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

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

We aimed to efficiently enhance plant Hg(II) tolerance by the transgenic approach utilizing a bacterial mercury transporter MerC, an Arabidopsis mesophyll specific promoter pRBCS1A, and a vacuolar membrane targeting syntaxin AtVAM3/SYP22. We generated two independent homozygous Arabidopsis pRBCS1A-TCV lines expressing mT-Sapphire-MerC-AtVAM3 under the control of pRBCS1A. Quantitative RT-PCR showed that the transgene was expressed specifically in shoots of pRBCS1A-TCV lines. Confocal analyses further demonstrated the leaf mesophyll specific expression of mT-Sapphire-MerC-AtVAM3. Confocal observation of the protoplast derived from the F1 plants of the pRBCS1A-TCV line and the tonoplast marker line p35S-GFP-δTIP showed the tonoplast colocalization of mT-Sapphire-MerC-AtVAM3 and GFP-δTIP. These results clearly demonstrated that mT-Sapphire-MerC-AtVAM3 expression in Arabidopsis is spatially regulated as designed at the transcript and the membrane trafficking levels. We then examined the Hg(II) tolerance of the pRBCS1A-TCV lines as well as the p35S-driven MerC-AtVAM3 expressing line p35S-CV under the various Hg(II) stress conditions. Short-term (12 d) Hg(II) treatment indicated the enhanced Hg(II) tolerance of both pRBCS1A-TCV and p35S-CV lines. The longer (3 weeks) Hg(II) treatment highlighted the better shoot growth of the transgenic plants compared to the wild-type Col-0 and the pRBCS1A-TCV lines were more tolerant to Hg(II) stress than the p35S-CV line. These results suggest that mesophyll-specific expression of MerC-AtVAM3 is sufficient or even better to enhance the Arabidopsis Hg(II) tolerance. The Hg accumulation in roots and shoots did not differ between the wild-type Col-0 and the MerC-AtVAM3 expressing plants, suggesting that the boosted Hg(II) tolerance of the transgenic lines would be attributed to vacuolar Hg-sequestration by the tonoplast-localized MerC. Further perspectives of the MerC-based plant engineering are also discussed.

Related collections

Most cited references 33

Record: found
Abstract: found
Article: not found

Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana

Steven J Clough, Andrew Bent, Kazuhiro Oiwa (2000)

The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.

0 comments Cited 2141 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: found

Is Open Access

Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

Fu-Hui Wu, Shu-Chen Shen, Lan-Ying Lee … (2009)

Background Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments. Results In this report, we present a new procedure that we call the Tape-Arabidopsis Sandwich. This is a simple and fast mesophyll protoplast isolation method. Two kinds of tape (Time tape adhered to the upper epidermis and 3 M Magic tape to the lower epidermis) are used to make a "Tape-Arabidopsis Sandwich". The Time tape supports the top side of the leaf during manipulation, while tearing off the 3 M Magic tape allows easy removal of the lower epidermal layer and exposes mesophyll cells to cell wall digesting enzymes when the leaf is later incubated in an enzyme solution. The protoplasts released into solution are collected and washed for further use. For TEAMP, plasmids carrying a gene expression cassette for a fluorescent protein can be successfully delivered into protoplasts isolated from mature leaves grown under optimal conditions. Alternatively, these protoplasts may be used for bimolecular fluorescence complementation (BiFC) to investigate protein-protein interactions in vivo, or for Western blot analysis. A significant advantage of this protocol over the current method is that it allows the generation of protoplasts in less than 1 hr, and allows TEAMP transfection to be carried out within 2 hr. Conclusion The protoplasts generated by this new Tape-Arabidopsis Sandwich method are suitable for the same range of research applications as those that use the current method, but require less operator skill, equipment and time.

0 comments Cited 340 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Gene limiting cadmium accumulation in rice.

Daisei Ueno, Naoki Yamaji, Izumi Kono … (2010)

Intake of toxic cadmium (Cd) from rice caused Itai-itai disease in the past and it is still a threat for human health. Therefore, control of the accumulation of Cd from soil is an important food-safety issue, but the molecular mechanism for the control is unknown. Herein, we report a gene (OsHMA3) responsible for low Cd accumulation in rice that was isolated from a mapping population derived from a cross between a high and low Cd-accumulating cultivar. The gene encodes a transporter belonging to the P(1B)-type ATPase family, but shares low similarity with other members. Heterologous expression in yeast showed that the transporter from the low-Cd cultivar is functional, but the transporter from the high-Cd cultivar had lost its function, probably because of the single amino acid mutation. The transporter is mainly expressed in the tonoplast of root cells at a similar level in both the low and high Cd-accumulating cultivars. Overexpression of the functional gene from the low Cd-accumulating cultivar selectively decreased accumulation of Cd, but not other micronutrients in the grain. Our results indicated that OsHMA3 from the low Cd-accumulating cultivar limits translocation of Cd from the roots to the above-ground tissues by selectively sequestrating Cd into the root vacuoles.

0 comments Cited 234 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Contributors

Shimpei Uraguchi: URI : http://loop.frontiersin.org/people/99791/overview

Yuka Ohshiro: URI : http://loop.frontiersin.org/people/1922106/overview

Journal

Journal ID (nlm-ta): Front Plant Sci

Journal ID (iso-abbrev): Front Plant Sci

Journal ID (publisher-id): Front. Plant Sci.

Title: Frontiers in Plant Science

Publisher: Frontiers Media S.A.

ISSN (Electronic): 1664-462X

Publication date (Electronic): 12 August 2022

Publication date Collection: 2022

Volume: 13

Electronic Location Identifier: 986600

Affiliations

Department of Public Health, School of Pharmacy, Kitasato University , Tokyo, Japan

Author notes

Edited by: Tomoko Nozoye, Meiji Gakuin University, Japan

Reviewed by: Akira Yoshinari, Nagoya University, Japan; Jie Zhang, Foshan University, China

*Correspondence: Masako Kiyono kiyonom@ 123456pharm.kitasato-u.ac.jp

This article was submitted to Plant Membrane Traffic and Transport, a section of the journal Frontiers in Plant Science

Article

DOI: 10.3389/fpls.2022.986600

PMC ID: 9412105

PubMed ID: 36035696

SO-VID: 27d62cb5-cb2d-40bc-a4bd-cde2c1335510

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

History

Date received : 05 July 2022

Date accepted : 26 July 2022

Page count

Figures: 7, Tables: 0, Equations: 0, References: 34, Pages: 10, Words: 6325

Funding

Funded by: Japan Society for the Promotion of Science, doi 10.13039/501100001691;

Award ID: 15H02839

Award ID: 18H03401

Comments

Comment on this article

scite_

Smart Citations

Citing PublicationsSupportingMentioningContrasting

View Citations

See how this article has been cited at scite.ai

scite shows how a scientific paper has been cited by providing the context of the citation, a classification describing whether it supports, mentions, or contrasts the cited claim, and a label indicating in which section the citation was made.