In planta evidence that the HAK transporter OsHAK2 is involved in Na+ transport in rice.

HAK family transporters primarily function as K+ transporters and play major roles in K+ uptake and translocation in plants, whereas several HAK transporters exhibit Na+ transport activity. OsHAK2, a rice HAK transporter, was shown to mediate Na+ transport in Escherichia coli in a previous study. In this study, we investigated whether OsHAK2 is involved in Na+ transport in the rice plant. Overexpression of OsHAK2 increased Na+ translocation from the roots to the shoots of transgenic rice. It also increased both root and whole-plant Na+ content, and enhanced shoot length under low Na+ and K+ conditions. Meanwhile, OsHAK2 overexpression increased salt sensitivity under a long-term salt stress condition, indicating that OsHAK2 is not involved in salt tolerance, unlike in the case of ZmHAK4 in maize. These results suggest that OsHAK2 is permeable to Na+ and contributes to shoot growth in rice plants under low Na+ and K+ conditions.

Identification of an H2 O2 permeable PIP aquaporin in barley and a serine residue promoting H2 O2 transport.

A barley (Hordeum vulgare) plasma membrane type aquaporin, HvPIP2;5, was identified as an H2 O2 permeable aquaporin among 21 barley and rice PIPs examined in the heterologous expression system using Saccharomyces cerevisiae. Four TIPs were also detected as H2 O2 -transporting aquaporins among 15 barley and rice TIPs. Influx of H2 O2 into yeast cells expressing HvPIP2;5 was determined with a florescent-dye-based assay. Indirect immunofluorescence indicated that the expression of HvPIP2;5 protein was ubiquitous in root tissues, and was also weakly observed in leaf epidermal cells and cells in the vascular bundle. Point mutated variants of HvPIP2;5 were generated by the site-directed mutagenesis. Growth assays of yeast cells expressing these mutated HvPIP2;5 proteins suggested that Ser-126 in HvPIP2;5 has a large impact on H2 O2 transport with a minor influence on the HvPIP2;5-mediated water transport.

CO2 transport by PIP2 aquaporins of barley.

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.

Mechanisms of water transport mediated by PIP aquaporins and their regulation via phosphorylation events under salinity stress in barley roots.

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.