Nitrate Acts as a Signal to Induce Organic Acid Metabolism and Repress Starch Metabolism in Tobacco.

Nia30(145) transformants with very low nitrate reductase activity provide an in vivo screen to identify processes that are regulated by nitrate. Nia30(145) resembles nitrate-limited wild-type plants with respect to growth rate and protein and amino acid content but accumulates large amounts of nitrate when it is grown on high nitrate. The transcripts for nitrate reductase (NR), nitrite reductase, cytosolic glutamine synthetase, and glutamate synthase increased; NR and nitrite reductase activity increased in leaves and roots; and glutamine synthetase activity increased in roots. The transcripts for phosphoenolpyruvate carboxylase, cytosolic pyruvate kinase, citrate synthase, and NADP-isocitrate dehydrogenase increased; phosphoenolpyruvate carboxylase activity increased; and malate, citrate, isocitrate, and [alpha]-oxoglutarate accumulated in leaves and roots. There was a decrease of the ADP-glucose pyrophosphorylase transcript and activity, and starch decreased in the leaves and roots. After adding 12 mM nitrate to nitrate-limited Nia30(145), the transcripts for NR and phosphoenolpyruvate carboxylase increased, and the transcripts for ADP-glucose pyrophosphorylase decreased within 2 and 4 hr, respectively. Starch was remobilized at almost the same rate as in wild-type plants, even though growth was not stimulated in Nia30(145). It is proposed that nitrate acts as a signal to initiate coordinated changes in carbon and nitrogen metabolism.

Antisense Repression of Both ADP-Glucose Pyrophosphorylase and Triose Phosphate Translocator Modifies Carbohydrate Partitioning in Potato Leaves.

Previous experiments have shown that carbohydrate partitioning in leaves of potato (Solanum tuberosum L.) plants can be modified by antisense repression of the triose phosphate translocator (TPT), favoring starch accumulation during the light period, or by leaf-specific antisense repression of ADP-glucose pyrophosphorylase (AGPase), reducing leaf starch content. These experiments showed that starch and sucrose synthesis can partially replace each other. To determine how leaf metabolism acclimates to an inhibition of both pathways, transgenic potato (S. tuberosum L. cv Desiree) plants, with a 30% reduction of the TPT achieved by antisense repression, were transformed with an antisense cDNA of the small subunit of AGPase, driven by the leaf-specific ST-LS1 promoter. These double-transformed plants were analyzed with respect to their carbohydrate metabolism, and starch accumulation was reduced in all lines of these plants. In one line with a 50% reduction of AGPase activity, the rate of CO2 assimilation was unaltered. In these plants the stromal level of triose phosphate was increased, enabling a high rate of triose phosphate export in spite of the reduction of the TPT protein by antisense repression. In a second line with a 95% reduction of AGPase activity, the amount of chlorophyll was significantly reduced as a consequence of the lowered triose phosphate utilization capacity.

Association of plant K+(in) channels is mediated by conserved C-termini and does not affect subunit assembly.

Inward rectifying potassium (K+(in)) channels play an important role in turgor regulation and ion uptake in higher plants. Here, we report a previously unrecognized feature of these proteins: K+(in) channel C-terminal polypeptides mediate channel protein interactions. Using a C-terminal fragment of potato guard cell K+(in) channel KST1 in a yeast two-hybrid screen two novel putative K+(in) channel proteins (SKT2 and SKT3) were identified by interaction of their C-termini which contained a conserved domain (K(HA)). Interactions were confirmed by Western blot-related assays utilizing K+(in) channel C-termini fused to green fluorescence protein. Although deletion of the K(HA)-domain abolished these interactions, K+(in) currents were still detectable by patch-clamp measurements of insect cells expressing these KST1 mutants, indicating that formation of a functional channel does not depend on this C-terminal domain.

The role of transient starch in acclimation to elevated atmospheric CO2.

Although increased concentrations of CO2 stimulate photosynthesis, this stimulation is often lost during prolonged exposure to elevated carbon dioxide, leading to an attenuation of the potential gain in yield. Under these conditions, a wide variety of species accumulates non-structural carbohydrates in leaves. It has been proposed that starch accumulation directly inhibits photosynthesis, that the rate of sucrose and starch synthesis limits photosynthesis, or that accumulation of sugars triggers changes in gene expression resulting in lower activities of Rubisco and inhibition of photosynthesis. To distinguish these explanations, transgenic plants unable to accumulate transient starch due to leaf mesophyll-specific antisense expression of AGP B were grown at ambient and elevated carbon dioxide. There was a positive correlation between the capacity for starch synthesis and the rate of photosynthesis at elevated CO2 concentrations, showing that the capability to synthesize leaf starch is essential for photosynthesis in elevated carbon dioxide. The results show that in elevated carbon dioxide, photosynthesis is restricted by the rate of end product synthesis. Accumulation of starch is not responsible for inhibition of photosynthesis. Although transgenic plants contained increased levels of hexoses, transcripts of photosynthetic genes were not downregulated and Rubisco activity was not decreased arguing against a role of sugar sensing in acclimation to high CO2.

Contribution of adenosine 5'-diphosphoglucose pyrophosphorylase to the control of starch synthesis is decreased by water stress in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch and cell-wall synthesis in tissue slices of growing potato (Solanum tuberosum L. cv. Desiree) tubers. Based on the analysis of fluxes and metabolites, Geigenberger et al. (1997, Planta 201: 502-518) proposed that water deficits up to -0.72 MPa stimulate sucrose synthesis, leading to decreased starch synthesis as a result of the resulting decline of phosphorylated metabolite levels, whereas more-severe water deficits directly inhibit the use of ADP-glucose. Potato plants with decreased expression of adenosine 5'-diphosphoglucose pyrophosphorylase (AGPase) have been used to test the prediction that the contribution of AGPase to the control of starch synthesis should decrease in severely water-stressed tuber material. Freshly cut slices from wild-type and antisense tubers were incubated at a range of mannitol concentrations (20, 300 and 500 mM) and the metabolism of [(14)C]glucose was analysed. A 86-97% reduction of AGPase activity led to a major but non-stoichiometric inhibition of starch accumulation in intact growing tubers attached to the plant (40-85%), and an inhibition of starch synthesis in non-stressed tuber slices incubated in 20 mM mannitol (60-80%). The inhibition of starch synthesis was accompanied by a 2- to 8-fold increase in the levels of sugars in intact tubers and a 2- to 3-fold stimulation of sucrose synthesis in tuber slices, whereas respiration and cell-wall synthesis were not significantly affected. The strong impact of AGPase on carbon partitioning in non-stressed tubers and tuber slices was retained in slices subjected to moderate water deficit (300 mM mannitol, corresponding to -0.72 MPa). In discs incubated in 500 mM mannitol (corresponding to -1.2 MPa) this response was modified. A 80-97% reduction of AGPase resulted in only a 0-40% inhibition of starch synthesis. Further, the water stress-induced stimulation of sucrose synthesis was abolished in the transformants. The results provide direct evidence that the contribution of AGPase to the control of starch synthesis can be modified by environmental factors, leading to a lower degree of control during severe water deficits. There was also a dramatic decrease in the labelling of cell-wall components in wild-type tuber slices incubated with 300 or 500 mM mannitol. The water stress-induced inhibition of cell-wall synthesis occurred independently of AGPase expression and the accompanying changes in starch and sucrose metabolism, indicating a direct inhibition of cell-wall synthesis in response to water stress.

The contribution of adenosine 5'-diphosphoglucose pyrophosphorylase to the control of starch synthesis in potato tubers

The aim of this work was to investigate the extent to which starch synthesis in potato (Solanum tuberosum L.) tubers is controlled by the activity of ADPglucose pyrophosphorylase (EC 2.7.7.27; AGPase). In order to do this, fluxes of carbohydrate metabolism were measured in tubers that had reduced AGPase activity as a result of the expression of a cDNA encoding the B subunit in the antisense orientation. Reduction in AGPase activity led to a reduction in starch accumulation, and an increase in sucrose accumulation. The control coefficient of AGPase on starch accumulation in intact plants was estimated to be around 0.3. The fluxes of carbohydrate metabolism were measured in tuber discs from wild-type and transgenic plants by investigating the metabolism of [U-(14)C]glucose. In tuber discs, the control coefficient of AGPase over starch synthesis was estimated as 0.55, while the control coefficient of the enzyme over sucrose synthesis was -0.47. The values obtained suggest that AGPase activity exerts appreciable control over tuber metabolism in potato.

The influence of alterations in ADP-glucose pyrophosphorylase activities on starch structure and composition in potato tubers.

In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway affected various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K(m) for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found, but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed.Key words: ADP-glucose pyrophosphorylase. Amylopectin structure. Amylose. Solanum (starch. tuber). Starch granule size. Starch phosphorylation

Cloning and expression analysis of an ADP-ribosylation factor from Solanum tuberosum L.

A cDNA clone encoding an ADP-ribosylation factor from potato (Solanum tuberosum L.) was isolated. The nucleotide and deduced amino acid sequences show high homology to known ADP-ribosylation factor sequences from Arabidopsis, yeast, cow and man. In northern blot experiments, all tissues analysed showed expression of the corresponding mRNA. Strongest expression was found, however, in potato tubers.

Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli.

The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells.

Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes.

Transgenic potato plants were created in which the expression of ADP-glucose pyrophosphorylase (AGPase) was inhibited by introducing a chimeric gene containing the coding region of one of the subunits of the AGPase linked in an antisense orientation to the CaMV 35S promoter. Partial inhibition of the AGPase enzyme was achieved in leaves and almost complete inhibition in tubers. This resulted in the abolition of starch formation in tubers, thus proving that AGPase has a unique role in starch biosynthesis in plants. Instead up to 30% of the dry weight of the transgenic potato tubers was represented by sucrose and up to 8% by glucose. The process of tuber formation also changed, resulting in significantly more tubers both per plant and per stolon. The accumulation of soluble sugars in tubers of antisense plants resulted in a significant increase of the total tuber fresh weight, but a decrease in dry weight of tubers. There was no significant change in the RNA levels of several other starch biosynthetic enzymes, but there was a great increase in the RNA level of the major sucrose synthesizing enzyme sucrose phosphate synthase. In addition, the inhibition of starch biosynthesis was accompanied by a massive reduction in the expression of the major storage protein species of potato tubers, supporting the idea that the expression of storage protein genes is in some way connected to carbohydrate formation in sink storage tissues.

Ion channels in plant signaling.

Plant ion channel activities are rapidly modulated in response to several environmental and endogenous stimuli such as light, pathogen attack and phytohormones. Electrophysiological as well as pharmacological studies provide strong evidence that ion channels are essential for the induction of specific cellular responses, implicating their tight linkage to signal transduction cascades. Ion channels propagate signals by modulating the membrane potential or by directly affecting cellular ion composition. In addition, they may also be effectors at the end of signaling cascades, as examplified by ion channels which determine the solute content of stomatal guard cells. Plant channels are themselves subject to regulation by a variety of cellular factors, including calcium, pH and cyclic nucleotides. In addition, they appear to be regulated by (de)-phosphorylation events as well as by direct interactions with cytoskeletal and other cellular proteins. This review summarizes current knowledge on the role of ion channels in plant signaling.

Cloning and expression analysis of the cytosolic NADP(+)-dependent isocitrate dehydrogenase from potato. Implications for nitrogen metabolism.

A full-length cDNA (icdh-1) encoding a cytosolic NADP(+)-dependent isocitrate dehydrogenase (ICDH-1) from potato (Solanum tuberosum L.) has been isolated. Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other eukaryotes such as tobacco, alfalfa, soybean, cattle, pig, and yeast. The gene was transcribed in all tissues tested, with the highest amount of icdh-1 transcript being found in green tissues, in flowers, and in roots. In leaves, enzyme activities were dependent on the age, with fully mature leaves showing the highest level of RNA expression and enzyme activity. This observation may indicate that NADP(+)-dependent ICDH is not only involved in amino acid biosynthesis via the glutamine synthetase/glutamine oxoglutarate aminotransferase cycle but also in cycling, redistribution, and export of amino acids. The latter assumption has been strengthened by our finding of a preferential expression of NADP(+)-dependent ICDH in leaf veins. Under in vivo conditions, the expression pattern paralleled the enzyme activity, indicating coarse control on the RNA level. Experiments carried out with detached leaves revealed an influence of light, nitrate, and sucrose on icdh-1 transcript levels and in some cases also on NADP(+)-dependent ICDH activity. In darkness, nitrate or sucrose induced icdh-1 mRNA expression. Leaves kept under starvation conditions exhibited a decrease of their protein content, whereas icdh-1 expression and ICDH activity increased significantly.

Mitochondrial citrate synthase from potato: predominant expression in mature leaves and young flower buds.

A cDNA clone encoding mitochondrial citrate synthase (EC 4.1.3.7), the first enzyme of the tricarboxylic-acid cycle, was isolated from potato (Solanum tuberosum L.) and expression of the enzyme analyzed. The deduced amino-acid sequence of the potato mitochondrial citrate synthase showed high similarity to known citrate synthases from fungi, mammals and Arabidopsis thaliana. The expression pattern of this clone was determined by Northern blot analysis. Expression was detected in all tissues analyzed. The highest level of expression was found in green flower buds. In photosynthetic tissues, stronger mRNA expression was detected in mature than in immature leaves. This rise in expression with leaf age was accompanied by an increase in citrate-synthase activity. Within flowers, expression was severalfold stronger in anthers than in ovaries, indicating a role of mitochondrial citrate synthase during anther or pollen development. A comparatively low level of transcript was detected in underground heterotrophic tissues, such as stolons, tubers and roots. When tubers were stored at low temperature (4 degrees C), mitochondrial citrate-synthase gene expression increased slightly. From the data obtained, we conclude that expression of the mitochondrial citrate-synthase gene is regulated by developmental and environmental factors. The relatively high expression in leaves is in line with the assumption that mitochondria play an important role in photosynthetically active tissues.

Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers.

The tricarboxylic acid (TCA) cycle constitutes a major component of the mitochondrial metabolism of eucaryotes, including higher plants. To analyze the importance of this pathway, we down-regulated mitochondrial citrate synthase (mCS; EC 4.1.3.7), the first enzyme of the TCA cycle, in transgenic potato plants using an antisense RNA approach. Several transformants were identified with reduced citrate synthase activity (down to approximately 6% of wild-type activity). These plants were indistinguishable from wild-type plants in the greenhouse during vegetative growth. A major change, however, was seen upon initiation of the generative phase (flower formation). In the case of transgenic plants with a strong reduction in citrate synthase activity (< 30% of wild-type levels), flower buds formed > 2 weeks later as compared with wild-type plants. Furthermore, flower buds from these plants did not develop into mature flowers but rather were aborted at an early stage of development. Microscopic analysis showed that in these cases ovaries disintegrated during flower development. We conclude that the TCA cycle is of major importance during the transition from the vegetative to the generative phase.

Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggest that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.

Isolation and expression analysis of cDNA clones encoding a small and a large subunit of ADP-glucose pyrophosphorylase from sugar beet.

The cDNA cloning of a small and a large subunit of ADP-glucose pyrophosphorylase (AGPase) from sugar beet is reported. The deduced amino acid sequences are highly homologous to previously identified AGPase polypeptides from other plant species. Both subunits are encoded by low copy genes. When RNA gel blot experiments were performed, strongest expression was detected in sink and source leaves of greenhouse-grown sugar beet plants. A lower expression was found in other tissues tested, i.e. in the hypocotyl, the tap root and roots. In these tissues, slightly higher transcript levels were found for the small subunit gene than for the large subunit gene.

New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency.

Potassium (K+) channels mediating important physiological functions are characterized by a common pore-forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of 'two-pore' K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium-binding EF-hand motifs. Heterologous expression of KCO1 in baculovirus-infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+-selective currents elicited by depolarizing voltage pulses in whole-cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to <150 nM. However, KCO1 strongly activated at increasing [Ca2+]cyt, with a saturating activity observed at approximately 300 nM [Ca2+]cyt. KCO1 single channel analysis on excised membrane patches, resulting in a single channel conductance of 64 pS, confirmed outward rectification as well as Ca2+-dependent activation. These data suggest a direct link between calcium-mediated signaling processes and K+ ion transport in higher plants. The identification of KCO1 as the first plant K+ outward channel opens a new field of structure-function studies in plant ion channels.

Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates.

Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229-1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and 13C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyro-phosphorylase antisense plants was observed.

Complementary DNAs encoding eukaryotic-type cytidine-5'-diphosphate-diacylglycerol synthases of two plant species.

Cytidine diphosphate (CDP)-diacylglycerol synthase (cytidine triphosphate:phosphatidate cytihyltransferase, EC 2.7.7.41) catalyzes the formation of CDP-diacylglycerol, which is the precursor of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We report the first cloning, to our knowledge, of two plant cDNAs, StCDS1 and AtCDS1, coding for CDP-diacylglycerol synthase from potato (Solanum tuberosum) and Arabidopsis thaliana, respectively. The two proteins belong to the eukaryotic type of CDP-diacylglycerol synthase and contain eight predicted transmembrane-spanning domains. We analyzed gene expression in shoot and root tissues of potato plants and demonstrated enzyme activity by expression of N-terminally truncated, recombinant StCDS1 in Escherichia coli.

Potato guard cells respond to drying soil by a complex change in the expression of genes related to carbon metabolism and turgor regulation.

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+ cotransporter, 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.