Expression of vacuolar H+-pyrophosphatase (OVP3) is under control of an anoxia-inducible promoter in rice

Vacuolar H⁺-pyrophosphatase (V-PPase) expression increases in a number of abiotic stresses and is thought to play a role in adaptation to abiotic stresses. This paper reports on the regulation of six V-PPase genes in rice (Oryza sativa L.) coleoptiles under anoxia, using flood tolerant and intolerant cultivars to test our hypothesis. Quantitative PCR analysis showed that one vacuolar H⁺-pyrophosphatase (OVP3) was induced by anoxia, particularly in flood-tolerant rice. Regulation of OVP3 expression under anoxia was investigated by analysing putative OVP promoters. The putative OVP3 promoter contained more previously identified anoxia-inducible motifs than the putative promoters of the other five OVP genes. GUS activity in transgenic rice plants containing the OVP3 promoter region linked to the GUS reporter gene was induced only by anoxia. Salt and cold treatments had little effect on OVP3 promoter-driven GUS expression when compared to the anoxic treatment.     

Plasma membrane localization of the type I H-PPase AVP1 in sieve element-companion cell complexes from Arabidopsis thaliana

Previous literature has shown the presence of a plasma membrane (PM) localized type I H⁺-PPase in sieve elements of Ricinus communis. Unfortunately, the physiological relevance of these findings remains obscure due to the lack of genetic and molecular reagents to study R. communis. The availability of H⁺-PPase gain and loss-of-function mutants in Arabidopsis thaliana makes this plant an attractive genetic model to address the question, but data on the PM localization of this H⁺-PPase in A. thaliana are limited to two proteomic approaches. Here we present the first report on the localization of the type I H⁺-PPase AVP1 in sieve element-companion cell complexes (SE-CCc) from A. thaliana. Double epifluorescence and immunogold labeling experiments are consistent with the co-localization of AVP1 and PIP1 (a bona fide PM maker) in PM of SE-CCc from A. thaliana.     

Membrane-bound pyrophosphatase of human gut microbe Clostridium methylpentosum confers improved salt tolerance in Escherichia coli,Saccharomyces cerevisiae and tobacco

Membrane-bound pyrophosphatases (PPases) are involved in the adaption of organisms to stress conditions, which was substantiated by numerous plant transgenic studies with H⁺-PPase yet devoid of any correlated evidences for other two subfamilies, Na⁺-PPase and Na⁺,H⁺-PPase. Herein, we demonstrate the gene cloning and functional evaluation of the membrane-bound PPase (CmPP) of the human gut microbe Clostridium methylpentosum. The CmPP gene encodes a single polypeptide of 699 amino acids that was predicted as a multi-spanning membrane and K⁺-dependent Na⁺,H⁺-PPase. Heterologous expression of CmPP could significantly enhance the salt tolerance of both Escherichia coli and Saccharomyces cerevisiae, and this effect in yeast could be fortified by N-terminal addition of a vacuole-targeting signal peptide from the H⁺-PPase of Trypanosoma cruzi. Furthermore, introduction of CmPP could remarkably improve the salt tolerance of tobacco, implying its potential use in constructing salt-resistant transgenic crops. Consequently, the possible mechanisms of CmPP to underlie salt tolerance are discussed.     

Functional Investigation of Transmembrane Helix 3 in H+-Translocating Pyrophosphatase

H⁺-translocating pyrophosphatase (H⁺-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PP_i) hydrolysis. Vigna radiata H⁺-PPase (VrH⁺-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H⁺-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H⁺-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH⁺-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K⁺-mediated stimulation of H⁺-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PP_i hydrolysis, proton transport, expression, and K⁺ stimulation of H⁺-PPase.     

Identification of a new 130 bp <Emphasis Type="Italic">cis</Emphasis>-acting element in the <Emphasis Type="Italic">TsVP1</Emphasis> promoter involved in the salt stress response from <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Background  

Salt stress is one of the major abiotic stresses affecting plant growth and productivity. Vacuolar H⁺-pyrophosphatase (H⁺-PPase) genes play an important role in salt stress tolerance in multiple species.     

Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors

The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H⁺-PPase) from oat (Avena sativa L.) roots to changes in Mg²⁺ and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indicate that the substrate for the oat root vacuolar H⁺-PPase is Mg₂PPi and that this complex is also a non-competitive inhibitor. In addition, the enzyme is activated by free Mg²⁺ and competitively inhibited by free PPi. This conclusion differs from that reached in previous studies, in which it was proposed that MgPPi is the substrate for plant vacuolar H⁺-PPases. However, models incorporating MgPPi as a substrate were unable to describe the kinetics of the oat H⁺-PPase. It is demonstrated that models incorporating Mg₂PPi as the substrate can describe some of the published kinetics of the Kalanchoë daigremontiana vacuolar H⁺-PPase. Calculations of the likely concentrations of Mg₂PPi in plant cytoplasm suggest that the substrate binding site of the oat vacuolar H⁺-PPase would be about 70% saturated in vivo.     

Functional and fluorescence analyses of tryptophan residues in H+-pyrophosphatase of Clostridium tetani

Homodimeric proton-translocating pyrophosphatase (H⁺-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PP_i) hydrolysis and proton translocation. H⁺-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PP_i binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H⁺-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-P_i binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H⁺-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H⁺-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis.     

Oligomerization of H-pyrophosphatase and its structural and functional consequences

The H⁺-pyrophosphatase (H⁺-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H⁺-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H⁺-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H⁺-PPase is present as an oligomer made up of at least two or three sets of dimers.     

Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding

Homodimeric proton-translocating pyrophosphatase (H⁺-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PP_i to proton translocation across the membrane. H⁺-PPase consists of 14–16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H⁺-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H⁺-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H⁺-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H⁺-PPase upon substrate binding.     

Roles of the Hydrophobic Gate and Exit Channel in Vigna radiata Pyrophosphatase Ion Translocation

Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes pyrophosphate to drive ion (H⁺ and/or Na⁺) translocation. We determined crystal structures and functions of Vigna radiata M-PPase (VrH⁺-PPase), the VrH⁺-PPase–2P_i complex and mutants at hydrophobic gate (residue L555) and exit channel (residues T228 and E225). Ion pore diameters along the translocation pathway of three VrH⁺-PPases complexes (P_i-, 2P_i- and imidodiphosphate-bound states) present a unique wave-like profile, with different pore diameters at the hydrophobic gate and exit channel, indicating that the ligands induced pore size alterations. The 2P_i-bound state with the largest pore diameter might mimic the hydrophobic gate open. In mutant structures, ordered waters detected at the hydrophobic gate among VrH⁺-PPase imply the possibility of solvation, and numerous waters at the exit channel might signify an open channel. A salt-bridge, E225–R562 is at the way out of the exit channel of VrH⁺-PPase; E225A mutant makes the interaction eliminated and reveals a decreased pumping ability. E225–R562 might act as a latch to regulate proton release. A water wire from the ion gate (R-D-K-E) through the hydrophobic gate and into the exit channel may reflect the path of proton transfer.     

Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots

A H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) was associated with low density membranes enriched in tonoplast vesicles of oat roots. The H⁺-PPase catalyzed the electrogenic transport of H⁺ into the vesicles, generating a pH gradient, inside acid (quinacrine fluorescence quenching), and a membrane potential, inside positive (Oxonol V fluorescence quenching). Transport activity was dependent on cations with a selectivity sequence of Rb⁺ = K⁺ > Cs⁺; but it was inhibited by Na⁺ or Li⁺. Maximum rates of transport required at least 20 millimolar K⁺ and the K_m for this ion was 4 millimolar. Fluoride inhibited both ΔpH formation and K⁺-dependent PPase activity with an I₅₀ of 1 to 2 millimolar. Inhibitors of the anion-sensitive, tonoplast-type H⁺-ATPase (e.g. a disulfonic stilbene or NO₃⁻) had no effect on the PPase activity. Vanadate and azide were also ineffective. H⁺-pumping PPase was inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide, but its sensitivity to N,N′-dicyclohexylcarbodiimide was variable. The sensitivity to ions and inhibitors suggests that the tonoplast H⁺-PPase and the H⁺-ATPase are distinct activities and this was confirmed when they were physically separated after Triton X-100 solubilization and Sepharose CL-6B chromatography. H⁺ pumping activity was strongly affected by Mg²⁺ and pyrophosphate (PPi) concentrations. At 5 millimolar Mg²⁺, H⁺ pumping showed a K_maPP for PPi of 15 micromolar. The rate of H⁺ pumping at 60 micromolar PPi was often equivalent to that at 1.5 millimolar ATP. The results suggest PPi hydrolysis could provide another source of a proton motive force used for solute transport and other energy-requiring processes across the tonoplast and other membranes with H⁺-PPase.     

Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast

Nitric oxide (NO), an endogenous signaling molecule in animals and plants, mediates responses to abiotic and biotic stresses. Our previous work demonstrated that 100 μM sodium nitroprusside (SNP, an NO donor) treatment of maize seedlings increased K⁺ accumulation in roots, leaves and sheathes, while decreasing Na⁺ accumulation (Zhang et al. in J Plant Physiol Mol Biol 30:455–459, 2004b). Here we investigate how NO regulates Na⁺, K⁺ ion homeostasis in maize. Pre-treatment with 100 μM SNP for 2 days improved later growth of maize plants under 100 mM NaCl stress, as indicated by increased dry matter accumulation, increased chlorophyll content, and decreased membrane leakage from leaf cells. An NO scavenger, methylene blue (MB-1), blocked the effect of SNP. These results indicated that SNP-derived NO enhanced maize tolerance to salt stress. Further analysis showed that NaCl induced a transient increase in the NO level in maize leaves. Both NO and NaCl treatment stimulated vacuolar H⁺-ATPase and H⁺-PPase activities, resulting in increased H⁺-translocation and Na⁺/H⁺ exchange. NaCl-induced H⁺-ATPase and H⁺-PPase activities were diminished by MB-1. 1-Butanol, an inhibitor of phosphatidic acid (PA) production by phospholipase D (PLD), reduced NaCl- and NO-induced H⁺-ATPase activation. In contrast, applied PA stimulated H⁺-ATPase activity. These results suggest that NO acts as a signal molecule in the NaCl response by increasing the activities of vacuolar H⁺-ATPase and H⁺-PPase, which provide the driving force for Na⁺/H⁺ exchange. PLD and PA play an important role in this process.     

Isolation and Characterization of a Conserved Domain in the Eremophyte H+-PPase Family

H⁺-translocating inorganic pyrophosphatases (H⁺-PPase) were recognized as the original energy donors in the development of plants. A large number of researchers have shown that H⁺-PPase could be an early-originated protein that participated in many important biochemical and physiological processes. In this study we cloned 14 novel sequences from 7 eremophytes: Sophora alopecuroid (Sa), Glycyrrhiza uralensis (Gu), Glycyrrhiza inflata (Gi), Suaeda salsa (Ss), Suaeda rigida (Sr), Halostachys caspica (Hc), and Karelinia caspia (Kc). These novel sequences included 6 ORFs and 8 fragments, and they were identified as H⁺-PPases based on the typical conserved domains. Besides the identified domains, sequence alignment showed that there still were two novel conserved motifs. A phylogenetic tree was constructed, including the 14 novel H⁺-PPase amino acid sequences and the other 34 identified H⁺-PPase protein sequences representing plants, algae, protozoans and bacteria. It was shown that these 48 H⁺-PPases were classified into two groups: type I and type II H⁺-PPase. The novel 14 eremophyte H⁺-PPases were classified into the type I H⁺-PPase. The 3D structures of these H⁺-PPase proteins were predicted, which suggested that all type I H⁺-PPases from higher plants and algae were homodimers, while other type I H⁺-PPases from bacteria and protozoans and all type II H⁺-PPases were monomers. The 3D structures of these novel H⁺-PPases were homodimers except for SaVP3, which was a monomer. This regular structure could provide important evidence for the evolutionary origin and study of the relationship between the structure and function among members of the H⁺-PPase family.     

Identification and Characterization of New Members of Vacuolar H<Superscript>+</Superscript>-Pyrophosphatase Family from <Emphasis Type="Italic">Oryza sativa</Emphasis> Genome

The vacuolar H⁺-pyrophosphatase (V-PPase) is an electrogenic H⁺ pump localized in the plant vacuolar membrane. V-PPase from many species has been characterized previously and the corresponding genes/cDNAs have been cloned. Cloning of the V-PPase genes from many plant species has revealed conserved motifs that may correspond to catalytic sites. The completion of the entire DNA sequence of Oryza sativa (430 Mb) presented an opportunity to study the structure and function of V-PPase proteins, and also to identify new members of this family in Oryza sativa. Our analysis identified three novel V-PPase proteins in the Oryza sativa genome that contain functional domains typical of V-PPase. We have designated them as OVP3 to OVP5. The new predicted OVPs have chromosomal locations different from previously characterized V-PPases (OVP1 and OVP2) located on chromosome 6. They all contain three characteristic motifs of V-PPase and also a conserved motif [DE]YYTS, specific to type I V-PPases and involved in coupling PP_i hydrolysis to H⁺ translocation.     

Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue

Vacuolar proton pumping pyrophosphatase (H⁺-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP_i hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H⁺-translocation of vacuolar H⁺-PPase in a concentration-dependent manner. Absorbance of vacuolar H⁺-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PP_i hydrolysis activity as well. The pK _a of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H⁺-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H⁺-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H⁺-PPase. Inhibition of vacuolar H⁺-PPase by DEPC changes V _max but not K _m values. Moreover, DEPC inhibition of vacuolar H⁺-PPase could be substantially protected against by its physiological substrate, Mg²⁺-PP_i. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H⁺-PPase by DEPC. Taken together, we suggest that vacuolar H⁺-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by DEPC, and this histidine residue may located in a domain sensitive to the modification of Cys-629 by NEM.     

Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer

Homodimeric H⁺-pyrophosphatase (H⁺-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H⁺-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PP_i), for H⁺ translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PP_i binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H⁺-PPase are 49.3 ± 4.0 and 67.2 ± 5.7 Å, respectively. Furthermore, putative PP_i binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H⁺-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H⁺-PPase upon substrate binding.     

Common identity of substrate binding subunit of vacuolar h-translocating inorganic pyrophosphatase of higher plant cells

There have been conflicting reports in the literature concerning the polypeptide composition of the vacuolar H⁺-translocating inorganic pyrophosphatase (tonoplast H⁺-PPase) of plant cells. The major subunit(s) of the enzyme have been attributed to polypeptides of relative molecular weight (M_r) 64,500 (Beta vulgaris), 67,000 (Beta vulgaris), 73,000 (Vigna radiata), and 37,000 to 45,000 (Zea mays). Here, we reconcile these differences to show, through the combined application of independent purification, affinity-labeling, sequencing, and immunological procedures, that the major polypeptide associated with the H⁺-PPase from all of these organisms, and Arabidopsis thaliana, corresponds to the same moiety. The principal polypeptide components of the H⁺-PPase purified from Beta and Vigna by independent procedures have similar apparent subunit masses when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under identical conditions (M_r(Beta) = 64,500; M_r(Vigna) = 66,000) and exhibit identical kinetics of irreversible inhibition and ligand-modified labeling by [¹⁴C]-N-ethylmaleimide. Similarly, the M_r 64,500 and 67,000 polypeptides isolated from Beta by independent methods (cf. C.J. Britten, J.C. Turner, P.A. Rea [1989] FEBS Lett 256: 200-206 versus V. Sarafian and R.J. Poole [1989] Plant Physiol 91: 34-38) are indistinguishable: the two polypeptides comigrate when electrophoresed under the same conditions and yield tryptic fragments with identical overlapping sequences. Because both the N-terminal sequence of the M_r 66,000 subunit of the H⁺-PPase isolated from Vigna and the direct sequence data from Beta align precisely with the deduced amino acid sequence of cDNAs encoding the H⁺-PPase of Arabidopsis, all three enzymes are inferred to be highly conserved structurally. Accordingly, immunoblots of membranes prepared from Arabidopsis, Beta, Vigna, and Zea, probed with antibody affinity purified against the magnesium inorganic pyrophosphate-binding, M_r 66,000 polypeptide of Vigna, reveal a single immunoreactive band at M_r 64,500 to 67,000 in all four preparations. The M_r 66,000 polypeptide of Zea membranes is, however, prone to proteolysis during membrane fractionation and selective aggregation during sample denaturation for SDS-PAGE. The anomalous M_r 37,000 to 45,000 subunit pattern previously ascribed to the H⁺-PPase from Zea (A. Chanson and P.E. Pilet [1989] Plant Physiol 90: 934-938) is attributed to loss of the M_r 66,000 subunit and the appearance of polypeptide fragments of M_r 44,700 and 39,000 through the combined effects of sample aggregation before SDS-PAGE and proteolysis, respectively. It is, therefore, concluded that the substrate-binding subunit of the tonoplast H⁺-PPase has a common identity in all four organisms.     

Heterologous expression of Arabidopsis H+‐pyrophosphatase enhances salt tolerance in transgenic creeping bentgrass (Agrostis stolonifera L.)

The Arabidopsis vacuolar H⁺‐pyrophosphatase (AVP1), when over‐expressed in transgenic (TG) plants, regulates root and shoot development via facilitation of auxin flux, and enhances plant resistance to salt and drought stresses. Here, we report that TG perennial creeping bentgrass plants over‐expressing AVP1 exhibited improved resistance to salinity than wild‐type (WT) controls. Compared to WT plants, TGs grew well in the presence of 100 mm NaCl, and exhibited higher tolerance and faster recovery from damages from exposure to 200 and 300 mm NaCl. The improved performance of the TG plants was associated with higher relative water content (RWC), higher Na⁺ uptake and lower solute leakage in leaf tissues, and with higher concentrations of Na⁺, K⁺, Cl^‐ and total phosphorus in root tissues. Under salt stress, proline content was increased in both WT and TG plants, but more significantly in TGs. Moreover, TG plants exhibited greater biomass production than WT controls under both normal and elevated salinity conditions. When subjected to salt stress, fresh (FW) and dry weights (DW) of both leaves and roots decreased more significantly in WT than in TG plants. Our results demonstrated the great potential of genetic manipulation of vacuolar H⁺‐pyrophosphatase expression in TG perennial species for improvement of plant abiotic stress resistance.     

Characterization and expression analyses of the H<Superscript>+</Superscript>-pyrophosphatase gene in rye

The H⁺-pyrophosphatase (H ⁺-PPase) gene plays an important role in maintaining intracellular proton gradients. Here, we characterized the full-length complementary DNA (cDNA) and DNA of the H ⁺-PPase gene ScHP1 in rye (Secale cereale L. ‘Qinling’). We determined the subcellular localization of this gene and predicted the corresponding protein structure. We analysed the evolutionary relationship between ScHP1 and H ⁺?PPase genes in other species, and did real-time quantitative polymerase chain reaction to explore the expression patterns of ScHP1 in rye plants subjected to N, P and K deprivation and to cold, high-salt and drought stresses. ScHP1 cDNA included a 2289 bp open reading frame (ORF) encoding 762 amino acid residues with 14 transmembrane domains. The genomic ScHP1 DNA was 4354 bp and contained eight exons and seven introns. ScHP1 was highly homologous with other members of the H ⁺-PPase gene family. When the full-length ORF was inserted into the expression vector pA7-YFP, the fluorescent microscopy revealed that ScHP1-YFP fusion protein was located in the plasma membrane. Rye plants that were subjected to N deprivation, cold and high-salt stresses, ScHP1 expression was higher in the leaves than roots. Conversely, plants subjected to P and K deprivation and drought stress, ScHP1 expression was higher in the roots than leaves. Under all the investigated stress conditions, expression of ScHP1 was lower in the stem than in the leaves and roots. Our results imply that ScHP1 functions under abiotic stress response.