ABSTRACT
For researchers in the plant metal field, the agar reagent used for the solid plate medium is a problematic factor because application of different agar types and even a different lot of the same agar type can mask the plant metal-related phenotypes and impair the reproducibility. In this study, we systematically assessed effects of different agar reagents on metal(loid) sensitivity and element accumulation of the Arabidopsis metal sensitive mutants. Three established mutants (cad1-3, cad1-6, and abcc1/2), and three different types of purified agar reagents (Type A, Type E, and Nacalai) with two independent batches for each reagent were subjected to the analyses. First, we found that element concentrations in the agar reagents largely varied among the agar types. Then the effects of agar reagents on the mutant metal(loid)-sensitivity were examined under As(III), Hg(II), Cd(II), and excess Zn(II) conditions. A significant variation of the mutant metal(loid)-sensitivity was observed among the different agar plates but the variation depended on the combination of metal(loid) stress and agar reagents. Briefly, the type-dependent variation was more evident under As(III) and Hg(II) than Cd(II) or excess Zn(II) conditions. A lot-dependent variation was also observed for Type A and Type E but not for Nacalai: hypersensitive phenotypes of cad1-3, cad1-6, and abcc1/2 under As(III) or Hg(II) treatments were diminished when different batches of the Type A or Type E agar types were used. We also found a significant variation of As and Hg accumulation in the wild-type and cad1-3. Plant As and Hg concentrations were remarkably higher and the difference between the genotypes was more evident when grown with Type A agar plates. We finally analyzed ionomic profiles in the plants exposed to As(III) stress. Agar-type specific ionomic changes in cad1-3 were more observed with the Type A plates than with the Nacalai plates. The presented results overall suggest that suitability of agar reagents for metal(loid)-related phenotyping depends on the experimental design, and an inappropriate selection of agar reagents can mask even very clear phenotypes of the established mutant like cad1-3. We also discuss perspectives on the agar problem in the plant metal study.
ABSTRACT
For mercury phytoextraction, we previously demonstrated in Arabidopsis thaliana that a constitutive and ubiquitous promoter-driven expression of a bacterial mercury transporter MerC fused with SYP121, a plant SNARE for plasma membrane protein trafficking increases plant mercury accumulation. To advance regulation of ectopic expression of the bacterial transporter in the plant system, the present study examined whether merC-SYP121 expression driven by a root epidermis specific promoter (pEpi) is sufficient to enhance mercury accumulation in plant tissues. We generated five independent transgenic Arabidopsis plant lines (hereafter pEpi lines) expressing a transgene encoding MerC-SYP121 N-terminally tagged with a fluorescent protein mTRQ2 under the control of pEpi, a root epidermal promoter. Confocal microscopy analysis of the pEpi lines showed that mTRQ2-MerC-SYP121 was preferentially expressed in lateral root cap in the root meristematic zone and epidermal cells in the elongation zone of the roots. Mercury accumulation in shoots of the pEpi lines exposed to inorganic mercury was overall higher than the wild-type and comparable to the over-expressing line. The results suggest that cell-type specific expression of the bacterial transporter MerC in plant roots sufficiently enhances mercury accumulation in shoots, which could be a useful phenotype for improving efficiency of mercury phytoremediation.
