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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 wildtype 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.

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

Cytidine diphosphate (CDP)-diacylglycerol synthase (cytidine triphosphate:phosphatidate cytidyltransferase, 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 synthases from potato (Solanum tuberosum) and Arabidopsis thaliana, respectively. The two proteins belong to the eukaryotic type of CDP-diacylglycerol synthases 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

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

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.

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-trisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 micromolar Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner

During stomatal opening potassium uptake into guard cells and K+ channel activation is tightly coupled to proton extrusion. The pH sensor of the K+ uptake channel in these motor cells has, however, not yet been identified. Electrophysiological investigations on the voltage-gated, inward rectifying K+ channel in guard cell protoplasts from Solanum tuberosum (KST1), and the kst1 gene product expressed in Xenopus oocytes revealed that pH dependence is an intrinsic property of the channel protein. Whereas extracellular acidification resulted in a shift of the voltage-dependence toward less negative voltages, the single-channel conductance was pH-insensitive. Mutational analysis allowed us to relate this acid activation to both extracellular histidines in KST1. One histidine is located within the linker between the transmembrane helices S3 and S4 (H160), and the other within the putative pore-forming region P between S5 and S6 (H271). When both histidines were substituted by alanines the double mutant completely lost its pH sensitivity. Among the single mutants, replacement of the pore histidine, which is highly conserved in plant K+ channels, increased or even inverted the pH sensitivity of KST1. From our molecular and biophysical analyses we conclude that both extracellular sites are part of the pH sensor in plant K+ uptake channels

A cDNA encoding a novel, inwardly rectifying K+ ($\\text{K}^{+}{}_{\\text{in}}$) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of $\\text{K}^{+}{}_{\\text{in}}$ channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda) cells, SKT1 was identified as a $\\text{K}^{+}{}_{\\text{in}}$ channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than -60 mV. The pharmacological inhibitor Cs+, when applied externally, inhibited SKT1-mediated $\\text{K}^{+}{}_{\\text{in}}$ currents half-maximally with an inhibitor concentration (IC50) of 105 μM. An almost identical high Cs+ sensitivity (IC50 = 90 μM) was found for the potato guard-cell $\\text{K}^{+}{}_{\\text{in}}$ channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type $\\text{K}^{+}{}_{\\text{in}}$ channels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.

Plant ion channel activities are rapidly modulated in response to several environmental and endogenous stimuli. Electrophysiological as well as pharmacological studies provide strong evidence that ion channels are essential for the induction of specific cellular responses and that they are themselves subject to regulation by a variety of cellular factors. Genes and cDNAs of several plant ion channels have been identified in recent years giving access to molecular mechanisms of such regulatory processes. Cloned inwardly rectifying potassium channels have been investigated in various heterologous expression systems. Two other ion channel classes, namely members of the 'two-pore' K(+) channel family as well as of the anion-conducting chloride channel (ClC) family, have been cloned, but a direct link to corresponding plasma membrane or endomembrane ion channel conductances has not been demonstrated yet. Analysis of cellular expression patterns of plant ion channel genes in combination with transgenic approaches now gives access to a detailed ex planta/in planta correlation of channel function, as has recently been demonstrated for proteins of the K(in)(+) channel family. This review summarizes current knowledge on molecular structures and some features of structure-function relationships of plant ion channels.