Abstract
Many physiological processes are limited to specific tissues or even specific cell types. Analysing entire plants or organs results in averaged data of all cell types contained in the sample; thus, specific metabolic functions cannot be assigned to individual cell types. A higher spatial resolution is required. By microdissecting plant organs, homogeneous material can be obtained. If a suitable amount of material is collected, standard analytical methods can be applied to elucidate cell type-specific processes. The collection of sufficient quantities of homogeneous material can be done by means of mechanical microdissection. This technique is a low-cost alternative to laser-coupled microdissection techniques. Here we describe a protocol for chisel-assisted mechanical microdissection of embedded plant material and demonstrate that the collected material is suitable to obtain nucleic acids and proteins.
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Abbreviations
- LCM:
-
laser capture microdissection
- m/z:
-
mass/charge
- PPMD:
-
Piezo Power Microdissection
- Q-TOF:
-
quadrupole-time-of-flight
- RT-PCR:
-
reverse transcription-polymerase chain reaction
- RuBisCo:
-
ribulose-1,5-bisphosphate carboxylase
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
References
Arlt K, Brandt S, and Kehr J (2001) Amino acid analysis in five pooled single plant cell samples using capillary electrophoresis coupled to laser-induced fluorescence detection. J Chomatogr A 926: 319–325.
Asano T, Masumura T, Kusano H, Kikuchi S, Kurita A, Shimada H, and Kadowaki KI (2002) Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem. Plant J 32: 401–408.
Brandt S, Kehr J, Walz C, Imlau A, Willmitzer L, and Fisahn J (1999) A rapid method for detection of plant gene transcripts from single epidermal, mesophyll and companion cells of intact leaves. Plant J 20: 245–250.
Brandt S, Kloska S, Altmann T, and Kehr J (2002) Using array hybridization to monitor gene expression at the single cell level. J Exp Bot 53, 2315–2323.
Emmert-Buck M, Bonner R, Smith P, Chuaqui R, Zhuang Z, Goldstein S, Weiss R, and Liotta L (1996) Laser capture microdissection. Science 274: 998–1001.
Fricke W, Leigh R, and Tomos D (1994) Concentrations of inorganic and organic solutes in extracts from individual epidermal, mesophyll and bundle-sheath cells of barley leaves. Planta 192: 310–316.
Jones M, Outlaw W, and Lowry O (1977) Enzymic assay of 10−7 to 10−14 moles of sucrose in plant tissue. Plant Physiol 60: 379–383.
Kehr J, Wagner C, Willmitzer L, and Fisahn J (1999) Effect of modified carbon allocation on turgor, osmolality, sugar and potassium content, and membrane potential in the epidermis of transgenic potato (Solanum tuberosum L.) plants. J Exp Bot 50: 565–571.
Kehr J (2001) High resolution spatial analysis of plant systems. Curr Opin Plant Biol 4: 197–201.
Kehr J (2003) Single cell technology. Curr Opin Plant Biol 6: 1–5.
Kerk NM, Ceserani T, Tausta SL, Sussex IM, and Nelson TM (2003) Laser capture microdissection of cells from plant tissues. Plant Physiol 132: 27–35.
Lochmann H, Bazzanella A, and Bächmann K (1998) Analysis of solutes and metabolites in single plant cell vacuoles by capillary electrophoresis. J Chromatogr A 817: 337–343.
Nakazono M, Qiu F, Borsuk LA, and Schnable PS (2003) Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell 15: 583–596.
Outlaw W and Fisher D (1975) Compartimentation inVicia faba leaves. I. Kinetics of14C in the tissues following pulse labelling. Plant Physiol 55: 699–703.
Outlaw W and Lowry O (1977) Organic acid and potassium acculmulation in guard cells during stomatal opening. Proc Natl Acad Sci USA 74: 4434–4438.
Outlaw W and Lowry O (1979) Measurement of 10−7 to 10−12 mol of potassium by stimulation of pyruvate kinase. Anal Biochem 92: 370–379.
Outlaw W (1980) A descriptive evaluation of quantitative histochemical methods based on pyridine nucleotides. Ann Rev Plant Physiol 31: 299–311.
Rekhter MD and Chen J (2001) Molecular analysis of complex tissues is facilitated by laser capture microdissection. Cell Biochem Biophys 35: 103–113.
Shevchenko A, Jensen O, Podtelejnikov A, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, and Mann M (1996) Linking genome and proteome by mass spectrometry-large scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci USA 93: 14440–14445.
Tomos D, Hinde P, Richardson P, Pritchard J, and Fricke W (1994) Microsampling and measurements of solutes in single cells. In: Harris N and Oparka K (eds) Plant Cell Biology-A practical approach, pp 297–314, Oxford University Press Oxford.
Tomos D and Sharrock R (2001) Cell sampling and analysis (SiCSA): metabolites measured at single cell resolution. J Exp Bot 52: 623–630.
Wittliff J, Kunitake S, Chu S, and Travis J (2000) Applications of laser capture microdissection in genomics and proteomics. J Clin Ligand Assay 23: 66–73.
Ziegler B, Lamping C, Thoma S, and Thomas C (1992) Single cell cDNA- PCR: Removal of contaminating genomic DNA from total RNA using immobilized DNAse I. BioTechniques 13: 726–729.
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Brandt, S., Walz, C., Schad, M. et al. A simple, chisel-assisted mechanical microdissection system for harvesting homogenous plant tissue suitable for the analysis of nucleic acids and proteins. Plant Mol Biol Rep 21, 417–427 (2003). https://doi.org/10.1007/BF02772591
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DOI: https://doi.org/10.1007/BF02772591