Plant Molecular Biology Reporter 17: 371–383, 1999. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. Protocols Isolation of RNA and Protein from Guard Cells of Nicotiana glauca LAWRENCE B. SMART 1,∗ , NICOLE M. NALL 1 and ALAN B. BENNETT 2 1 Faculty of Environmental and Forest Biology, State University of NewYork College of Environmental Science and Forestry, 6 Illick Hall, Syracuse, NY 13210, USA; 2 Department of Vegetable Crops, Mann Laboratory, University of California-Davis, Davis, CA 95616, USA Abstract. Stomatal guard cells are critical for maintenance of plant homeostasis and represent an interesting cell type for studies of leaf cell differentiation and patterning. Here we describe techniques for the isolation of guard cell RNA and protein from blended epidermal peels of Nicotiana glauca. The RNA isolation procedure is a modification of the hot borate method, which is particularly well-suited for recalcitrant tissues. Protein was extracted by disrupt- ing guard cell-enriched epidermis with a French  press. This system offers the following advantages: relatively high yield, low or no contamination by other cell types, fresh tissue as a source of RNA and protein rather than protoplasts, and a plant species that is readily transformable. These techniques will allow for cloning and analysis of genes expressed in guard cells, application of traditional biochemical techniques to guard cell proteins, as well as characterization of genetic manipulation of guard cell function in transgenic plants. Key words: epidermal peel, extraction, gene expression, stomata, tree tobacco Abbreviations: DEPC, diethylpyrocarbonate; DTT, dithiothreitol; EGTA, ethylene glycol- bis(β-aminoethylether)-N,N,N ′ ,N ′ -tetraacetic acid; PMSF, phenylmethylsulfonyl fluoride. Introduction Guard cells are highly differentiated cells in the epidermis that perform the specialized function of regulating stomatal aperture to control gas exchange between the leaf and the atmosphere (Kearns and Assmann, 1993). Guard cells arise from the L1 layer of the meristem and differentiate from guard mother cells in the protoderm (Larkin et al., 1997). The differentiation of guard cells includes the formation of thickened cell walls with radially ar- ∗ Author for correspondence: e-mail: lbsmart@syr.edu; fax: (315) 470-6934; ph: (315) 470-6737