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.

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 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

A plant 126-kDa phosphatidylinositol 4-kinase with a novel repeat structure. Cloning and functional expression in baculovirus-infected insect cells.

Phosphatidylinositol metabolism plays a central role in signaling pathways in animals and is also believed to be of importance in signal transduction in higher plants. We report here the molecular cloning of a cDNA encoding a previously unidentified 126-kDa phosphatidylinositol (PI) 4-kinase (AtPI4Kbeta) from the higher plant Arabidopsis thaliana. The novel protein possesses the conserved domains present in animal and yeast PI 4-kinases, namely a lipid kinase unique domain and a catalytic domain. An additional domain, approximately 300 amino acids long, containing a high percentage (46%) of charged amino acids is specific to this plant enzyme. Recombinant AtPI4Kbeta expressed in baculovirus-infected insect (Spodoptera frugiperda) cells phosphorylated phosphatidylinositol exclusively at the D4 position of the inositol ring. Recombinant protein was maximally activated by 0.6% Triton X-100 but was inhibited by adenosine with an IC50 of approximately 200 microM. Wortmannin at a concentration of 10 microM inhibited AtPI4Kbeta activity by approximately 90%. AtPI4Kbeta transcript levels were similar in all tissues analyzed. Light or treatment with hormones or salts did not change AtPI4Kbeta transcript levels to a great extent, indicating constitutive expression of the AtPI4Kbeta gene.

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.

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.

Characterization of SKT1, an inwardly rectifying potassium channel from potato, by heterologous expression in insect cells.

A cDNA encoding a novel, inwardly rectifying K+ (K+in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K+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 K+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 K+in currents half-maximally with an inhibitor concentration (IC50) of 105 microM. An almost identical high Cs+ sensitivity (IC50 = 90 microM) was found for the potato guard-cell K+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 K+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.

Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato.

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 microM 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.

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.

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.

Plant K+ channel alpha-subunits assemble indiscriminately.

In plants a large diversity of inwardly rectifying K+ channels (K(in) channels) has been observed between tissues and species. However, only three different types of voltage-dependent plant K+ uptake channel subfamilies have been cloned so far; they relate either to KAT1, AKT1, or AtKC1. To explore the mechanisms underlying the channel diversity, we investigated the assembly of plant inwardly rectifying alpha-subunits. cRNA encoding five different K+ channel alpha-subunits of the three subfamilies (KAT1, KST1, AKT1, SKT1, and AtKC1) which were isolated from different tissues, species, and plant families (Arabidopsis thaliana and Solanum tuberosum) was reciprocally co-injected into Xenopus oocytes. We identified plant K+ channels as multimers. Moreover, using K+ channel mutants expressing different sensitivities to voltage, Cs+, Ca2+, and H+, we could prove heteromers on the basis of their altered voltage and modulator susceptibility. We discovered that, in contrast to animal K+ channel alpha-subunits, functional aggregates of plant K(in) channel alpha-subunits assembled indiscriminately. Interestingly, AKT-type channels from A. thaliana and S. tuberosum, which as homomers were electrically silent in oocytes after co-expression, mediated K+ currents. Our findings suggest that K+ channel diversity in plants results from nonselective heteromerization of different alpha-subunits, and thus depends on the spatial segregation of individual alpha-subunit pools and the degree of temporal overlap and kinetics of expression.

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.

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.

Molecular basis of plant-specific acid activation of K+ uptake channels.

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.

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.

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.

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.