics of the effect of ZmSYP121-Sp2 on

newly synthesized mYFP:ZmPIP2;5 that reaches the plasmamembrane via anterograde transport, we compared the intensityof mYFP signal according to time in mesophyll protoplastsexpressing mYFP:ZmPIP2;5 alone or coexpressing mYFP:ZmPIP2;5 with mCFP:ZmSYP121-Sp2. One image acquisitionwas performed every 30 min during 16 h after cell transfection(see Supplemental Figure 3 online with associated SupplementalMovie 1 and Supplemental Movie Legend 1 online). The quanti-fication of the global (plasma membrane and intracellular) mYFPfluorescent signals in protoplasts expressing mYFP:ZmPIP2;5alone or in combination with mCFP:ZmSYP121-Sp2 is shownin Supplemental Figure 4 online. The mYFP and mCFP signalswere detected from 5 to 8 h after transfection. During the first 8h, the increase in mYFP signal intensity (integrated density)was slightly lower but not significantly different in cells coex-pressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2 com-pared with protoplasts expressing mYFP:ZmPIP2;5 alone,indicating that the protein synthesis rate was not affected.However, 12 h after transfection, while the mYFP signal wasstill increasing in protoplasts expressing mYFP:ZmPIP2;5alone, it plateaued in cells coexpressing mCFP:ZmSYP121-Sp2 and mYFP:ZmPIP2;5. During this time range, the signal ofmCFP:ZmSYP121-Sp2 was still rising, demonstrating thatprotein synthesis was not affected by mCFP:ZmSYP121-Sp2expression.To monitor the cycling of Zm-PIP2;5 from intracellular com-

partments to the plasma membrane or within the membranesurface, we performed fluorescence recovery after photo-bleaching (FRAP) experiments in tobacco epidermal cells tran-siently expressing mYFP:ZmPIP2;5 alone or together withmCFP:ZmSYP121-Sp2 (see Supplemental Figure 5 online). Incells expressing mYFP:ZmPIP2;5 alone, a recovery of 20% of

Figure 1. (continued).

(A) Plasma membrane abundance of mYFP:ZmPIP2;5 in maize mesophyll protoplasts. (a) to (c) Protoplast expressing mYFP:ZmPIP2;5 (a). (b) Channelshowing the FM4-64 fluorescence and chlorophyll a autofluorescence. (c) Merged image. (d) to (f) Protoplast coexpressing mYFP:PIP2;5 (d) andmCFP:PMA2 (e). (f) Merged image. (g) to (i) Protoplast coexpressing mYFP:PIP2;5 (g) and mCFP:AtSYP121-Sp2 (h). (i) Merged image. Bars = 5 µm.(B) Quantification of mYFP:ZmPIP2;5 fluorescence intensity in the protoplast plasma membrane (PM). Acquisitions were performed with the samesettings for all samples. Each bar represents the mean of five independent experiments (10 protoplasts per experiment). Error bars show the 95%confidence interval. Student’s t test, P < 0.05 was used to compare the data. Means that are significantly different (P < 0.05) are indicated by differentletters. a.u., arbitrary units.(C) Plasma membrane abundance of Zm-PIP2;5 in maize mesophyll protoplasts. (a) and (b) Protoplast expressing mYFP:ZmPIP2;5 (b). (a) Chlorophylla autofluorescence. (c) and (d) Protoplast coexpressing the soluble mCFP (c) and mYFP:ZmPIP2;5 (d). (e) and (f) Protoplast coexpressing mCFP:ZmSYP121 (e) and mYFP:ZmPIP2;5 (f). (g) and (h) Protoplast coexpressing mCFP:ZmSYP121-Sp2 (g) and mYFP:ZmPIP2;5 (h). All the pictures wereacquired with the same settings. In panel (a), the chloroplast autofluorescence is shown, whereas, in panels (c), (e), and (f), it is subtracted. Bars = 5µm.(D) Quantification of mYFP:ZmPIP2;5 fluorescence intensity in the protoplast plasma membrane. Each bar represents the mean of three independentexperiments (10 protoplasts by experiments). Error bars show 95% confidence interval. Statistical differences were inferred by analysis of variance(ANOVA) followed by Tukey’s honestly significant difference test. Means that are significantly different (P < 0.05) are indicated by different letters.(E) Plasma membrane abundance of mCFP:PMA2 in maize mesophyll protoplasts. (a) to (c) Protoplast expressing mCFP:PMA2 (a). (b) YFP channel. (c)Chlorophyll a channel. (d) to (f) Protoplast coexpressing mCFP:PMA2 (d) and mYFP:ZmSYP121-Sp2 (e). (f) Chlorophyll a channel. All the images wereacquired with the same settings. Bars = 5 µm.(F) Quantification of mCFP:PMA2 fluorescence intensity in the protoplast plasma membrane. Each bar represents the mean of three independentexperiments (10 protoplasts per experiment). Error bars show the 95% confidence interval. Means are not significantly different (Student’s t test, P =0.90).









the initial plasma membrane fluorescence was detected 150 safter photobleaching. These data are in agreement with previousfindings on the KAT1 K+ channel in Arabidopsis (Honsbein et al.,2009) and on At-PIP2;1 (Sorieul et al., 2011; Luu et al., 2012). Incells coexpressing mCFP:ZmSYP121-Sp2 and mYFP:ZmPIP2;5,the recovery reached only 5% of the initial plasma membranesignal.

The Inhibition of Zm-PIP2;5 Delivery to the PlasmaMembrane Is SYP121-Sp2 Specific

To test whether the delivery of Zm-PIP2;5 to the plasma mem-brane was specifically dependent on SYP121, mYFP:ZmPIP2;5was coexpressed in maize protoplasts with the Sp2 truncatedforms of At-SYP71, At-SYP122, or At-SYP21 syntaxins, and itsabundance in the plasma membrane was measured (Figure 2).SYP71 and SYP122 are syntaxins localized in the plasmamembrane (Leyman et al., 2000; Borner et al., 2005; Suwastikaet al., 2008), while SYP21 was found in the prevacuolar com-partment membrane (da Silva Conceição et al., 1997; Sanderfootet al., 2001; Tse et al., 2004). None of these Sp2 fragmentsaffected the plasma membrane accumulation of mYFP:ZmPIP2;5 (Figures 2A and 2B), although AtSYP21-Sp2 has beenshown to be active in suppressing traffic to the (pre)vacuolarcompartment (Tyrrell et al., 2007). The plasma membrane fluo-rescent signal intensity detected in cells coexpressing mCFP-tagged Sp2 fragments of At-SYP71, At-SYP122, or At-SYP21with mYFP:ZmPIP2;5 was similar to the intensity found in pro-toplasts expressing mYFP:ZmPIP2;5 alone or in combinationwith mCFP:ZmSYP121. As previously mentioned, protoplastscoexpressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2showed a strong reduction of the plasma membrane signal.These data demonstrate that the regulation of Zm-PIP2;5 traf-ficking to the plasma membrane is specifically dependent onZm-SYP121.

Coexpression of Zm-PIP2;5 and the ZmSYP121-Sp2Fragment Leads to a Decrease in Maize Protoplast Pf

and Reproduces the Cell Water Permeability Phenotypeof the syp121-1 Arabidopsis Mutant

To determine whether the reduction in Zm-PIP2;5 delivery to theplasma membrane was correlated with a decrease in the mem-brane water permeability coefficient, we analyzed the swelling ofprotoplasts expressing mYFP:ZmPIP2;5 or mCFP:ZmSYP121singly or coexpressing mYFP:ZmPIP2;5 with mCFP:SYP121 ormCFP:ZmSYP121-Sp2. Mock protoplasts were used as a neg-ative control. For each protoplast population, the Pf values wereobtained as described (Moshelion et al., 2004; Volkov et al.,2007), plotted on a frequency diagram (Figure 3A), and themedians were calculated (Figure 3B). Expression of mYFP:ZmPIP2;5 alone induced an important increase in the Pf valuescompared with the mock protoplasts. Its coexpression withmCFP:ZmSYP121 did not alter this high Pf. For both protoplastpopulations, the Pf distributions were similar, with the presenceof cells having a Pf higher than 10 µm s21. By contrast, proto-plasts coexpressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2 exhibited similar Pf values as mock or mCFP:ZmSYP121expressing protoplasts.

A detailed analysis of the swelling curves obtained with pro-toplasts expressing mYFP:ZmPIP2;5 or mCFP:ZmSYP121-Sp2and mYFP:ZmPIP2;5 was performed as previously reported(Moshelion et al., 2004; Volkov et al., 2007; Moshelion et al.,2009). While no difference in the lag phase preceding theswelling event was detected, the Pf values determined 15, 30,and 45 s after the solution exchange increased during theswelling event but were always significantly lower for proto-plasts coexpressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2 compared with their respective controls (see SupplementalFigure 6 online). In addition, the Pf differences between thesetwo cell populations increased with the swelling time, probablyreflecting differences in the amount of active aquaporins reachingthe plasma membrane (Moshelion et al., 2004).To further characterize the role of SYP121 in regulating the

membrane water permeability, leaf protoplasts isolated from thewild type (Columbia-0) and syp121-1 Arabidopsis mutated line(Collins et al., 2003) were isolated and subjected to an osmoticchallenge, and the Pf was determined. Interestingly, syp121-1protoplasts had a lower Pf than wild-type ones (Figures 3C and3D), phenocopying the permeability of maize protoplasts ex-pressing the SYP121-Sp2 fragment. These data demonstratea clear link between SYP121 function in Arabidopsis and theregulation of the plasma membrane water permeability con-trolled by aquaporins.Finally, we note that no significant increase in Pf was measured

in maize protoplasts coexpressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2 compared with mock cells (Figure 3B), even ifsome mYFP fluorescence was detected in the plasma membrane(Figures 1Ch and 2Af). These observations led us to questionwhether ZmSYP121-Sp2 might regulate Zm-PIP2;5 activity in-dependently of any effect mediated through trafficking.

The Water Channel Activity of Zm-PIP2;5 Is Regulatedby ZmSYP121-Sp2 in Xenopus Oocytes

We tested the impact of Zm-SYP121 and ZmSYP121-Sp2 onthe water channel activity of Zm-PIP2;5 expressed in Xenopusoocytes. Venus:ZmPIP2;5 was expressed alone or togetherwith Zm-SYP121 or the ZmSYP121-Sp2 fragment fused to a 6His tag at the N terminus. The Pf values calculated from oocyteswelling experiments were plotted in Figure 4A. In contrastwith 6His:ZmSYP121, 6His:ZmSYP121-Sp2 induced a signifi-cant decrease in the Pf of Zm-PIP2;5 expressing oocytes, butthe Pf was significantly higher than the Pf of mock oocytes andoocytes expressing 6His:ZmSYP121 or 6His:ZmSYP121-Sp2alone. The Pf decrease might be due to impairment of thedelivery of Venus:ZmPIP2;5 into the oocyte plasma membrane.To determine the amount of protein in the cell membrane, theVenus fluorescence was detected in fixed oocytes using CLSMand quantified (Figures 4B and 4C). When coexpressedwith either 6His:ZmSYP121 or 6His:ZmSYP121-Sp2, Venus:ZmPIP2;5 was still localized in the plasma membrane (Figures4Ba, 4Bb, and 4Bc) and the plasma membrane Venus fluores-cence intensity was similar to the intensity measured in theplasma membrane of oocytes expressing Venus:ZmPIP2;5alone (Figure 4C). Immunodetection analysis of the microsomalfraction showed that the Zm-PIP2;5 amount was similar in




oocytes expressing Zm-PIP2;5 alone or together with Zm-SYP121 or Zm-SYP121-Sp2 (Figures 4D and 4E). As the Pf

values measured in these experiments were around 0.4 to 0.6µm s21, far below the maximum values reported when higheramounts of Zm-PIP cRNAs were injected, we can exclude thepossibility that the protein expression was saturated (Chaumontet al., 2000; Fetter et al., 2004). Altogether, these data areconsistent with a direct effect of Sp2 on Zm-PIP2;5 activity thatis independent of any action on Zm-PIP2;5 trafficking to theplasma membrane.

Zm-PIP2;5 and Zm-SYP121 Physically Interact in XenopusOocytes and in Plant Cells

The regulation of the water channel activity of Zm-PIP2;5 by theZmSYP121-Sp2 fragment might be due to a direct physical in-teraction between both proteins. To evaluate this possibility, weextracted the microsomes of oocytes expressing 6His-taggedZm-PIP2;5 alone or together with Venus:ZmSYP121 and purified6His:ZmPIP2;5 on a nickel–nitrilotriacetic acid affinity column.The presence of 6 His:ZmPIP2;5 and Venus:ZmSYP121 in theelution fraction was checked by immunodetection using anti-bodies raised against ZmPIP2;5 and green fluorescent protein(GFP), respectively (Chaumont et al., 2000; Hachez et al., 2006)(Figures 5A and 5B). A signal corresponding to the Zm-PIP2;5 dimerwas detected in the eluted fractions coming from oocytes ex-pressing 6His:ZmPIP2;5 alone or together with Venus:ZmSYP121(Figure 5A, lanes a and b). No signal was observed in eluted frac-tions from mock oocytes or oocytes expressing Venus:ZmSYP121alone (Figure 5A, lanes c and d). The 6His:ZmPIP2;5 dimeric formwas due to the presence of a disulfide bond linking two Zm-PIP2;5monomers (Bienert et al., 2012) and was detected as no reducingagent was added in the solubilization buffer. Interestingly, a YFPsignal corresponding to Venus:ZmSYP121 was observed in thefraction coming from oocytes coexpressing 6His:ZmPIP2;5 andVenus:ZmSYP121 (Figure 5B, lane b), demonstrating that bothproteins were copurified.

Figure 2. Inhibition of Zm-PIP2;5 Delivery to the Plasma Membrane IsSpecific for ZmSYP121-Sp2.

(A) Plasma membrane abundance of mYFP:ZmPIP2;5 in maize meso-phyll protoplasts. (a) and (b) Protoplast expressing mYFP:ZmPIP2;5 (b).(a) Chlorophyll a autofluorescence. (c) and (d) Protoplast coexpressingmCFP:ZmSYP121 (c) and mYFP:ZmPIP2;5 (d). (e) and (f) Protoplastcoexpressing mCFP:ZmSYP121-Sp2 (e) and mYFP:ZmPIP2;5 (f). (g)and (h) Protoplast coexpressing mCFP:AtSYP71-Sp2 (g) and mYFP:ZmPIP2;5 (h). (i) and (j) Protoplast coexpressing mCFP:AtSYP21-Sp2 (i)and mYFP:ZmPIP2;5 (j). (k) and (l) Protoplast coexpressing mCFP:At-SYP122-Sp2 (k) and mYFP:ZmPIP2;5 (l). All images were acquired withthe same settings. In panel (a), autofluorescence of the chlorophyll a isshown, whereas in panels (c) to (k), chlorophyll a autofluorescence issubtracted. Bars = 5 µm.(B) Quantification of mYFP:ZmPIP2;5 fluorescence signal intensity in theprotoplast plasma membrane (PM). Each bar represents the mean ofthree independent experiments (10 protoplasts per experiment). Errorbars show the 95% confidence interval. Statistical differences were in-ferred by ANOVA, followed by the Tukey’s honestly significant differencetest. Means that are significantly different (P < 0.05) are indicated bydifferent letters. a.u., arbitrary units.


Figure 3. Maize Mesophyll Protoplasts Coexpressing mCFP:ZmSYP121-Sp2 and mYFP:ZmPIP2;5 Phenocopies the Lower Plasma Membrane WaterPermeability (Pf) of Arabidopsis syp121-1 Mesophyll Protoplasts.


The interaction between Zm-PIP2;5 and Zm-SYP121 wasalso checked in plant cells using bimolecular fluorescencecomplementation (BiFC) experiments in maize epidermal cellstransfected by biolistic bombardment. We used split Venus(ADE48838.1) fusion constructs (Nour-Eldin et al., 2006; Shyuet al., 2006). First, the plasma membrane localization of tran-siently expressed mYFP:ZmPIP2;5 was confirmed by colocali-zation with the styryl dye FM4-64 (Figures 5Ca to 5Cc). WhenmCFP:ZmSYP121 and mYFP:ZmPIP2;5 were coexpressed,both proteins colocalized at the cell periphery (Figures 5Cd to5Cg), as previously observed in maize protoplasts (Figures 1Ceto 1Cf). As we previously showed that Zm-PIP2;5 and Zm-PIP1;2 interact in maize cells (Zelazny et al., 2007), we usedthem as a positive control in the BiFC experiments. When Ve-nusN:ZmPIP2;5 and VenusC:ZmPIP1;2 were coexpressed,a fluorescent signal was detected mostly in the cell periphery(Figure 5Ch), validating the method. When VenusN:ZmPIP2;5and VenusC:ZmSYP121 were coexpressed, a Venus signal wasobserved in the cell plasma membrane (Figure 5Ci), demon-strating an interaction between the two proteins. Fluorescentsignals were also detected in the plasma membrane and/or in-ternal membranes when these proteins were coexpressed inmaize mesophyll protoplasts (Figures 5Cl to 5Co). The spectralspecificity of the signal was validated by determination of itsemission spectrum (Figure 5D). Cells expressing VenusN:PMA2and VenusC:ZmSYP121 were used as negative controls for in-teraction (Figures 5Cj and 5Ck or 5Cp and 5Cr). Similar BiFCresults were obtained when the proteins were expressed in to-bacco epidermal cells (see Supplemental Figure 7 online).

To further validate the direct interaction between Zm-PIP2;5and Zm-SYP121, FRET experiments were performed on maizemesophyll protoplasts and tobacco epidermal cells coexpress-ing mCFP:ZmPIP1;2 and mYFP:ZmPIP2;5 or mCFP:ZmSYP121and mYFP:ZmPIP2;5. The FRET efficiency was determined bymeasuring the fluorescence intensity of the donor fluorochromebefore and after acceptor photobleaching (Bhat et al., 2005). Arepresentative experiment is given in Supplemental Figure 8online for each cell type, and quantitative results are reported inTable 1. Coexpression of mCFP:ZmPIP1;2 and mYFP:ZmPIP2;5or mCFP:ZmSYP121 and mYFP:ZmPIP2;5 in maize protoplastsgave a FRET efficiency of ;26 and 15%, respectively, valuessignificantly higher than those of the negative controls consti-tuted by the coexpression of mCFP:ZmPIP2;5 and mYFP:ROP6(Zelazny et al., 2007). These values were validated by assessingFRET efficiency by acceptor-sensitized emission on the samecells. Data obtained by the two methods showed 3% divergence,

which is comparable to the stochastic variations observed in theacceptor photobleaching experiments (see Supplemental Figure 9online).Additionally, we tested the potential for SYP121 interaction

with Zm-PIP2;5 using a mating-based, split-ubiquitin assay,much as previously described in the analysis of the Arabi-dopsis SYP121 interaction with the K+ channels KC1 and KAT1(Honsbein et al., 2009, 2011; Grefen et al., 2010). In this case,we used a ZmPIP2;5-Cub-PLV fusion protein as bait and testedits ability to rescue growth of diploid yeast carrying the preyfusions of Zm-SYP121 and At-SYP121 with Nub, the N-terminalhalf of ubiquitin. For purposes of comparison, we tested forinteractions with a homolog of Zm-PIP2;5, the Arabidopsisaquaporin At-PIP2;2, and we performed each assay with boththe maize SNARE and the Arabidopsis SNARE SYP121. Figures6A and 6B summarize the results of these experiments, with thecolumn on the left in each frame showing the control for growthon mating at 24 h and the subsequent three columns showinggrowth on increasing concentrations of Met that repress theexpression of the bait. Figure 6C shows the immunoblot analysisfor the two experiments, confirming the expression of all fourproteins and the presence of bands at the correct molecularweights. From these results, it is clear that Zm-PIP2;5 interactswith the maize SYP121, and even more strongly with At-SYP121, in high concentrations of Met. The Arabidopsis aqua-porin also interacted with At-SYP121 but showed only a weakassociation with its maize homolog. We note that the negativecontrol (NubG) also showed a low level of growth rescue withthe ZmPIP2;5-Cub-PLV fusion, which may suggest some leak-age, possibly a low level of proteolytic turnover in this casesufficient to promote growth under the nonstringent, low Metconditions. Nonetheless, a comparison shows a clear in-teraction of Zm-PIP2;5 with Zm-SYP121, consistent with ourcopurification data. Taken together, these data demonstratea direct interaction between Zm-SYP121 and Zm-PIP2;5, con-sistent with a regulation of the water channel activity of Zm-PIP2;5 by SYP121.

DISCUSSION

Aquaporins constitute an important selective pathway for waterand small neutral solutes to move across cellular membranes.However, to exert their function, aquaporins have to reach theirtarget membrane and be active. PIP aquaporins are synthesized atthe ER and traffic through the secretory pathway before reachingthe plasma membrane (Paciorek et al., 2005; Dhonukshe et al.,

Figure 3. (continued).

(A) Relative distribution of Pf values of maize mesophyll protoplasts untransfected or (co)expressing mYFP:ZmPIP2;5, mYFP:ZmPIP2;5 and mCFP:ZmSYP121, mYFP:ZmPIP2;5 and mCFP:SYP121-Sp2, or mCFP:ZmSYP121.(B) Median Pf values for each protoplast subpopulation shown in (A).(C) Relative distribution of Pf values of mesophyll protoplasts from wild-type (WT) and syp121 Arabidopsis plants.(D) Median Pf values for both Arabidopsis protoplast subpopulations.Each bar represents the mean of 20 measurements. Error bars show 95% confidence interval. Statistical differences were inferred by the Kruskall-Wallistest followed by a Dunns test or Mann-Whitney U test. Medians that are significantly different (P < 0.05) are shown with different letters.





2007; Jaillais et al., 2007; Zelazny et al., 2007; Sorieul et al.,2011). The fusion of vesicles coming from the endomembranecompartments to the plasma membrane is generally dependenton the activities of SNARE complexes in all eukaryotic cells(Pratelli et al., 2004). Among the SNARE proteins, the plasmamembrane syntaxin SYP121 was demonstrated to regulate thedelivery but also the gating of plant K+ channels through directphysical interaction, indicating that this protein may play animportant role in the control of ion transport and cellular volume(Sutter et al., 2006b; Honsbein et al., 2009).As aquaporin-facilitated water movement through the plasma

membrane is an inherent process controlling the cell water ho-meostasis, we questioned whether SNAREs also regulate thetraffic and activity of these channels. Our results now addressthis question using Zm-PIP2;5 as a model aquaporin and theplasma membrane–resident syntaxin SYP121. We demon-strate that the plasma membrane delivery of ZmPIP2;5, but notthe H+-ATPase PMA2, is reduced by dominant-negative Sp2fragments of SYP121 from Arabidopsis and maize. This regu-lation is SYP121 specific, as Sp2 fragments of At-SYP71, At-SYP122, and At-SYP21 syntaxins had no effect on Zm-PIP2;5traffic. We also show that the membrane osmotic water per-meability coefficient of plant cells and oocytes coexpressingZm-PIP2;5 and SYP121-Sp2 is significantly reduced. However,

Figure 4. ZmSYP121-Sp2 Decreases the Pf of Xenopus Oocytes WhenCoexpressed with Zm-PIP2;5.

(A) Pf of Xenopus oocytes (co)expressing Venus:ZmPIP2;5, Venus:ZmPIP2;5 and 6His:ZmSYP121, Venus:ZmPIP2;5 and 6His:ZmSYP121-Sp2, or 6His:ZmSYP121 and 6His:ZmSYP121-Sp2 or oocytes injectedwith water (Mock). Each bar is the mean of 10 replicates. Error bars are95% confidence intervals. Values were subjected to ANOVA and Tukeyposthoc analysis. Means that are significantly different (P < 0.05) areindicated by different letters.(B) Confocal images of oocytes (co)expressing Venus:ZmPIP2;5 (a),Venus:ZmPIP2;5 and 6His:ZmSYP121 (b), (c) Venus:ZmPIP2;5 and 6His:ZmSYP121-Sp2, Venus:ZmSYP121 (d), or Venus:ZmSYP121-Sp2 (e),water-injected oocytes (f), or oocytes expressing Venus (g). The oocyteswere fixed in 4% (w/v) paraformaldehyde and analyzed by CLSM. Notethat all proteins mainly accumulated in the plasma membrane, exceptVenus:ZmSYP121-Sp2, which showed a localization pattern similar tothat of Venus. Bars = 100 µm.(C) Relative Venus fluorescence signal intensity in the plasma membrane(M) compared with the cytoplasm (C) of oocytes expressing Venus:ZmPIP2;5, Venus:ZmPIP2;5, and 6His:ZmSYP121, Venus:ZmPIP2;5 and6His:ZmSYP121-Sp2, Venus:ZmSYP121, Venus:ZmSYP121-Sp2, orVenus. Each bar is the mean of 20 replicates. Error bars are 95% con-fidence intervals. Values were subjected to ANOVA and Tukey posthocanalysis. Means that are significantly different (P < 0.05) are indicated bydifferent letters.(D) Oocyte total protein extracts were probed with antibodies raisedagainst AtSYP121. Protein extracts from oocytes expressing Venus:ZmPIP2;5 and 6His:ZmSYP121-Sp2 (a), 6His:ZmSYP121-Sp2 (b), Ve-nus:ZmPIP2;5 and 6His:ZmSYP121 (c), 6His:ZmSYP121 (d), Venus:ZmSYP121 (e), or mock-injected sample (f).(E) Oocyte total protein extracts were probed with antibodies raisedagainst Zm-PIP2;5. Protein extracts from oocytes expressing Venus:ZmPIP2;5 (a), Venus:ZmPIP2;5 and 6His:ZmSYP121 (b), and Venus:ZmPIP2;5 and 6His:ZmSYP121-Sp2 (c), or mock-injected sample (d). NoDTT was added to the samples, meaning that only dimers of Venus:ZmPIP2;5 (;125 kD) are detected.


in contrast with what is observed in plant cells, the Sp2 frag-ment did not affect the plasma membrane localization of Zm-PIP2;5 in oocytes, suggesting a role of SYP121 in regulatingaquaporin activity in specific conditions. We now also dem-onstrate a direct interaction of maize SYP121 with Zm-PIP2;5.These findings suggest an important role for SNAREs in theregulation of the traffic and activity of plasma membraneaquaporins.

The Syntaxin SYP121 Is a Specific Regulator of Zm-PIP2;5Plasma Membrane Trafficking

Coexpression of mYFP:ZmPIP2;5 with the soluble ZmSYP121-Sp2 fragment (cotransfected plasmid ratio of 1:1) leads toa decrease in its plasma membrane abundance and, to an ex-tent, its accumulation in intracellular structures. This intracellularaccumulation is further increased in cells cotransfected with theplasmids encoding mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2at a ratio of 1:3 (see Supplemental Figure 4 online). The totalamount of mYFP:ZmPIP2;5 fluorescence (plasma membrane andinternal signals) in these protoplasts was similar to the one foundin cells expressing mYFP:ZmPIP2;5 alone. These data are in ac-cordance with the observation that the soluble SYP121-Sp2fragment competes with the endogenous full-length SYP121 andbehaves as a dominant-negative (competitor) protein (Geelenet al., 2002). From the time-lapse experiments, we found that

Figure 5. Zm-SYP121 and Zm-PIP2;5 Interact Both in Oocytes and inPlant Cells.

(A) and (B) Copurification of solubilized proteins extracted from Xenopusoocyte microsomal fractions on nickel–nitrilotriacetic acid resin. Elutionfractions were probed with antibodies raised against Zm-PIP2;5 in (A)and against GFP in (B). Eluted fractions containing proteins extractedfrom oocytes expressing 6His:ZmPIP2;5 (a), 6His:ZmPIP2;5 and Venus:ZmSYP121 (b), and Venus:ZmSYP121 (c) or water-injected oocytes (d).(C) BiFC in maize epidermal cells and mesophyll protoplasts. (a) Con-focal images of maize epidermal cells transiently expressing mYFP:ZmPIP2;5 and (b) stained with the plasma membrane stain, styryl dyeFM4-64. (c) Colocalization was determined as the relative fluorescenceintensities of mYFP and FM4-64 along the white dotted line. The blackdotted line above the spectra represents the white dotted lines shown in(a) and (b). Note the matches between peaks for the two channelscorresponding to plasma membranes. (d) to (g) Maize epidermal cellscoexpressing mCFP:ZmSYP121 (d) and mYFP:ZmPIP2;5 (e). Theplasma membrane was stained with FM4-64 (f). Merged image of mCFP:ZmSYP121, mYFP:ZmPIP2;5, and FM4-64 (g). (h) to (k) BiFC experi-ments in maize epidermal cells transfected by biolistic bombardment.Confocal images of epidermal cells expressing VenusN:ZmPIP2;5 andVenusC:ZmPIP1;2 (h), and VenusN:ZmPIP2;5 and VenusC:ZmSYP121 (i).(j) Bright field. (k) YFP channel of cells expressing VenusN:PMA2 andVenusC:ZmSYP121. (l) to (r) BiFC experiments in maize mesophyll pro-toplasts. Confocal images of protoplasts transiently coexpressing Ve-nusN:ZmPIP2;5 and VenusC:ZmPIP1;2 ([l] and [m]) or VenusN:ZmPIP2;5and VenusC:ZmSYP121 ([n] and [o]). (p) to (r) Protoplasts coexpressingVenusN:PMA2 and VenusC:ZmSYP121. (p) Chlorophyll a auto-fluorescence. (q) YFP channel. (r) Bright field. Bars = 20 mm in (a) to (k)and 5 mm in (l) to (r). a.u., arbitrary units.(D) Emission spectra after excitation at 514 nm of mYFP:ZmPIP2;5 (blacksolid line) and reconstituted Venus (gray dashed line). Fluorescent signalwas collected from 517 to 683 nm with an interval of 9.8 nm. mYFP andreconstituted Venus share a similar emission pattern.



mYFP:ZmPIP2;5 accumulates in the plasma membrane within 6 hof transfection (see Supplemental Figure 3, Supplemental Movie1, and Supplemental Movie Legend 1 online). However, in cellscoexpressing mYFP:ZmPIP2;5 and ZmSYP121-Sp2, the quanti-fication of fluorescence intensity provides unequivocal evidencethat the traffic dynamics of mYFP:ZmPIP2;5 are affected primarilyat time points after the protein had reached the plasma mem-brane, suggesting an action of the Sp2 fragments on the vesiclescontaining newly synthesized and recycling PIPs from the plasmamembrane. This is in agreement with the results obtained usingthe vesicle-trafficking inhibitor brefeldin A, which prevents thetraffic from the Golgi apparatus to the plasma membrane andinhibits the recycling of membrane protein to the plasma mem-brane (Geldner et al., 2001; Dhonukshe et al., 2007). Brefeldin Ablocks the plasma membrane trafficking of At-PIP2;1 in Arab-idopsis roots (Paciorek et al., 2005) and phenocopies the KAT1 K+

channel trafficking phenotype in the syp121 mutant (Eisenachet al., 2012). Indeed, SYP121 cycles actively between the TGNand the plasma membrane (Reichardt et al., 2011). In addition, themCFP:ZmSYP121-Sp2 signal continuously rose during our ex-periments, demonstrating that the global protein synthesis is notinhibited and did not constitute the origin of the decrease in mYFP:ZmPIP2;5 plasma membrane fluorescence (see SupplementalFigure 4 online).

The short-term FRAP kinetic experiments showed that only20% 6 4.48% of YFP:ZmPIP2;5 is mobile at the cell periphery.Similar results were obtained in Arabidopsis plants expressingAtPIP2;1:GFP (Li et al., 2011; Sorieul et al., 2011; Luu et al., 2012).Interestingly, coexpression with the dominant-negative SYP121-Sp2 fragment reduced the mobile fraction to 7.02%6 3.62% butthe fitted time constants were not affected (20.7 6 7.69 s and19.64 6 6.58 s, respectively). We interpret this result as an in-hibition of mYFP:ZmPIP2;5 recycling to the plasma membrane inthese cells. We did not detect any fluorescence loss in theneighborhood of the bleached area, suggesting that little lateraldiffusion of ZmPIP2;5 occurred (see Supplemental Figure 4 on-line). While very low lateral membrane mobility was observed byFRAP in normal conditions for several fluorescent tagged chan-nels, including Arabidopsis KAT1, PIP2;1, and Nodulin 26-likeintrinsic protein NIP5;1, a boric acid channel, the mobility of At-KAT1 in the presence of the Sp2 fragment increased significantly,indicating a role of the SYP121 in KAT1 distribution and behaviorat the membrane surface (Sutter et al., 2006b; Takano et al., 2010;Sorieul et al., 2011; Luu et al., 2012). This was not observed forZm-PIP2;5, suggesting either that SYP121 is required for dockingthis channel in the plasma membrane but not for its anchoring or

that in vivo interactions are more transient than they are for the K+

channel and therefore do not constitute the overriding factordetermining mobility.The syntaxin SNAREs are well conserved among all eukar-

yotes (Yoshizawa et al., 2006). In plants, sequence comparisonallowed the identification of rice orthologs for most of the Arab-idopsis Q-SNAREs (Sutter et al., 2006a). Our observation thatSYP121-Sp2 fragments from Arabidopsis and maize decreasedthe plasma membrane trafficking of mYFP:ZmPIP2;5 in tobaccoepidermal cells and maize mesophyll protoplasts demonstratesthat the mechanism of SYP121-mediated plasma membraneanchoring of PIP2 aquaporins is conserved between monocotsand dicots. In addition, the localization of the human AQP2 at theapical membrane of epithelial cells of the collecting duct is alsospecifically regulated by the syntaxin-3, SNARE-associated pro-tein Snapin and SNAP33 complex (Procino et al., 2008; Mistryet al., 2009), suggesting that the relative abundance of plasmamembrane aquaporins is regulated by SNARE complexes in alleukaryotes.The specificity of SYP121 in regulating plasma membrane

Zm-PIP2;5 trafficking is indicated by the fact that AtSYP122-Sp2, AtSYP71-Sp2, and AtSYP21-Sp2 do not significantly al-ter the plasma membrane Zm-PIP2;5 abundance. In silicosearch in the Gramene database (http://www.gramene.org/)showed that At-SYP122, At-SYP71, and At-SYP21 have closehomologs in maize, indicating a possible conservation of thetrafficking regulation. Protein trafficking to the plasma mem-brane in Arabidopsis is defined by at least seven of the nineQa-SNAREs of the SYP1 subfamily (Grefen et al., 2011). At-SYP121 and its close homolog At-SYP122 localize to theplasma membrane and their function is partially redundant(Leyman et al., 2000; Collins et al., 2003; Uemura et al., 2004).However, a coexpression study with a dominant-negativemutant of Rab11, a GTPase required for plasma membraneand cell plate trafficking, indicate that SYP121 and SYP122drive two independent secretory events (Rehman et al., 2008).Interestingly, similarly to what has been observed for At-SYP121-Sp2 in tobacco (Sutter et al., 2006b), ZmSYP121-Sp2does not affect the plasma membrane delivery of the mCFP:NpPMA2 H+ATPase in maize protoplasts.

SYP121 Affects the Membrane Osmotic Water Permeabilityby Direct Interaction with Zm-PIP2;5

A first link between the trafficking and the regulation of Zm-PIP2;5 activity by Zm-SYP121 derives from protoplast swelling

Table 1. FRET Efficiencies for the PIP/SYP121 Interaction Studies

Protein FRET Efficiency 6 SD (%) No. of Measurements Cell Type

mCFP:ZmPIP1;2 + mYFP:ZmPIP2;5 25.7 6 9.56 69 Maize protoplastsmCFP:ZmSYP121 + mYFP:ZmPIP2;5 14.8 6 3.12 35 Maize protoplastsmCFP:ZmPIP2;5 + mYFP:ROP6 6.86 6 9.26 20 Maize protoplastsmCFP:ZmPIP1;2 + mYFP:ZmPIP2;5 23.9 6 5.42 35 Tobacco epidermal cellsmCFP:ZmSYP121 + mYFP:ZmPIP2;5 14.1 6 7.96 50 Tobacco epidermal cells

For each analysis, the average value of the FRET efficiency was determined as described in Methods.








http://www.gramene.org/

Figure 6. PIP2 Proteins Interact with SYP121 in Mating-Based Spilt-Ubiquitin Assay.

(A) and (B) Mating-based split-ubiquitin assays were performed using At-PIP2;2 (A) and Zm-PIP2;5 (B) as baits, in each case with Zm-SYP121 and theArabidopsis ortholog At-SYP121 as the prey. Diploid yeast containing the water channels as bait and the SNAREs as prey or Nub-moiety peptides(NubG = negative; NubI = positive control) were dropped in a dilution series (OD 1.0 and 0.1) onto the yeast synthetic media CSM-L-W- to verify matingand on CSM-L-, W-, Ura-, H-, M-, and Ade- containing increasing Met levels to repress expression of the bait. Yeast growth was recorded afterincubation for 24 h (mating test) and for 48 h (interaction test) at 30°C.(C) Expression of bait and prey fusion proteins was verified using anti-SYP121 antibody (Tyrrell et al., 2007) and VP16 (Abcam) antibodies. Molecularmasses of the primary bands in the protein gel blots are indicated below. Ponceau S staining (above) was recorded to monitor equal loading. The doublebands are characteristic of the SNAREs (Geelen et al., 2002).


assays. A significant decrease in Pf was measured in protoplastscoexpressing mYFP:ZmPIP2;5 and the ZmSYP121-Sp2 frag-ment. The most plausible hypothesis is that this Pf alteration re-sults from a decrease in mYFP:ZmPIP2;5 abundance in theplasma membrane. However, this interpretation does not pre-clude additional effects of ZmSYP121-Sp2 in directly modifyingthe water channel activity of ZmPIP2;5. We observed that oocytescoexpressing mYFP:ZmPIP2;5 and ZmSYP121-Sp2 were char-acterized by a lower Pf compared with oocytes expressing mYFP:ZmPIP2;5 singly or in combination with Zm-SYP121. Significantly,in these oocytes, no difference in the mYFP:ZmPIP2;5 expressionlevel and plasma membrane fluorescence intensity was detected,suggesting that in this heterologous system, mYFP:ZmPIP2;5trafficking could not explain the difference in activity.

In mammalian cells, the plasma membrane delivery and gatingof the cAMP-gated chloride channel and the inward rectifierpotassium channels (Kir) are regulated by syntaxin and otherexocytotic proteins (Naren et al., 1997; Leung et al., 2007). Inplants, At-SYP121 directly interacts with the KC1 K+ regulatorysubunit and modulates the activity of the AKT1/KC1 potassiumchannel (Honsbein et al., 2009; Grefen et al., 2010). Similarly,a direct interaction between Zm-PIP2;5 and Zm-SYP121 isdemonstrated by affinity chromatography copurification aftertheir expression in oocytes, yeast mating-based split-ubiquitinassays, and microscopy-based techniques (BiFC and FRET) inplant cells. These imaging approaches showed an interactionbetween Zm-PIP2;5 and Zm-SYP121 in both the plasmamembrane and ER. This latter localization was predominant inmaize protoplasts. Considering that Zm-PIP2;5 and Zm-SYP121use the secretory pathway to reach the plasma membrane, thislocalization may simply reflect the lack of sufficient coordinatepartners necessary to correctly process and target the proteinsto the plasma membrane. Validation of in vivo interactionscomes from FRET measurements by acceptor photobleaching.In our hands, this FRET methodology produces similar results tothe FRET measured by acceptor-sensitized emission. Interactionsbetween mYFP:ZmPIP2;5 and mCFP:ZmSYP121 are detected inthe plasma membrane, but the FRET efficiency was 10% weakerthan the efficiency measured in cells coexpressing mCFP:ZmPIP1;2 and mYFP:ZmPIP2;5. Since no FRET was detected inprotoplasts coexpressing mCFP:ZmPIP2;5 and mYFP:ROP6,a plasma membrane–localized GTPase, which does not interactwith Zm-PIP2;5 (Bischoff et al., 2000; Zelazny et al., 2007), weconcluded that FRET by acceptor photobleaching could be usedto investigate membrane protein interactions in tobacco epidermalcells and maize protoplasts. Altogether, the microscopy-basedinteraction data demonstrate that SYP121 physically interacts withZm-PIP2;5 in plant cells. These observations were further sup-ported by yeast split-ubiquitin assays that demonstrated aninteraction of Zm-PIP2;5 with Zm-SYP121 or At-SYP121 in therescue of diploid yeast growth.

These data allow us to propose hypothetical models (Figure7). In plant cells, the plasma membrane delivery of Zm-PIP2;5during both constitutive recycling and anterograde transportfrom the TGN vesicles is mediated by SYP121, either by a Qa-SNARE/VAMP vesicle recognition independent of Zm-PIP2;5and/or by a direct docking interaction between Zm-PIP2;5,SYP121, and VAMP (Figure 7A). In cells coexpressing Zm-

PIP2;5 and ZmSYP121-Sp2, the truncated SNARE interactswith Zm-PIP2;5 and VAMP, preventing the formation of theSNARE complex with native Zm-SYP121 and, therefore, Zm-PIP2;5 plasma membrane delivery (Figure 7B). In addition, ex-periments in Xenopus oocytes indicate that SYP121-Sp2 candirectly affect the activity of Zm-PIP2;5. In this system, the ve-sicular secretion of heterologously expressed protein appears

Figure 7. Hypothetical Models Summarizing the Regulation of Zm-PIP2;5 Trafficking and Activity by Zm-SYP121.

(A) Regulation of Zm-PIP2;5 trafficking in plant cells coexpressing Zm-PIP2;5 and full-length Zm-SYP121. Constitutive recycling and delivery ofnewly synthesized Zm-PIP2;5 to the plasma membrane are regulated bySYP121 through Qa-SNARE/VAMP and direct interactions. SYP121 al-lows a proper docking of Zm-PIP2;5 at the plasma membrane and reg-ulates its amount.(B) Regulation of Zm-PIP2;5 trafficking in plant cells coexpressing Zm-PIP2;5 and the ZmSYP121-Sp2 fragment. (a) The dominant-negativemutant SYP121-Sp2 might bind to Zm-PIP2;5 proteins, altering theirwater channel activity and preventing their interaction with full-lengthSYP121. (b) Constitutive recycling and delivery of newly synthesizedZm-PIP2;5 to the plasma membrane are altered by the binding ofSYP121-Sp2 to VAMP and Zm-PIP2;5. This interaction does not allowSNARE complex formation and the fusion of vesicles carrying Zm-PIP2;5cargo to the plasma membrane. Additionally, SYP121-Sp2 might bindresident ZmPIP2;5 proteins and inhibit their water transport activity. Seetext for additional explanations.


not to be specific (Mohun et al., 1981), explaining the fact thatSYP121-Sp2 does not affect Zm-PIP2;5 trafficking to theplasma membrane. In cells (co)expressing Zm-PIP2;5 and Zm-SYP121, the two partners interact at the plasma membrane andpossibly in the vesicle without affecting the trafficking or waterchannel activity of Zm-PIP2;5. By contrast, in oocytes coex-pressing Zm-PIP2;5 and ZmSYP121-Sp2, the plasma mem-brane delivery of ZmPIP2;5 is not affected, but its water channelactivity is significantly decreased. The observation that no sig-nificant increase in Pf was measured in maize protoplasts coex-pressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121-Sp2 comparedwith mock cells, even if some mYFP fluorescence was detected inthe plasma membrane, suggest that, in plant cells, the Sp2 sol-uble fragment also inhibits Zm-PIP2;5 activity (Figure 7B). Onehypothesis is that the Sp2-soluble fragment interacts with Zm-PIP2;5 cytosolic loops or extremities and modifies its conforma-tion and gating (Törnroth-Horsefield et al., 2006). A modification ofAQP1-mediated water entry in pancreatic zymogen granules wasshown to be blocked after incubation with antibodies raisedagainst the C-terminal end of the protein (Cho et al., 2002). Be-cause of steric constraint for accessibility, this kind of confor-mational change would not occur with membrane-anchoredSYP121, which would rather act to increase PIP stability in theplasma membrane.

Is SYP121 a Stress-Responsive SNARE Involvedin the Maintenance of the Cell Turgor and WaterBalance in Plants?

It is striking to observe that the SNARE SYP121 regulates thetrafficking and activity of plasma membrane K+ channels andaquaporins, transporters involved in the regulation of cell waterhomeostasis. In addition, all of these components are regulatedby the stress hormone abscisic acid (Leyman et al., 1999;Geelen et al., 2002; Parent et al., 2009). An attractive hypothesisis that SYP121 acts as an essential component to coregulate ionand water movement through the cell membrane.

Besides modulating trafficking and anchoring of KAT1,SYP121 can directly modulate K+ channel activity. Split-ubiquitin,BiFC, and coimmunoprecipitation experiments demonstrated thatSYP121, but not its closest homolog SYP122, directly interactswith the KC1 regulatory subunit to form a tripartite SYP121-KC1-AKT1 complex. Mutations in any of the three proteins selectivelysuppressed the inward-rectifying K+ current in Arabidopsis rootepidermal protoplasts as well as K+ acquisition and growth inseedlings when channel-mediated K+ uptake was limiting(Honsbein et al., 2009). Further characterization showed thatSYP121 can also interact with KAT1 (Grefen et al., 2010) andthat the FxRF motif located in the first 12 amino acid residues ofSYP121 was required for the selective interaction with the K+

channels (Grefen et al., 2010). These data indicate an essentialrole of SYP121 in the connection between ion transport andmembrane traffic (Grefen et al., 2011; Honsbein et al., 2011). Ourresults suggest that SYP121 could also regulate water homeo-stasis through modulation of aquaporin docking and possiblyactivity. We showed here that SYP121 can physically interactwith Zm-PIP2;5 and acts as a regulator of its plasma membranedelivery. The absence of direct modulation of Zm-PIP2;5 water

channel activity by full-length SYP121 led us to suggest that itcan rather play a role in coordinating water and K+ ion fluxes. Wepropose that SYP121 acts to coordinate the plasma membranedensity of both PIP and K+ channels by regulating their mem-brane delivery and recycling. The next challenge will be tomonitor the dynamics of PIPs and K+ channels in the plasmamembrane of cells subjected to osmotic stress. However, asSYP121 physically interacts with both PIPs and KC1 subunits,we cannot exclude at this stage that SYP121 also plays a role asa physical bridge between both proteins, resulting in the for-mation of a higher complex. It was suggested that PIPs couldact as turgor sensors in the plasma membrane that modulate theconductance of K+ ion channels (Hill et al., 2004). The PIP/SYP121/K+ channel tripartite regulation could coordinate theturgor sensing with a tight tuning of ions and water movementand content in growing cells, guard cells, or cells submitted todrought stress. A direct link between ion transport, stomatalaperture, and optimization of water use efficiency has recentlybeen established in the Arabidopsis syp121 loss-of-functionmutant growing in high light intensity and low relative humidity(Eisenach et al., 2012). Importantly, we showed that protoplastsisolated from syp121 leaves have a lower membrane waterpermeability than do wild-type protoplasts, indicating a role ofSYP121 in aquaporin regulation. Altogether, our data add evi-dence to the concept of SNAREs being a molecular governor(Grefen and Blatt, 2008; Honsbein et al., 2011) that coordinatesmembrane transporter traffic and activity with the regulation ofwater and solute transport in plant cells.

METHODS

Isolation and Transfection of Maize and Arabidopsis Protoplasts

Maize (Zea mays) B73 seedlings were grown, and mesophyll protoplastisolation and transfection were performed as described previously (Ze-lazny et al., 2007). Arabidopsis thaliana Columbia-0 wild type and syp121(syp121-1/pen1-1) mutants (Collins et al., 2003) were grown in soil under250mmol m22 s21 in long days (16:8 light [L]:dark [D]) at 22:21°C (L:D) witha relative humidity of 70%. Mesophyll protoplasts were isolated as de-scribed previously (Ramahaleo et al., 1999).

Protoplast Swelling Assay

Maize and Arabidopsis protoplast swelling experiments were performedas described previously (Moshelion et al., 2004; Volkov et al., 2007). Thesolutions used for Arabidopsis protoplast swelling have been described(Postaire et al., 2010). The selection of protoplasts (co)expressing mYFP:ZmPIP2;5 and mCFP:ZmSYP121 or mCFP:ZmSYP121-Sp2 was achieveddue to their fluorescence emission.

Phylogenetic Analysis

The multiple alignment in Supplemental Figure 1 online was obtainedusing the ClustalW algorithm with a Blosum62 scoring matrix, open gappenalty of 10, and gap extension penalty of 0.5. The neighbor-joiningmethod was used to build the phylogenetic tree. Nodes’ significance wasinferred by 1000 bootstrap iterations.

Molecular Biology

PCR products encoding Np-PMA2, Zm-PIP2;5, Zm-SYP121, ZmSYP121-Sp2, AtSYP121-Sp2, AtSYP122-Sp2, AtSYP21-Sp2, and AtSYP71-Sp2



cDNAs were directionally subcloned using a uracil excision-based im-proved high-throughput USER cloning technique (Nour-Eldin et al., 2006)into the USER-compatible plant expression vectors pCAMBIA2300 35SuNterm mYFP and pCAMBIA2300 35Su Nterm mCFP (Bienert et al., 2011).This allowed the expression and in vivo visualization of the protein ofinterest tagged with either mCFP or mYFP at the N terminus. For BiFCexperiments, new vectors were generated by adding a linker coding forGSGGSGGS before the USER cassette in the pCAMBIA2300 35Su NtermY155N and pCAMBIA2300 35Su Nterm Y155C vectors and after theUSER cassette in the pCAMBIA2300 35Su Cterm Y155N and pCAM-BIA2300 35Su Nterm C155C vectors. This vector set was used to expressZm-PIP2;5, Zm-PIP1;2, Zm-SYP121, and Np-PMA2 fused to the splitVenus. All cloning products were checked by sequencing. cDNAs en-coding Zm-PIP2;5, Zm-SYP121, Zm-SYP121-Sp2, and Zm-PIP1;2 weresubcloned into the USER-compatible Xenopus laevis expression pNB1u,pNBRGS-HISu, and pNB1YFPu vectors containing the YFP gene ora sequence encoding a RGS-6 His tag 59 of the insertion site (Nour-Eldinet al., 2006).

Particle Bombardment Transfection

Gold beads (0.6-µm diameter, 480 µg) were coated with 1 µg of plasmidDNA. The median parts of the third leaf of 7-d-old etiolated maizeseedlings were placed in a Petri dish, which was positioned 3 cm belowthe microprojectile stopping plate. Samples were bombarded once under948 kPa partial vacuum at a rupture pressure of 1100 p.s.i. (7600 kPa)using a PDS-1000/He Biolistic device (Bio-Rad). After transfection, leafexplants were incubated overnight at 25°C in the dark on solid half-strength Hoagland medium.

Confocal Microscopy

Detection of mCFP and mYFP fusion proteins was performed usinga Zeiss LSM710 confocal microscope equipped with a spectral detector.Imaging of the maize mesophyll protoplasts was achieved with a Plan-Apochromat 363/1.40 oil immersion objective. mCFP and mYFP wereexcited with the 445- and 514-nm laser lines, respectively. Emitted lightwas collected through a dichroic mirror on detectors 450 to 510 nm(mCFP) and 520 to 600 nm (mYFP). In each experiment, calibration of thelaser beam intensity, gain, and offset parameters were achieved on cellsexpressing mYFP:ZmPIP2;5. The same parameters were then used inimage acquisitions performed on coexpressing cells, allowing a sub-sequent calculation of the plasma membrane fluorescence intensity. Thefluorescence intensity in the protoplast plasma membrane and in-tracellular compartments was quantified using an in-house developedmacro for the ImageJ software. First, the average thickness of the plasmamembrane was determined using the colocalized signals arising frommYFP:ZmPIP2;5 and the steryl dye FM4-64 by averaging 100 meas-urements (10 measurements performed on 10 independent cells). Themean value of the optical thickness of the protoplast plasma membranewas 0.35 mm. Computation of the images was then performed in threesteps. First, the image was binarized, smoothed by the application ofa median filter, and, finally, converted into traces using the outline functionof the software. Second, the periphery trace was converted into the regionof interest (ROI-1), which thus contains the overall cell fluorescence. Third,the ROI-1 was narrowed 0.35 mm in diameter to obtain ROI-2 that re-flected the intracellular fluorescence. The fluorescence in the plasmamembrane is given by the difference between the integrated signaldensities (product of the surface area and the intensity of the signal)measured in ROI-1 and ROI-2.

Time-lapse acquisitions were performed using a combination of thetime-lapse, tile scan, position, and Z-stack modules of the Zen 2009software (Carl Zeiss MicroImaging). Image acquisition was done every 30

min for 16 h, using a C-Apochromat340/1.20 water immersion objectiveto maximize the size of the acquisition field.

FRAP experiments were performed as follows. Photobleaching (80iterations, 514-nm laser beam at 100% [i.e., 7.5 mW]) was performed ina defined ROI and recovery of fluorescence was monitored by acquisitionof one image every 5 s for 200 s. FRAP data were analyzed as previouslydescribed (Sprague et al., 2004). The best fitting curve was obtainedaccording to a single exponential association using the “bottom to span”algorithm in Prism software (GraphPad Software). The following equationwas used: y = span3 (12 e-kt) + bottom, where the bottom is defined bythe minimal fluorescence intensity recorded after photobleaching, span isthe difference between the bottom and the calculated plateau, and k is thebinding constant defined by the half time of recovery, such as t1/2 = 0.69/k.

The BiFC assay was performed in tobacco (Nicotiana tabacum) epi-dermal cells transformed by Agrobacterium tumefaciens infiltration (Batokoet al., 2000) or in polyethylene glycol–transfected maize protoplasts. In cellsexhibiting a fluorescence signal after excitation at 514 nm, an emissionspectrum was determined to validate the signal specificity. The subcellularlocalization was assessed by Z-stack acquisition.

FRET was measured using either the acceptor photobleaching oracceptor-sensitized emission methods. Acquisitions were performed ontwo optical tracks, in sequential line mode. For the photobleachingmethod, three images were acquired for both the mCFP and mYFPchannels, and then the mYFP was bleached by 80 iterations with full-power laser illumination, while the fluorescence intensity of themCFPwassimultaneously monitored along time. The FRET efficiency (E) was de-termined according the equation E = 12 IDA/ID, where IDA is the intensity ofthe donor fluorescence before photobleaching and ID the fluorescenceintensity of the donor after photobleaching, as described elsewhere(Kwaaitaal et al., 2010). The sensitized emission method was used ona subpopulation of tobacco epidermal cells and protoplasts to confirm thedata obtained by acceptor photobleaching. One acquisition track wascomposed of two acquisition channels to detect the mCFP and FRETsignals. mCFP was excited using the 445-nm laser beam, and the emittedlight was collected between 447 and 517 nm and between 523 nm and630 nm for mCFP and FRET detection, respectively. The acceptor wasexcited at 514 nm, and emitted light was collected between 523 and 628nm. FRET efficiency was calculated according to the Youvan method(Youvan et al., 1997) using the FRET module of Zen 2009 software (CarlZeiss MicroImaging).

Heterologous Expression in Xenopus Oocytes

In vitro cRNA synthesis, oocyte isolation, microinjection, and measure-ment of Pf and microsome isolation were performed as described byFetter et al. (2004). Oocytes were injected with 2 or 4 ng of cRNA encodingthe 6 His-tagged or YFP-tagged proteins, respectively. The cRNA wasquantified with a Nanodrop 1000 spectrophotometer (NanoDrop Tech-nologies) and its integrity checked on an agarose gel. Oocytes expressingthe YFP-tagged constructs were fixed and sliced as described previously(Sayers et al., 1997). Fluorescence was visualized by CLSM using a Plan-Neofluar 310/0.30 objective as described above and quantified withImageJ software.

Mating-Based Split-Ubiquitin Assays

The haploid yeast strains THY.AP4 and THY.AP5 (Obrdlik et al., 2004;Grefen et al., 2007) were transformed as described previously (Grefenet al., 2009). Approximately 15 single colonies of bait (Cub-PLV) and prey(Nub) constructs were selected following 3 d of growth on plates andinoculated in vector-selective media for overnight growth. Liquid cultureswere harvested at an OD600 of 2 to 3 and resuspended in yeast-extractpeptone dextrose medium. Equal volumes (20 µL) of each bait and prey


were mixed and dropped on YPD plates. After 6 to 8 h incubation at 30°C,yeast was scraped off and streaked on vector-selective media (CSM-L-,W-, and Ura-). After ;1 to 2 d of growth, the diploid yeast was used toinoculate liquid vector-selective media and grown overnight. The nextday, serial dilutions at OD600 1.0 and 0.1 in water were dropped, 7 mL perspot, onto plates without and with the addition of 5, 50, and 500 µM Meton interaction-selective media (CSM-L-, W-, Ura-, Ade-, H-, and M-).Growth was monitored after 2 or 3 d at 30°C. Control plates on vector-selective media were incubated for 24 h only to verify that an equalamount of yeast had been dropped. To verify expression, haploid yeastwas harvested prior to mating and analyzed via immunoblotting usingpolyclonal antibodies against SYP121 and VP16, as described previously(Grefen et al., 2009; Honsbein et al., 2009).

Solubilization and Purification of His:ZmPIP2;5and Immunodetection

Microsomes were resuspended in 13 PBS buffer. Three hundred mi-crograms of proteins were solubilized with 1% (w/v) octyl-b-D-gluco-pyranoside in a final volume of 400 mL and incubated for 2 h on a rotarywheel at room temperature. Unsolubilized material was removed bycentrifugation at 169,000g for 20 min, and the supernatant was added to100 mL of Ni2+-nitriloacetic acid agarose matrix (Qiagen) preequilibratedwith 13 PBS buffer containing 1% [w/v] octyl-b-D-glucopyranoside and10 mM imidazole. The mixture was incubated for 2 h on a rotary wheel atroom temperature, loaded on a column, andwashed three timeswith 1mLof 13 PBS buffer containing 10 mM imidazole and 0.05% Tween 20.Bound proteins were eluted with 100 mL Laemmli buffer. Proteins (50 µL/well) were electrophoresed by SDS-PAGE, transferred to a nitrocellulosemembrane, and immunodetected with antibodies directed against GFP(Duby et al., 2001), SYP121 (Tyrrell et al., 2007), and Zm-PIP2;5 (Hachezet al., 2006) by the enhanced bioluminescence method.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL andmaize sequencing project data libraries (http://www.maizesequence.org/index.html; Schnable et al., 2009) under the following accession numbers:At-SYP111, NP_172332; At-SYP112, NP_179418.2; At-SYP121,NP_187788; At-SYP122, NP_190808; At-SYP123, NP_192242; At-SYP124, NP_176324; At-SYP125, NP_172591; At-SYP131, NP_187030;At-SYP132, NP_568187; Os-SYP111, ABB22783; Os-SYP112, ABA96047;Os-SYP121, ABF99241; Os-SYP124, NP_001172852; Os-SYP131,NP_001058959; Os-SYP132, NP_001056925; Zm-SYP111, NP_001149999;Zm-SYP112, GRMZM2G168017; Zm-SYP121, NP_001150776, Zm-SYP124, GRMZM2G411561; Zm-SYP123, GRMZM2G416733; Zm-SYP132, NP_001149376; Zm-SYP131, GRMZM2G330772; Np-PMA2,AAA34052; and Zm-PIP2;5, AF130975.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Unrooted Phylogenetic Tree of the PlasmaMembrane–Localized Qa SNAREs.

Supplemental Figure 2. Intracellular Accumulation of mYFP:ZmPIP2;5 in Protoplasts Transfected with the Plasmids EncodingmCFP:ZmSYP121-Sp2 and mYFP:ZmPIP2;5 at a 3:1 Ratio.

Supplemental Figure 3. Still Images of Supplemental Movie 1,Showing Time-Lapse Acquisition of mCFP:ZmSYP121-Sp2 andmYFP:ZmPIP2;5 Accumulation in Maize Mesophyll Protoplasts.

Supplemental Figure 4. ZmSYP121-Sp2 Affects Plasma MembraneDelivery but Not Synthesis of mYFP:ZmPIP2;5.

Supplemental Figure 5. Fluorescence Recovery after Photobleaching.

Supplemental Figure 6. Pf Values Measured after 15, 30, and 45 s ofProtoplast Swelling.

Supplemental Figure 7. Zm-SYP121 and Zm-PIP2;5 Interact inTobacco Epidermal Cells.

Supplemental Figure 8. FRET Experiments.

Supplemental Figure 9. Comparison of Sensitized Emission andAcceptor Photobleaching FRET Efficiencies.

Supplemental Data Set 1. Multiple Alignments of the Full-LengthProtein Sequences of Qa SNARE Used to Build the Phylogenetic Treeof Supplemental Figure 2 Online.

Supplemental Movie 1. Movie of Time-Lapse Acquisition of mCFP:ZmSYP121-Sp2 and mYFP:ZmPIP2;5 Accumulation in Maize Meso-phyll Protoplasts.

Supplemental Movie Legend 1. Legend for Supplemental Movie 1.

ACKNOWLEDGMENTS

We thank the imaging platform IMABIOL. This work was supported bygrants from the Belgian National Fund for Scientific Research (FNRS), theInteruniversity Attraction Poles Programme–Belgian Science Policy, the“Communauté française de Belgique–Actions de Recherches Concert-ées”, and by grants BB/F001630/1 and BB/H0009817/1 to M.R.B. fromthe UK Biotechnology and Biological Sciences Research Council. A.B.was a postdoctoral research fellow at the FNRS. G.P.B. was supportedby an individual Marie Curie European fellowship and by the FNRS. A.S.C.was a research fellow at the “Fonds pour la Formation à la Recherche dansl’Industrie et dans l’Agriculture.”

AUTHOR CONTRIBUTIONS

A.B. designed and performed research, analyzed data, developed newanalytic tools, and wrote the article. E.B. performed research andanalyzed data. G.P.B. designed the research, provided molecular tools,and analyzed data. A.S.C. provided molecular tools, performed research,and analyzed the data. A.E. provided technical assistance. C.G.performed research, analyzed data, and wrote the article. F.C. designedthe research and, together with M.R.B., analyzed data and wrote thearticle.

Received June 19, 2012; revised July 23, 2012; accepted August 1,2012; published August 31, 2012.

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DOI 10.1105/tpc.112.101758; originally published online August 31, 2012;Plant Cell

Errachid, Christopher Grefen, Michael R. Blatt and François ChaumontArnaud Besserer, Emeline Burnotte, Gerd Patrick Bienert, Adrien S. Chevalier, Abdelmounaim

SNARE SYP121Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the

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