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Rapid and simple isolation of vascular, epidermal and mesophyll cells from plant leaf tissue

Nature Protocols volume 11, pages 1388–1395 (2016)Cite this article

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Abstract

To understand physiological phenomena at the tissue level, elucidation of tissue-specific molecular functions in vivo is required. As an example of the current state of affairs, many genes in plants have been reported to have discordant levels of expression between bulk tissues and the specific tissues in which the respective gene product is principally functional. The principal challenge in deciphering such tissue-specific functions lies in separating tissues with high spatiotemporal resolution to evaluate accurate gene expression profiles. Here, we provide a simple and rapid tissue isolation protocol to isolate all three major leaf tissues (mesophyll, vasculature and epidermis) from Arabidopsis within 30 min with high purity. On the basis of the different cell-to-cell connectivities of tissues, the mesophyll isolation is achieved by making protoplasts, and the vasculature and epidermis isolation is achieved through sonication and enzymatic digestion of leaves. We have successfully tested the protocol on several other plant species, including crop plants such as soybean, tomato and wheat. Furthermore, isolated tissues can be used not only for tissue-specific transcriptome assays but also potentially for tissue-specific proteome and methylome assays.

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Figure 1: Workflow and processing time of mesophyll, vasculature and epidermis isolation.
Figure 2: Preparation of cotyledons for tissue isolation.
Figure 3: Mesophyll protoplast isolation.
Figure 4: Vasculature and epidermis isolation.

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Acknowledgements

We thank T. Koto for technical assistance and J.A. Hejna for English proofreading. This work was supported by JST PRESTO grant 888067 (to M.E.), JSPS KAKENHI grants 16H01240, 25650097 and 15H05958 (to M.E.), grants from the Nakatani Foundation (to M.E.) and the Mitsubishi Foundation (to M.E.), and Grants-in-Aid for Scientific Research on Priority Areas 19060012 and 19060016 (to T.A.).

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Authors and Affiliations

  1. Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan

    Motomu Endo, Hanako Shimizu & Takashi Araki

  2. Japan Science and Technology Agency, PRESTO, Saitama, Japan

    Motomu Endo

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  1. Motomu Endo
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  2. Hanako Shimizu
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  3. Takashi Araki
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Contributions

M.E. and H.S. conceived and designed the protocol and experimental procedures. M.E. wrote the manuscript. M.E. and T.A. supervised the project.

Corresponding author

Correspondence to Motomu Endo.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Isolated mesophyll, vasculature, and epidermis from crop plants.

Isolated mesophyll (a, d, g), vasculature (b, e, h), and epidermis (c, f, i) from a cotyledon or primary leaf of soybean (a-c), tomato (d-f) and wheat (g-i). Bars = 250 µm.

Supplementary Figure 2 RNA purification and cDNA amplification from other crops.

Semi-quantitative RT-PCR detection for beta-tubulin genes in leaf tissue of Arabidopsis, tomato, soybean, and wheat. Non-RT controls were run to confirm that there was no false positive amplification from genomic DNA. W; whole leaf, M; mesophyll, V; vasculature, E; epidermis.

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Supplementary Figures 1 and 2 (PDF 379 kb)

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Endo, M., Shimizu, H. & Araki, T. Rapid and simple isolation of vascular, epidermal and mesophyll cells from plant leaf tissue. Nat Protoc 11, 1388–1395 (2016). https://doi.org/10.1038/nprot.2016.083

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