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.

Arabidopsis ALF5, a multidrug efflux transporter gene family member, confers resistance to toxins.

The Arabidopsis genome contains many gene families that are not found in the animal kingdom. One of these is the multidrug and toxic compound extrusion (MATE) family, which has homology with bacterial efflux transporters. Arabidopsis has at least 54 members of this family, which often are found in tandem repeats. Analysis of ALF5, one member of this Arabidopsis family, suggests that its function is required for protection of the roots from inhibitory compounds. Loss of ALF5 function results in the sensitivity of the root to a number of compounds, including a contaminant of commercial agar. Moreover, expression of the Arabidopsis ALF5 cDNA in yeast confers resistance to tetramethylammonium. These phenotypes are consistent with a role for ALF5 as an efflux transporter. Both transcriptional and translational fusions of ALF5 to the beta-glucuronidase reporter gene show that ALF5 is expressed strongly in the root epidermis, a tissue in direct contact with the external environment. The distinct requirement for ALF5 function is remarkable because of the large number of MATE gene family members in Arabidopsis, one of which is adjacent to ALF5 and 83% identical to ALF5 at the amino acid level.

EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana.

The EIR1 gene of Arabidopsis is a member of a family of plant genes with similarities to bacterial membrane transporters. This gene is expressed only in the root, which is consistent with the phenotypes of the eir1 mutants-the roots are agravitropic and have a reduced sensitivity to ethylene. The roots of eir1 mutants are also insensitive to the excess auxin produced by alf1-1 and fail to induce an auxin-inducible gene in the expansion zone. Although they fail to respond to internally generated auxin, they respond normally to externally applied auxin. Expression of the EIR1 gene in Saccharomyces cerevisiae confers resistance to fluorinated indolic compounds. Taken together, these data suggest that the EIR1 protein has a root-specific role in the transport of auxin.

The yeast CLC chloride channel functions in cation homeostasis.

A defect in the yeast GEF1 gene, a CLC chloride channel homolog leads to an iron requirement and cation sensitivity. The iron requirement is due to a failure to load Cu2+ onto a component of the iron uptake system, Fet3. This process, which requires both Gef1 and the Menkes disease Cu2+-ATPase yeast homolog Ccc2, occurs in late- or post-Golgi vesicles, where Gef1 and Ccc2 are localized. The defects of gef1 mutants can be suppressed by the introduction of Torpedo marmorata CLC-0 or Arabidopsis thaliana CLC-c and -d chloride channel genes. The functions of Gef1 in cation homeostasis provide clues to the understanding of diseases caused by chloride channel mutations in humans and cation toxicity in plants.

The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast.

Overexpression of the Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1) confers salt tolerance to the salt-sensitive ena1 mutant of Saccharomyces cerevisiae. Suppression of salt sensitivity requires two ion transporters, the Gef1 Cl- channel and the Nhx1 Na+/H+ exchanger. These two proteins colocalize to the prevacuolar compartment of yeast and are thought to be required for optimal acidification of this compartment. Overexpression of AtNHX1, the plant homologue of the yeast Na+/H+ exchanger, suppresses some of the mutant phenotypes of the yeast nhx1 mutant. Moreover, the level of AtNHX1 mRNA in Arabidopsis is increased in the presence of NaCl. The regulation of AtNHX1 by NaCl and the ability of the plant gene to suppress the yeast nhx1 mutant suggest that the mechanism by which cations are detoxified in yeast and plants may be similar.

Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump.

Transgenic plants overexpressing the vacuolar H(+)-pyrophosphatase are much more resistant to high concentrations of NaCl and to water deprivation than the isogenic wild-type strains. These transgenic plants accumulate more Na(+) and K(+) in their leaf tissue than the wild type. Moreover, direct measurements on isolated vacuolar membrane vesicles derived from the AVP1 transgenic plants and from wild type demonstrate that the vesicles from the transgenic plants have enhanced cation uptake. The phenotypes of the AVP1 transgenic plants suggest that increasing the vacuolar proton gradient results in increased solute accumulation and water retention. Presumably, sequestration of cations in the vacuole reduces their toxic effects. Genetically engineered drought- and salt-tolerant plants could provide an avenue to the reclamation of farmlands lost to agriculture because of salinity and a lack of rainfall.