Plant proton pumping pyrophosphatase: the potential for its pyrophosphate synthesis activity to modulate plant growth.

Cellular pyrophosphate (PPi) homeostasis is vital for normal plant growth and development. Plant proton-pumping pyrophosphatases (H+ -PPases) are enzymes with different tissue-specific functions related to the regulation of PPi homeostasis. Enhanced expression of plant H+ -PPases increases biomass and yield in different crop species. Here, we emphasise emerging studies utilising heterologous expression in yeast and plant vacuole electrophysiology approaches, as well as phylogenetic relationships and structural analysis, to showcase that the H+ -PPases possess a PPi synthesis function. We postulate this synthase activity contributes to modulating and promoting plant growth both in H+ -PPase-engineered crops and in wild-type plants. We propose a model where the PPi synthase activity of H+ -PPases maintains the PPi pool when cells adopt PPi-dependent glycolysis during high energy demands and/or low oxygen environments. We conclude by proposing experiments to further investigate the H+ -PPase-mediated PPi synthase role in plant growth.

CAX-ing a wide net: Cation/H(+) transporters in metal remediation and abiotic stress signalling.

Cation/proton exchangers (CAXs) are a class of secondary energised ion transporter that are being implicated in an increasing range of cellular and physiological functions. CAXs are primarily Ca(2+) efflux transporters that mediate the sequestration of Ca(2+) from the cytosol, usually into the vacuole. Some CAX isoforms have broad substrate specificity, providing the ability to transport trace metal ions such as Mn(2+) and Cd(2+) , as well as Ca(2+) . In recent years, genomic analyses have begun to uncover the expansion of CAXs within the green lineage and their presence within non-plant species. Although there appears to be significant conservation in tertiary structure of CAX proteins, there is diversity in function of CAXs between species and individual isoforms. For example, in halophytic plants, CAXs have been recruited to play a role in salt tolerance, while in metal hyperaccumulator plants CAXs are implicated in cadmium transport and tolerance. CAX proteins are involved in various abiotic stress response pathways, in some cases as a modulator of cytosolic Ca(2+) signalling, but in some situations there is evidence of CAXs acting as a pH regulator. The metal transport and abiotic stress tolerance functions of CAXs make them attractive targets for biotechnology, whether to provide mineral nutrient biofortification or toxic metal bioremediation. The study of non-plant CAXs may also provide insight into both conserved and novel transport mechanisms and functions.

Plant cation/H+ exchangers (CAXs): biological functions and genetic manipulations.

Inorganic cations play decisive roles in many cellular and physiological processes and are essential components of plant nutrition. Therefore, the uptake of cations and their redistribution must be precisely controlled. Vacuolar antiporters are important elements in mediating the intracellular sequestration of these cations. These antiporters are energized by the proton gradient across the vacuolar membrane and allow the rapid transport of cations into the vacuole. CAXs (for CAtion eXchanger) are members of a multigene family and appear to predominately reside on vacuoles. Defining CAX regulation and substrate specificity have been aided by utilising yeast as an experimental tool. Studies in plants suggest CAXs regulate apoplastic Ca(2+) levels in order to optimise cell wall expansion, photosynthesis, transpiration and plant productivity. CAX studies provide the basis for making designer transporters that have been used to develop nutrient enhanced crops and plants for remediating toxic soils.

The expression of the open reading frame of Arabidopsis CAX1, but not its cDNA, confers metal tolerance in yeast.

The biochemical properties and regulation of several plant CAX (CAtion eXchanger)-type vacuolar Ca2+/H+ exchangers have been extensively analysed in yeast expression assays. In the present study, we compare and contrast the phenotypes of yeast cells expressing the CAX1 cDNA and open reading frame (ORF). We report that the CAX1 ORF, but not the cDNA containing the 3'-untranslated region (UTR), was able to confer Ca2+ tolerance when expressed in a Ca2+-sensitive yeast mutant. Additionally, only yeasts expressing the N-terminal truncated CAX1 ORF were able to grow on high Mn2+ media, suggesting that removal of the 3'-UTR altered activity. However, removal of the 3'-UTR from another CAX did not alter the yeast phenotypes. Expression studies demonstrated that expressing the CAX1 ORF in yeast elevates CAX1 RNA and protein levels. Our results suggest that the 3'-UTR modulates expression of CAX1 in yeast.

Identification of three distinct phylogenetic groups of CAX cation/proton antiporters.

Ca(2+)/cation antiporter (CaCA) proteins are integral membrane proteins that transport Ca(2+) or other cations using the H(+) or Na(+) gradient generated by primary transporters. The CAX (for CAtion eXchanger) family is one of the five families that make up the CaCA superfamily. CAX genes have been found in bacteria, Dictyostelium, fungi, plants, and lower vertebrates, but only a small number of CAXs have been functionally characterized. In this study, we explored the diversity of CAXs and their phylogenetic relationships. The results demonstrate that there are three major types of CAXs: type I (CAXs similar to Arabidopsis thaliana CAX1, found in plants, fungi, and bacteria), type II (CAXs with a long N-terminus hydrophilic region, found in fungi, Dictyostelium, and lower vertebrates), and type III (CAXs similar to Escherichia coli ChaA, found in bacteria). Some CAXs were found to have secondary structures that are different from the canonical six transmembrane (TM) domains-acidic motif-five TM domain structure. Our phylogenetic tree indicated no evidence to support the cyanobacterial origin of plant CAXs or the classification of Arabidopsis exchangers CAX7 to CAX11. For the first time, these results clearly define the CAX exchanger family and its subtypes in phylogenetic terms. The surprising diversity of CAXs demonstrates their potential range of biochemical properties and physiologic relevance.

Diverse functions and molecular properties emerging for CAX cation/H+ exchangers in plants.

Steep concentration gradients of many ions are actively maintained, with lower concentrations typically located in the cytosol, and higher concentrations in organelles and outside the cell. The vacuole is an important storage organelle for many ions. The concentration gradient of cations is established across the plant tonoplast, in part, by high-capacity cation/H+ (CAX) exchange activity. While plants may not be green yeast, analysis of CAX regulation and substrate specificity has been greatly aided by utilizing yeast as an experimental tool. The basic CAX biology in ARABIDOPSIS has immediate relevance toward understanding the functional interplay between diverse transport processes. The long-range applied goals are to identify novel transporters and express them in crop plants in order to "mine" nutrients out of the soil and into plants. In doing so, this could boost the levels of essential nutrients in plants.

Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.).

We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.

Regulation of CAX1, an Arabidopsis Ca(2+)/H+ antiporter. Identification of an N-terminal autoinhibitory domain.

Regulation of Ca(2+) transport determines the duration of a Ca(2+) signal, and hence, the nature of the biological response. Ca(2+)/H+ antiporters such as CAX1 (cation exchanger 1), play a key role in determining cytosolic Ca(2+) levels. Analysis of a full-length CAX1 clone suggested that the CAX1 open reading frame contains an additional 36 amino acids at the N terminus that were not found in the original clone identified by suppression of yeast (Saccharomyces cerevisiae) vacuolar Ca(2+) transport mutants. The long CAX1 (lCAX1) could not suppress the yeast Ca(2+) transport defects despite localization to the yeast vacuole. Calmodulin could not stimulate lCAX1 Ca(2+)/H+ transport in yeast; however, minor alterations in the 36-amino acid region restored Ca(2+)/H+ transport. Sequence analysis suggests that a 36-amino acid N-terminal regulatory domain may be present in all Arabidopsis CAX-like genes. Together, these results suggest a structural feature involved in regulation of Ca(2+)/H+ antiport.

Phenotypic changes in Arabidopsis caused by expression of a yeast vacuolar Ca2+/H+ antiporter.

In plants, cytosolic Ca2+ levels are tightly regulated, and changes in cytosolic Ca2+ have been implicated in converting numerous signals into adapted responses. Vacuolar ion transporters are thought to be key mediators of cytosolic Ca2+ concentrations. In an attempt to interpret the role of vacuolar Ca2+ transport in plant processes, we have expressed the yeast vacuolar Ca2+/H+ antiporter, VCX1, in Arabidopsis and tobacco. This transporter localizes to the plant vacuolar membrane. VCX1-expressing Arabidopsis plants displayed increased sensitivity to sodium and other ions. These ion sensitivities could be suppressed by addition of calcium to the media. VCX1-expressing plants demonstrated increased tonoplast-enriched Ca2+/H+ antiport activity as well as increased Ca2+ accumulation. These results suggest that VCX1 expression in Arabidopsis could be a valuable tool with which to experimentally dissect the role of Ca2+ transport around the plant vacuole.

Molecular approaches to studying nutrient metabolism and function: an array of possibilities.

Genomics promises to revolutionize the study of nutrient function and requirements and, thereby, solidify the connection of this field to basic sciences, such as molecular genetics. In this short review, we address the general concepts and techniques used in high throughput measurements of gene expression. We also speculate on how these technologies can be used to further our understanding of basic metabolism and nutrient regulation of gene expression in developmental and pathological conditions.

Expression of arabidopsis CAX2 in tobacco. Altered metal accumulation and increased manganese tolerance.

Metal transport from the cytosol to the vacuole is thought to be an important component of ion tolerance and of a plant's potential for use in phytoremediation. The Arabidopsis antiporter CAX2 (calcium exchanger 2) may be a key mediator of this process. CAX2 expression in yeast suppressed both Ca(2+) and Mn(2+) growth defects. A peptide-specific antibody to the antiporter reacted with a 39-kD protein from plant vacuolar membranes. Tobacco (Nicotiana tabacum) plants expressing CAX2 accumulated more Ca(2+), Cd(2+), and Mn(2+) and were more tolerant to elevated Mn(2+) levels. Expression of CAX2 in tobacco increased Cd(2+) and Mn(2+) transport in isolated root tonoplast vesicles. These results suggest that CAX2 has a broad substrate range and modulation of this transporter may be an important component of future strategies to improve plant ion tolerance.

Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity.

Calcium (Ca(2)+) efflux from the cytosol modulates Ca(2+) concentrations in the cytosol, loads Ca(2+) into intracellular compartments, and supplies Ca(2+) to organelles to support biochemical functions. The Ca(2+)/H(+) antiporter CAX1 (for CALCIUM EXCHANGER 1) of Arabidopsis is thought to be a key mediator of these processes. To clarify the regulation of CAX1, we examined CAX1 RNA expression in response to various stimuli. CAX1 was highly expressed in response to exogenous Ca(2+). Transgenic tobacco plants expressing CAX1 displayed symptoms of Ca(2+) deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium concentrations, and to cold shock, but increasing the Ca(2+) in the media abrogated these sensitivities. Tobacco plants expressing CAX1 also demonstrated increased Ca(2+) accumulation and altered activity of the tonoplast-enriched Ca(2+)/H(+) antiporter. These results emphasize that regulated expression of Ca(2+)/H(+) antiport activity is critical for normal growth and adaptation to certain stresses.

Overexpression of Erg11p by the regulatable GAL1 promoter confers fluconazole resistance in Saccharomyces cerevisiae.

The contribution of the dosage of target enzyme P-450 14alpha-demethylase (14alphaDM) to fluconazole resistance in both Candida albicans and Saccharomyces cerevisiae remains unclear. Here, we show that overexpression of Saccharomyces P-450 14alphaDM in S. cerevisiae, under the control of the regulatable promoter GAL1, results in azole resistance.

CAX1, an H+/Ca2+ antiporter from Arabidopsis.

Reestablishment of the resting state after stimulus-coupled elevations of cytosolic-free Ca2+ requires the rapid removal of Ca2+ from the cytosol of plant cells. Here we describe the isolation of two genes, CAX1 and CAX2, from Arabidopsis thaliana that suppress a mutant of Saccharomyces cerevisiae that has a defect in vacuolar Ca2+ accumulation. Both genes encode polypeptides showing sequence similarities to microbial H+/Ca2+ antiporters. Experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing CAX1 or CAX2 demonstrate that these genes encode high efficiency and low efficiency H+/Ca2+ exchangers, respectively. The properties of the CAX1 gene product indicate that it is the high capacity transporter responsible for maintaining low cytosolic-free Ca2+ concentrations in plant cells by catalyzing pH gradient-energized vacuolar Ca2+ accumulation.

Identification of DNA sequences involved in regulating Bacillus subtilis glnRA expression by the nitrogen source.

The DNA binding protein, GlnR, encoded by glnR, is believed to be directly responsible for regulating glnRA expression in Bacillus subtilis. Identification of cis-acting loci involved in glnRA control is the focus of this study. Analysis of glnRA-lacZ transcriptional fusions harboring deletions extending into the promoter region demonstrated that sequences upstream from position -35, relative to the transcription start-point, were necessary for nitrogen source regulation. These sequences included a 21 base-pair (bp) element, from positions -40 to -60, having 2-fold symmetry; the element shares homology to certain binding sites utilized by proteins having the alpha-helix-turn-alpha-helix motif, of which GlnR is a member. Involvement of this element in regulation was examined by using synthetic DNA fragments containing the promoter and upstream sequences driving lacZ expression. Fragments extending from positions -63 to -8 and from positions -52 to -8 yielded full and partial regulation, respectively. Regulation from a fragment containing a 5 bp insertion between positions -36 and -37 was impaired. A T.A to A.T transversion mutation at position -41 did not have any detectable effect on regulation, whereas a T.A to C.G transition mutation at the same site resulted in constitutive expression. Using a gel electrophoresis mobility shift assay, it was found that purified GlnR bound to a glnRA restriction fragment that extended from positions -104 to +83; binding was abolished after digestion with HinfI, which cleaves between positions -52 and -48. Furthermore, HinfI digestion was inhibited by the presence of GlnR. Thus, the GlnR binding site extends from the vicinity of position -35 upstream to position -63. We suggest that the glnRA operator is the 21 bp sequence lying within this region.

Regulation of Bacillus subtilis glutamine synthetase gene expression by the product of the glnR gene.

Transcription of the Bacillus subtilis gene coding of glutamine synthetase (glnA) is regulated by the nitrogen source. The glnA gene lies in an operon in which it is preceded by an open reading frame with the potential to encode a polypeptide of approximately 16,000 Mr. We have now shown that this open reading frame is utilized in vivo, that its product (GlnR) acts as a diffusible, negative regulator of gln transcription, and that GlnR is likely to be a DNA-binding protein. Certain mutations in glnR, including a large, in-frame deletion and a start codon mutation, led to high-level constitutivity of the operon; other mutations caused low-level constitutivity. These latter mutations, which affected the C terminus of GlnR, seemed to disrupt response to the nitrogen source without eliminating the ability of GlnR to bind to DNA. Wild-type GlnR by itself, however, did not impose nitrogen-dependent regulation; such regulation also required the product of glnA. A model is presented in which glutamine synthetase monitors the availability of nitrogen and imposes negative regulation by interaction with or modification of GlnR.