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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 reduces the time to publication by several weeks. Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase Anne Kriegel,a Zaida Andrés,a Anna Medzihradszky,b Falco Krüger,a Stefan Scholl,a Simon Delang,a M. Görkem Patir-Nebioglu,a Gezahegn Gute,c Haibing Yang,d Angus S. Murphy,c Wendy Ann Peer,c,e Anne Pfeiffer,b Melanie Krebs,a Jan U. Lohmann,a and Karin Schumachera,1 a Department of Plant Developmental Biology, Centre for Organismal Studies, Heidelberg University, 69120 Heidelberg, Germany of Stem Cell Biology, Centre for Organismal Studies, Heidelberg University, 69120 Heidelberg, Germany c Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland 20742 d Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907 e Environmental Science and Technology, University of Maryland, College Park, Maryland 20742 b Department ORCID IDs: 0000-0002-5418-6248 (H.Y.); 0000-0001-5649-7413 (A.S.M.); 0000-0003-0046-7324 (W.A.P.); 0000-0001-6484-8105 (K.S.) The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H+-pyrophosphatase (V-PPase) and the vacuolar H+-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH. INTRODUCTION The evolutionary success of higher plants is in large part due to their unique cell architecture. Their often large cell volumes are filled with a central vacuole containing mostly water and solutes that allow plants to maximize collection of solar energy and mineral nutrients by increasing the surface of their photosynthesizing and nutrient-absorbing organs at minimal cost. Besides being lowcost space fillers, vacuoles are the main store for solutes and serve as a hydrostatic skeleton that provides the driving force for cell growth and reversible volume changes. They allow plants to adapt to the fluctuating availability of essential nutrients, to detoxify the cytosol when challenged by harmful molecules, and serve as lysosome-like organelles in which endocytic and autophagic cargo are digested (Marty, 1999; Martinoia et al., 2012). Although vacuoles are generally multifunctional, a single function, like protein storage, tends to prevail in a particular cell type or developmental stage (Zheng and Staehelin, 2011), and coexistence of vacuoles with different functions in a single cell has been demonstrated only in very few cases (Epimashko et al., 2004; Frigerio et al., 2008). All vacuolar functions require massive fluxes of molecules across the tonoplast, which are assumed to be 1 Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Karin Schumacher (karin. [email protected]). www.plantcell.org/cgi/doi/10.1105/tpc.15.00733 energized by the proton gradient and membrane potential created by the combined activity of two proton pumps, the vacuolar H+-ATPase (V-ATPase) and the vacuolar H+-pyrophosphatase (V-PPase). Both proton pumps are highly abundant tonoplast proteins, underlining that the amount of energy invested into vacuolar transport is substantial (Gaxiola et al., 2007; Schumacher, 2014). Pyrophosphate (PPi) is a by-product of many biosynthetic processes, and the V-PPase could thus be the predominant vacuolar proton pump in young, growing cells. However, the V-PPase has also been discussed as a backup system for the V-ATPase under ATP-limiting conditions like anoxia or cold stress, and it is generally assumed that the combined action of V-ATPase and V-PPase enables plants to maintain transport into the vacuole even under stressful conditions (Maeshima, 2000). V-ATPases are multisubunit proton pumps found in all eukaryotes that consist of the peripheral V1 complex responsible for ATP hydrolysis and the membrane-integral Vo complex responsible for proton translocation. Localization of the V-ATPase is determined by the isoforms of the membrane-integral Vo subunit VHA-a with VHA-a1 mediating targeting to the trans-Golgi network/early endosome (TGN/EE) and VHA-a2 and VHA-a3 conferring tonoplast localization (Dettmer et al., 2006). Arabidopsis thaliana mutants that lack both tonoplast-localized isoforms are viable but show a strong reduction in growth and nutrient storage capacity (Krebs et al., 2010). The fact that the vha-a2 vha-a3 mutant still maintains a 10-fold proton gradient across the tonoplast (vacuolar pH 6.4 versus cytosolic pH 7.4) argues that the V-PPase, a homodimer of a single polypeptide chain, plays a more important role in vacuolar acidification than the V-ATPase. Arabidopsis The Plant Cell Preview, www.aspb.org ã 2015 American Society of Plant Biologists. All rights reserved. 1 of 14 2 of 14 The Plant Cell plants carrying a T-DNA insertion in the only gene encoding a K+-stimulated Arabidopsis vacuolar PPase 1AVP1 or Arabidopsis vacuolar H+-PPase VHP1 were reported to show severe developmental phenotypes caused by defects in auxin transport (Li et al., 2005). However, additional independent alleles of AVP1/ VHP1, uncovered by the analysis of fugu5 mutants that failed to support heterotrophic growth after germination, did not show auxin-related phenotypes. Importantly, vacuolar pH in the fugu5 mutants was only mildly affected, and compensation by increased V-ATPase activity was ruled out (Ferjani et al., 2011). Moreover, the mild postgermination growth defect of fugu5 seedlings resulting in a slightly different cotyledon shape could be rescued by expression of a soluble yeast pyrophosphatase (Ferjani et al., 2011), highlighting a so far undiscovered role of the V-PPase in the removal of PPi required to avoid accumulation of inhibitory concentrations of PPi (Ferjani et al., 2012). These findings are also highly relevant as overexpression of AVP1 in Arabidopsis, as well as in a number of crop plants, results in improved drought and salt tolerance (Gaxiola et al., 2001; Pasapula et al., 2011; Gamboa et al., 2013; Schilling et al., 2014). AVP1 overexpression has also been reported to result in increased cell division at the onset of organ formation and increased auxin transport, which appeared to be a consequence of increased DpHPM (visualized as cytosolic alkalinization) resulting from altered distribution and abundance of the plasma membrane (PM) H+-ATPase and the PIN-FORMED1 auxin efflux facilitator (Li et al., 2005). Although AVP1 clearly is an abundant tonoplast protein (Segami et al., 2014), it has been reported to be localized at the PM in sieve element companion cells and upon overexpression also in other cell types (Langhans et al., 2001; Paez-Valencia et al., 2011; Pizzio et al., 2015). The mechanistic base of the beneficial traits achieved by overexpression of AVP1 is thus unclear, and it remains to be determined if and to what extent increased vacuolar solute accumulation due to increased proton pumping by the V-PPase is involved. By combining loss- and gain-of-function approaches, we have addressed how V-ATPase and V-PPase share the job of vacuolar acidification. Here, we show that lack of the V-ATPase cannot be compensated for by increased V-PPase activity but also that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we show that a mutant lacking both tonoplast V-ATPase and V-PPase is viable and retains significant vacuolar acidification, revealing the presence of a so far unnoticed contribution of the TGN/EE-localized V-ATPase. RESULTS Lack of Tonoplast V-ATPase Activity Cannot Be Compensated for by Increased V-PPase Activity To investigate if a lack of tonoplast V-ATPase activity can be compensated for by increased V-PPase activity, the vha-a2 vha-3 double mutant was crossed with AVP1-1, a transgenic line expressing AVP1 under the control of the double 35S promoter (Gaxiola et al., 2001). In the segregating F2 generation, vha-a2 vha-a3 AVP1-1 plants were identified by genotyping and showed a small increase in size compared with vha-a2 vha-a3 (Supplemental Figure 1A). Although it was reported previously that AVP1 protein levels are increased in AVP1-1 plants (Gaxiola et al., 2001), we could not detect ubiquitous AVP1 overexpression based on qPCR, RNA in situ hybridization, immunocytochemistry, and immunoblot analysis in the vha-a2 vha-a3 mutant background as well as in the progeny of the original transgenic AVP1-1 line (Supplemental Figures 1B to 1K). Lack of AVP1 overexpression is most likely due to transgene silencing, as indicated by the complete absence or patchy kanamycin resistance in AVP1-1 and AVP1-2 in subsequent generations (Supplemental Figures 1M and 1N). We thus generated transgenic lines expressing AVP1 under the control of the UBQ10 promoter, which leads to constitutive and robust overexpression (Grefen et al., 2010; Behera et al., 2015) and resulted in 2- to 3-fold higher V-PPase activity (Figures 1A and 1B). Notably, constitutive overexpression of AVP1 did not correlate with an increase in rosette size and fresh weight (Figures 1C and 1D) or with altered auxin transport and content as reported previously (Li et al., 2005; Supplemental Tables 1 and 2). Importantly, overexpression of AVP1 in the vha-a2 vha-a3 mutant background did not cause an increase in rosette size (Figures 2A to 2C) and did not affect leaf cell sap pH (Figure 2D) or root vacuolar pH (Figure 2E). Taken together, these results show that constitutive overexpression of the tonoplast V-PPase AVP1 does not necessarily cause an increase in biomass in wild-type plants and that enhanced V-PPase activity cannot compensate for a lack of tonoplast V-ATPase activity under standard growth conditions. A Second T-DNA Insertion in GNOM Is Responsible for the avp1-1 Phenotype The apparent discrepancy between the severe phenotype of avp1-1 seedlings and the mild cotyledon shape phenotype of vhp1-1 and fugu5 seedlings led us to test the hypothesis that the avp1-1 allele is linked to a second-site mutation (Ferjani et al., 2012). Therefore, whole-genome sequencing of the avp1-1 line (GABI-Kat 005D04) was performed and identified a second T-DNA insertion in the 39 end of At1g13980, the gene encoding the ARFGEF GNOM that is genetically linked to the insertion in AVP1 (At1g15690; Figure 3A; Supplemental Figure 2). GNOM is required for polar auxin transport (Geldner et al., 2003) and weak alleles cause phenotypes that are highly similar to the one observed in avp1-1 (Geldner et al., 2004). To test if the additional T-DNA insertion in GNOM is indeed responsible for the avp1-1 phenotype, we performed allelism tests among the avp1/vhp1/fugu alleles and the gnom allele emb30-1 (Mayer et al., 1993). The gnom phenotype was observed in 25% of seedlings in the F1 progeny from a cross between the avp1-1/+ and emb30/+ lines, whereas crosses of homozygous vhp1-1 and fugu5-1 plants to avp1-1/+ resulted in a 1:1 ratio of the wild type and fugu5-1/vhp1-1-phenotype seedlings (Figures 3B and 3C). Further comparison among the AVP1/VHP1 loss-of-function alleles showed that auxin transport and free indole-3-acetic acid (IAA) levels in the fugu5 alleles, unlike avp1-1, were similar to those in the wild type (Supplemental Tables 3 and 4). Based on the combined results, we concluded that the avp1-1 phenotype is caused by a linked second site mutation in GNOM, whereas the fugu5/vhp1 phenotype reflects the lack of AVP1/VHP1 activity. Importantly, although V-PPase activity is not detectable in both vhp1-1 and fugu5-1, we observed only a marginal increase of cell sap pH in Job Sharing between V-ATPase and V-PPase 3 of 14 Figure 1. Increased Biomass Does Not Require Constitutive AVP1 Overexpression. (A) Increased AVP1 protein amounts in UBQ:AVP1 lines. Microsomal membrane extracts of 4-week-old wild-type, 35S:AVP1, and UBQ:AVP1 plants were separated by SDS-PAGE and subsequently immunoblotted with anti-V-PPase antibody. Equal protein loading is indicated by anti-VHA-C detection. (B) Elevated K+-stimulated PPase activity in UBQ:AVP1 but not in 35S:AVP1 lines. Plants were grown for 4 weeks under LD conditions. Wild type activity was set to 100%. Graph shows result of one representative experiment of three biological replicates. Error bars indicate SD of n = 3 technical replicates. (C) and (D) Phenotypes (C) and rosette fresh weight (D) of 4-week-old 35S:AVP1 plants compared with UBQ:AVP1 lines grown under LD conditions. Error bars indicate SE of n = 8 to 11 plants. FW, fresh weight. Bar = 3.5 cm. both mutants (Supplemental Figure 3B). However, under our growth conditions, the rosette size of vhp1-1 mutants was reduced, whereas fugu5-1 was indistinguishable from the wild type (Supplemental Figure 3A). Based on these findings, we used both mutants, fugu5-1 and vhp1-1, in our further studies. Upregulation of V-ATPase during Cold Acclimation Depends on the Presence and Activity of V-PPase Under standard growth conditions and based on steady state pH, the V-PPase does not seem to make a major contribution to vacuolar acidification (Ferjani et al., 2011), and we thus next investigated its role during cold acclimation, a process previously reported to require increased tonoplast proton-pumping activity (Schulze et al., 2012). In the wild type, cold acclimation for 4 d at 4°C led to a slight reduction of leaf cell sap pH from 5.8 to 5.7 (Figure 4A), consistent with increased amounts and activities of both V-ATPase and V-PPase (Figures 4B to 4E). In both fugu5-1 and vhp1-1, vacuolar pH was slightly higher than in the wild type under control conditions but the cold-induced drop of cell sap pH was reduced. Conversely, overexpression of AVP1 induced an even stronger pH decrease (Figure 4A). Surprisingly, the cold-induced increase in V-ATPase amount and activity was reduced in the V-PPase loss-of-function mutants and enhanced in the overexpression line (Figure 4D), indicating that upregulation of the V-ATPase during cold acclimation depends on the presence and activity of the V-PPase. A Triple Mutant Lacking Both Tonoplast Proton Pumps Is Viable A triple mutant lacking V-ATPase and V-PPase would allow us to address two important questions. First, if the presence of proton pumps at the tonoplast is essential and, second, if the V-PPase does indeed not contribute to vacuolar acidification under nonstress conditions, in which case we would expect no vacuolar pH difference between vha-a2 vha-a3 and the triple mutant. In contrast, the pH gradient at the tonoplast would be even more reduced in the triple mutant compared with vha-a2 vha-a3 if the lack of proton pumping in vhp1-1/fugu5-1 single mutants is masked by a compensatory mechanism. We thus crossed the tonoplast V-ATPase-deficient vha-a2 vha-a3 mutant with plants homozygous for vhp1-1 or fugu5-1. In the segregating F2 progeny, we were able to identify vhp1-1 vha-a2 vha-a3 and fugu5-1 vha-a2 vha-a3 seedlings; however, the vegetative growth of both triple mutants was strongly impaired and seedlings only survived when germinated and grown in sterile culture for a few days before transfer to soil (Figure 5A). Flowers of the triple mutant had smaller petals and shorter stamen filaments as well as nondehiscent anthers, leading to strongly reduced fertility (Figure 5B). Siliques of fugu5-1 vha-a2/+ vha-a3 plants were slightly shorter and contained ;10% aborted ovules, indicating that gametophyte development was impaired. This became more evident as vhp1-1 vha-a2 vha-a3 mutants did not produce any seeds, whereas fugu5-1 vha-a2 vha-a3 plants produced few but viable seeds (Figure 5C). We next measured cell sap pH using leaf material of 3-week-old plants grown on soil under standard long-day (LD) growth and found it to be strongly increased to pH 7.1 in the fugu5-1 vha-a2 vha-a3 triple mutant, whereas cell sap pH for the fugu5-1 mutant was slightly increased (pH5.8) and the vha-a2 vha-a3 mutant showed the expected increase to pH 6.4 (Figure 5D). Cell sap pH can be used as a robust approximation of vacuolar pH, as the vacuolar lumen normally occupies ;90% of the cell volume. To determine if this assumption is also true for the triple mutant, we used BCECF staining to show that although in the fugu5-1 vha-a2 vha-a3 mutant the cell size of both epidermis and mesophyll cells was reduced, a large central vacuole was clearly present (Figures 5E to 5J). Based on these findings, the 4 of 14 The Plant Cell Figure 2. Overexpression of AVP1 Does Not Complement the Tonoplast V-ATPase Double Mutant vha-a2 vha-a3. (A) Arabidopsis wild-type and vha-a2 vha-a3 mutant plants expressing AVP1 under the UBQ promoter have higher AVP1 protein level. Microsomal membrane proteins of 3-week-old plants were extracted, separated by SDS-PAGE, and subsequently immunoblotted with anti-V-PPase antibody. Equal protein loading is indicated by TIP1;1 detection. A quantification of AVP1 protein levels is shown below and each bar corresponds with the band in the blot immediately above it. Error bars indicate SE of n = 3 technical replicates. (B) UBQ:AVP1 cannot restore wild type growth in vha-a2 vha-a3. Plants were grown for 3 weeks under LD conditions at 22°C. Bar = 1.75 cm. (C) AVP1 overexpression lines show increased V-PPase activity. K+-stimulated PPase activity was determined using microsomal membranes extracted from 3-week-old plants. Wild type activity was set to 100%. Error bars represent SE of n = 3 biological replicates. (D) Overexpression of AVP1 has no effect on cell sap pH. Plants for cell sap pH measurements were grown for 3 weeks under LD conditions at 22°C. Error bars show SD of n = 3 biological replicates. (E) Vacuolar pH in root epidermal cells is not changed upon AVP1 overexpression. Vacuolar pH was measured in roots of 6-d-old plants. Error bars represent SD of n = 3 biological replicates. V-PPase AVP1/VHP1 acts as a proton pump and is responsible for the remaining vacuolar acidification observed in leaves of the vha-a2 vha-a3 mutant. However, these results also raise the important question of if and how cell expansion and vacuole formation can occur in the absence of the two proton pumps. Lack of V-ATPase and V-PPase Does Not Abolish Cell Expansion and Vacuole Acidification in Hypocotyl and Root Cells Hypocotyl length of etiolated seedlings is solely based on cellular expansion (Gendreau et al., 1997), and we found it to be reduced by 50% in the fugu5-1 vha-a2 vha-a3 mutant (Supplemental Figure 4A). Similarly, root length was also reduced by ;50% due to reduced cell expansion as the size of the meristematic zone was found to be not significantly different between wild-type and fugu5-1 vha-a2 vha-a3 seedlings (Supplemental Figures 4B and 4C). To visualize vacuolar morphology and to measure vacuolar pH in the three developmental zones, roots were stained with BCECF and analyzed by confocal laser scanning microscopy (CLSM) (Viotti et al., 2013). Meristematic root cells of wild-type roots contained a complex tubular vacuolar network surrounding the nucleus (Figure 6A, bottom). In the root elongation zone, cells start to rapidly expand accompanied by the inflation of vacuoles Job Sharing between V-ATPase and V-PPase Figure 3. T-DNA Insertion in GNOM Causes Phenotypic Defects in avp1-1. (A) Position of the two T-DNA Insertions on chromosome 1 in the GABI-Kat 005D04 line. RB, right border; LB, left border. Bars = 500 bp. (B) Comparison of the phenotypes of 5-d-old wild-type, fugu5-1, avp1-1, and gnomR5 seedlings. Bar = 1 cm. (C) Allelism test between V-PPase and gnom mutants. V-PPase mutants (vhp1-1 and fugu5-1) were crossed with avp1-1/+ or with gnom/+ (emb301/+) mutants. Phenotypes of 6-d-old F1 seedlings (n = 80 to 190) were counted. Numbers given in the table are percentages (%). (Figure 6A, middle). Finally, in the differentiation zone, cells reach their final size and are almost completely filled by the large central vacuole (Figure 6A, top). No strong differences in vacuole morphology between the wild type and vha-a2 vha-a3 or fugu5-1 mutants were observed (Figures 6B and 6C). Only in the elongation and transition zone of fugu5-1 vha-a2 vha-a3 roots was vacuole morphology severely altered (Figure 6D, middle and top). Here, vacuoles appeared as multiple spheres of different sizes distributed within the cell, whereas vacuoles in fully elongated cells of the triple mutant have regained a completely normal shape. For pH measurements, CLSM images of stained roots were analyzed and the obtained emission ratios were converted to pH values using an in situ calibration curve (Krebs et al., 2010). In general, all genotypes showed an increased vacuolar pH in the elongation zone, but surprisingly the vacuolar pH in the different regions of fugu5-1 vha-a2 vha-a3 roots was not significantly increased when compared with vha-a2 vha-a3 (Figures 6E to 6G). Vacuolar Acidification Is Abolished by the V-ATPase Inhibitor Concanamycin A The fact that the triple mutant is able to maintain a 10-fold proton gradient across the tonoplast of root cells points to the contribution of an additional source of proton import into the vacuole. In recent years, it has become clear that P-type H+-ATPases can be found at the tonoplast in some cell types (Verweij et al., 2008; 5 of 14 Appelhagen et al., 2015), and we thus assessed the effect of ortho-vanadate, an inhibitor of P-type ATPases on media acidification (Supplemental Figure 5A), and vacuolar pH in roots using BCECF. Although a slight increase of vacuolar pH was detected in the roots of wild-type, vha-a2 vha-a3, and fugu5-1 plants, vacuolar pH in the triple mutant fugu5-1 vha-a2 vha-a3 was not affected by ortho-vanadate (Figure 7A). Moreover, although transcripts of AHA10, the only tonoplast-localized P-type H+-ATPase of Arabidopsis identified to date (Appelhagen et al., 2015), are detectable in roots, transcript levels are not increased in any of the V-ATPase and/or V-PPase mutants (Supplemental Figure 5B). In contrast, the V-ATPase inhibitor concanamycin A (ConcA) abolished vacuolar acidification in all genotypes tested, including vha-a2 vha-a3 and fugu5-1 vha-a2 vha-a3 (Figure 7B), leading to the accumulation of autophagic bodies without affecting BCECF accumulation or vacuolar morphology (Figure 7C). Importantly, TGN/EE-localized V-ATPase complexes containing VHA-a1 are the only remaining targets of ConcA in vha-a2 vha-a3 and fugu5-1 vha-a2 vha-a3. We cannot rule out that a minor proportion of VHA-a1 is relocated to the tonoplast in vha-a2 vha-a3; however, we could not detect VHA-a1-GFP at the tonoplast using highly sensitive hybrid detection CLSM (Supplemental Figures 5C to 5F). We thus conclude that the V-ATPase in the TGN/EE contributes to vacuolar acidification either by controlling trafficking of a yet unknown proton pump or by vesicular transport of protons. DISCUSSION How Does AVP1 Overexpression Enhance Crop Yield? Although constitutive overexpression of AVP1 was reported for the two transgenic lines AVP1-1 and AVP1-2 (Gaxiola et al., 2001), we could not detect increased AVP1 RNA or protein levels in leaves and several other tissues. Given that a vector containing several copies of the 35S promoter was used in the original construct (Yoo et al., 2005) and that multiple copies of the T-DNA have been detected in the transgenic lines (Gaxiola et al., 2001), it is not unexpected that expression of the transgene was lost over time presumably due to 35S-siRNA-mediated transgene silencing (Daxinger et al., 2008; Mlotshwa et al., 2010). Silencing of transgenes expressed under the control of the 35S promoter is common in Arabidopsis; however, it is widely used in crop plants, as it has been shown to cause constitutive and stable overexpression of many transgenes, including AVP1. 35S-driven overexpression of AVP1 is a successful strategy to improve crop performance. Increased biomass, higher stress tolerance, and enhanced P solubilization are highly desirable traits that have been achieved in many crop plants including rice (Oryza sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), cotton (Gossypium hirsutum), sugarcane (Saccharum officinarum) (Yang et al., 2007; Pasapula et al., 2011; Kumar et al., 2014; Li et al., 2014), and tomato (Solanum lycopersicum), for which stable expression of the transgene up to the T8 generation has been reported (Park et al., 2005; Yang et al., 2014). Despite the fact that constitutive overexpression of AVP1 in Arabidopsis was lost due to transgene silencing in the original lines, the progeny still display the significant increase in rosette 6 of 14 The Plant Cell Figure 4. Increase of V-ATPase Activity after Cold Acclimation Depends on V-PPase. (A) Lack of V-PPase activity prevents full acidification upon cold acclimation. Plants for cell sap pH measurements were grown for 6 weeks under short-day conditions at 22°C. Error bars represent SD of n = 3 biological replicates. Asterisk indicates significant difference at P < 0.05 (Student’s t test). (B) V-PPase activity affects the induction of V-ATPase activity upon cold acclimation. KNO3-inhibited ATPase activity was determined with microsomal membranes extracted from 6-week-old, short day-grown plants that were cold-acclimated for 4 d at 4°C. Error bars show SD of n = 3 biological replicates. Asterisk indicates significant difference at P < 0.05 (Student’s t test). (C) V-PPase activity of the wild type and UBQ:AVP1 increases after cold exposure. K+-stimulated PPase activity was determined from microsomal membranes extracted from 6-week-old, short day-grown plants that were cold-acclimated for 4 d at 4°C. Error bars represent SD of n = 3 biological replicates. Asterisk indicates significant difference at P < 0.05 (Student’s t test). (D) and (E) Protein levels of V-ATPase and V-PPase increase upon cold exposure in the wild type and UBQ:AVP1. Microsomal membrane proteins of 6-week-old plants were extracted, separated by SDS-PAGE, and subsequently immunoblotted with VHA-C antibody (D) and anti-V-PPase antibody (E). TIP1;1 detection was used as loading control. Protein levels were measured using ImageJ and normalized to TIP1;1. Protein levels at 22°C were set to 100%. Bar charts represent quantification of one representative immunoblot. biomass that was originally reported. In contrast, the lines overexpressing AVP1 established in this study only showed a marginal increase in biomass, suggesting that constitutive overexpression is neither necessary nor sufficient for improved growth and possibly also stress tolerance. One possible scenario to explain this is that overexpression is maintained in particular cell types or tissues of AVP1-1 and AVP1-2 plants. It was shown recently that guard cell-specific overexpression of the plasma membrane H+ATPase AHA2 causes enhanced stomatal aperture and increased photosynthetic activity, ultimately resulting in larger leaves (Wang et al., 2014). Similarly, based on the fact that AVP1 has been proposed to function as a PPi synthase at the PM of sieve element companion cell (Pizzio et al., 2015), restricted overexpression of AVP1 in the phloem could be causative. The positive effects of AVP1 overexpression were initially assumed to be solely due to the increased capacity for cation transport into the vacuole, but it is now clear that other roles of AVP1, e.g., in PPi homeostasis, need to be considered. Under stress conditions such as salinity or phosphate starvation, endogenous AVP1 expression is induced along with an increase in rhizosphere acidification in the case of phosphate starvation (Yang et al., 2007). In support of the acid growth hypothesis, for the AVP1-1 line, auxin transport was reported to display increased acidification of the apoplast caused by the higher presence and activity of P-type H+-ATPase at the PM (Li et al., 2005). Importantly, no correlation between increased AVP1 expression and altered auxin transport and content was detected for the transgenic lines expressing AVP1 under control of the UBQ10 promoter. This is in line with the fact that, based on DR5:GUS expression and its response to exogenous IAA, the fugu5-1 mutant has been shown to have a normal auxin distribution and response (Ferjani et al., 2011). In addition, we have shown here that auxin transport and auxin content are indistinguishable from that of the wild type in all fugu5 alleles and that the severe developmental phenotype of avp1-1 is due to the presence of a second T-DNA insertion close to the 39 end of the gene encoding the ARF-GEF GNOM that is required for PIN cycling. Taken together, these results show that AVP1 is not required for auxin transport and that auxin might thus not be directly involved in and responsible for the enlarged root systems observed in Arabidopsis and different crop species. Clearly, further insight into the mechanisms underlying the beneficial traits is urgently required and could lead to better-defined strategies for AVP1 expression in crop improvement. Does AVP1 Act as a Proton Pump? Based on the facts that high concentrations of PPi, a by-product of many biosynthetic reactions, are present and that the V-PPase is Job Sharing between V-ATPase and V-PPase 7 of 14 Figure 5. Vacuolar Proton Pump Triple Mutant Is Viable and Has a Neutral Cell Sap pH. (A) fugu5-1 vha-a2 vha-a3 growth is even more inhibited than vha-a2 vha-a3. Wild-type and proton pump mutant plants were grown for 26 d under LD conditions at 22°C. Bar = 1.75 cm. (B) Proton pump triple mutants exhibit defects in flower development. Bright-field micrographs of dissected flowers are shown. Stamen filaments reached the pistil in the wild type and the segregating proton pump triple mutant flower, whereas they are shortened in the fugu5-1 vha-a2 vha-a3 flower. Bar = 1 mm. (C) Silique size of fugu5-1 vha-a2 vha-a3 is strongly reduced. Excised siliques of the wild type, fugu5-1 vha-a2/+ vha-a3, and fugu5-1 vha-a2 vha-a3 are shown. Bar = 3 mm. (D) Cell sap pH of fugu5-1 vha-a2 vha-a3 is nearly neutral. Plants were grown for 3 weeks under LD conditions at 22°C. Error bars show SD of n = 3 biological replicates. (E) to (J) Vacuoles of epidermal cells ([E] to [G]) and mesophyll cells ([H] to [J]) of rosette leaves of 5-week-old wild-type and vacuolar proton pump mutants stained with BCECF (green). Autofluorescence of chloroplasts is shown in magenta. Bars = 25 µm. more abundant than the V-ATPase in young tissues, the V-PPase has been considered to be the main tonoplast proton pump in such tissues (Maeshima, 2000). This notion was supported by our previous work in which we found that the tonoplast V-ATPase- deficient mutant vha-a2 vha-a3 maintained a 10-fold proton gradient across the tonoplast (vacuolar pH 6.4 versus cytosolic pH 7.4; Krebs et al., 2010). In contrast, the role of the V-PPase as a major proton pump was challenged by the finding that loss of 8 of 14 The Plant Cell Figure 6. Root Vacuole Morphology in Tonoplast Proton Pump Mutants. (A) to (D) The shape of vacuoles was monitored in the root differentiation zone, elongation zone, and meristematic zone of 6-d-old wild-type (A), vha-a2 vhaa3 (B), fugu5-1 (C), and fugu5-1 vha-a2 vha-a3 (D) seedlings. Roots were stained with BCECF (green) and FM4-64 (red). Bars = 20 µm. (E) to (G) Vacuolar pH measurement in three different root zones. pH values were determined in the root differentiation zone (E), elongation zone (F), and meristematic zone (G) of 6-d-old wild-type and vacuolar proton pump mutant seedlings. Error bars represent SD of n = 3 biological replicates. V-PPase activity in the fugu5-1 and vhp1-1 mutants only had a mild effect on vacuolar pH. Whereas the vacuolar pH in seedlings of fugu5-1 and vhp1-1 was shown to be 0.25 pH units more alkaline than in the wild type (Ferjani et al., 2011), we showed here that the cell sap pH of mature rosette leaves of the V-PPase mutants is only marginally changed (<0.1 pH units). The contribution of AVP1 to vacuole acidification, at least under standard growth conditions, thus seemed questionable. However, it is important to keep in mind that due to the flexible ATP/H+-coupling rate of the V-ATPase, the lack of V-PPase activity might be masked by increased V-ATPase H+-pumping activity. Although it has been shown that in the fugu5 mutants the ATP hydrolysis rate was not increased, proton-pumping activity was not determined. Moreover, we demonstrate here that overexpression of AVP1 does not ameliorate the vha-a2 vha-a3 mutant phenotype, which also argues against an important role of the V-PPase in vacuolar acidification. Interestingly, the fact that in Arabidopsis lack of tonoplast V-ATPase activity cannot be compensated for by increased V-PPase activity stands in contrast to the fact that expression of AVP1 rescues vph1, a yeast mutant lacking the vacuolar isoform of the V-ATPase (Pérez-Castiñeira et al., 2011; Coonrod et al., 2013). This is most easily reconciled by assuming that the activities of V-ATPase and V-PPase depend on each other and our results concerning cold acclimation support such a scenario. The massive increase in the concentration of sugars and organic acids in the vacuole, one of the most prominent metabolic changes that reaches a maximum after 4 to 5 d of cold acclimation, goes along with an increase in V-ATPase amount and activity, whereas the V-PPase remained largely unaffected (Schulze et al., 2012). Surprisingly, we could show here that cold-induced upregulation of the V-ATPase depends on the presence of the V-PPase. Coregulation of V-ATPase and V-PPase has previously been proposed based on the observation that in the cassulacean acid metabolism plant Kalanchoe blossfeldiana H+-pumping by the V-ATPase could be increased by ;300% when vesicles were preenergized by a short incubation with PPi (Fischer-Schliebs et al., 1997). The underlying mechanism remains to be elucidated, but a direct physical interaction as suggested previously (Fischer-Schliebs et al., 1997) seems likely to be involved. Although the membrane potential difference between the vacuolar lumen and the cytosol is generally reported to be rather low (#30 mV), it will be important to determine how this physiological parameter is affected in mutants lacking one or both vacuolar H+-pumps. Determination of the tonoplast membrane potential is challenging as it can currently only be measured via impalement with double-barreled microelectrodes (Miller et al., 2001; Wang et al., 2015) and novel tools that allow in vivo imaging of tonoplast membrane potential based on voltagesensitive fluorescent proteins are highly desirable (Baker et al., 2008). Job Sharing between V-ATPase and V-PPase 9 of 14 Figure 7. Inhibition of V-ATPase Abolishes Vacuolar Acidification. (A) Inhibition of P-type ATPases has minor effect on vacuolar pH. Vacuolar pH was measured in roots of 6-d-old seedlings after treatment with (+) or without (2) 2 mM vanadate for 18 h. Error bars represent SD of n = 3 biological replicates. (B) ConcA-mediated inhibition of V-ATPase blocks vacuolar acidification. Vacuolar pH was measured in roots of 6-d-old plants. Seedlings of Arabidopsis wild-type and vacuolar proton pump mutants were incubated for 20 h in 1 µM ConcA (+) or DMSO (2) prior to pH measurements. Error bars represent SD of n = 3 biological replicates. (C) Vacuolar morphology after ConcA treatment. Wild-type seedlings were incubated for 20 h in 1 µM ConcA or DMSO. Roots were stained with BCECF (green) and FM4-64. Bars = 20 µm. What Do We Learn from a Mutant Lacking Both Vacuolar H+- Pumps? Given that all vacuolar functions rely on transport across the tonoplast that is either energized by the proton gradient or the membrane potential generated by the two tonoplast proton pumps, it is surprising that the fugu5-1 vha-a2 vha-a3 triple mutant is viable. The analysis of this important genetic resource allowed us to draw several important conclusions. First of all, the nearly neutral cell sap pH in leaves of the triple mutant is most easily explained by a significant contribution of the V-PPase to vacuolar acidification that is not revealed in the V-PPase single mutants. Although mesophyll cells of the triple mutant are substantially smaller, they develop a large central vacuole, and it remains to be determined if vacuolar pH in these cells is always high or if an initial pH gradient is established but then consumed by secondary active transport processes. Second, our finding that cell expansion in the hypocotyl and in the root is reduced but not abolished demonstrates that vacuolar functions can be maintained in the absence of both pumps. In contrast to the situation in leaves, vacuolar pH in roots was largely unaffected by the additional lack of the V-PPase in the triple mutant. This indicates either that the V-PPase only plays a minor role in the root or that a compensatory mechanism for vacuolar acidification acts in roots but not in leaves. However, we have also shown here that in the root elongation zone, vacuole morphology is affected in the triple mutant but not in the vha-a2 vha-a3 double mutant, indicating a function of the V-PPase in roots, the nature of which remains to be identified. How Can Vacuoles Be Acidic without V-ATPase and V- PPase? V-ATPase and V-PPase have long been considered to be the only tonoplast proton pumps, but it is by now clear that members of the P3A subgroup of P-type ATPases previously assumed to be exclusively present at the plasma membrane can also be found at the tonoplast (Verweij et al., 2008; Faraco et al., 2014). In Arabidopsis, AHA10 is required for the vacuolar accumulation of proanthocyanidins (Baxter et al., 2005) and has recently been shown to be localized at the tonoplast of seed coat endothelial cells (Appelhagen et al., 2015). In agreement with its preferential expression in the integument layers of developing seeds, no other phenotypes than the altered seed color have been reported for aha10 mutants. Although we could detect expression of AHA10 in roots, it is not upregulated in the triple mutant. Given that vacuolar pH was not affected by vanadate treatment, compensation by a P3A-type ATPase is unlikely to be responsible for the vacuolar 10 of 14 The Plant Cell acidification observed in the triple mutant. In contrast, inhibition of the remaining activity of V-ATPase complexes containing VHA-a1 by ConcA in the double and triple mutant led to complete loss of vacuolar acidification. Relocalization of Stv1p, the otherwise strictly Golgi-localized isoform of yeast subunit a, to the vacuole has been reported for yeast mutants lacking Vph1p (Perzov et al., 2002). A similar relocalization of VHA-a1 to the tonoplast could explain both the remaining vacuolar acidification and its ConcA sensitivity. Within the detection limit of the method, we could not observe a shift in VHA-a1 localization to the tonoplast and thus favor an alternative hypothesis in which the TGN/EE-localized V-ATPase contributes directly or indirectly to vacuolar pH. ConcA specifically inhibits V-ATPase activity; as a consequence, secretory and endocytic trafficking from the TGN/EE to the vacuole are impaired (Dettmer et al., 2006; Viotti et al., 2010). Assuming the existence of a so far unidentified proton pump that is delivered via the TGN/EE to the tonoplast, the effect of ConcA could be explained by reduced tonoplast levels of this unknown protein. An alternative explanation would be that vesicular trafficking from the TGN/EE to the vacuole delivers a substantial amount of protons and/or organic acids to the vacuolar lumen. The TGN/EE is the most acidic compartment of the plant endomembrane system (Martinière et al., 2013; Shen et al., 2013), and using a pH-sensor with suitable resolution at low pH, we have recently shown that in Arabidopsis roots pH in the TGN/EE is indeed lower than the vacuolar pH (Luo et al., 2015). At least three independent, parallel trafficking pathways exist from the TGN/EE to the vacuole (Pedrazzini et al., 2013; Rojas-Pierce, 2013), and it remains to be determined if the combined flux of protons delivered to the vacuolar lumen would be sufficient to maintain a proton gradient even in the absence of tonoplast-localized proton pumps. METHODS Plant Materials and Growth Conditions Arabidopsis thaliana Col-0 ecotype was used in all experiments as control. The transgenic 35S:AVP1 lines (AVP1-1 and AVP1-2) were previously established (Gaxiola et al., 2001). The two V-PPase mutant lines fugu5-1 and vhp1-1 were previously described (Ferjani et al., 2007, 2011). The loss-of-function mutant avp1-1 (GK_005D04) was obtained from the GABI-KAT collection (www. gabi-kat.de/). The GNOM mutants gnomR5 and emb30-1 were described previously (Mayer et al., 1993). The tonoplast V-ATPase double mutant vha-a2 vha-a3 has been previously characterized (Krebs et al., 2010). Standard growth medium for plate experiments contained 0.53 Murashige and Skoog (MS), 0.5% sucrose, 0.5% phyto agar, and 10 mM MES, and the pH was set to 5.8 using KOH. Agar and MS basal salt mixture were purchased from Duchefa. Seeds were surface sterilized with ethanol and stratified for 48 h at 4°C. Plants were grown under LD conditions (16 h light/8 h dark). Plant material used for in situ hybridization, immunohistochemistry, immunoblotting, qRT-PCR, V-PPase activity assay, cell sap pH, leaf vacuolar morphology, and fresh weight determination was grown in soil at 22°C under LD conditions. For auxin transport assays and free IAA quantification, plants were grown as previously described (Kubeš et al., 2012). Briefly, plants were grown on 0.253 MS media, pH 5.7, 0.5% sucrose, and 1% phyto agar for 14 h under 100 µmol m22 s21 light unless otherwise indicated. Seedlings for root vacuolar pH measurements, vacuolar morphology, root zone determination, and AHA10 RNA level were grown for 6 d on plates containing standard growth medium. Etiolated seedlings were grown on medium containing 10 mM MESKOH, pH 5.8, solidified with 1% phyto agar. Seeds were exposed to light for 4 h and then grown in the dark at 22°C for 4 d. For root length measurements, single seeds were placed on standard growth medium solidified with 0.8% phyto agar and then grown vertically for 10 d. Seedlings for external media acidification were cultivated for 5 d on medium containing 0.53 MS, 0.5% sucrose, and 2.5 mM MES-KOH, pH 5.8, solidified with 0.6% phyto agar before they were transferred to a 96well plate containing 0.53 MS and 0.5% sucrose with the pH set to 6.4. Construct Design and Plant Transformation To generate transgenic plants expressing AVP1 under the control of the UBQ10 promoter, the plant binary vector UBQ:AVP1 was generated. First, a 2331-bp fragment of AVP1 was amplified from cDNA using AVP1-XbaI-Fw and AVP1-SalI-Rv (Supplemental Table 3). After subcloning into pJET1.2blunt (Thermo Scientific), the fragment was released using the introduced XbaI and SalI restriction sites and inserted into the backbone of the binary vector pTKan (Krebs et al., 2012) to generate pTKan-AVP1. In addition, a 661-bp fragment of the Arabidopsis UBQ10 promoter was amplified using UBQ10-KpnI-Fw and UBQ10-KpnI-Rv. After subcloning of UBQ10 into pJET1.2blunt, the fragment was released via the KpnI site and inserted into pTKan-AVP1 to finally yield UBQ:AVP1. The UBQ:AVP1 construct was introduced into the Agrobacterium tumefaciens strain GV3101:pMP90 and selected on 5 mg/mL rifampicin, 10 mg/mL gentamycin, and 100 mg/mL spectinomycin. Arabidopsis ecotype Col-0 and vha-a2 vha-a3 plants were used for transformation using standard procedures. Transgenic plants were selected on MS medium containing 50 µg/mL kanamycin. For creating the template for in situ hybridization, the cDNA of AVP1 was amplified from the pJET1.2blunt construct using AVP1-SalI-Fw and AVP1-XbaI-Rv primers and cloned into pGEM-T Easy. Genome Sequencing For genome sequencing, avp1-1 seedlings showing strong shoot and root abnormalities were collected and used for DNA preparation. DNA was isolated according to the manufacturer’s instructions using the DNeasy Plant Mini Kit (Qiagen). The library was produced at the CellNetworks Deep Sequencing Core Facility (Heidelberg) and sequenced at the EMBL Genomics Core Facility (Heidelberg) as 50-bp single-end reads, multiplexed on a HiSeq2000 (Illumina). Sequences were mapped to the TAIR10 genome using bowtie2 (version 2.0.5) (Langmead and Salzberg, 2012) with the fast-local option, and 21.7 mio reads mapped for the avp1 mutant line. The avp1 mutant was additionally mapped against the GABI-KAT plasmid pAC106 (Kirik et al., 2006; Kleinboelting et al., 2012). The mappings were converted to bam format, sorted and indexed using samtools (version 0.1.16) (Li et al., 2009), and displayed on the Integrative Genomics Viewer (Robinson et al., 2011; Thorvaldsdóttir et al., 2013). Reads that showed partial mapping to the T-DNA borders were compared with gaps in the genomic alignment to identify the insertion sites. RNA Isolation and cDNA Synthesis For the analysis of AVP1 and AHA10 transcript levels, RNA was isolated according to the manufacturer’s instructions using the RNeasy Plant Mini Kit (Qiagen). cDNA was synthesized from 2 µg of total RNA using M-MuLV reverse transcriptase (Thermo) and an oligo(dT) primer. Real-Time RT-PCR Real-time PCR reactions were performed using the DNA Engine Opticon System (DNA Engine cycler and Chromo4 detector; Bio-Rad) and Absolute qPCR SYBR Green Mix (Thermo Scientific). The real-time PCR reaction Job Sharing between V-ATPase and V-PPase mixture with a final volume of 20 mL contained 0.5 µM of each forward and reverse primer, 10 mL of SYBR Green Mix, 4 mL of cDNA, and 4 mL of RNase-free water. The thermal cycling conditions were composed of an initial denaturation step at 95°C for 15 min followed by 40 cycles at 95°C for 15 s, 59°C for 30 s, and 72°C for 15 s and ended with a melting curve. For the analysis of each sample, three analytical replicas were used. Target genes were normalized to the expression of Actin2. Primer sequences for AVP1, AHA10, and Actin2 are listed in Supplemental Table 3. 11 of 14 analyze whether avp1-1 mutants also carry a T-DNA insertion in GNOM, a T-DNA right border primer (GN.T-DNA) and a GNOM-specific reverse primer (GN.REV) were used. The GNOM wild-type allele was identified using GNOM forward and reverse primer (GN.For and GN.Rev; Supplemental Table 3). Amplification of VHA-a3 using VHA-a3-specific forward (S029786.for) and reverse (S029786.rev) primer served as a control. Pharmacological Treatments and Stains In Situ Hybridization In situ hybridizations were performed as previously described (Medzihradszky et al., 2014). For AVP1 probe preparations, AVP1-pGEM-T Easy (see constructs) was used. The template for the transcription was PCR amplified with T7 and SP6 primers from these vectors. Immunohistochemistry on Tissue Sections Meristems were fixed and embedded in Steedman’s wax according to a previously described protocol (Paciorek et al., 2006) and sectioned at 8 mm by a microtome (Leica CM3050S). Sections were mounted on slides (X-Tra; Leica) and dried overnight at room temperature. Antibody binding was performed as described (Paciorek et al., 2006), with the anti-AVP1 antiserum (Cosmo Bio Co.; 1:10,000 in 2% BSA-PBS) covered with Parafilm at room temperature for 4 h and an AP-conjugated secondary antibody [goat anti-rabbit IgG (H+L)-alkaline phosphatase; Dianova 111-055-045; 1:2500 in 2% BSA-PBS] for 1 h. After washing the sections for 90 min in microtubulestabilizing buffer (50 mM PIPES-KOH, pH 6.8, 6.5 mM EDTA, and 5 mM MgSO4) and equilibrating in the substrate buffer, staining was developed with NBT-BCIP solution (Roche) at room temperature for 1 h. Arabidopsis seedlings were incubated in liquid 0.53 MS medium with 0.5% sucrose, pH 5.8, containing 1 µM ConcA, 1 µM FM4-64, 10 µM BCECF, or the equivalent amount of DMSO in control samples for the indicated time at room temperature. Stock solutions were prepared in DMSO. Sodium orthovanadate (Sigma-Aldrich) was applied in a concentration of 2 mM and liquid 0.53 MS without any supplement served as the mock control. Confocal Microscopy Images were recorded using a Leica TCS SP5II microscope equipped with a Leica HCX PL APO lambda blue 63.0 3 1.20 UV water immersion objective. GFP as well as BCECF was excited at 488 nm using a VIS-argon laser, and FM4-64 was excited at 561 nm with a VIS-DPSS 561 laser diode. Fluorescence emission of GFP and BCECF was detected between 500 and 555 nm, and the emission of FM4-64 was detected between 615 and 676 nm. The Leica Application Suite Advanced Fluorescence software was used for image acquisition. Post processing and measurements of images were performed using Fiji (based on ImageJ 1.50a). SDS-PAGE and Immunoblotting pH Measurements Microsomal membrane proteins were analyzed by SDS-PAGE and subsequent immunoblotting. Upon gel electrophoresis, the proteins were transferred to a nitrocellulose membrane (Whatman). The primary antibody against the V-PPase was purchased from Cosmo Bio (for details, see immunohistochemistry section); the primary antibody against VHA-C (Schumacher et al., 1999) and anti-TIP1;1 were previously described (Jauh et al., 1998). Antigen on the membrane was visualized with horseradish peroxidase-coupled anti-rabbit IgG (Promega) and chemiluminescent substrate (Peqlab). Immunostained bands were analyzed using a cooled CCD camera system (Intas). Vacuolar pH measurements were performed as previously described (Viotti et al., 2013) with minor modifications. BCECF fluorescence was detected using a Leica SP5II confocal laser scanning microscope equipped with a HCX PL APO CS 20.0 3 0.70 IMM UV water immersion objective. All images were exclusively recorded within the root differentiation zone except when root vacuolar pH was determined additionally in the root meristematic and elongation zone (Figure 6). In situ calibration was performed separately for each root zone. Cell sap pH measurements were conducted as previously described (Krebs et al., 2010). Microsomal Membrane Preparation and Enzyme Assay Extracellular Acidification Assay Extraction of microsomal membranes and V-PPase enzyme activity measurements were performed as described (Krebs et al., 2010). Media acidification was measured according to Haruta et al. (2010) with minor modifications. Five-day-old single seedlings were transferred to a 96-well plate containing 200 mL of 0.53 MS and 0.5% sucrose, pH 6.4, supplemented with 30 µg/mL fluorescein dextran (molecular weight = 10.000; Life Technologies) and 2 mM vanadate. Before and after an incubation time of 18 h, fluorescence emission was detected at 521 nm after excitation at 494 and 435 nm using a microplate reader (Tecan Infinite M1000). The media pH was calculated using a calibration curve ranging from pH 3.5 to 6.5. Auxin Transport Assays and Free IAA Determinations Auxin transport assays and free IAA determinations were conducted as described (Kubeš et al., 2012). Statistics were performed with SigmaStat, using one-way ANOVA followed by Dunnett’s post-hoc analyses set to P < 0.05. Data shown are means 6 SD of two or three independent replicates, as indicated in the figure legends. Accession Numbers Genotyping Genomic DNA was extracted as described before (Edwards et al., 1991). To check for the presence of the T-DNA in the AVP1 gene, PCR was performed using a T-DNA left border-specific primer (LB-JL202) and an AVP1-specific reverse primer (AVP1.Rev). To identify the AVP1 wild-type allele, two gene-specific primers were used (AVP1.For and AVP1.Rev). To Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: AVP1/VHP1 (At1g15690), GNOM (At1g13980), VHA-a1 (At2g28520), VHA-a2 (At2g21410), VHA-a3 (At4g39080), VHA-C (At1g12840), AHA10 (At1g17260), TIP1;1 (At2g36830), CNX (At5g61790), and ACT2 (At3g18780). 12 of 14 The Plant Cell Supplemental Data Supplemental Figure 1. AVP1 transgene is silenced in 35S:AVP1 lines. Supplemental Figure 2. T-DNA insertions in AVP1 and GNOM. Supplemental Figure 3. Phenotypes of the T-DNA insertion line vhp1-1 and the point mutation allele fugu5-1. Supplemental Figure 4. Cell expansion in fugu5-1 vha-a2 vha-a3 is independent of the tonoplast V-ATPase and V-PPase. Supplemental Figure 5. Transcript level of tonoplast P-type ATPase AHA10. Supplemental Table 1. Rootward and shootward auxin transport Supplemental Table 2. Free IAA content Supplemental Table 3. Primer list. ACKNOWLEDGMENTS We thank B. Schöfer and K. Piiper for excellent technical assistance and R. Gaxiola, A. Ferjani, and G. Jürgens for providing published materials. This work was supported by the Deutsche Forschungsgemeinschaft within FOR1061 to K.S. and the ERC by a starting grant to J.U.L. Funding to W.A.P. was provided by the Maryland Agricultural Research Station. AUTHOR CONTRIBUTIONS A.K., Z.A., A.M., J.U.L., and K.S. designed experiments. A.K., Z.A., A.M., F.K., S.S., S.D., and M.G.P.-N. performed experiments and analyzed data. M.K. established UBQ:AVP1 lines. A.P. analyzed sequencing data. G.G., H.Y., A.S.M., and W.A.P. contributed with auxin content and transport data. 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Lohmann and Karin Schumacher Plant Cell; originally published online November 20, 2015; DOI 10.1105/tpc.15.00733 This information is current as of June 18, 2017 Supplemental Data /content/suppl/2015/11/13/tpc.15.00733.DC1.html /content/suppl/2015/12/16/tpc.15.00733.DC2.html Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY