The internal conductance for CO(2) diffusion (g(i)) and CO(2) assimilation rate were measured and the related anatomical characteristics were investigated in transgenic rice leaves that overexpressed barley aquaporin HvPIP2;1. This study was performed to test the hypothesis that aquaporin facilitates CO(2) diffusion within leaves. The g(i) value was estimated for intact leaves by concurrent measurements of gas exchange and carbon isotope ratio. The leaves of the transgenic rice plants that expressed the highest levels of Aq-anti-HvPIP2;1 showed a 40% increase in g(i) as compared to g(i) in the leaves of wild-type rice plants. The increase in g(i) was accompanied by a 14% increase in CO(2) assimilation rate and a 27% increase in stomatal conductance (g(s)). The transgenic plants that had low levels of Aq-anti-HvPIP2;1 showed decreases in g(i) and CO(2) assimilation rate. In the plants with high levels of Aq-anti-HvPIP2;1, mesophyll cell size decreased and the cell walls of the epidermis and mesophyll cells thickened, indicating that the leaves had become xeromorphic. Although such anatomical changes could partially offset the increase in g(i) by the aquaporin, the increase in aquaporin content overcame such adverse effects.

The release of organic anions from roots can protect plants from aluminum (Al) toxicity and help them overcome phosphorus (P) deficiency. Our previous findings showed that Al treatment induced malate and citrate efflux from rape (Brassica napus) roots, and that P deficiency did not induce the efflux. Since this response is similar to the malate efflux from wheat (Triticum aestivum) that is controlled by the TaALMT1 gene, we investigated whether homologs of TaALMT1 are present in rape and whether they are involved in the release of organic anions. We isolated two TaALMT1 homologs from rape designated BnALMT1 and BnALMT2 (B. napus Al-activated malate transporter). The expression of these genes was induced in roots, but not shoots, by Al treatment but P deficiency had no effect. Several other cations (lanthanum, ytterbium, and erbium) also increased BnALMT1 and BnALMT2 expression in the roots. The function of the BnALMT1 and BnALMT2 proteins was investigated by heterologous expression in cultured tobacco (Nicotiana tabacum) cells and in Xenopus laevis oocytes. Both transfection systems showed an enhanced capacity for malate efflux but not citrate efflux, when exposed to Al. Smaller malate fluxes were also activated by ytterbium and erbium treatment. Transgenic tobacco cells grew significantly better than control cells following an 18 h treatment with Al, indicating that the expression of BnALMT1 and BnALMT2 increased the resistance of these plant cells to Al stress. This report demonstrates that homologs of the TaALMT1 gene from wheat perform similar functions in other species.Aluminum (Al) toxicity is the primary factor limiting crop production on acidic soils. When the soil pH falls below 5.0 the soluble Al in the soil solution exists predominantly as the toxic trivalent cation Al 31 that can inhibit root growth in many species at micromolar concentrations . Some species have developed mechanisms to overcome Al-related stresses by either excluding Al from the root cells (resistance mechanisms) or by increasing their tolerance to Al once these cations have been absorbed by the roots (tolerance mechanisms). Exclusion mechanisms might involve the exudation of Al-chelating ligands, the binding of Al within the cell wall or mucilage, plant-induced pH changes in the rhizosphere that reduce the local concentration of Al 31 relative to other hydrolysis products, the selective permeability of the plasma membrane, or perhaps the efflux of Al itself from the root cells. Tolerance mechanisms might include the chelation of Al in the cytosol, the sequestration of Al in the vacuole or other organelles, or modifications to metabolism that allow cellular function to continue normally in the presence of Al (Foy et al., 1978;Taylor, 1991;Delhaize and Ryan, 1995;Horst, 1995;Kochian, 1995;Ma et al., 2001;Matsumoto, 2002).The mechanism of Al resistance that has been observed most commonly in monocotyledonous and dicotyledonous species involves the release of organic anions such as malate, citrate, and oxalate from roots (Miyasaka et a...

Water homeostasis is crucial to the growth and survival of plants under water-related stress. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. Here we report the water transport activity and mechanisms for the regulation of barley (Hordeum vulgare) PIP aquaporins. HvPIP2 but not HvPIP1 channels were found to show robust water transport activity when expressed alone in Xenopus laevis oocytes. However, the co-expression of HvPIP1 with HvPIP2 in oocytes resulted in significant increases in activity compared with the expression of HvPIP2 alone, suggesting the participation of HvPIP1 in water transport together with HvPIP2 presumably through heteromerization. Severe salinity stress (200 mM NaCl) significantly reduced root hydraulic conductivity (Lp(r)) and the accumulation of six of 10 HvPIP mRNAs. However, under relatively mild stress (100 mM NaCl), only a moderate reduction in Lp(r) with no significant difference in HvPIP mRNA levels was observed. Sorbitol-mediated osmotic stress equivalent to 100 and 200 mM NaCl induced nearly identical Lp(r) reductions in barley roots. Furthermore, the water transport activity in intact barley roots was suggested to require phosphorylation that is sensitive to a kinase inhibitor, staurosporine. HvPIP2s also showed water efflux activity in Xenopus oocytes, suggesting a potential ability to mediate water loss from cells under hypertonic conditions. Water transport via HvPIP aquaporins and the significance of reductions of Lp(r) in barley plants during salinity stress are discussed.

We identified three genes homologous to water channels in the plasma membrane type subfamily from roots of barley seedlings. These genes were designated HvPIP2;1, HvPIP1;3, and HvPIP1;5 after comparison to Arabidopsis aquaporins. Competitive reverse transcription (RT)-PCR was applied in order to distinguish and to quantify their transcripts. The HvPIP2;1 transcript was the most abundant among the three in roots. Salt stress (200 mM NaCl) down-regulated HvPIP2;1 (transcript and protein), but had almost no effect on the expressions of HvPIP1;3, or HvPIP1;5. Approximately equal amounts of the transcripts of the three were detected in shoots, and salt stress enhanced the expression of HvPIP2;1 but not of HvPIP1;3, or HvPIP1;5. HvPIP2;1 protein was confirmed to be localized in the plasma membrane. Functional expression of HvPIP2;1 in Xenopus oocytes confirmed that HvPIP2;1 encoded an aquaporin that transports water. This water permeability was reduced by HgCl(2), which is a typical water channel inhibitor. This activity was not modified by some inhibitors against protein kinase and protein phosphatase.

Barley HvPIP2;1 is a plasma membrane aquaporin and its expression was down-regulated after salt stress in barley [Katsuhara et al. (2002) Plant Cell Physiol. 43: 885]. We produced and analyzed transgenic rice plants over-expressing barley HvPIP2;1 in the present study. Over-expression of HvPIP2;1 increased (1) radial hydraulic conductivity of roots (Lp(r)) to 140%, and (2) the mass ratio of shoot to root up to 150%. In these transgenic rice plants under salt stress of 100 mM NaCl, growth reduction was greater than in non-transgenic plants. A decrease in shoot water content (from 79% to 61%) and reduction of root mass or shoot mass (both less than 40% of non-stressed plants) were observed in transgenic plants under salt stress for 2 weeks. These results indicated that over-expression of HvPIP2;1 makes rice plants sensitive to 100 mM NaCl. The possible involvement of aquaporins in salt tolerance is discussed.

CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2;4 that is highly homologous to HvPIP2;3 did not. The isoleucine residue at position 254 of HvPIP2;3 was conserved in PIP2 aquaporins of barley, except HvPIP2;4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2;3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity.

A novel SmtB/ArsR family metalloregulator, denoted BxmR, has been identified and characterized from the cyanobacterium Oscillatoria brevis. Genetic and biochemical evidence reveals that BxmR represses the expression of both bxa1, encoding a CPx-ATPase metal transporter, as well as a divergently transcribed operon encoding bxmR and bmtA, a heavy metal sequestering metallothionein. Derepression of the expression of all three genes is mediated by both monovalent (Ag(I) and Cu(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions, a novel property among SmtB/ArsR metal sensors. Electrophoretic gel mobility shift experiments reveal that apoBxmR forms multiple resolvable complexes with oligonucleotides containing a single 12-2-12 inverted repeat derived from one of the two operator/promoter regions with similar apparent affinities. Preincubation with either monovalent or divalent metal ions induces disassembly of both the BxmR-bxa1 and BxmR-bxmR/ bmtA operator/promoter complexes. Interestingly, the temporal regulation of expression of bxa1 and bmtA mRNAs is different in O. brevis with bxa1 induced first upon heavy metal treatment, followed by bmtA/bxmR. A dynamic interplay among Bxa1, BmtA, and BxmR is proposed that maintains metal homeostasis in O. brevis by balancing the relative rates of metal storage and efflux of multiple heavy metal ions.Transition metal ions such as zinc, copper, iron, and manganese are essential trace elements that play integral catalytic functions in myriad metalloenzymes and electron transfer in all organisms (1-3). However, they are required only in trace amounts and, when present in excess in the environment, even essential metals can be cytotoxic, like heavy metal pollutants (4 -6). All organisms have evolved a range of mechanisms that govern metal homeostasis, defined as maintaining the intracellular bioavailable concentrations of essential metal ions within a range compatible with cell viability (7-9). Multiple lines of evidence from the past decade reveal that heavy metal homeostasis is maintained in all organisms by a small number of critical processes that include metal sensing, chelation, and transport (10 -18).Two distinct mechanisms play prominent roles in governing metal ion homeostasis and resistance in many organisms. One involves the uptake or efflux of specific heavy metal ions across biomembranes, mediated by ATP-coupled high affinity metal ion transporters such as those derived from the CPx-ATPase family (19 -21). Another mechanism involves the specific chelation of metal ions by intracellular chelators, e.g. metallothioneins (MTs), 1 now known to be widely distributed in nature (13,22,23). To meet the diverse biological requirements of specific metal ions, various strategies have evolved to regulate the transcription of genes encoding these heavy metal homeostasis proteins. In prokaryotes, the expression of these genes is tightly controlled by specific metalloregulators or "metal-sensing" transcriptional regulators (12,24,25). One such family of homologous metal sensor pro...

A novel gene related to heavy-metal transport was cloned and identified from the filamentous cyanobacterium Oscillatoria brevis. Sequence analysis of the gene (the Bxa1 gene) showed that its product possessed high homology with heavy-metal transport CPx-ATPases. The CPC motif, which is proposed to form putative cation transduction channel, was found in the sixth transmembrane helix. However, instead of the CXXC motif that is present in the N termini of most metal transport CPx-ATPases, Bxa1 contains a unique Cys-Cys (CC) sequence element and histidine-rich motifs as a putative metal binding site. Northern blotting and real-time quantitative reverse transcription-PCR showed that expression of Bxa1 mRNA was induced in vivo by both monovalent (Cu ؉ and Ag ؉ ) and divalent (Zn 2؉ and Cd 2؉ ) heavy-metal ions at similar levels. Experiments on heavy-metal tolerance in Escherichia coli with recombinant Bxa1 demonstrated that Bxa1 conferred resistance to both monovalent and divalent heavy metals. This is the first report of a CPx-ATPase responsive to both monovalent and divalent heavy metals.Organisms usually utilize emergency mechanisms such as reduced uptake, facilitated efflux, sequestration, and modification to achieve resistance to the toxicity of heavy metals (5, 22, 27, 37). Heavy-metal transport P-type ATPases (or CPx-ATPases) are thought to act as a potential key heavy-metal transporter involved not only in metal ion homeostasis but also in the overall strategy for heavy-metal tolerance (3, 37).CPx-ATPases are a new subgroup of P-type ATPases and possess all the common characteristics of P-type ATPases (25, 37). One significant difference, however, is the presence of a conserved intramembranous cysteine-proline-cysteine/histidine/serine (CPx) motif. P-type ATPases with this unique characteristic are therefore termed CPx-ATPases. Another unique feature of CPx-ATPases is the N-terminal metal binding domain. In most cases, the heavy-metal binding domain consists of one or several cysteine-rich (CXXC) motifs. In certain cases, the cysteine-rich N terminus is replaced by a short sequence containing histidine.There is currently great interest in the N-terminal-cysteinerich CPx-ATPases (1, 6, 9, 17, 31); however, little interest has been paid to the N-terminal-histidine-rich CPx-ATPases. Recently, genomic DNA programs have revealed that the Nterminal-histidine-rich CPx-ATPases are also distributed widely among organisms (8,10,11,37,38), especially plants, although the true roles of these CPx-ATPases are not yet known.Although the detailed characterization of CPx-ATPases is still awaited, the main physiological function of these enzymes is thought to be the transport of heavy-metal ions across biological membranes to maintain the intracellular homeostasis of essential or nonessential heavy metals and deliver specific metal ions to target enzymes. CPx-ATPases have been found to have high specificity for the heavy-metal ions they transport. It has been demonstrated that the type of transport substrate is restricted by he...