Phytochelatin-mediated metal detoxification pathway is crucial for an organomercurial phenylmercury tolerance in Arabidopsis.

KEY MESSAGE: An organomercurial phenylmercury activates AtPCS1, an enzyme known for detoxification of inorganic metal(loid) ions in Arabidopsis and the induced metal-chelating peptides phytochelatins are essential for detoxification of phenylmercury. Small thiol-rich peptides phytochelatins (PCs) and their synthases (PCSs) are crucial for plants to mitigate the stress derived from various metal(loid) ions in their inorganic form including inorganic mercury [Hg(II)]. However, the possible roles of the PC/PCS system in organic mercury detoxification in plants remain elusive. We found that an organomercury phenylmercury (PheHg) induced PC synthesis in Arabidopsis thaliana plants as Hg(II), whereas methylmercury did not. The analyses of AtPCS1 mutant plants and in vitro assays using the AtPCS1-recombinant protein demonstrated that AtPCS1, the major PCS in A. thaliana, was responsible for the PheHg-responsive PC synthesis. AtPCS1 mutants cad1-3 and cad1-6, and the double mutant of PC-metal(loid) complex transporters AtABCC1 and AtABCC2 showed enhanced sensitivity to PheHg as well as to Hg(II). The hypersensitivity of cad1-3 to PheHg stress was complemented by the own-promoter-driven expression of AtPCS1-GFP. The confocal microscopy of the complementation lines showed that the AtPCS1-GFP was preferentially expressed in epidermal cells of the mature and elongation zones, and the outer-most layer of the lateral root cap cells in the meristematic zone. Moreover, in vitro PC-metal binding assay demonstrated that binding affinity between PC and PheHg was comparable to Hg(II). However, plant ionomic profiles, as well as root morphology under PheHg and Hg(II) stress, were divergent. These results suggest that PheHg phytotoxicity is different from Hg(II), but AtPCS1-mediated PC synthesis, complex formation, and vacuolar sequestration by AtABCC1 and AtABCC2 are similarly functional for both PheHg and Hg(II) detoxification in root surficial cell types.

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.

Potentiality of Sustainable Maize Production under Rainfed Conditions in the Tropics by Triggering Agro-Physio-Biochemical Traits Ascertained from a Greenhouse.

A major portion of maize is produced under rainfed conditions in the tropics with relatively poor yield because of the unpredictable and irregular distribution of seasonal rainfall, as well as a decline in pre-rainy season rainfall due to climate change, so identification of sustainable production options is utmost needed. Thus, the present studies were conducted in a greenhouse (GH) to ascertain the water stress-tolerant traits of maize and at the field level in the tropical environment of Thailand to see the stimulating possibility of the ascertained traits in a locally popular cultivar using ethephon. Depending on tolerance level, three maize genotypes (Suwan 2301 > Suwan 4452 > S 7328) were tested under different water conditions-well-watered, short-term, and long-term water stress-in the GH. At the field level, the locally popular maize cultivar Suwan 5819 was examined with six ethephon levels (doses in g a.i. ha-1 of ethephon, i.e., T1, 281 at V6 stage; T2, 281 at V6 + 281 at V10 stage; T3, 281 at V10 stage; T4, 562 at V6 stage; T5, 562 at V6 + 562 at V10 stage; T6, 562 at V10 stage) against no ethephon application (T0) under rainfed conditions. Maize suffered from the scarcity of sufficient rainfall during 26-39 days after planting (DAP) and 43-63 DAP in the field. The yield index (YI) was identified from biplot analysis as one of the suitable standards for drought tolerance checks for maize at GH as well as at field level in the tropics. The YI value of observed agro-physio-biochemical traits of maize in GH showed that relative water content (RWC, 1.23), stem base diameter (SBD, 1.21), total soluble sugar (TSS, 1.15), proline (Pr, 1.13), aboveground plant biomass (APB, 1.13), root weight (RW, 1.13), relative growth rate (RGR, 1.15), specific leaf weight (SLW, 1.12), and net assimilation rate (NAR, 1.08) were the most desirable. Efforts were made to stimulate these traits under water stress at the field level. Ethephon application as T1 helped to gain higher kernel yield (KY) (5.26 t ha-1) with the support of higher RWC (90.38%), proline (24.79 µmol g-1 FW), TSS (1629 mg g-1 FW), SBD (24.49 mm), APB (271.34 g plant-1), SLW (51.71 g m-2), RGR (25.26 mg plant-1 day-1), and NAR (0.91 mg cm-2 day-1) compared to others, especially no ethephon application. Furthermore, the attributes SLW, SBD, Pr, heat utilization efficiency (HUE), 100-kernel weight, TSS, electrolyte leakage, and lodging percentage showed a substantial direct effect and significant correlation with KY. Aside from higher KY, ethephon application as T1 tactics resulted in higher values of energy efficiency (1.66), HUE (2.99 kg ha-1 °C days-1), gross margin (682.02 USD ha-1), MBCR (3.32), and C absorption (6.19 t C ha-1), indicating that this practice may be a good option for maize sustainable production under rainfed conditions.

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.

Foliar-applied glutathione activates zinc transport from roots to shoots in oilseed rape.

Glutathione is a tripeptide involved in diverse aspects of plant metabolism. We investigated how the reduced form of glutathione, GSH, applied site-specifically to plants, affects zinc (Zn) distribution and behavior in oilseed rape plants (Brassica napus) cultured hydroponically. Foliar-applied GSH significantly increased the Zn content in shoots and the root-to-shoot Zn translocation ratio; furthermore, this treatment raised the Zn concentration in the cytosol of root cells and substantially enhanced Zn xylem loading. Notably, microarray analysis revealed that the gene encoding pectin methylesterase was upregulated in roots following foliar GSH treatment. We conclude that certain physiological signals triggered in response to foliar-applied GSH were transported via sieve tubes and functioned in root cells, which, in turn, increased Zn availability in roots by releasing Zn from their cell wall. Consequently, root-to-shoot translocation of Zn was activated and Zn accumulation in the shoot was markedly increased.

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.