Abstract
Laser microdissection (LMD) is a powerful tool to isolate pure cell populations from heterogeneous tissues. This system has been successfully used for animal research; however, the reports of its application to plant tissues remain limited. One of the challenges of LMD for plant material is the tissue preparation. Although cryosectioning is commonly used for animal tissues, this is not a desirable method for fragile plant material with large central vacuoles. While paraffin preparation provides high histological quality and stability, the procedure is highly time consuming and may result in degradation of molecules of interest. In addition, conventional fixation and paraffin preparation methods do not preserve the structural integrity of very delicate plant tissues such as mature Arabidopsis thaliana leaves. Here, we used the rapid microwave paraffin preparation method with no fixative for preparation of Arabidopsis leaf tissue for LMD. This method resulted in Arabidopsis leaf sections with excellent preservation of leaf internal structure as evidenced by well-defined vascular bundles, phloem, and chloroplasts, and expanded and rounded epidermal cells. RNA extracted from leaf epidermal and mesophyll cells was of sufficient yield and specificity to use in downstream applications such as microarray analysis of the amplified mRNA. We employed the mesophyll cell-specific molecular marker, chloroplastic carbonic anhydrase, and developed an epidermal cell-specific marker, the very-long-chain fatty acid-condensing enzyme, CUT1, to assess specificity of harvested Arabidopsis leaf cell types by reverse transcription polymerase chain reaction. The described method is also likely to be superior for the preparation of other fragile botanical tissue for LMD and downstream applications.
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Abbreviations
- DEPC:
-
Diethylpyrocarbonate
- DMSO:
-
Dimethylsulfoxide
- RT-PCR:
-
Reverse transcription polymerase chain reaction
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Acknowledgements
We thank Drs. Denise Schichnes and Steven Ruzin of the Biological Imaging Facility, UC Berkeley, for technical advice, useful discussions, and review of this manuscript. We also thank Drs. Keni Jiang, Lewis Feldman, and Sharon Marr (Department of Plant and Microbial Biology, UC Berkeley) for their valuable input and discussions. Finally, we thank Andy Lee (Leica Microsystems, Inc.) and Dr. Thea Tlsty (UCSF Comprehensive Cancer Center, San Francisco, CA, USA) for the use of Leica AS Laser Microdissection systems. This work was funded in part by NSF Arabidopsis 2010 Award MCB-0420267.
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Inada, N., Wildermuth, M.C. Novel tissue preparation method and cell-specific marker for laser microdissection of Arabidopsis mature leaf. Planta 221, 9–16 (2005). https://doi.org/10.1007/s00425-004-1427-y
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DOI: https://doi.org/10.1007/s00425-004-1427-y