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      Tape- Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

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          Abstract

          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.

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          Most cited references14

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          A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants.

          Genome sequencing has resulted in the identification of a large number of uncharacterized genes with unknown functions. It is widely recognized that determination of the intracellular localization of the encoded proteins may aid in identifying their functions. To facilitate these localization experiments, we have generated a series of fluorescent organelle markers based on well-established targeting sequences that can be used for co-localization studies. In particular, this organelle marker set contains indicators for the endoplasmic reticulum, the Golgi apparatus, the tonoplast, peroxisomes, mitochondria, plastids and the plasma membrane. All markers were generated with four different fluorescent proteins (FP) (green, cyan, yellow or red FPs) in two different binary plasmids for kanamycin or glufosinate selection, respectively, to allow for flexible combinations. The labeled organelles displayed characteristic morphologies consistent with previous descriptions that could be used for their positive identification. Determination of the intracellular distribution of three previously uncharacterized proteins demonstrated the usefulness of the markers in testing predicted subcellular localizations. This organelle marker set should be a valuable resource for the plant community for such co-localization studies. In addition, the Arabidopsis organelle marker lines can also be employed in plant cell biology teaching labs to demonstrate the distribution and dynamics of these organelles.
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            Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.

            Networks of protein interactions coordinate cellular functions. We describe a bimolecular fluorescence complementation (BiFC) assay for determination of the locations of protein interactions in living cells. This approach is based on complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are brought together by interactions between proteins fused to each fragment. BiFC analysis was used to investigate interactions among bZIP and Rel family transcription factors. Regions outside the bZIP domains determined the locations of bZIP protein interactions. The subcellular sites of protein interactions were regulated by signaling. Cross-family interactions between bZIP and Rel proteins affected their subcellular localization and modulated transcription activation. These results attest to the general applicability of the BiFC assay for studies of protein interactions.
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              Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.

              Plant membrane compartments and trafficking pathways are highly complex, and are often distinct from those of animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context, and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all of these cases a central role is played by the endosomal membrane system, which, however, is the most divergent and ill-defined aspect of plant cell compartmentation. We have designed a new vector series, and have generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns, and stable, non-toxic expression. We demonstrate the power of this multicolor 'Wave' marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live-imaging and immunoelectron microscopy. Among other findings, our systematic co-localization analysis revealed that a class of plant Rab1-homologs has a much more extended localization than was previously assumed, and also localizes to trans-Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available, and will promote rapid progress in diverse areas of plant cell biology.
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                Author and article information

                Journal
                Plant Methods
                Plant Methods
                BioMed Central
                1746-4811
                2009
                24 November 2009
                : 5
                : 16
                Affiliations
                [1 ]Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan
                [2 ]Scientific Instrument Center, Academia Sinica, Taipei, 115, Taiwan
                [3 ]Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA
                Article
                1746-4811-5-16
                10.1186/1746-4811-5-16
                2794253
                19930690
                4550ef3a-01f6-4222-8be7-84bac9639f9d
                Copyright ©2009 Wu et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 7 October 2009
                : 24 November 2009
                Categories
                Methodology

                Plant science & Botany
                Plant science & Botany

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