Phloem-specific overexpression of AtOPT6 in Arabidopsis enhances Zn transport into shoots.

The Arabidopsis oligopeptide transporter AtOPT6 is membrane transport protein that mediated transport of glutathione in both the reduced (GSH) and oxidized (GSSG) forms. In this study, the role of AtOPT6 in glutathione distribution throughout the plant was investigated. We found that transgenic Arabidopsis overexpressing AtOPT6 under the control of a phloem-specific promoter of sucrose-proton symporter 2 (pSUC2), remarkably increased AtOPT6 transcript levels, ranging from 30- to 40-fold in shoots and 6- to 10-fold in roots, relative to the wild type. AtOPT6-overexpressing lines could elevate the foliar glutathione content; however, glutathione content in the phloem did not change. We observed that the ratio of shoot glutathione content to total glutathione content increased in AtOPT6-overexpressing lines, but not in transgenic Arabidopsis with elevated foliar GSH synthesis. These results indicate the possibility that loading and unloading of glutathione in phloem tissues are enhanced in AtOPT6-overexpressing lines under the control of pSUC2. The results of heavy metal analysis revealed that transgenic Arabidopsis overexpressing AtOPT6 under the control of pSUC2 could promote the transport of Zn into shoots as effectively as transgenic Arabidopsis with elevated foliar GSH synthesis, or wild-type plants with exogenous foliar application of GSH.

Elevated glutathione synthesis in leaves contributes to zinc transport from roots to shoots in Arabidopsis.

Glutathione (GSH) is a vital compound involved in several plant metabolic pathways. Our previous study indicated that foliar GSH application can increase zinc (Zn) levels in leafy vegetables. The objective of this study was to determine the mode of action of GSH as it relates to Zn transport from roots to shoots. Two types of transgenic Arabidopsis plants with genes for GSH synthesis, including StGCS-GS or AtGSH1 driven by the leaf-specific promoter of chlorophyll a/b-binding protein (pCab3) gene were generated. Both types of transgenic Arabidopsis plants showed significant increases in shoot GSH concentrations compared to the wild type (WT). Monitoring 65Zn movement by positron-emitting tracer imaging system (PETIS) analysis indicated that the 65Zn amount in the shoots of both types of transgenic Arabidopsis plants were higher than that in the WT. GSH concentration in phloem sap was increased significantly in WT with foliar applications of 10 mM GSH (WT-GSH), but not in transgenic Arabidopsis with elevated foliar GSH synthesis. Both types of transgenic Arabidopsis with elevated foliar GSH synthesis and WT-GSH exhibited increased shoot Zn concentrations and Zn translocation ratios. These results suggest that enhancement of endogenous foliar GSH synthesis and exogenous foliar GSH application affect root-to-shoot transport of Zn.

AtOPT6 Protein Functions in Long-Distance Transport of Glutathione in Arabidopsis thaliana.

The involvement of the Arabidopsis oligopeptide transporter AtOPT6, which was previously shown to take up glutathione (GSH) when expressed in yeast cells or in Xenopus laevis oocytes, in GSH transport was analyzed using opt6 knockout mutant lines. The concentration of GSH in flowers or siliques was lower in opt6 mutants relative to wild-type plants, suggesting involvement of AtOPT6 in long-distance transport of GSH. The GSH concentration in phloem sap was similar between opt6 mutants and wild-type plants. These results, combined with earlier reports showing expression of AtOPT6 in the vascular bundle, especially in the cambial zone, suggest that AtOPT6 functions to transport GSH into cells surrounding the phloem in sink organs. The opt6 mutant plants showed delayed bolting, implying the importance of AtOPT6 for regulation of the transition from vegetative to reproductive growth. After cadmium (Cd) treatment, the concentration of the major phytochelatin PC2 was lower in flowers in the opt6 mutants and Cd was accumulated in roots of opt6 mutant plants compared with wild-type plants. These results suggest that AtOPT6 is likely to be involved in transporting GSH, PCs and Cd complexed with these thiols into sink organs.

Phenotypic and genetic characterization of rhizobia associated with alfalfa in the Hokkaido and Ishigaki regions of Japan.

Twenty five rhizobial isolates were obtained from root nodules of Medicago sativa inoculated with soil samples collected from the Sapporo region and Ishigaki Island in Japan. To study their diversity and characterize them in relation to the climatic conditions of their soils of origin, a polyphasic approach analyzing stress tolerance, symbiotic and genetic properties was used. Stress tolerance assays revealed marked variations in salinity, pH and temperature tolerance. Isolates originating from a sub-tropical climate in alkaline soil (Ishigaki Island) tolerated high temperature, salinity and pH levels. Moreover, isolates recovered from a temperate climate in acidic soil (Sapporo) were sensitive to high temperature and salinity, and tolerated acidic pH. Phylogenetic analysis of conserved 16S rRNA and recA genes, and symbiotic nodA and nifDK revealed 25 isolates to be closely related to Ensifer meliloti. Furthermore, the branch patterns of phylogenetic trees constructed from different genes revealed the existence of at least two E. meliloti types in the soils studied. These results may be relevant to programs directed towards improving crop productivity through biofertilization with locally adapted and genetically defined strains.

Two-dimensional analysis of spurious modes in aluminum nitride film resonators.

In this paper, a hybrid method, which combines the traditional concept of guided waves and the finite element method (FEM), is proposed to analyze the spurious modes of aluminum nitride (AIN) film with electrodes. First, the guided wave modes in the plated area are obtained by 1-D FEM. Second, a mode-match method is used to satisfy the boundary conditions. The vibration of the film resonator is a superposition of all of the guided modes. With respect to an A1N film resonator, which is a thickness-stretch mode resonator, we have identified three families of spurious modes: extension, thickness-stretch, and thickness-shear. The spectrum of spurious modes is calculated and the influence of the spurious modes is discussed.

Isolation and molecular characterization of a Spotted leaf 18 mutant by modified activation-tagging in rice.

A lesion mimic mutant that we designated Spotted leaf 18 (Spl18) was isolated from 13,000 activation-tagging lines of rice produced by our modified activation-tagging vector and further characterized. Spl18 was dominant and its phenotype was linked to the T-DNA insertion. An ORF was located about 500 bp downstream of the inserted T-DNA, and the deduced protein, designated OsAT1, showed sequence similarity to an acyltransferase whose expression is induced by hypersensitive reaction in tobacco. The transcriptional level of OsAT1 was very low in the WT leaf blade but high in Spl18 leaf blade. In wild-type rice, OsAT1 was transcribed mainly in the young panicle, in the panicle just after heading, and in the leaf sheath. In addition, transcription of the genes for PR protein was upregulated in Spl18, accumulation of phytoalexins (both momilactone A and sakuranetin) was increased, and resistance to blast disease was improved. We then combined OsAT1 genomic DNA downstream of the modified 35S promoter and re-transformed it into rice. Lesion mimic and blast resistance phenotypes were detected in the transgenic lines produced, clearly indicating that overexpression of OsAT1 caused the Spl18 phenotypes. In addition, plants overexpressing OsAT1 showed resistance to bacterial blight.