Plant Molecular Biology 42: 857–869, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Isolation of genes predominantly expressed in guard cells and epidermal
cells of Nicotiana glauca
Lawrence B. Smart
Faculty of Environmental and Forest Biology, State University of New York College of Environmental Science
and Forestry, 6 Illick Hall, Syracuse, NY 13210, USA (
author for correspondence; e-mail: firstname.lastname@example.org; fax:
+1 315 470 6934);
Department of Vegetable Crops, Mann Laboratory, University of California-Davis, Davis,
CA 95616, USA
Received 27 August 1999; accepted in revised form 8 February 2000
Key words: epidermis, gene expression, glycine-rich protein, lipid transfer protein, proline-rich protein, stomata
Guard cells are specialized and metabolically active cells which arise during the differentiation of the epidermis.
Using Nicotiana glauca epidermal peels as a source of puriﬁed guard cells, we have constructed a cDNA library
from guard cell RNA. In order to isolate genes that are predominantly expressed in guard cells, we performed
a differential screen of this library, comparing the hybridization of a radiolabeled cDNA probe synthesized from
guard cell RNA to that from a mesophyll cell cDNA probe. Sixteen clones were isolated based on their greater
level of hybridization with the guard cell probe. Of these, eight had high homology to lipid transfer protein (LTP),
two were similar to glycine-rich protein (GRP), and one displayed high homology to proline-rich proteins from
Arabidopsis thaliana (AtPRP2, AtPRP4) and from potato guard cells (GPP). Northern analysis conﬁrmed that
one or more NgLTP genes, NgGRP1,andNgGPP1 are all differentially expressed, with highest levels in guard
cells, and low or undetectable levels in mesophyll cells and in roots. In addition, all are induced to some degree
in drought-stressed guard cells. NgLTP and NgGRP1 expression was localized by in situ hybridization to the
guard cells and pavement cells in the epidermis. NgGRP1 expression was also detected in cells of the vasculature.
Genomic Southern analysis indicated that LTP is encoded by a family of highly similar genes in N. glauca.This
work has identiﬁed members of a subset of epidermis- and guard cell-predominant genes, whose protein products
are likely to contribute to the unique properties acquired by guard cells and pavement cells during differentiation.
Stomata serve the unique function in plants of reg-
ulating the exchange of carbon dioxide and water
vapor across the leaf-atmosphere interface, because
the other epidermal cells arecoveredby a waxy cuticle
that is largely impermeable to those gases. Stomatal
conductance is regulated by the guard cells, which
swell to effect opening of the pore and shrink dur-
ing closure by a chemiosmotic mechanism (Tallman,
1992). The regulation of those functions by environ-
mental, physiological, and hormonal signals has been
The nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the ac-
cession numbers AF151214, AF151215, AF151216 and AF151217.
well characterized, but there is still much that we do
not know about how those signals are perceived and
transduced (Assmann, 1993).
Guard cells also represent a unique cell type within
the plant and differentiatefromthe L1 protodermlayer
together with subsidiary cells, pavement cells (ground
epidermal cells), and trichomes (Larkin et al., 1997).
The two attached guard cells in a stomatal complex
arise from the symmetric division of a guard mother
cell, which then differentiate to acquire their unique
structural and biochemical features. These include
characteristics of the cell wall that are important for
stomatal function, such as the radial arrangement of
cellulose microﬁbrils, differential thickening of the
inner and outer walls, and localized cutinization to