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Altering stomatal function by a guard cell-targeted transgenic approach with the aim of increased stress tolerance and crop yield requires knowledge of the natural fluctuations of stomatal gene expression under stress conditions. We developed a fast method for the isolation of RNA from epidermal fragments of potato leaves (Solanum tuberosum L. cv. Désirée), demonstrated that this RNA preparation is highly enriched in guard cell transcripts and used this method to investigate the response of gene expression in guard cells to mild drought stress. Drought was applied in planta by withholding water over a period of 2–4 days. In the following work responses observed under these conditions are called ‘long-term’ in contrast to immediate (short-term) stomatal opening and closing responses to environmental stress. We observed both gene-specific increases and decreases of steady-state transcript levels. In particular, the mRNA levels of sucrose synthase and sucrose-phosphate synthase were elevated 5.5-fold and 1.4-fold, respectively. In contrast, expression of an inwardly rectifying K+ channel from guard cells (kst1) and of a plasma membrane H+-ATPase (pha2) was reduced to 26% and 36%, respectively, of the expression in watered controls. In addition, expression of vacuolar invertase, UDP-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase (large subunit), cytosolic glyceraldehyde-3-phosphate dehydrogenase, a sucrose/H+ co-transporter, and a novel isoform of phosphoenolpyruvate carboxylase were also reduced. Other genes exhibited unaltered expression. Compared with the response in whole leaves, the transcript levels of phosphoenolpyruvate carboxylase, vacuolar invertase, and cytosolic glyceraldehyde-3-phosphate dehydrogenase were regulated guard cell specifically. Most importantly, changes in steady-state transcript levels were complete before the onset of a decrease in leaf water potential, when drought-induced stomatal closure was already obvious. These data support the hypothesis that a systemic drought-stress signal acts not only on short-term stomatal movements but also on long-term gene expression in guard cells. Such long-term changes in gene expression might contribute to the fine-tuning of guard cell responses to environmental stimuli.