Selection of Agar Reagents for Medium Solidification Is a Critical Factor for Metal(loid) Sensitivity and Ionomic Profiles of Arabidopsis thaliana

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 typedependent 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. Agartype 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.

Mesophyll specific expression of a bacterial mercury transporter-based vacuolar sequestration machinery sufficiently enhances mercury tolerance of Arabidopsis

“…This point is significant for further molecular breeding towards phytoremediation which primarily requires higher metal accumulation in shoots. Vacuolar sequestration of toxic metals in the root cells can limit root-to-shoot translocation of toxic elements (Ueno et al, 2010;Miyadate et al, 2011;Uraguchi et al, 2018Uraguchi et al, , 2020, which is not desirable for efficient phytoremediation. Although the ubiquitous expression of MerC-AtVAM3 did not inhibit shoot Hg accumulation under Hg(II) treatment (Figure 7), Hg was more retained in the roots of the p35S-CV line when exposed to methylmercury (Sone et al, 2017).…”

“…It is very likely that the tonoplast-targeted expression of MerC in the mesophyll cells facilitates vacuolar sequestration of Hg and reduces its phytotoxicity (Figure 1A). The importance of the vacuolar sequestration in Hg-detoxification is demonstrated for the Arabidopsis endogenous ABC transporters AtABCC1 and AtABCC2: the loss-of-function mutant abcc1/abcc2 exhibits the hypersensitivity to mercurials (Park et al, 2012;Uraguchi et al, 2020Uraguchi et al, , 2021. In the present study, we successfully introduced the bacterial mercury transporter to the tonoplast of the mesophyll cells and this vacuolar sequestration machinery was suggested to protect cytosol and other organelles from Hg-toxicity.…”

Mesophyll specific expression of a bacterial mercury transporter-based vacuolar sequestration machinery sufficiently enhances mercury tolerance of Arabidopsis

“…A4550-500G) was used for medium solidification. We previously demonstrated that this agar reagent is better suited for evaluating Arabidopsis responses to Hg(II) stress compared to the other agar reagents (Uraguchi et al, 2020). The concentration of agar reagents used for medium solidification was 1% (w/v) for the vertical plate assay and 0.6% (w/v) for the horizontal plate assay.…”

“…This suggests that MerC on the tonoplast would be able to sequester Hg from cytosol to vacuoles through active transport. The importance of the vacuolar sequestration in Hgdetoxification is demonstrated for the Arabidopsis endogenous ABC transporters AtABCC1 and AtABCC2: the loss-offunction mutant abcc1/abcc2 exhibits the hypersensitivity to mercurials (Park et al, 2012;Uraguchi et al, 2020Uraguchi et al, , 2022. In the present study, we successfully introduced the bacterial mercury transporter to the tonoplast of the mesophyll cells and this vacuolar sequestration machinery was suggested to protect cytosol and other organelles from Hg-toxicity, although we do not have direct evidence for MerC-mediated enhanced Hg sequestration in the vacuoles, due to technical difficulties.…”

“…This point is significant for further molecular breeding toward phytoremediation which primarily requires higher metal accumulation in shoots. Vacuolar sequestration of toxic metals in the root cells can limit root-to-shoot translocation of toxic elements (Ueno et al, 2010;Miyadate et al, 2011;Uraguchi et al, 2018Uraguchi et al, , 2020, which is not desirable for efficient phytoremediation. Although the ubiquitous expression of MerC-AtVAM3 did not inhibit shoot Hg accumulation under Hg(II) treatment (Figure 7), Hg was more retained in the roots of the p35S-CV line when exposed to methylmercury (Sone et al, 2017).…”