Vacuolar H⁺-Pyrophosphatase AVP1 is Involved in Amine Fungicide Tolerance in Arabidopsis thaliana and Provides Tridemorph Resistance in Yeast

Yeast Strains, Plasmids, and Growth Conditions

All strains are derivatives of S. cerevisiae strain W303-1a (MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15). Relevant genotypes are described in Table 1. Yeast soluble PPase IPP1 or TcGFP-AVP1, a chimera consisting of the main H⁺-PPase vacuolar isoform AVP1 from A. thaliana fused to the N-terminal sequence of the H⁺-PPase TcVP from the protist Tripanosoma cruzi and the sequence of the GFP from Aequorea victoria, were expressed from a pRS699 multicopy plasmid as described (Drake et al., 2010). Introduction of plasmids into yeast cells was done by the lithium-acetate method (Gietz and Woods, 2002). Cells were routinely grown on standard YP or synthetic media supplemented with appropriate carbon sources (Sherman, 1991). Unless otherwise stated, all determinations were done on exponentially growing cells (0.5 < A₆₀₀ < 0.8). For drop tests, cells were grown to early stationary phase; A₆₀₀ of cultures was adjusted to 0.4 (ca 4 × 10⁶ cells/ml) with water and three 10-fold serial dilutions in water prepared from them. Two-point-five microliter aliquots from each dilution were placed onto appropriate agar plates, resulting in ca 10⁴, 10³, 10², or 10 cells per spot, accordingly. Liquid cultures were routinely treated with 3 μM tridemorph for 3 h (annexin V experiments) or 5 h (cell cycle analysis) prior analysis.

Vacuole Vesicles and ATPase Assays

Intact vacuoles were isolated essentially as described (Hernandez et al., 2009) with the only modification of using a single 8% (w/w) Ficoll gradient. After isolation, vacuoles were vesiculated by resuspension in TKE buffer [10 mM Tris/HCl pH 7.5, 2 mM KCl, 1 mM EDTA and protease inhibitor cocktail (Sigma-Aldrich)]; vesicles were recovered after centrifugation at 100,000 × g for 30 min and resuspended in TKE. The formation of a ΔpH was evaluated as the ACMA fluorescence quenching produced by the activity of the tonoplast H⁺-ATPase using the following reaction mixture: 1 μM ACMA, 20 mM MOPS-Tris pH 7.2, 25 mM KCl, 2 mM MgCl₂, and vacuolar membrane preparation (up to 50 μg of protein). The assay was initiated with the addition of 1.5 mM ATP. Recovery of fluorescence, as a proof of gradient formation, was induced by adding 3 μM gramicidin D. ATPase hydrolytic activities were assayed as described (Hernandez et al., 1994).

Flow Cytometry

Annexin-V-FITC and PI staining test were done essentially as described in Madeo et al. (1997). Briefly, after treatment, cells were collected, washed once in distilled water, once in SPMB (1.2 M sorbitol, 50 mM phosphate buffer pH 7.5, 1 mM MgCl₂) and resuspended in SPMB with 30 units of lyticase (Sigma, St. Louis, MO, USA) per 10⁷ cells. After protoplasting was complete (0.5–1 h), protoplasts were collected by centrifugation for 5 min at 500 × g and washed once with fresh SPMB and a second time with annexin-V labeling buffer (1.2 M sorbitol, 10 mM HEPES-NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂). Annexin-V-FITC and PI (final concentrations 1 μg/ml and 2.5 μg/ml, respectively) were incubated with protoplasts in labeling buffer for 20 min prior to flow cytometry. For DNA content analysis, cells were stained with PI essentially as described by Sazer and Sherwood (1990), and analyzed on a Coulter Epics XL apparatus as previously described (Hernandez et al., 2008). Although the extent of death induction by tridemorph was somewhat batch-dependent, the phenomena were consistent and reproducible within each batch.

Plant Materials and Growth Conditions

Plant mutant genotypes are described in Table 2. All A. thaliana wild-type and mutants were from Columbia ecotype. Seeds were sterilized using vapor phase-chlorine gas for 5 h (Clough and Bent, 1998) and sown individually using sterile forceps on 1X Murashige and Skoog (MS) plates containing 1% sucrose and 3.5 g l^-1 Phytagel (Sigma-Aldrich).

Homozygous mutant lines for gene At1g15690 encoding the vacuole-localized H⁺-PPase AVP1 isoform (fugu 5-1 and fugu 5-3) were a kind gift of Dr Ferjani (Tokyo Gakugei University, Tokyo, Japan). Mutants for gene At1g78920 encoding the AVP2 isoform were obtained from the collection held at the Salk Institute Genomic Analysis Laboratory (USA) and named for this work as sm1 and sm2. Homozygosis of the T-DNA insertion was confirmed by PCR. Details on mutants are listed on Table 2.

Histological Determination of Gene Expression

Promotor sequences from genes AVP1 (At1g15690, 1,930 bp upstream of ATG), AVP2 (At1g78920, 1,100 bp), and AVP3 (At1g16780, 900 bp) were amplified by PCR from genomic DNA and cloned into vector pMDC162 (Curtis and Grossniklaus, 2003). Columbia wild-type A. thaliana plants were transformed by the floral dipping method (Clough and Bent, 1998). Plants carrying gene insertions were selected on kanamycin-supplemented plates. All experiments were performed with F3 seeds. The transgene insertions in all lines were sequenced to check for integrity of the inserted constructs. Staining was done by immersing plants or seedlings in GUS buffer [2 mM 5-bromo-4-chloro-3-indol-β-D-glucuronic acid, 50 mM potassium phosphate buffer (pH 7.2), 2 mM potassium ferrocyanide, 2 mM potassium ferrycyanide, 0.2% (w/v) triton X-100] for 1–3 days. Chlorophyll was washed-off using several 96% (v/v) ethanol washes at 37°C. Images were collected using a Nikon Eclipse 80i microscope coupled to a Nikon Digital Sight DS L1 camera or with a Leica MZFLIII binocular coupled to a Leica DC camera.

Semi-quantitative Reverse Transcription-PCR

Total RNA (1 μg) was subjected to cDNA synthesis using a “QuantiTect^® Reverse Transcription kit” (Qiagen) according to manufacturer’s instructions. Final cDNA (1 μl) was subjected to PCR using primers amplifying 300–400 bp fragments at the 3′ UTR regions of the cDNA corresponding to genes At1g15690 (AVP1), At1g78920 (AVP2), At1g16780 (AVP3), and At4g05320 (UBIQUITIN10, UBQ10). PCR was run for a total of 20 cycles. Primers annealing on the UBQ10 cDNA were used to normalize equal cDNA loading onto PCR mixes. Saturation of the reaction and specificity of primers were checked using a five-fold excess of cDNA.

Plant Extracts

Seedlings were frozen in liquid nitrogen and kept at -80°C until use. Seedlings were ground with pestle and mortar in liquid nitrogen. The powder was resuspended in 1 ml of extraction buffer containing 50 mM MOPS adjusted to pH 7.5 with NaOH, 330 mM sorbitol, 2 mM MgCl₂, 1 mM DTT, 1 mM PMSF and Plant Protease Inhibitor Cocktail (Sigma-Aldrich). The mixture was centrifuged for 15 min at 16,000 rpm at 4°C, the supernatant transferred to a new tube and reserved for Western blot analysis.

Hypocotyl and Root Length Measurements

To measure hypocotyl and root elongation in the darkness, seeds were stratified for 5 days at 4°C in the dark before being placed vertically in a growth chamber equipped with fluorescence light (light intensity 100 μE m^-2 s^-1) at 22°C for 4 h. To ensure dark conditions after that period, plates were wrapped in four layers of aluminum foil and plates incubated vertically at 22°C for 6 days. When appropriate, plates were supplemented with 10 μM tridemorph. Fungicide treatments and mock controls were carried out simultaneously in three replicate experiments. Hypocotyl and root length were quantified using Image J software (Schneider et al., 2012).

Protein Determination and Western Blotting

Protein determination was done using a dye-binding based assay from Bio-Rad (Hercules, CA, USA), according to manufacturer instructions or a modified Lowry assay (Thermo Scientific, Rockford, IL, USA), using ovoalbumin as a standard. Proteins were separated in SDS-PAGE gels using standard procedures (Sambrook et al., 1989). Proteins were then transferred to nitrocellulose filters and probed with polyclonal antibodies raised in rabbit against VHA-A (Cosmobio, Tokyo) and AVP1 (Agrisera, Vånnås) at a 1:1000 dilution. Blots were visualized on X-ray films using horseradish peroxidase-coupled secondary antibodies and a chemiluminescence kit (Millipore, Madrid). Quantification of blot band intensity was done using a Bio-Rad GS-800 densitometer.

The Amine Fungicide Tridemorph Induces Metacaspase-Independent Apoptosis

We aimed at determining if inhibition of ERG2 gene product was fungistatic or if it induced cell death, and in the latter case, which type of cell death. Therefore, we treated W303-1a cells with 3 μM tridemorph for 5 h prior to annexin V-FITC/PI flow cytometric tests (Figure 1A). Both annexin-V-FITC (AV) and PI are membrane impermeable molecules; AV is a protein that binds phosphatidyl serine, while PI dyes nucleic acids. Briefly, cells dying from apoptosis expose phosphatidyl serine on the outer leaflet of the plasma membrane early in the process and only become permeable to PI at late stages, while necrotic cells are characterized by early breakage of the plasma membrane permeability barrier and little or no translocation of phosphatidyl serine (Madeo et al., 1997). Yeast cells treated with a growth inhibitory dose of tridemorph for 3 h displayed a differential staining with AV but little staining with PI in non-permeabilized protoplasts, compared with an untreated control (Figure 1A and Table 3). In comparison, cells treated with 160 mM acetic acid, a condition known to induce both apoptosis and necrosis in yeast (Ludovico et al., 2001) yielded a significant fraction of the protoplasts permeable to PI (Figure 1A). Accumulation of abnormal sterols has been associated to cell cycle perturbations (Marcireau et al., 1990; Fernández et al., 2002); hence, we also investigated if cell cycle arrest occurred concomitantly to apoptosis. Experiments using PI staining of permeabilised cells previously treated with tridemorph revealed that genomic DNA fragmentation takes place upon fungicide treatment with an increase in cells at the G1 phase of the cell cycle (Figure 1B). On their turn, erg2Δ (JRY7773) cells showed no differences with respect to a wild-type strain in subG₁ extent (below 4%) and only a slight accumulation of cells in G₂/M phases of the cell cycle, compared with W303-1a. Deletion of YCA1, the only yeast metacaspase, on a W303-1a background yielded cells with no differential sensitivity toward tridemorph as assessed by drop test assays (Figure 1C) or PI-flow cytometry of permeabilised cells (data not shown).

FIGURE 1

Cell death and cell cycle arrest induction in tridemorph treated yeast cells. (A) Determination of early apoptosis and necrosis in tridemorph-treated yeast. Externalization of phosphatydilserine and plasma membrane permeability to propidium iodide (Annexin V-FITC/PI assays) assessed by flow cytometry in non-permeabilised yeast protoplasts obtained from cells treated with 3 μM tridemorph for 3 h. Acetic acid treatment is included as a control inducing both Annexin V-FITC and PI fluorescence. (B) Cell cycle profiles in tridemorph treated yeast cells; quantitation of cell death. Flow cytometry determination of DNA contents by propidium iodide staining in ethanol-permeabilised yeast cells. Percentages of cells in every stage are shown in each panel. Total cells analyzed per case: 10,000. (C) Tridemorph-induced apoptosis dependence from metacaspases. Early stationary yeast cultures were diluted and spotted onto YPD agar plates (control, left) and YPD plus 1 μm tridemorph (right) at densities distributing ca 10⁴, 10³, 10², and 10 cells per spot, from left to right, respectively.

Tridemorph Causes V-ATPase Dysfunction

Tridemorph is a fungicide that phenocopies a defect on ERG2, which causes V-ATPase dysfunction (Hernandez et al., 2015). Bearing in mind the differences found in terms of cell death induction between tridemorph treatment and an erg2Δ mutation, we were interested in ascertaining if inhibition of V-ATPase proton pumping was reproduced by tridemorph (Figure 2). Vacuole vesicles extracted from cells treated with 3 μM tridemorph for 5 h were not capable to form proton gradients (Figure 2A). This impairment was similar using erg2Δ-derived vesicles (Hernandez et al., 2015). Also in agreement with previous results, hydrolytic activity assays showed that V-ATPase activity was inhibited to a minor extent (435 ± 97 vs. 341 ± 8 μmol Pi min^-1 mg^-1 protein ± SEM for vesicles obtained from control and tridemorph-treated yeast cells respectively).

FIGURE 2

Vacuolar V-ATPase inhibition by tridemorph treatment. (A) Proton pumping assays using vacuolar vesicles. Proton transport in vacuole vesicles isolated from control yeasts (lower trace) and treated with 3 μM tridemorph for 5 h (top trace) followed by quenching of ACMA fluorescence in the presence of Mg⋅ATP (B) Enhancement of tridemorph tolerance in yeast by deletion of the gene corresponding to Proteinase A (PEP4). Early stationary yeast cultures were diluted and spotted onto control (upper panels) or 2 μm tridemorph-supplemented (lower panels) YPD (Glucose) or YPGlycerol agar plates at densities distributing ca 10⁴, 10³, 10², and 10 cells per spot, up to down, respectively. (C) Quantitation of cell death in pep4Δ mutant cells by flow cytometry. Flow cytometry determination of DNA contents by propidium iodide staining in ethanol-permeabilised pep4Δ mutant (left) and W303-1a (right) yeast cells in control (upper panels) and 3 μm tridemorph-treated cultures (lower panels). Arrows point to dead cell populations (Sub-G₁). A total of 10,000 cells were analyzed per case per experiment.

In a previous report (Hernandez et al., 2015), we described that the presence of 8-dehydrosterols in yeast membranes induced a change in subunit a of the V-ATPase that made it more susceptible to proteolytic attack. We hypothesized that limiting the proteolytic turnover of vacuolar proteins might help yeast to withstand tridemorph treatment. To evaluate this hypothesis, we deleted PEP4, the gene encoding vacuolar protease A, since this deletion is known to affect a wide range of proteolytic activities in this organelle (Ammerer et al., 1986; Woolford et al., 1986). In addition, defects on vacuole acidification are associated to deficiencies to grow on non-fermentable carbon sources (Kane, 2006). Spot tests using W303-1a and its pep4Δ derivative showed that deletion of this protease provided a proliferative advantage when cells were challenged with tridemorph on agar plates, both on glucose and on glycerol carbon sources (Figure 2B). We also tested if cell death induction or cell cycle were affected in these cells. Analysis of PI-stained permeabilised yeast cells by flow cytometry showed again a modest accumulation of cells in G₁/G₀ phases, irrespectively of the genotype (Figure 2C, upper panels). However, while proportions of cells in subG₁ raised from 4.5 ± 0.4% to 47.6 ± 0.5% in the case of W303-1a cells treated with vehicle or tridemorph, respectively, pep4Δ cells showed only 4.9 ± 0.3% and 7.3 ± 0.4% for vehicle and fungicide-treatments, respectively (Figure 2C, lower panels).

A Heterologously Expressed Plant H⁺-PPase Gene Alleviates Tridemorph Growth Inhibition and Cell Death in Yeast

Since tridemorph induced cell death in yeast cells but deletion of ERG2 did not reproduce this effect, we were interested to know if an abnormal vacuolar acidification could be involved in the stimulation of cell death by tridemorph. To this end, YPC3 cells were transformed with pTcGFP-AVP1 or pIPP1 plasmids and kept on glucose medium to repress the expression of the genomic IPP1 gene encoding a soluble pyrophosphatase (Figure 3). Overexpression of the A. thaliana H⁺-PPase chimera TcGFP-AVP1 conferred resistance to concentrations of this amine fungicide above those able to restrict growth to wild-type cells (Figure 3A). This resistance could also be observed and was slightly greater in strains lacking a vacuole-localized V-ATPase (Figure 3A, rightmost panel). Under the present conditions, tridemorph treatment did not affect substantially the H⁺-pumping ability of TcGFP-AVP1 (Supplementary Figure S1).

FIGURE 3

Influence of the heterologous expression of a H⁺-PPase gene on tridemorph tolerance in yeast cells. (A) Evaluation of tolerance to tridemorph in wild-type type (YPC3) and V-ATPase (SAH6) mutants. Early stationary yeast cultures were diluted and spotted onto control (left), 3 μm tridemorph-supplemented (middle) and 5 μm tridemorph-supplemented (right) YPD plates at densities distributing ca 10⁴, 10³, 10², and 10 cells per spot, respectively. (B) Quantitation of cell death in yeasts expressing a functional plant H⁺-PPase gene by flow cytometry. Flow cytometry determination of DNA contents by propidium iodide staining in ethanol-permeabilised yeast cells expressing episomally IPP1 (left panels) and plant AVP1 (right panels). Arrows point to dead cell populations (Sub-G₁). A total of 10,000 cells were analyzed per case per experiment.

In order to obtain a more direct and quantifiable measurement of apoptosis, YPC3 cells transformed with pTcGFP-AVP1 or pIPP1 plasmids were treated with tridemorph in liquid culture for 5 h. Cell cycle was then analyzed by PI-flow citometry (Figure 3B). Tridemorph treatment induced changes in cell cycle profiles leading to an accumulation of cells in the G₁/G₀ phase of the cell cycle, irrespectively of the plasmid borne. This was akin to what was observed in W303-1a cells (Figure 1B), although reduced in extent. In particular, the proportion of cells in G₁/G₀ was 42.8 ± 1.8% and 55.3 ± 0.8% for IPP1-expressing cells untreated or treated with 3 μM tridemorph, respectively. Cells expressing TcGFP-AVP1 showed similar proportions: 45.7 ± 2.2% vs. 52.4 ± 0.1% in G₁/G₀ for untreated and tridemorph-treated cells, respectively. As previously shown, tridemorph induced a dramatic increase in cell death as assessed by accumulation of cells with subG₁ complements of genomic DNA. Thus, pIPP1-transformed YPC3 strain showed 3.7 ± 0.2% of cells in subG₁ when untreated but this proportion raised up to 29.0 ± 2.8% upon tridemorph treatment. However, presence of an H⁺-PPase as an alternative proton pump in YPC3 alleviated this cell death induction (4.2 ± 0.2% vs. 14.3 ± 2.8% in untreated and tridemorph-treated pTcGFP-AVP1 YPC3 cells, respectively).

Expression of H⁺-PPases Gene Isoforms in Arabidopsis thaliana

Arabidopsis thaliana genome includes three different genes encoding membrane-bound H⁺-translocating pyrophosphatases (AVPs), with gene products named AVP1 (type I or K⁺-stimulated isoform), and AVP2, AVP3 (type II, K⁺-independent isoforms). Bearing in mind the observations made in yeast, we hypothesized that these proteins could play a major role in fungicide tolerance in this model plant. However, in order to make meaningful functional assignments, a previous comparative study on the expression of these genes was necessary. Although some studies have already been published (Mitsuda et al., 2001a,b; Segami et al., 2010; Pizzio et al., 2015), none of them made a comparative study, nor included AVP3 expression. We met this task by making transgenic lines with AVP-promoter-β-glucuronidase (GUS) reporter system, where the different AVPs promoters were genomic fragments encompassing 1930, 1100, and 930 bp upstream the ATG codon of AVP1, AVP2, and AVP3 genes, respectively. Qualitative results are summarized on Table 4.

In adult plants (Figure 4), AVP1 was expressed mostly at the vascular tissues but at low levels in leaf mesophyll. In inflorescences, its expression was evident in pistils, stamen pedicels and the vascular system of sepals, while its expression in siliqua was only significantly observed at its base (Figures 4A–D). GUS staining corresponding to AVP2 (Figures 4E–H) was also present in the vascular system, albeit to a smaller degree than AVP1 (Figure 4E). Leaf expression was again very low in mesophyll and, in addition with vascular bundles, only clearly observed in guard cells (Figure 4F). In reproductive tissues, its signal was perceptible at the top and bottom parts of the pistil, in stamen pedicels and vascular tissue of sepals (Figure 4G), while in siliqua, the signal was nearly undetectable (Figure 4H). GUS signal corresponding to AVP3 expression was very weak throughout the plant and down to undetectable levels in many tissues. However, some signal was apparent in reproductive tissues, particularly a weak expression was observed in pistils, stamen pedicels and sepals (Figure 4K).

FIGURE 4

Expression of H⁺-PPase genes in adult organs and tissues of Arabidopsis thaliana. (A,E,I) Cauline leaves; (B,F,J) detail of cauline leaves (mesophyll); (C,G,K) inflorescences; (D,H,L) silique. Transgenic plants expressing β-glucuronidase as a reporter of the expression driven from a 1.9, 1.1, or 0.9 kb fragment from the AVP1, AVP2, or AVP3 promotor sequence, respectively. Bars correspond to 0.5 mm (A,C–E,G–I,K,L) or 50 μm (B,F,J).

In general, expression was more evident in developing seedlings for all three isoforms (Figure 5). AVP1 was robustly expressed in roots and stems and, especially, at the root columella and apical meristem (Figures 5A–D). In roots, the signal was easily detectable up to the transition zone with the shoot (Figure 5D). On the other hand, developing leaves and meristems showed high levels of GUS staining, particularly at the hydathodes (Figure 5B). AVP2 signal (Figures 5E–H) was found in leaf and root apices, although at a lower intensity to AVP1 and often showing intermissions in non-dividing tissue (Figure 5E). Also, it was detected in developing apical meristems (Figure 5G). Leaf expression was again observed in developing organs, remarkably at the hydathodes and in comparable levels to that of AVP1 (compare Figures 5B–F). In the case of AVP3 expression (Figures 5I–L), a clear signal was only observed in the apical meristem (Figure 5K) and in the root vascular system (Figure 5L); in leaves, no clear signal was detected (Figure 5J).

FIGURE 5

Expression of H⁺-PPase genes in seedling organs and tissues of A. thaliana. (A,E,I) Whole seedling; (B,F,J) detail of rosette leaves showing hydathodes; (C,G,H) shoot apical meristems; (D,H,L) root. Transgenic plants expressing β-glucuronidase as a reporter of the expression driven from a 1.9, 1.1, or 0.9 kb fragment from AVP1 (upper panel), AVP2 (middle panel), or AVP3 (lower panel) promotor sequences, respectively. Plants were incubated with X-Gluc to develop the signal prior to being photographed. Bars correspond to 0.1 mm (A,D,E,H,I,L) or 50 μm (B,C,F,G,J,K).

Since GUS staining indicated some clear differences in the expression of the three AVPs, and in order to confirm the data, semi-quantitative RT-PCR experiments were used (Figure 6). First, we confirmed that there were large differences in the expression of the isoforms. We chose tissues of adult plants grown on soil where the most diverse differences in GUS staining had been observed and, in particular, two tissues where the AVPs expression mostly differed: roots and stems (Table 4). The expression of the gene UBQ10 encoding Ubiquitin-10 was used as a loading control. AVP1 mRNA was more abundant than any of the other two isoforms in roots, but AVP1 and AVP2 transcripts showed comparable levels in stems. On the other hand, AVP3 mRNA was nearly undetectable in both tissues (Figure 6A).

FIGURE 6

Relative expression of the different H⁺-PPase isoforms. (A) Expression of AVP1-3 in adult root and stem tissues. Open bars: AVP1; Shaded bars: AVP2; Black bars: AVP3. (B) Relative expression of AVP1 in different plant organs. (C) Relative expression of AVP2 in different plant organs. (D) Relative expression of AVP3 in different plant organs. Semi-quantitation of mRNA abundance estimated by RT-PCR. Data were normalized against the expression of UBQ10 gene. (B–D) Average expression in stems was taken as 100%. Expression (AVPx/UBQ10) in stems were AVP1: 0.52 ± 0.21; AVP2: 0.48 ± 0.36; AVP3: 0.04 ± 0.00. Data are mean ± SE of three independent experiments.

We next wanted to confirm that the expression of each AVP gene varied widely between organs. On the whole, AVP1 mRNA relative amount estimates agreed with GUS data. Thus, it showed a high level of expression in roots, stems and influorescences and minimal in siliques and rosette leaves. Curiously, cauline leaves showed a strong AVP1 expression, in contrast to GUS data. All in all, little differences in expression were found among organs and tissues with values near those found in stems (Figure 6B). AVP2 mRNA showed a relatively robust expression in stems and inflorescences and again in cauline leaves. Minimal levels of expression were observed in roots, rosette leaves and siliques. In this case, the differences between high- and low-expression tissues were greater than in the case of AVP1 (Figure 6C). Regarding AVP3 expression, it was only found unambiguously in roots, while levels in other tissues were nearly negligible (Figure 6D).

Role of H⁺-PPases in Plant Tridemorph Tolerance

Once the expression of the three AVP isoforms was settled, we met the question of whether H⁺-PPases were involved in A. thaliana fungicide tolerance (Figure 7). We found that tridemorph produced a dose-dependent growth retardation effect in developing seedlings grown in the dark. This effect was noticeable in both hypocotyl and root lengths as reductions in both parameters nearing ca 40% at a concentration of 10 μM tridemorph (Figure 7A). To answer which of the H⁺-PPases was involved in fungicide tolerance, we treated wild-type (Columbia ecotype) and two null alleles of either avp1 (fugu 5-1 and fugu 5-3) or avp2 (sm1 and sm2) mutants with 10 μM tridemorph. Plants mutant for AVP3 were cast-off from this study since expression levels of this gene, even in developing tissues, was nearly undetectable. As shown on Figure 7B, all seedlings displayed a remarkable reduction in both root and hypocotyl length upon tridemorph treatment. However, fugu 5-1 and 5-3 mutants showed a significantly greater reduction in root length compared with either wild-type or avp2 mutants, while these latter ones showed no differences between them (Figure 7B). In the case of hypocotyl length, none of the mutants showed any differences compared with a wild-type.

FIGURE 7

Effect of tridemorph addition on seedling root and hypocotyl elongation. (A) Length of roots and hypocotyls on wild-type A. thaliana as a function of fungicide concentration. Circles, root length; squares, hypocotyl length. (B) Length of roots and hypocotyls on wild-type and H⁺-PPase mutants of A. thaliana. Solid bars, roots; empty bars, hypocotyls. (C) Polypeptide levels of AVP1 and V-ATPase in tridemorph treated wild-type and pyrophosphatase mutants. Seeds were germinated and grown in the dark in phytagel-MS medium supplemented with 1% sucrose for 14 days prior analysis. Length measurements done by image analysis using NIH-ImageJ. Asterisks denote significant differences at p ≤ 0.05 using unpaired t-tests. Equal amounts of whole plant extracts were separated by SDS-PAGE, electroblotted onto nitrocellulose and decorated with antibodies recognizing the subunit A from V-ATPase (VHA-A) or AVP1. The protein band corresponding to RUBISCO large subunit (arrow) and the whole lanes stained with Ponceau S were used as loading controls with similar results. Those obtained with whole lanes are shown.

Next, we wanted to find out if H⁺ PPase or V-ATPase polypeptides levels varied as a response to tridemorph treatment. For this purpose, we measured the levels of polypeptides in whole seedling extracts by Western blot compared to levels of the small subunit of the ribulose 1,5 biphosphate carboxilase-oxygenase (RuBisCO; Figure 7C). Unfortunately, we were unable to perceive detectable levels of AVP2 even in avp1 null backgrounds, despite the use of two different polyclonal rabbit antibodies recognizing H⁺-PPases (Figure 7C, upper panel and data not shown). Effects on V-ATPases, as measured using the signal from the polypeptide corresponding to their catalytic subunit A as a marker of the holoenzyme, were apparent but modest in all mutants (Figure 7C, lower panel). Similarly, H⁺-PPase abundance increased discreetly in the case of wild-type plants treated with tridemorph; however, the accumulation of AVP1 augmented distinctly in the case of avp2 null mutants (Figure 7C, upper panel).