Different responses of tonoplast proton pumps in cucumber roots to cadmium and copper

Journal of Plant Physiology 167 (2010) 1328–1335

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Different responses of tonoplast proton pumps in cucumber roots to cadmium and copper ˙ Katarzyna Kabała ∗ , Małgorzata Janicka-Russak, Grazyna Kłobus Department of Plant Physiology, Institute of Plant Biology, University of Wrocław, Kanonia 6/8, 50-328 Wrocław, Poland

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Article history: Received 6 December 2009 Received in revised form 29 March 2010 Accepted 29 March 2010 Keywords: Heavy metals Proton pump Tonoplast V-ATPase V-PPase

a b s t r a c t Cadmium (Cd) and copper (Cu) effects on the two tonoplast proton pumps were compared in cucumber roots. Different alterations of vacuolar H+ transporting ATPase (V-ATPase) (EC 3.6.3.14) and vacuolar H+ transporting pyrophosphatase (V-PPase) (EC 3.6.1.1) activities under heavy metal stress were investigated. ATP-dependent proton transport and ATP hydrolysis increased after exposure of seedlings to Cu, whereas both decreased in plants stressed with Cd. PPi hydrolysis was relatively insensitive to both heavy metals. However, cadmium, but not copper, clearly inhibited PPi -driven H+ transport. Changes in enzyme activities were not due to the metal action on the expression of CsVHA-A, CsVHA-c and CsVP genes encoding V-ATPase subunit A and c, and V-PPase, respectively, in cucumber roots. Moreover, immunoblot analysis using specific antibodies against V-ATPase holoenzyme, phosphoserine and phosphothreonine suggested that the phosphorylation at Ser residue in regulatory subunit B of cucumber V-ATPase was not regulated by metals. Oxidative alterations of membrane lipids were measured as malondialdehyde (MDA) content. Cu ions, in contrast to Cd, visibly enhanced the lipid peroxidation in the root tonoplast fractions. Because ATP and PPi are absolutely required by V-ATPase and V-PPase, respectively, for proton transport, their contents were determined in the control roots and roots treated with cadmium and copper. Both ATP and pyrophosphate amounts decreased under heavy metal stress. © 2010 Elsevier GmbH. All rights reserved.

Introduction The central vacuole is known as a multifunctional compartment of mature plant cells. Because the plant vacuole accumulates many inorganic ions and metabolites, it participates in the generation of turgor, osmoregulation, and cytosolic pH regulation as well as in Ca2+ -dependent signal transduction. This organelle serves as a store for reserve pools of anions, including nitrates and phosphates, which are released under their deficit in the environment. On the other hand, by deposition of toxic compounds, the plant vacuole is involved in the cytosol detoxification under abiotic stress (Krebs et al., 2010; Martinoia et al., 2007). Ion compartmentalization within the vacuole is an energydependent process mediated by active antiporters operating at the tonoplast. The vacuolar membrane possesses two proton pumping

Abbreviations: BSA, bovine serum albumin; DAB, 3,3 -diaminobenzidine; DTT, dithiothreitol; EDTA, ethylene diamine tetraacetic acid; FW, fresh weight; MDA, malondialdehyde; MES, 2-(N-morpholino)ethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; PPi , pyrophosphate; SD, standard deviation; SDS, dodecylsulfate sodium salt; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TCA, trichloroacetic acid; TRIS, hydroxymethylaminomethane; V-ATPase, vacuolar H+ transporting ATPase; V-PPase, vacuolar H+ transporting pyrophosphatase. ∗ Corresponding author. Tel.: +48 071 375 4107; fax: +48 071 375 4118. E-mail address: [email protected] (K. Kabała). 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.03.020

enzymes, vacuolar H+ transporting ATPase (V-ATPase) and vacuolar H+ transporting pyrophosphatase (V-PPase), generating proton motive force which energizes secondary active transport systems (Schumacher, 2006; Serrano et al., 2007). Vacuolar ATPase is the oldest and most complicated proton pump found in plant cells. Enzyme subunits are divided into two major domains, the catalytic peripheral V1 domain responsible for ATP hydrolysis and the membrane-integral V0 domain responsible for H+ translocation. V1 sector consists of eight subunits, named A, B, C, D, E, F, G and H whereas V0 complex is composed of a, c, c , c , d and e subunits. In contrast, vacuolar pyrophosphatase is a single-subunit protein forming 14–17 highly hydrophobic transmembrane ␣-helices and functions as a homodimer in the tonoplast (Cipriano et al., 2008; Gaxiola et al., 2007). Many studies have demonstrated that stress factors induce expression and/or activity of both enzymes, confirming an essential role of the tonoplast proton pumps in plant tolerance to salinity (Fukuda et al., 2004; Golldack and Dietz, 2001; Guo et al., 2006; Lehr et al., 1999; Parks et al., 2002; Vera-Estrella et al., 2005; Wang et al., 2001), mineral deficiency (Kasai et al., 1998; Palma et al., 2000; Yang et al., 2007), chilling and anoxia (Carystinos et al., 1995; Darley et al., 1995). It has been postulated that, under environmental stress, the V-ATPase functions as a stress response enzyme, undergoing moderate changes in expression of subunits and modulation of enzyme structure and activity (Dietz et al., 2001). When environmental stress generates an energy deficiency caused by reduction

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in the cellular ATP level, the vacuolar pyrophosphatase seems to be an important element of the plant survival strategy (Carystinos et al., 1995). Research of metal transport suggests that the tonoplast proton pumps may also play a significant role in the plant adaptation to heavy metal stress. The active metal/proton antiporters operating at the tonoplast have been identified and characterized. These proteins belong to the Cation Diffusion Facilitator (CDF) and Cation Exchanger (CAX) families of metal transporters, which translocate metal ions from the cytoplasm to the vacuolar lumen (Delhaize et al., 2003; Shigaki and Hirschi, 2006). Moreover, studies employing bafilomycin, a specific inhibitor of V-type ATPases, have clearly indicated that zinc transport across the tonoplast in Zn-tolerant Silene vulgaris depends on V-ATPase activity (Chardonnens et al., 1999). However, little information is available on the functioning and regulation of the two tonoplast enzymes in plants exposed to heavy metals. We have demonstrated previously that Cd, Cu and Ni had no significant effect on the activity of vacuolar ATPase in cucumber roots when applied at 10 ␮M concentration for 2 h (Kabała et al., 2008). Unaffected activity of V-ATPase suggested that the proton electrochemical gradient across the tonoplast is maintained by enzymes at similar levels in control plants and plants grown under mild heavy metal stress. To date, studies of heavy metal effects on proton pumping enzymes have examined the H+ -ATPases, whereas the plant vacuolar pyrophosphatase has not been examined under such stress conditions. Therefore, in this work, we aimed to explain the mechanism of cadmium and copper action on both tonoplast proton pumps, V-ATPase and V-PPase, in cucumber roots under stronger heavy metal stress. We measured the hydrolytic and transporting activities of the tonoplast proton pumps in parallel with the expression of genes encoding both enzymes. Moreover, the levels of lipid peroxidation, as an indicator of metal-induced oxidative membrane modification, were determined in the tonoplast fractions prepared from untreated and Cd- or Cu-treated roots. Using specific antibodies against phosphoserine and phosphothreonine, immunoblot analyses of V-ATPase were performed to verify whether modulation of enzyme activity, observed under heavy metal stress, resulted from changes in phosphorylation of some subunits. Because the level of substrate may be a limiting factor for enzyme activity in vivo, the contents of ATP and pyrophosphate (PPi ) were also assayed in cucumber roots. Materials and methods Plant material Cucumber seeds (Cucumis sativus L. var. Krak), after germination in darkness (2 days at 25 ◦ C), were grown in the nitrate-containing medium composed of 1.7 mM KNO3 , 1.7 mM Ca(NO3 )2 × 4H2 O, 0.33 mM KH2 PO4 , 0.33 mM MgSO4 × 7H2 O and micronutrients: 75 ␮M ferric citrate, 10 ␮M MnSO4 × 5H2 O, 5 ␮M H3 BO4 , 1 ␮M CuSO4 × 5H2 O, 0.01 ␮M ZnSO4 × 7H2 O, 0.05 ␮M Na2 MoO4 × 2H2 O (pH 6.5). After 5 days, cucumber seedlings were transferred to 0.33 mM MES-NaOH solution (pH 5.5), containing 0.2 mM CaSO4 , without (control) or with 10 or 100 ␮M CdCl2 or CuCl2 for 2 or 24 h. The plants were grown hydroponically under a 16-h photoperiod (180 ␮mol m−2 s−1 ) at 25 ◦ C by day and 22 ◦ C by night. The relative humidity in the light and dark was 70%. Isolation of tonoplast fractions Tonoplast fractions were prepared from cucumber roots according to the method of Kabała and Kłobus (2001). Highly purified tonoplast vesicles were obtained after the separation of micro-

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somes on a discontinuous sucrose density gradient consisting of 20, 28, 32 and 42% (w/w) sucrose. High purity of tonoplast fractions was confirmed by determination of enzymes used as markers for subcellular organelles including mitochondria, the ER and the plasma membrane (Kabała and Kłobus, 2001). Enzyme assays The hydrolytic activity of vacuolar ATPase (EC 3.6.3.14), sensitive to nitrate, was determined according to the method described by Gallagher and Leonard (1982). The reaction mixture contained about 50 ␮g of tonoplast protein, 33 mM TRIS-MES (pH 7.5), 3 mM ATP, 3 mM MgSO4 , 50 mM KCl, 1 mM NaN3 , 0.1 mM Na2 MoO4 , 200 ␮M Na3 VO4 ± 50 mM NaNO3 and 0.02% Triton X-100. V-ATPase activity was expressed as the difference between the activities measured in the absence and presence of NaNO3 . The hydrolytic activity of V-PPase (EC 3.6.1.1) was measured according to the modified method of Maeshima and Yoshida (1989) in reaction medium containing about 50 ␮g of tonoplast protein, 30 mM TRIS-MES (pH 7.2), 1 mM sodium pyrophosphate (Na4 P2 O7 ), 1 mM MgSO4 , 50 mM KCl, 1 mM Na2 MoO4 and 0.02% Triton X-100. The Pi released during the reactions was determined according to Ames (1966). Proton transport was measured spectrophotometrically as a drop in acridine orange absorbance at 495 nm (A495 ). Tonoplast vesicles (about 50 ␮g protein) were incubated for 3 min at 25 ◦ C with 20 mM TRIS-MES (pH 7.2), 250 mM sucrose, 50 mM KCl and 10 ␮M acridine orange, according to Kabała and Kłobus (2001). The reaction was initiated by the addition of 3 mM Mg-ATP (determination of ATP-dependent proton transport) or 1 mM MgSO4 and 0.1 mM Na4 P2 O7 (determination of PPi -dependent proton transport). For each combination, passive proton movement through the membrane was determined without ATP and PPi in the reaction medium. The protein level was estimated by the method of Bradford (1976) in the presence of 0.02% Triton X-100 with bovine serum albumin (BSA) as the standard. Isolation of RNA and gene expression Total RNA was isolated from 50 mg of root tissue using Tri Reagent (Sigma). The concentration and the purity of isolated RNA were determined spectrophotometrically measuring the absorbance at 260 and 280 nm. To evaluate the expression of vacuolar ATPase subunit A (CsVHA-A) and subunit c (CsVHA-c), vacuolar pyrophosphatase (CsVP) and actin (internal standard) genes, semi-quantitative RT-PCR analyses (Titan one tube RT-PCR system, Roche) with specific primers for each gene were performed. The following forward and reverse primers were constructed on the basis of the sequence published in GenBank and used for amplifications: for the vacuolar ATPase subunit A (CsVHA-A, GenBank accession no. AY580162) 5 -ATTCACCATGCTTCAGAGCTGGCC-3 and 5 -GCCATCATACTGACATTGTAACCC-3 ; for the vacuolar ATPase subunit c (CsVHA-c, GenBank accession no. EF373537) 5 -TGCAC TCGTCTTCTCCTGTATGGG-3 and 5 -CTGCTGTGCATTGGCTCTAACAC C-3 ; for the vacuolar PPase (CsVP, GenBank accession no. EF373538) 5 -GCAGCCATTGGAAAGGGTTTTGCC-3 and 5 -CGAT GTACTTCTTAGCGTTATCCC-3 ; for actin 5 -CCGTTCTGTCCCTCT ACGCTAGTG-3 and 5 -GGAACTGCTCTTTGCAGTCTCGAG-3 . cDNA was synthesized using 150 ng of total RNA with TthDNA polymerase. Twenty-four amplification cycles composed of 30 s at 94 ◦ C (denaturation), 30 s at 58 ◦ C (annealing), 3 min at 68 ◦ C (extension) were performed for CsVHA-A and actin genes. Twenty-five amplification cycles consisting of 30 s at 94 ◦ C, 30 s at 59 ◦ C, 1 min and 30 s at 68 ◦ C were performed for CsVHA-c gene. Twenty-five amplification cycles consisting of 30 s at 94 ◦ C, 30 s at 55 ◦ C, 1 min and 30 s at 68 ◦ C were performed for CsVP gene. A 2 min denaturation

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at 94 ◦ C at the beginning and a 7 min final extension at 68 ◦ C at the end were performed for all reactions. Under these conditions, transcripts were amplified in a linear range. A UNO II (Biometra) thermocycler was used to run the reactions. The PCR products were subjected to electrophoresis in 1.5% (w/v) agarose gels and stained with ethidium bromide. The gel images were digitally captured with a Sony XC-ST50CE camera and analyzed using the Biocapt version 99 program. Determination of metal level The level of metals was determined spectrophotometrically (Perkin-Elmer 3300) in fresh root tissues digested with concentrated HNO3 in microwave system. Determination of ATP and PPi contents 5 g of root material was ground in liquid nitrogen and 5 cm3 of 4.5% perchloric acid was added and mixed until it thawed. The mixture was supplemented with 0.125 cm3 2 mM TRIS and centrifuged for 5 min at 5000 × g. The pH of the supernatant was adjusted to 7.4–7.6 with 5 M K2 CO3 . After recentrifugation, the supernatant was used for analyses. ATP content was measured with the ‘firefly’ luciferin-luciferase assay using a TD-20/20 Luminometer (Turner Designs) according to Glaab and Kaiser (1999). The PPi level was determined using Pyrophosphate Reagent (P 7275, Sigma). Lipid peroxidation assays The lipid peroxidation was expressed as malondialdehyde (MDA) content and determined as thiobarbituric acid reactive substances (TBARSs) formation according to Sairam et al. (2002) with some modifications. A volume of 0.8 ml of 0.5% thiobarbituric acid (TBA) in 20% trichloroacetic acid (TCA) was added to 0.3 ml of aliquot of tonoplast fraction (75 ␮g of protein). The mixture was heated at 95 ◦ C for 30 min and then quickly cooled in ice. After centrifugation at 10 000 × g for 10 min, the absorbance of the supernatant was measured at 532 nm. Correction of nonspecific turbidity was made by subtracting the absorbance of the same at 600 nm. The TBARS content was calculated using an extinction coefficient of 155 mmol−1 cm−1 . Immunoblot analysis Tonoplast proteins (20 ␮g) were incubated in buffer containing 2% (w/v) dodecylsulfate sodium salt (SDS), 80 mM dithiothreitol (DTT), 40% (w/v) glycerol, 5 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM TRIS, 1 mM ethylene diamine tetraacetic acid (EDTA) and 0.05% (w/v) bromophenol blue for 30 min at room temperature, and separated on 10% SDS-polyacrylamide gel for 60 min at room temperature (140 V, 20 mA). After electrophoresis, proteins were electrotransferred for 80 min at room temperature (60 V, 220 mA) to nitrocellulose membrane using a SV10-EB10 blotting apparatus (Sigma–Aldrich). Transfer buffer contained 25 mM TRIS, 150 mM glycine and 10% (v/v) methanol. To identify the vacuolar ATPase, the blots were incubated overnight at 8 ◦ C with primary antibody against V-ATPase holoenzyme (Haschke II) kindly provided by Dr. E. Fischer-Schliebs (University of Darmstadt, Germany), diluted 1:1500. To detect phosphorylation of vacuolar ATPase, the blots were incubated overnight at 8 ◦ C with the primary antibody against phosphothreonine i phosphoserine (Abcam), used at concentration of 2 ␮g cm−3 . After repeated washing, the nitrocellulose membranes were incubated for 1 h at room temperature with secondary antibody (conjugated to horseradish peroxidase,

Abcam), diluted 1:15,000, and visualized by staining with 3,3 diaminobenzidine (DAB).

Results The effect of heavy metals on the functioning of two vacuolar proton pumps was analyzed in roots of cucumber seedlings grown in medium without metals (control plants) or containing 10 or 100 ␮M Cd and Cu for 2 or 24 h. Activities of both enzymes, V-ATPase and V-PPase, were measured as proton pumping across the membrane (driven by ATP or PPi , respectively) and substrate (ATP or PPi , respectively) hydrolysis in tonoplast vesicles. Tonoplast proton pumps had different sensitivity to Cd and Cu (Fig. 1). The presence of Cu ions in the nutrient solution significantly stimulated V-ATPase activity, measured as both ATP-dependent H+ transport and ATP hydrolysis, whereas Cd ions diminished it (Fig. 1A and B). Cu-induced stimulation of enzymes depended on metal concentration and time of exposure, and reached the highest level in roots treated with 100 ␮M CuCl2 for 24 h – more than 200% in comparison to the control. Cadmium inhibited the hydrolytic and transporting activity of V-ATPase to a similar extent under all treatment conditions, by 25 and 35% (approximately), respectively. Functioning of vacuolar pyrophosphatase was not modified by Cu and Cd as clearly as the functioning of V-ATPase (Fig. 1C and D). PPi hydrolysis catalyzed by V-PPase seemed to be relatively insensitive to both metals. The only exception was 24-h exposure of roots to 100 ␮M copper, which caused an increase of approximately 40% in the hydrolytic enzyme activity (Fig. 1D). On the other hand, heavy metal ions affected PPi -driven proton transport in tonoplast vesicles. Treatment of cucumber roots with Cd inhibited pumping activity of pyrophosphatase (Fig. 1C). Because the effect of heavy metals on the cellular processes, including action of proton pumps, depends on the efficiency of absorption, the contents of Cd and Cu in roots of untreated and stressed plants were determined. Accumulation of both metals increased in root tissues in a concentration and time-dependent manner (Fig. 2). Cadmium and copper ions were accumulated with similar efficiency, reaching the highest levels in roots after 24-h exposure to 100 ␮M metal concentration, 3.3 ␮mol of Cd g−1 FW (Fig. 2A) and 4.8 ␮mol of Cu g−1 FW (Fig. 2B). The alterations of the tonoplast proton pump activities could involve the transcriptional level and/or the protein level. The expression of genes encoding both enzymes in cucumber roots was analyzed in control roots and roots treated with 10 or 100 ␮M Cd and Cu for 2 or 24 h. The levels of specific transcripts for subunits A and c of vacuolar ATPase (CsVHA-A and CsVHA-c) and vacuolar PPase (CsVP) were assayed using the RT-PCR approach. The constitutively expressed gene encoding actin was used as an internal standard. Results are presented as a gel image (Fig. 3A) and as a ratio of the proton pump gene signal in the selected line to the actin gene signal (Fig. 3B). Treatment of cucumber seedlings with cadmium and copper had no significant influence on the mRNA amounts for tonoplast H+ pumps in roots, indicating that heavy metal-dependent inhibition or the activation of enzyme activities is not related to the gene expression. Unchanged expression of genes encoding V-ATPase subunits was confirmed by results obtained from immunoblot analysis. Hybridization with a specific antibody against the vacuolar ATPase holoenzyme showed that protein amounts of subunit A, B and C were similar in the tonoplast fractions isolated from untreated and heavy metal treated roots (Fig. 4A). To examine whether alteration in enzyme activity under heavy metals was caused by changes in phosphorylation of its subunits, antibodies against phosphoserine and phosphothreonine were used. As demonstrated in Fig. 4B and C, the phosphorylation occurred at the Ser residue in regulatory

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Fig. 1. Effect of Cd and Cu treatment on the vacuolar ATPase (A and B) and PPase (C and D) activities in the tonoplast vesicles isolated from cucumber roots. The tonoplast vesicles were prepared from control roots and roots treated with 10 and 100 ␮M Cd and Cu for 2 or 24 h. V-ATPase activity was measured as ATP hydrolysis (grey bars) and ATP-dependent proton transport (striped bars). V-PPase activity was measured as PPi hydrolysis (grey bars) and PPi -dependent proton transport (striped bars). Presented results are means ± SD of three independent experiments run in triplicate. Hydrolytic activity of V-ATPase and V-PPase reached value 97 ␮g Pi and 190 ␮g PPi h−1 mg−1 protein, respectively, in control samples (100%). Mean ATP and PPi -dependent proton transport of control samples was 0.186 and 0.212 A495 min−1 mg−1 protein, respectively (100%).

subunit B of V-ATPase, but no significant differences in phosphorylation intensity were found between the V-ATPase of control and metal stressed plants. Changes in activities of membrane-bound enzymes may arise from metal-induced modification of membrane structure. Oxidative alterations of membrane lipids were demonstrated by determination of the MDA content. Lipid peroxidation increased in tonoplast fractions isolated from cucumber roots exposed to copper, but remained unaffected in tonoplast fractions obtained from cadmium-treated roots (Fig. 5). Because one of the factors limiting the in vivo activity of enzyme proteins in plant tissues is substrate availability, ATP and PPi contents were determined in cucumber roots of control plants and plants treated with 10 or 100 ␮M Cd and Cu for 2 or 24 h. The results revealed that ATP decreased after exposure of seedlings to cadmium as well as after exposure to copper (Fig. 6A). Reduction in the ATP level was dependent on the concentration of metal and

time of the treatment. As shown in Fig. 6B, pyrophosphate content was also affected by heavy metals. The 100 ␮M concentrations of Cd and Cu lowered PPi amount more markedly than the ATP level.

Discussion A number of cellular mechanisms including phytochelatin and metallothionein biosynthesis are believed to be involved in the detoxification of heavy metals entering the cytoplasm. However, a very important role seems to be reserved for transmembrane transport systems operating at the tonoplast (Clemens, 2006; Dietz et al., 2001). Several classes of tonoplast transporters, responsible for deposition of metal ions inside the vacuole, have been identified in the vacuolar membrane. All of these are energy-dependent proteins. While the primary active transporters use the energy from ATP hydrolysis, the secondary transporters are energized

Fig. 2. Accumulation of Cd (A) and Cu (B) ions in cucumber roots. Metal level was determined spectrophotometrically in roots of control plants and plants treated with 10 and 100 ␮M Cd and Cu for 2 or 24 h. Obtained values are represented as means (±SD) of nine replicates of three independent experiments.

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Fig. 3. Transcript level of genes encoding vacuolar ATPase subunit A (CsVHA-A) and c (CsVHA-c), and vacuolar PPase (CsVP) in cucumber roots treated with heavy metals. Total RNA was isolated with Tri Reagent from control roots (C) and roots treated with 10 and 100 ␮M Cd and Cu for 2 or 24 h. Semi-quantitative RT-PCR with specific primers was performed to evaluate the expression of genes. Ethidium bromide-stained bands for CsVHA-A, CsVHA-c and CsVP transcripts were quantified with respect to the bands of actin (internal standard). RT-PCR results are presented as a gel image (A) and as a ratio of specific transcript signal in the selected line to the actin transcript signal (B). All experiments were repeated tree times independently and comparable results (±SD) were obtained.

by the electrochemical proton gradient. Thus, the activity of the other transporters, operating as metal/proton antiporters, directly depends on the functioning of vacuolar ATPase and vacuolar PPase (Gaxiola et al., 2007). Enhanced activity and/or gene expression of the tonoplast proton pumps under heavy metals appears to be one of the mechanisms involved in plant stress tolerance, as previously confirmed for salinity, mineral deficiency, anoxia and cold.

Fig. 4. Immunoblotting of tonoplast proteins with antibodies against V-ATPase holoenzyme (Haschke II) (A), phosphoserine (B) and phosphothreonine (C). Tonoplast fractions were prepared from control roots and roots treated with 100 ␮M Cd and Cu for 24 h. Molecular mass standard (M) is indicated on the left. Presented values are representative for the results obtained from three independent tonoplast isolations.

On the other hand, membrane enzymes appear to be subjected to harmful heavy metal action. It is well documented that heavy metals may damage the secondary structure of proteins by oxidation and cross-linking of sylhydryl groups (Siedlecka and Krupa, 2002). Heavy metal-induced generation of reactive oxygen species and free radicals increases membrane lipid peroxidation and activates oxidative stress. Moreover, metals can produce qualitative and quantitative changes in membrane lipids. Such modifications of the lipid composition alter the membrane structure, functions, permeability and as a consequence, may affect the membrane-bound enzymes (Devi and Prasad, 1999; Gratão et al., 2005). In our study, cucumber seedlings were exposed to two heavy metals of different importance for plants. Copper, as a micronutrient, is absolutely required for plant growth and development. However, its elevated levels in environment are toxic to plants. Cadmium is a non-essential element for vascular plants (like lead or mercury) and is a potent inhibitor of growth (Hall, 2002). We have previously reported that vacuolar ATPase was not affected by Cd, Cu and Ni in cucumber roots under mild heavy metal stress (Kabała et al., 2008). In the present work, different effects of cadmium and copper on the tonoplast proton pumps, V-ATPase and V-PPase, were observed at higher metal concentrations and/or longer time of exposure. Cd ions inhibited ATP hydrolysis and ATP-driven proton transport, as well as PPi -dependent H+ pumping in tonoplast vesicles, but PPi hydrolysis remained unaffected (Fig. 1A and C). Inhibitory action of cadmium on the proton pump has been demonstrated in earlier studies of the plant plasma membrane ATPase (Astolfi et al., 2003, 2005; Janicka-Russak et al., 2008) and animal vacuolar ATPase (Herak-Kramberger et al., 1998, 2000). On the other hand, activities of V-ATPase were significantly stimulated in the tonoplast isolated from copper-treated roots (Fig. 1B), whereas V-PPase activities remained relatively unchanged (Fig. 1D). Other

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Fig. 5. Effect of Cd and Cu on the lipid peroxidation in tonoplast fractions isolated from cucumber roots. The content of MDA was estimated in the tonoplast fractions obtained from control plants and plants treated with 100 ␮M Cd and Cu for 24 h. Results are means (±SD) of three different experiments run in triplicate.

authors have reported different actions of Cu ions on the plasma ´ membrane proton pump, including stimulation (Burzynski and ´ Kolano, 2003) and inhibition (Burzynski and Kolano, 2003; JanickaRussak et al., 2008). In contrast to the cadmium, the effects of copper on both hydrolytic and transporting activity of V-ATPase depended on its concentration in the nutrient solution and correlated well with its accumulation in the root tissues (Fig. 2). Increased VATPase activity in cucumber roots treated with copper suggests that a higher proton electrochemical gradient across the tonoplast is generated to energize secondary active antiporters responsible for Cu2+ deposition inside the vacuole. It is well known that when heavy metals are introduced into the reaction medium (in vitro metal effect), the activities of the plasma ´ membrane ATPase are diminished (Astolfi et al., 2003; Burzynski

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and Kolano, 2003; Ros et al., 1992). Similar observations were made for the tonoplast proton pumps (data not shown), suggesting that different mechanisms are responsible for heavy metal action in vivo and in vitro. Ros et al. (1992) postulated that, while a direct effect of metals is observed in the in vitro assays, an indirect mechanism that might be involved in the response to stress is probably responsible for their effect in vivo. Inhibition of ATPase activity may be a result of complex formations between ATP and divalent metal cations, decreasing the level of enzyme substrate, or direct interactions of metals with enzyme functional ligands, such as –SH groups, negatively affecting its conformation (Ros et al., 1992). Cysteine residues (with sulfhydryl groups) are known to play an essential role in maintaining the functional conformation of the V-ATPase. One of the mechanisms controlling V-ATPase activity is reversible disulfide bond formation between Cys254 and Cys532 at the catalytic subunit A (Cipriano et al., 2008). Phospholipids are known to be required by V-ATPase for its activity. When the enzyme is delipidated, its activity almost disappears, but this activity may be restored by the exogenous application of phospholipids (Yamaguchi and Kasamo, 2001). Heavy metals can modulate membrane lipids, affecting their biosynthesis and changing lipid composition or increasing lipid peroxidation (Devi and Prasad, 1999). Most studies have demonstrated an increase in lipid peroxidation as a result of both Cd and Cu treatments (Chaoui and Ferjani, 2005; Groppa et al., 2001; Mazhoudi et al., 1997; Wu et al., 2003). However, unchanged levels of lipid peroxides under cadmium stress have also been reported (Astolfi et al., 2005; Guo et al., 2004; Pál et al., 2005; Sharma et al., 2004). Our results indicated that copper ions increased the lipid peroxidation in the tonoplast isolated from cucumber roots, whereas cadmium had no effect (Fig. 5). Different actions of the two metals may result from their chemical properties. Cu, in contrast to Cd, as a transition metal itself, can initiate the peroxidation

Fig. 6. ATP (A) and PPi (B) levels in Cd and Cu-treated cucumber roots. Cucumber seedlings were exposed to the solution without heavy metals (control plants) or with 10 and 100 ␮M Cd and Cu for 2 or 24 h. Obtained results represent an average (±SD) of three independent experiments run in triplicate.

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reactions. Thus, it has been suggested that excess copper causes stronger changes in lipid content and fatty acid composition, compared to an excess of cadmium (Quariti et al., 1997). The results presented here indicate that Cu affects the vacuolar membrane of cucumber root cells to a larger extent than Cd. Because V-ATPase activity is controlled by the lipid environment (especially by glycolipids/phospholipids ratio), it is possible that Cu-induced changes in tonoplast composition could be responsible for the observed increase in enzyme activity. Such a point of view, however, requires future studies to compare lipid content of tonoplast fractions isolated from roots untreated and treated with copper. Although plant responses to heavy metals have been extensively studied, it is still not completely understood how metals affect gene expression level. Changes in transcript levels upon Cd as well as Cu stress have been investigated in several plants. Transcriptional regulation of genes related to sulfur metabolism (to provide GSH for phytochelatin biosynthesis), signal transduction and antioxidative responses has been reported as a result of Cd exposure (Herbette et al., 2006; Yamaguchi et al., 2010). Cu treatment particularly affects genes involved in defense, photosynthesis and transport (Keinänen et al., 2007; Sudo et al., 2008). On the other hand, it is well known that expression of genes encoding both V-ATPase and V-PPase could be modulated under environmental stress, e.g. salinity. For this reason, it was hypothesized that modified proton pump activities, observed in our study, resulted at least partially from heavy metalinduced changes in expression of specific genes. Surprisingly, no differences in the mRNA levels for V-ATPase catalytic subunit A (CsVHA-A) and membrane subunit c (CsVHA-c) as well as for V-PPase (CsVP) in cucumber roots exposed to Cd and Cu were found (Fig. 3). Thus, we suggest that changes in the activity of the tonoplast H+ pumps in response to heavy metals were not dependent on changes in the transcription of the encoding genes. Protein expression is regulated not only at the transcriptional level, but also at the translational and post-translational levels. Since V-ATPase is a complex of several subunits, its regulation is likely to be very complicated. One of the posttranslational mechanisms regulating V-ATPase activity appears to be the phosphorylation of some subunits. Martiny-Baron et al. (1992) reported that stimulation of V-ATPase activity in zucchini plants was strongly correlated with the phosphorylation of subunit B by lysophospholipid-activated kinase. McCubbin et al. (2004) observed an activation of V-ATPase by the calciumdependent protein kinase HvCDPK1 in aleurone vacuoles isolated from barley. Ca2+ -dependent protein kinase was also found to stimulate tonoplast ATPase in cucumber roots exposed to NaCl (Kłobus and Janicka-Russak, 2004). It was proposed that subunit C of V-ATPase may be a target for WNK (with no lysine) kinases (HongHermesdorf et al., 2006). Liu et al. (2004) identified conserved serine (Ser525) residue in V-ATPase subunit A as a potent site of phosphorylation that was responsible for enzyme activation in maize roots. Moreover, 14-3-3 protein has recently been shown to interact specifically with subunit A in phosphorylation-dependent manner in barley (Klychnikov et al., 2007). To determine whether the metal-induced activation and/or inhibition of the vacuolar ATPase in cucumber roots were attributed to its post-translational modification, immunoblot analyses with specific antibodies against ATPase holoenzyme, phosphoserine and phosphothreonine were performed. Our results indicated that serine residues in subunit B of cucumber V-ATPase were subjected to phosphorylation, but this process was not clearly affected by cadmium and copper (Fig. 4). Thus, the mechanism responsible for modulation of V-ATPase activity under heavy metals seems to not involve the phosphorylation events in subunit A, B and C. Plant organelle proteomic studies have recently become a powerful tool for comparison of the quantity of specific proteins under various stress conditions, such as heavy metals (Ahsan et al., 2009;

Lanquar et al., 2007). Schneider et al. (2009) used a quantitative proteomic approach to determine the contribution of the tonoplast transporters to cadmium detoxification in barley. Only a few of identified transporters showed a Cd-dependent alteration in the protein ratio. Whereas the protein levels of the V-ATPase subunits were unchanged after cadmium treatments, one isoform of the vacuolar pyrophosphatase was up-regulated 2-fold under low (20 ␮M) Cd conditions (but not under 200 ␮M Cd). These results suggest the possibility that other V-ATPase subunits and/or other V-PPase isoforms may be modulated by cadmium and copper at the gene and/or protein levels in cucumber roots. It is well known that heavy metals, especially cadmium, cause the inhibition of oxidative phosphorylation in plant mitochondria, reducing cellular ATP content and diminishing ATP-dependent metabolic pathways (Kessler and Brand, 1994). Unlike ATP levels, cellular PPi concentrations seem to remain stable during marked changes in respiratory states. Under such energy stress conditions, some of the reactions consuming ATP can be replaced by reactions utilizing PPi including functioning of vacuolar PPase (Carystinos et al., 1995; Palma et al., 2000). Both ATP and pyrophosphate levels were lowered in cucumber roots treated with cadmium and copper (Fig. 6). Kasai et al. (1998) showed that PPi content significantly decreased in roots of rye plants grown under mineral deficiency. Under the same conditions, activity of V-PPase was markedly stimulated. The authors proposed the possibility that the actual in vivo activity of the root V-PPase is also higher in stressed plants than in those grown under normal conditions. Consequently, high enzyme activity is responsible for the reduction of pyrophosphate level. Our results do not show such dependence between the proton pump activity and substrate concentration in cucumber roots. Taken together, among two tonoplast H+ translocating pumps subjected to heavy metal stress, the V-ATPase is the only enzyme visibly stimulated by copper ions. Such data suggest that a high electrochemical proton gradient across the tonoplast, generated by V-ATPase, may be used by secondary active antiporters responsible for accumulation of Cu2+ excess inside the vacuole. Thus, the vacuolar ATPase seems to play an important role in cucumber adaptation to copper stress. However, the mechanism by which Cu ions acts on the V-ATPase complex has not been explained. One of the possibilities is indirect alteration via modification of membrane lipids. On the other hand, V-ATPase and V-PPase do not seem to be essential elements of cadmium tolerance in cucumber plants, as their activities are negatively regulated by Cd in the environment. Acknowledgement This work was supported by Polish Ministry of Science and Higher Education, grant no. N303 112 32/3841. References Ahsan N, Renaut J, Komatsu S. Recent developments in the application of proteomics to the analysis of plant responses to heavy metals. Proteomics 2009;9:2602–21. Ames BN. Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzym 1966;8:115–8. Astolfi S, Zuchia S, Chiania A, Passera C. In vivo and in vitro effects of cadmium on H+ -ATPase activity of plasma membrane vesicles from oat (Avena sativa L.) roots. J Plant Physiol 2003;160:387–93. Astolfi S, Zuchi S, Passera C. Effect of cadmium on H+ -ATPase activity of plasma membrane vesicles isolated from roots of different S-supplied maize (Zea mays L.) plants. Plant Sci 2005;169:361–8. Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principles of protein dye binding. Anal Biochem 1976;72:248–54. ´ Burzynski M, Kolano E. In vivo and in vitro effects of copper and cadmium on the plasma membrane H+ -ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots. Acta Physiol Plant 2003;25:39–45. Carystinos GD, MacDonald HR, Monroy AF, Dhindsa RS, Poole RJ. Vacuolar H+ translocating pyrophosphatase is induced by anoxia or chilling in seedlings of rice. Plant Physiol 1995;108:641–9.

K. Kabała et al. / Journal of Plant Physiology 167 (2010) 1328–1335 Chardonnens AN, Koevoets PLM, van Zanten A, Schat H, Verkleij JAC. Properties of enhanced tonoplast zinc transport in naturally selected zinc tolerant Silene vulgaris. Plant Physiol 1999;120:779–85. Chaoui A, Ferjani EE. Effects of cadmium and copper on antioxidant capacities, lignification and auxin degradation in leaves of pea (Pisum sativum L.) seedlings. C R Biol 2005;328:23–31. Cipriano DJ, Wang Y, Bond S, Hinton A, Jefferies KC, Qi J, et al. Structure and regulation of the vacuolar ATPases. Biochim Biophys Acta 2008;1777:599–604. Clemens S. Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 2006;88:1707–19. Darley CP, Davies JM, Sanders D. Chill-induced changes in the activity and abundance of the vacuolar proton-pumping pyrophosphatase from mung bean hypocotyls. Plant Physiol 1995;109:659–65. Delhaize E, Kataoka T, Hebb DM, White RG, Ryan RR. Genes encoding proteins of the cation diffusion facilitator family that confer manganese tolerance. Plant Cell 2003;15:1131–42. Devi SR, Prasad MNV. Membrane lipid alterations in heavy metal exposed plants. In: Prasad MNV, Hagemeyer J, editors. Heavy metal stress in plants. From molecules to ecosystems. Springer; 1999. p. 99–116. Dietz K-J, Tavakoli N, Kluge C, Mimura T, Sharma SS, Harris GC, et al. Significance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level. J Exp Bot 2001;52:1969–80. Fukuda A, Chiba K, Maeda M, Nakamura A, Maeshima M, Tanaka Y. Effect of salt and osmotic stresses of genes for the vacuolar H+ -pyrophosphatase, H+ -ATPase subunit A, and Na+ /H+ antiporter from barley. J Exp Bot 2004;55:585–94. Gallagher SR, Leonard RT. Effect of vanadate, molybdate and azide on membraneassociated ATPase and soluble phosphatase activities of corn roots. Plant Physiol 1982;70:1335–40. Gaxiola RA, Palmgren MG, Schumacher K. Plant proton pumps. FEBS Lett 2007;581:2204–14. Glaab J, Kaiser WM. Increased nitrate reductase activity in leaf tissues after application of the fungicide Kresoxim-methyl. Planta 1999;207:442–8. Golldack D, Dietz K-J. Salt-induced expression of the vacuolar H+ -ATPase in the common ice plant is developmentally controlled and tissue specific. Plant Physiol 2001;125:1643–54. Gratão PL, Polle A, Lea PJ, Azevedo RA. Making the life of heavy metal-stressed plants a little easier. Funct Plant Biol 2005;32:481–94. Groppa MD, Tomaro ML, Benavides MP. Polyamines as protectors against cadmium and copper-induced oxidative damage in sunflower leaf discs. Plant Sci 2001;161:481–8. Guo S, Yin H, Zhang X, Zhao F, Li P, Chen S, et al. Molecular cloning and characterization of a vacuolar H+ -pyrophosphatase gene, SsVP, from halophyte Suaeda salsa and its overexpression increases salt and drought tolerance of Arabidopsis. Plant Mol Biol 2006;60:41–50. Guo T, Zhang G, Zhou M, Wu F, Chen J. Effects of aluminum and cadmium toxicity on growth and antioxidant enzyme activities of two barley genotypes with different Al resistance. Plant Soil 2004;258:241–8. Hall JL. Cellular mechanisms for heavy metal detoxification and tolerance. J Exp Bot 2002;366:1–11. Herak-Kramberger CM, Brown D, Sabolic´ I. Cadmium inhibits vacuolar H+ -ATPase and endocytosis in rat kidney cortex. Kidney Int 1998;53:1713–26. Herak-Kramberger CM, Sabolic´ I, Blanuˇsa M, Smith PJS, Brown D, Breton S. Cadmium inhibits vacuolar H+ -ATPase-mediated acidification in the rat epididymis. Biol Reprod 2000;63:599–606. Herbette S, Taconnat L, Hugouvieux V, Piette L, Magniette M-LM, Cuine S, et al. Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots. Biochimie 2006;88:1751–65. Hong-Hermesdorf A, Brüx A, Grüber A, Grüber G, Schumacher K. A WNK kinase binds and phosphorylates V-ATPase subunit C. FEBS Let 2006;580:932–9. ´ Janicka-Russak M, Kabała K, Burzynski M, Kłobus G. Response of plasma membrane H+ -ATPase to heavy metal stress in Cucumis sativus roots. J Exp Bot 2008;59:3721–8. ´ Kabała K, Janicka-Russak M, Burzynski M, Kłobus G. Comparison of heavy metal effect on the proton pumps of plasma membrane and tonoplast in cucumber root cells. J Plant Physiol 2008;165:278–88. Kabała K, Kłobus G. Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells. Acta Physiol Plant 2001;23:55–63. Kasai M, Nakamura T, Kudo N, Sato H, Maeshima M, Sawada S. The activity of the root vacuolar H+ -pyrophosphatase in rye plants grown under conditions deficient in mineral nutrients. Plant Cell Physiol 1998;39:890–4. Keinänen SI, Hassinen VH, Kärenlampi SO, Tervahauta AI. Isolation of genes upregulated by copper in a copper-tolerant birch (Betula pendula) clone. Tree Physiol 2007;27:1243–52. Kessler A, Brand MD. Quantitative determination of the regulation of oxidative phosphorylation by cadmium in potato tuber mitochondria. Eur J Biochem 1994;225:923–35. Klychnikov OI, Li KW, Lill H, de Boer AH. The V-ATPase from etiolated barley (Hordeum vulgare L.) shoots is activated by blue light and interacts with 14-3-3 proteins. J Exp Bot 2007;58:1013–23. Kłobus G, Janicka-Russak M. Modulation by cytosolic components of proton pump activities in plasma membrane and tonoplast from Cucumis sativus L. roots during salt stress. Physiol Plant 2004;121:84–92.

1335

Krebs M, Beyhl D, Görlich E, Al-Rasheid KAS, Marten I, Stierhof Y-D, et al. Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation. PNAS 2010;107:3251–6. Lanquar V, Kuhn L, Lelièvre F, Khafif M, Espagne C, Bruley C, et al. 15 N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells. Proteomics 2007;7:750–4. Lehr A, Kirsch M, Viereck R, Schiemann J, Rausch T. cDNA and genomic cloning of sugar beet V-type H+ -ATPase subunit A and c isoforms: evidence for coordinate expression during plat development and coordinate induction in response to high salinity. Plant Mol Biol 1999;39:463–75. Liu GS, Chen S, Chen J, Wang XC. Identification of the phosphorylation site of the V-ATPase subunit A in maize roots. Acta Bot Sinica 2004;46:428–35. Maeshima M, Yoshida S. Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean. J Biol Chem 1989;264:20068–73. Martinoia E, Maeshima M, Neuhaus HE. Vacuolar transporters and their essential role in plant metabolism. J Exp Bot 2007;58:83–102. Martiny-Baron G, Manolson MF, Poole RJ, Hecker D, Scherer GFE. Proton transport and phosphorylation of tonoplast polypeptides from zucchini are stimulated by the phospholipid platelet-activating factor. Plant Physiol 1992;99:1635– 41. Mazhoudi S, Chaoui A, Ghorbal MH, Ferjani EE. Response of antioxidant enzymes to excess copper in tomato (Lycopersicon esculentum, Mill.). Plant Sci 1997;127:129–37. McCubbin AG, Ritchie SM, Swanson SJ, Gilroy S. The calcium-dependent protein kinase HvCDPK1 mediates the gibberellic acid response of barley aleurone through regulation of vacuolar function. Plant J 2004;39:206–18. Pál M, Horváth E, Janda T, Páldi E, Szalai G. Cadmium stimulates the accumulation of salicylic acid and its putative precursors in maize (Zea mays) plants. Physiol Plant 2005;125:356–64. Palma DA, Blumwald E, Plaxton WC. Upregulation of vacuolar H+ -translocating pyrophosphatase by phosphate starvation of Brassica napus (rapeseed) suspension cell cultures. FEBS Lett 2000;486:155–8. Parks GE, Dietrich MA, Schumaker KS. Increased vacuolar Na+ /H+ exchange activity in Salicornia bigelovii Torr. in response to NaCl. J Exp Bot 2002;53:1055– 65. Quariti O, Boussama N, Zarrouk M, Cherif A, Ghorbal MH. Cadmium- and copper-induced changes in tomato membrane lipids. Phytochemistry 1997;45:1343–50. Ros R, Morales A, Segura J, Picazo I. In vivo and in vitro effects of nickel and cadmium on the plasmalemma ATPase from rice (Oryza sativa L.) shoots and roots. Plant Sci 1992;83:1–6. Sairam RK, Rao KV, Srivastava GC. Differential response of wheat genotypes to long term salinity stress in relation to oxidative stress, antioxidant activity and osmolyte concentration. Plant Sci 2002;163:1037–46. Schneider T, Schellenberg M, Meyer S, Keller F, Gehrig P, Riedel K, et al. Quantitative detection of changes in the leaf-mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants. Proteomics 2009;9:2668–77. Schumacher K. Endomembrane proton pumps: connecting membrane and vesicle transport. Curr Opin Plant Biol 2006;9:595–600. Serrano A, Pérez-Castineira JR, Baltscheffsky M, Baltscheffsky H. H+ -PPases: yesterday, today and tomorrow. IUBMB Life 2007;59:76–83. Sharma SS, Kaul S, Metwally A, Goyal KC, Finkemeier I, Dietz K-J. Cadmium toxicity to barley (Hordeum vulgare) as affected by varying Fe nutritional status. Plant Sci 2004;166:1287–95. Shigaki T, Hirschi KD. Diverse functions and molecular properties emerging for CAX cation/H+ exchangers in plants. Plant Biol 2006;8:419–29. Siedlecka A, Krupa Z. Functions of enzymes in heavy metal treated plants. In: Prasad MNV, Strzałka K, editors. Physiology and biochemistry of metal toxicity and tolerance in plants. Kluwer Academic Publishers; 2002. p. 303–24. Sudo E, Itouga M, Yoshida-Hatanaka K, Ono Y, Sakakibara H. Gene expression and sensitivity in response to copper stress in rice leaves. J Exp Bot 2008;59:3465– 74. Vera-Estrella R, Barkla BJ, Garcia-Ramirez L, Pantoja O. Salt stress in Thellungiella halophila activates Na+ transport mechanisms required for salinity tolerance. Plant Physiol 2005;139:1507–17. Wang B, Lüttge U, Ratajczak R. Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of halophyte Suaeda salsa. J Exp Bot 2001;52:2355–65. Wu F, Zhang G, Dominy P. Four barley genotypes respond differentially to cadmium: lipid peroxidation and activities of antioxidant capacity. Environ Exp Bot 2003;50:67–78. Yamaguchi H, Fukuoka H, Arao T, Ohyama A, Nunome T, Miyatake K, et al. Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant Solanum torvum. J Exp Bot 2010;61:423–37. Yamaguchi M, Kasamo K. Modulation in the activity of purified tonoplast H+ -ATPase by tonoplast glycolipids prepared from cultured rice (Oryza sativa L. var. Boro) cells. Plant Cell Physiol 2001;42:516–23. Yang H, Knapp J, Koirala P, Rajagopal D, Peer WA, Silbart LK, et al. Enhanced phosphorus nutrition in monocots and dicots over-expressing a phosphorus-responsive type I H+ -pyrophosphatase. Plant Biotech J 2007;5:735–45.