Expression of an abscisic acid-binding single-chain antibody influences the subcellular distribution of abscisic acid and leads to developmental changes in transgenic potato plants.

Potato (Solanum tuberosum L. cv. Désirée) plants were transformed to express a single-chain variable-fragment antibody against abscisic acid (ABA), and present in the endoplasmic reticulum at to up to 0.24% of the soluble leaf protein. The resulting transgenic plants were only able to grow normally at 95% humidity and moderate light. Four-week-old plants accumulated ABA to high extent, were retarded in growth and their leaves were smaller than those of control plants. Leaf stomatal conductivity was increased due to larger stomates. The subcellular concentrations of ABA in the chloroplast, cytoplasm and vacuole, and the apoplastic space of leaves were determined. In the 4-week-old transgenic plants the concentration of ABA not bound to the antibody was identical to that of control plants and the stomates were able to close in response to lower humidity of the atmosphere. A detailed analysis of age-dependent changes in plant metabolism showed that leaves of young transformed plants developed in ABA deficiency and leaves of older plants in ABA excess. Phenotypic changes developed in ABA deficiency partly disappeared in older plants.

Metabolic response of potato plants to an antisense reduction of the P-protein of glycine decarboxylase.

Potato (Solanum tuberosum L. cv. Desiré) plants with reduced amounts of P-protein, one of the subunits of glycine decarboxylase (GDC), have been generated by introduction of an antisense transgene. Two transgenic lines, containing about 60-70% less P-protein in the leaves compared to wild-type potato, were analysed in more detail. The reduction in P-protein amount led to a decrease in the ability of leaf mitochondria to decarboxylate glycine. Photosynthetic and growth rates were reduced but the plants were viable under ambient air and produced tubers. Glycine concentrations within the leaves were elevated up to about 100-fold during illumination. Effects on other amino acids and on sucrose and hexoses were minor. Nearly all of the glycine accumulated during the day was metabolised during the following night. The data suggest that the GDC operates far below substrate saturation under normal conditions thus allowing a flexible and fast response to changes in the environment.

Ontogenetic changes of potato plants during acclimation to elevated carbon dioxide.

Transgenic potato plants (Solanum tuberosum cv. Desirée) with an antisense repression of the chloroplastic triosephosphate translocator were compared with wild-type plants. Plants were grown in chambers with either an atmosphere with ambient (400 mu bar) or elevated (1000 mu bar) CO2. After 7 weeks, the rate of CO2 assimilation between wild-type and transgenic plants in both CO2 concentrations was identical, but the tuber yield of both plant lines was increased by about 30%, when grown in elevated CO2. One explanation is that plants respond to the elevated CO2 only at a certain growth stage. Therefore, growth of wild-type plants was analysed between the second and the seventh week. Relative growth rate and CO2 assimilation were stimulated in elevated CO2 only in the second and the third weeks. During this period, the carbohydrate content of leaves grown with elevated CO2 was lower than that of leaves grown with ambient CO2. In plants grown in elevated CO2, the rate of CO2 assimilation started to decline after 5 weeks, and accumulation of carbohydrates began after 7 weeks. From this observation it was concluded that acclimation of potato plants to elevated CO2 is the result of accelerated development rather than of carbohydrate accumulation causing down-regulation of photosynthesis. For a detailed analysis for the cause of the stimulation of growth after 2 weeks, the contents of phosphorylated intermediates of wild-type plants and transgenics were measured. Stimulation of CO2 assimilation was accompanied by changes in the contents of phosphorylated intermediates, resulting in an increase in the amount of dihydroxyacetone phosphate, the metabolite which is exported from the chloroplast into the cytosol. An increase of dihydroxyacetone phosphate was found in wild-type plants in elevated CO2 when compared with ambient CO2 and in triosephosphate translocator antisense plants in ambient CO2, but not in the transgenic plants when grown in elevated CO2. These plants were not able to increase dihydroxyacetone phosphate further to cope with the increased CO2 supply. From these changes in phosphorylated intermediates in wild-type and transgenic plants it was concluded that starch and sucrose synthesis pathways can replace each other only at moderate carbon flux rates.

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.

Magnesium deficiency results in accumulation of carbohydrates and amino acids in source and sink leaves of spinach.

Accumulation of assimilates in source leaves of magnesium-deficient plants is a well-known feature. We had wished to determine whether metabolite concentrations in sink leaves and roots are affected by magnesium nutrition. Eight-week-old spinach plants were supplied either with a complete nutrient solution (control plants) or with one lacking Mg (deficient plants) for 12 days. Shoot and root fresh weights and dry weights were lower in deficient than in control plants. Mg concentrations in deficient plants were 11% of controls in source leaves, 12% in sink leaves and 26% in roots, respectively. As compared with controls, increases were found in starch and amino acids in source leaves and in sucrose, hexoses, starch and amino acids in sink leaves, whereas they were only slightly enhanced in roots. In phloem sap of magnesium-deficient and control plants no differences in sucrose and amino acid concentrations were found. To prove that sink leaves were the importing organs they were shaded, which did not alter the response to magnesium deficiency as compared with that without shading. Since in the shaded sink leaves the photosynthetic production of metabolites could be excluded, those carbohydrates and amino acids that accumulated in the sink leaves of the deficient plants must have been imported from the source leaves. It is concluded that in magnesium-deficient spinach plants the growth of sink leaves and roots was not limited by carbohydrate or amino acid supply. It is proposed that the accumulation of assimilates in the source leaves of Mg-deficient plants results from a lack of utilization of assimilates in the sink leaves.

Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast-derived invertase either in the apoplast, vacuole or cytosol.

Potato (Solanum tuberosum cv. Désirée) plants expressing yeast invertase directed either to the apoplast, vacuole or cytosol were biochemically and physiologically characterised. All lines of transgenic plants showed similarities to plants growing under water stress. Transformants were retarded in growth, and accumulated hexoses and amino acids, especially proline, to levels up to 40-fold higher than those of the wild types. In all transformants rates of CO2 assimilation and leaf conductance were reduced. From the unchanged intercellular partial pressure of CO2 and apoplastic cis-abscisic acid (ABA) content of transformed leaves it was concluded that the reduced rate of CO2 assimilation was not caused by a limitation in the availability of CO2 for the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). In the transformants the amount of Rubisco protein was not reduced, but both activation state and carboxylation efficiency of photosynthesis were lowered. In vacuolar and cytosolic transformants this inhibition of Rubisco might be caused by a changed ratio of organic bound and inorganic phosphate, as indicated by a doubling of phosphorylated intermediates. But in apoplastic transformants the pattern of phosphorylated intermediates resembled that of leaves of water-stressed potato plants, although the cause of inhibition of photosynthesis was not identical. Whereas in water-stressed plants increased contents of the phytohormone ABA are supposed to mediate the adaptation to water stress, no contribution of ABA to reduction of photosynthesis could be detected in invertase transformants.

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.

Comparison of the in vitro binding characteristics of the beta-carbolines harman and norharman in rat brain and liver and in bovine adrenal medulla.

The in vitro binding of the naturally occurring beta-carbolines harman and norharman in their tritium-labelled forms to cell membranes from the rat brain and liver and from bovine adrenal medulla was investigated. Displacement of the specific [3H]harman binding in bovine adrenal medulla and rat liver by several beta-carbolines and monoamine oxidase (MAO) inhibitors revealed the pharmacological profile of a single, high-affinity binding site (KD 4.92 +/- 0.43 nmol/l, Bmax 8.47 +/- 0.17 pmol/mg protein; adrenal medulla) which corresponded to the active site of MAO type A (MAO-A). Similar characteristics have previously been found for brain tissue from rat, marmoset and pig. In order to determine the temperature dependence of the [3H]harman binding, the KD and Bmax values for rat cerebral cortex were calculated from the results of saturation experiments at 5 temperatures (range: 0 degree C-37 degrees C). Whereas the Bmax values under all conditions were approximately 4 pmol/mg protein, the KD values, with increasing temperature, ranged from approximately 3 nmol/l to 30 nmol/l. The calculated linear van't Hoff plot (-ln KD against 1/T) suggested an enthalpy-driven binding of [3H]harman to MAO-A. At least three different [3H]norharman-binding sites were detected. In the rat forebrain, approximately 85% of the specific binding (at about 2 nmol/l of [3H]norharman) can be attributed to a MAO binding site of type B: the binding is displaceable, in nmol/l concentrations by the potent and selective MAO-B inhibitors MDL 72,974 A, R(-)-deprenyl and pargyline and, in mumol/l concentrations, by S(+)-deprenyl and the potent and selective MAO-A inhibitors clorgyline, harmine, harman, harmaline, brofaromine 5-F-alpha-methyltryptamine. After suppression of the MAO binding sites with 1 mumol/l clorgyline and 1 mumol/l R(-)-deprenyl, a second binding site was found. However, the binding at this site was biphasically displaceable by harman and norharman (Hill-slopes about 0.5 and 0.6, curvilinear Rosenthal plots) suggesting the presence of negative co-operativity or of two binding sites (states). A similar clorgyline/R(-)-deprenyl resistant single (Hill-slopes of displacement by norharman, harman and 6-hydroxy-beta-carboline about unity; linear Rosenthal plots) high affinity binding sites (KD 7.5 +/- 2 nmol/l, Bmax 130+/- 30 fmol/mg protein) was found in bovine adrenal medullary cell membranes. A third quite different clorgyline/R(-)-deprenyl resistant high-affinity (KD approximately 14 nmol/l) and high-density (Bmax 10-30 pmol/mg protein) binding site was detected in the liver.(ABSTRACT TRUNCATED AT 400 WORDS)

Antisense repression of the chloroplast triose phosphate translocator affects carbon partitioning in transgenic potato plants.

The major chloroplast envelope membrane protein E29 is central for the communication between chloroplasts and cytosol. It has been identified as the triose phosphate translocator (TPT) exporting the primary products of the Calvin cycle (i.e., triose phosphates and 3-phosphoglycerate) out of the chloroplast in a strict counter exchange for Pi. To study the in vivo role of the TPT, transgenic potato plants were constructed that have a reduced expression of the TPT at both the RNA and protein level due to antisense inhibition. Chloroplasts isolated from these plants show a 20-30% reduction with respect to their ability to import Pi. The reduced TPT activity leads to a reduction of maximal photosynthesis by 40-60%, to a change in carbon partitioning into starch at the expense of sucrose and amino acids, and to an increase of the leaf starch content by a factor of approximately 3. At early developmental stages the inhibited plants are retarded in growth compared to the wild type.

Apoplastic expression of yeast-derived invertase in potato : effects on photosynthesis, leaf solute composition, water relations, and tuber composition.

In potato plants (Solanum tuberosum), a chimeric yeast-derived invertase gene fused to a 35S cauliflower mosaic virus promoter has been expressed. The protein was targeted to the cell wall by using the signal peptide of proteinase inhibitor II fused to the amino terminus of the yeast invertase. The transformed plants had crinkled leaves, showed a reduced growth rate, and produced fewer tubers. Although in the apoplast of the leaves of the transformed plants the content of glucose and fructose rose by a factor of 20, and that of sucrose declined 20-fold, 98% of the carbohydrate in the phloem sap consisted of sucrose, demonstrating the strong specificity of phloem loading. In the leaf cells of the transformed plants, glucose, fructose, and amino acids, especially proline, were accumulated. Consequently, the osmolality of the cell sap rose from 250 to 350 mosmol/kg. Our results show that the observed 75% decrease of photosynthesis is not caused by a feedback regulation of sucrose synthesis and is accompanied by an increase in the osmotic pressure in the leaf cells. In the transformed plants, not only the amino acid to sucrose ratio in the phloem sap, but also the amino acid and protein contents in the tubers were found to be elevated. In the tubers of the transformed plants, the protein to starch ratio increased.

Amino Acid and sucrose content determined in the cytosolic, chloroplastic, and vacuolar compartments and in the Phloem sap of spinach leaves.

Amino acid and sucrose contents were analyzed in the chloroplastic, cytosolic, and vacuolar compartments and in the phloem sap of illuminated spinach leaves (Spinacia oleracea L.). The determination of subcellular metabolite distribution was carried out by nonaqueous fractionation of frozen and lyophilized leaf material using a novel three-compartment calculation method. The phloem sap was collected by aphid stylets which had been severed by a laser beam. Subcellular analysis revealed that the amino acids found in leaves are located mainly in the chloroplast stroma and in the cytosol, the sum of their concentrations amounting to 151 and 121 millimolar, respectively, whereas the amino acid concentrations in the vacuole are one order of magnitude lower. The amino acid concentrations in the phloem sap are found to be not very different from the cytosolic concentrations, whereas the sieve tube concentration of sucrose is found to be one order of magnitude higher than in the cytosol. It is concluded that the phloem loading results in a preferential extraction of sucrose from the source cells.

Redox Transfer across the Inner Chloroplast Envelope Membrane.

In leaves of spinach plants (Spinacia oleracea L.) grown in ambient CO(2) the subcellular contents of adenylates, pyridine nucleotides, 3-phosphoglycerate, dihydroxyacetone phosphate, malate, glutamate, 2-oxoglutarate, and aspartate were assayed in the light and in the dark by nonaqueous fractionation technique. From the concentrations of NADP and NADPH determined in the chloroplast fraction of illuminated leaves the stromal NADPH to NADP ratio is calculated to be 0.5. For the cytosol a NADH to NAD ratio of 10(-3) is calculated from the assay of the concentrations of NAD, malate, glutamate, aspartate, and 2-oxoglutarate on the assumption that the reactions catalyzed by the cytosolic glutamate oxaloacetate transaminase and malate dehydrogenase are not far away from equilibrium. For the transfer of redox equivalents from the chloroplastic NADPH to the cytosolic NAD two metabolite shuttles are operating across the inner envelope membrane: the triosephosphate-3-phosphoglycerate shuttle and the malate-oxaloacetate shuttle. Although both shuttles would have the capacity to level the redox state of the stromal and cytosolic compartment, this apparently does not occur. To gain an insight into the regulatory processes we calculated the free energy of the enzymic reactions and of the translocation steps involved. From the results it is concluded that the triosephosphate-3-phosphoglycerate shuttle is mainly controlled by the chloroplastic reaction of 3-phosphoglycerate reduction and of the cytosolic reaction of triosephosphate oxidation. The malate-oxaloacetate shuttle is found to be regulated by the chloroplastic NADP-malate dehydrogenase and also by the translocating step across the envelope membrane.

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate, and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts.

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg(2+)-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO2 fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.

Effects of inorganic phosphate on the light dependent thylakoid energization of intact spinach chloroplasts.

The light dependent energization of the thylakoid membrane was analyzed in isolated intact spinach (Spinacia oleracea L.) chloroplasts incubated with different concentrations of inorganic phosphate (Pi). Two independent methods were used: (a) the accumulation of [(14)C]5,5-dimethyl-2,4-oxazolidinedione and [(14)C] methylamine; (b) the energy dependent chlorophyll fluorescence quenching. The inhibition of CO(2) fixation by superoptimal medium Pi or by adding glyceraldehyde-an inhibitor of the Calvin cycle-leads to an increased energization of the thylakoid membrane; however, the membrane energization decreases when chloroplasts are inhibited by suboptimal Pi. This specific ;low phosphate' effect could be partially reversed by adding oxaloacetate, which regenerates the electron acceptor NADP(+) and stimulates linear electron transport. The energization seen in low Pi is, however, always lower than in superoptimal Pi, even in the presence of oxaloacetate. Energization recovers in the presence of low amounts of N,N'-dicyclohexylcarbodiimide, which reacts with proton channels including the coupling factor 1 ATP synthase. N,N'-Dicyclohexylcarbodiimide has no effect on energization of chloroplasts in superoptimal Pi. These results suggest there is a specific ;low phosphate' proton leak in the thylakoids, and its origin is discussed.

Limitation of Photosynthesis by Carbon Metabolism : II. O(2)-Insensitive CO(2) Uptake Results from Limitation Of Triose Phosphate Utilization.

The occurrence of O(2)-insensitive photosynthesis at high quantum flux and moderate temperature in Spinacia oleracea was characterized by analytical gas exchange measurements on intact leaves. In addition photosynthetic metabolite pools were measured in leaves which had been rapidly frozen under defined gas conditions. Upon switching to low O(2) in O(2)-insensitive conditions the ATP/ADP ratio fell dramatically within one minute. The P-glycerate pool increased over the same time. Ribulose bisphosphate initially declined, then increased and exceeded the pool size measured in air. The pools of hexose monophosphates and UDPglucose were higher at a partial pressure of O(2) of 21 millibars than at 210 millibars. These results are consistent with the hypothesis that the rate of sucrose synthesis limited the overall rate of assimilation under O(2)-insensitive conditions.

Pyruvate-Derived Amino Acids in Spinach Chloroplasts : Synthesis and Regulation during Photosynthetic Carbon Metabolism.

A probable carbon flow from the Calvin cycle to branched chain amino acids and lipids via phosphoenolpyruvate (PEP) and pyruvate was examined in spinach (Spinacia oleracea) chloroplasts. The interpendence of metabolic pathways in and outside chloroplasts as well as product and feedback inhibition were studied. It was shown that alanine, aromatic, and small amounts of branched chain amino acids were formed from bicarbonate in purified intact chloroplasts. Addition of PEP only favored formation of aromatic amino acids. Mechanisms of regulation remained unclear. Concentrations of PEP and pyruvate within the chloroplast impermeable space during photosynthetic carbon fixation were 15 times higher than in the reaction medium. A direct carbon flow to pyruvate was identified (0.1 micromoles per milligram chlorophyll per hour). Pyruvate was taken up by intact chloroplasts slowly, leading to the formation of lysine, alanine, valine, and leucine plus isoleucine (approximate ratios, 100-500:60-100:40-100:2-10). The K(m) for the formation of valine and leucine plus isoleucine was estimated to be 0.1 millimolar. Ten micromolar glutamate optimized the transamination reaction regardless of whether bicarbonate or pyruvate was being applied. Alanine and valine formation was enhanced by the addition of acetate to the reaction mixture. The enhancement probably resulted from an inhibition of pyruvate dehydrogenase by acetyl-S-coenzyme A formed from acetate, and resulting accumulation of hydroxyethylthiamine diphosphate and pyruvate. High concentrations of valine and isoleucine inhibited their own and each others synthesis and enhanced alanine formation. When pyruvate was applied, only amino acids were formed; when complemented with bicarbonate, fatty acids were formed as well. This is probably the result of a requirement of acetyl-S-coenzyme A-carboxylase for bicarbonate.