Ammonia assimilation by the plastidic glutamine synthetase/glutamate synthase system requires 2-oxoglutarate (2-OG) as a carbon precursor. Plastids depend on 2-OG import from the cytosol. A plastidic dicarboxylate translocator 1-[2-OG/malate translocator (DiT1)] has been identified and its substrate specificity and kinetic constants have been analyzed in vitro. However, the role of DiT1 in intact plants and its significance for ammonia assimilation remained uncertain. Here, to study the role of DiT1 in intact plants, its expression was antisense-repressed in transgenic tobacco plants. This resulted in a reduced transport capacity for 2-OG across the plastid envelope membrane. In consequence, allocation of carbon precursors to amino acid synthesis was impaired, organic acids accumulated and protein content, photosynthetic capacity and sugar pools in leaves were strongly decreased. The phenotype was consistent with a role of DIT1 in both, primary ammonia assimilation and the re-assimilation of ammonia resulting from the photorespiratory carbon cycle. Unexpectedly, the in situ rate of nitrate reduction was extremely low in alpha-DiT1 leaves, although nitrate reductase (NR) expression and activity remained high. We hypothesize that this discrepancy between extractable NR activity and in situ nitrate reduction is due to substrate limitation of NR. These findings and the severe phenotype of the antisense plants point to a crucial role of DiT1 at the interface between carbon and nitrogen metabolism.

Plant cells require a co-ordination of metabolism between their major compartments, the plastids and the cytosol, in particular as certain metabolic pathways are confined to either compartments. The inner envelope membrane of the plastids forms the major barrier for metabolite exchange and is the site for numerous transport proteins, which selectively catalyse metabolite exchanges characteristic for green and/or non-green tissues. This report is focused on the molecular biology, evolution and physiological function of the family of phosphate translocators (PT) from plastids. Until now, four distinct subfamilies have been identified and characterized, which all share inorganic phosphate as common substrate, but have different spectra of counter exchange substrates to fulfil the metabolic needs of individual cells and tissues. The PTs are named after their main transported substrate, triose phosphate (TPT), phosphoenolpyruvate (PPT), glucose 6-phosphate (GPT) and xylulose 5-P (XPT). All PTs belong to the TPT/nucleotide sugar transporter (NST) superfamily, which includes yet uncharacterized PT homologues from plants and other eukaryotes. Transgenic plants or mutants with altered transport activity of some of the PTs have been generated or isolated. The analysis of these plant lines revealed new insights in the co-ordination and flexibility of plant metabolism.

The Arabidopsis thalianachlorophyll a/b binding protein underexpressed (cue1) mutant, which has been isolated in a screen for chlorophyll a/b binding protein (CAB) underexpressors, exhibits a reticulate leaf phenotype combined with delayed chloroplast development and aberrant shape of the palisade parenchyma cells. The affected gene in cue1 is a phosphoenolpyruvate (PEP)/phosphate translocator (PPT) of the plastid inner envelope membrane. The proposed function of the PPT in C3-plants is the import of PEP into the stroma as one of the substrates for the shikimate pathway, from which aromatic amino acids and a variety of secondary plant products derive. The mutant phenotype could be: (i) complemented by constitutive overexpression of a heterologous PPT from cauliflower; and (ii) rescued by overexpression of a C4-type pyruvate,orthophosphate dikinase (PPDK). The latter approach indicates that PEP deficiency within plastids triggers developmental constraints in cue1. The impact of the mutation on aspects of primary and secondary metabolism was assessed in cue1 as well as in the individual transformant lines. The majority of the data obtained in this and an accompanying paper suggest that the mutant phenotype is not simply caused by a general restriction of the shikimate pathway because of a defect in a PPT.

SummaryThe Arabidopsis thaliana tpt‐1 mutant which is defective in the chloroplast triose phosphate/phosphate translocator (TPT) was isolated by reverse genetics. It contains a T‐DNA insertion 24 bp upstream of the start ATG of the TPT gene. The mutant lacks TPT transcripts and triose phosphate (TP)‐specific transport activities are reduced to below 5% of the wild type. Analyses of diurnal variations in the contents of starch, soluble sugars and phosphorylated intermediates combined with 14CO2 labelling studies showed, that the lack of TP export for cytosolic sucrose biosynthesis was almost fully compensated by both continuous accelerated starch turnover and export of neutral sugars from the stroma throughout the day. The utilisation of glucose 6‐phosphate (generated from exported glucose) rather than TP for sucrose biosynthesis in the light bypasses the key regulatory step catalysed by cytosolic fructose 1,6‐bisphosphatase. Despite its regulatory role in the feed‐forward control of sucrose biosynthesis, variations in the fructose 2,6‐bisphosphate content upon illumination were similar in the mutant and the wild type. Crosses of tpt‐1 with mutants unable to mobilise starch (sex1) or to synthesise starch (adg1‐1) revealed that growth and photosynthesis of the double mutants was severely impaired only when starch biosynthesis, but not its mobilisation, was affected. For tpt‐1/sex1 combining a lack in the TPT with a deficiency in starch mobilisation, an additional compensatory mechanism emerged, i.e. the formation and (most likely) fast turnover of high molecular weight polysaccharides. Steady‐state RNA levels and transport activities of other phosphate translocators capable of transporting TP remained unaffected in the mutants.

The process of photorespiration diminishes the efficiency of CO2 assimilation and yield of C-3-crops such as wheat, rice, soybean or potato, which are important for feeding the growing world population. Photorespiration starts with the competitive inhibition of CO2 fixation by O-2 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and can result in a loss of up to 50% of the CO2 fixed in ambient air. By contrast, C-4 plants, such as maize, sugar cane and Sorghum, possess a CO2 concentrating mechanism, by which atmospheric CO2 is bound to C-4-carbon compounds and shuttled from the mesophyll cells where the prefixation of bicarbonate occurs via phosphoenolpyruvate carboxylase (PEPC) into the gas-tight bundle-sheath cells, where the bound carbon is released again as CO2 and enters the Calvin cycle. However, the anatomical division into mesophyll and bundle-sheaths cells ('Kranz'-anatomy) appears not to be a prerequisite for the operation of a CO2 concentrating mechanism. Submerged aquatic macrophytes, for instance, can induce a C-4-like CO2 concentrating mechanism in only one cell type when CO2 becomes limiting. A single cell C-4-mechanism has also been reported recently for a terrestrial chenopod. For over 10 years researchers in laboratories around the world have attempted to improve photosynthesis and crop yield by introducing a single cell C-4-cycle in C-3 plants by a transgenic approach. In the meantime, there has been substantial progress in overexpressing the key enzymes of the C-4 cycle in rice, potato, and tobacco. In this review there will be a focus on biochemical and physiological consequences of the overexpression of C-4-cycle genes in C-3 plants. Bearing in mind that C-4-cycle enzymes are also present in C-3 plants, the pitfalls encountered when C-3 metabolism is perturbed by the overexpression of individual C-4 genes will also be discussed.

Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.

To improve the efficiency of CO(2) fixation in C(3) photosynthesis, C(4)-cycle genes were overexpressed in potato and tobacco plants either individually or in combination. Overexpression of the phosphoenolpyruvate carboxylase (PEPC) gene (ppc) from Corynebacterium glutamicum (cppc) or from potato (stppc, deprived of the phosphorylation site) in potato resulted in a 3-6-fold induction of endogenous cytosolic NADP malic enzyme (ME) and an increase in the activities of NAD-ME (3-fold), NADP isocitrate dehydrogenase (ICDH), pyruvate kinase (PK), NADP glycerate-3-P dehydrogenase (NADP-GAPDH), and PEP phosphatase (PEPP). In double transformants overexpressing cppc and chloroplastic NADP-ME from Flaveria pringlei (fpMe1), cytosolic NADP-ME was less induced and pleiotropic effects were diminished. There were no changes in enzyme pattern in single fpMe1 overexpressors. In cppc overexpressors of tobacco, the increase in endogenous cytosolic NADP-ME activity was small and changes in other enzymes were less pronounced. Determinations of the CO(2) compensation point (Gamma*) as well as temperature and oxygen effects on photosynthesis produced variational data suggesting that the desired decline in photorespiration occurred only under certain experimental conditions. Double transformants of potato (cppc/fpMe1) exhibited the most consistent attenuating effect on photorespiration. In contrast, photorespiration in tobacco plants appeared to be diminished most in single cppc overexpressors rather than in double transformants (cppc/fpMe1). In tobacco, introduction of the PEP carboxykinase (PEPCK) gene from the bacterium Sinorhizobium meliloti (pck) had little effect on photosynthetic parameters in single (pck) and double transformants (cppc/pck). In transgenic potato plants, increased PEPC activities resulted in a decline in UV protectants (flavonoids) in single cppc or stppc transformants, but not in double transformants (cppc/fpMe1). PEP provision to the shikimate pathway inside the plastids, from which flavonoids derive, might be restricted only in single PEPC overexpressors.

The inducible crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum accumulates malic acid during the night and converts it to starch during the day via a pathway that, because it is located in different subcellular compartments, depends on specific metabolite transport across membranes. The chloroplast glucose transporter (pGlcT) and three members of the phosphate translocator (PT) family were isolated. After induction of CAM, transcript amounts of the phosphoenolpyruvate (PEP) phosphate translocator (PPT) and the glucose-6-phosphate (Glc6P) phosphate translocator (GPT) genes were increased drastically, while triose phosphate (TP) phosphate translocator (TPT) and the pGlcT transcripts remained unchanged. PPT- and GPT-specific transcripts and transporter activities exhibited a pronounced diurnal variation, displaying the highest amplitude in the light. pGlcT transcripts were elevated towards the end of the light period and at the beginning of the dark period. These findings, combined with diurnal variations of enzyme activities and metabolite contents, helped to elucidate the roles of the PPT, GPT, TPT and pGlcT in CAM. The main function of the PPT is the daytime export from the stroma of PEP generated by pyruvate orthophosphate:dikinase (PPDK). The increased transport activity of GPT in the light suggests a higher requirement for Glc6P import for starch synthesis rather than starch mobilization. Most likely, Glc6P rather than 3-phosphoglycerate or triose phosphates is the main substrate for daytime starch biosynthesis in M. crystallinum plants in which CAM has been induced (CAM-induced), similar to non-green plastids. In the dark, starch is mobilized both phosphorylytically and amylolytically and the products are exported by the GPT, TPT and pGlcT. The transport activities of all three phosphate translocators and the transcript amounts of the pGlcT adapt to changing transport requirements in order to maintain high metabolic fluxes during the diurnal CAM cycle.

This article describes a method for the enzymatic detection of low-abundant metabolic intermediates in plant extracts via NAD(P)H fluorescence using a microtiter plate reader. The detection of changes in NAD(P)H fluorescence (excitation 340 nm, emission 465 nm) exhibits a high signal-to-noise ratio and is as sensitive (> or = 20 pmol per well) as absorbance measurements with dual-wavelength photometers. Since up to 96 reactions can be initiated, monitored, and evaluated simultaneously, this method might be suitable for high-throughput screening programs on metabolite profiles. However, in contrast to absorbance measurements, fluorescence detection of NAD(P)H yields relative data, which can be impaired by the quench characteristics and the basic fluorescence of the extracts. Hence, extensive calibration is required to gain reproducible results. Calibration of the assay system was performed using leaf or root material (equivalent to 2-35 mg of fresh weight per well) extracted with perchloric acid, chloroform/water/methanol, or hot ethanol. Extraction with perchloric acid was found to be superior for metabolite quantification. Examples of the kinetics of individual metabolite determinations are presented and the contents of 3-phosphoglycerate, hexose phosphates, triose phosphates, pyruvate, and phosphoenolpyruvate in illuminated and darkened spinach leaves as well as leaf rosettes of Arabidopsis thaliana and leaf segments of the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum were measured via NAD(P)H fluorescence and, where possible, compared to reported data determined with dual-wavelength photometers.

The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell–specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity–dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

The effect of decreased or increased phosphoenolpyruvate carboxylase (PEPC) activity on the CO2 com compensation point, respiration in the light or dark as well as the partitioning of carbon into starch, soluble sugars, organic acids, and amino acids was investigated gated using transgenic potato plants. Engineered PEPC activity ranged from 0.5-fold wild-type level in antisense plants to 5-fold wild-type levels in lines overexpressing the cppc gene of Corynebacterium glutamicum encoding for a PEPC not modulated by protein phosphorylation, The CO2 compensation point determined according to Brooks and Farquhar (1985) was lower in PEPC overexpressors (32 mu l l(-1) CO2) compared to control potato lines (38 mu l l(-1) CO2), but was increased in antisense PEPC plants (4% mu l l(-1) CO2). 3-fold overexpression of PEPC gave a minimum CO2 compensation point of 32 mu l l(-1) CO2. Increased PEPC activity resulted in enhanced respiration in the light and dark. Altered PEPC activity had no effect on the pattern of (CO2)-C-14 incorporation into leaf discs in the light. C-14 pulse-chase experiments in the dark, demonstrated that substantially more total label was lost in the leaf discs from PEPC overexpressors;. Metabolite levels were determined in 21 PEPC overexpressing lines after 8 h in the light. A 5-fold increase in PEPC over the wild-type increased malate (61%), starch (7.5%) and significantly increased sucrose contents (150%), Total amino acid contents were only marginally increased, From gas exchange characteristics and labelling experiments it was concluded that PEP carboxylation, followed by an increased rate of respiratory CO2 release, might work as a HCO3-/CO2 pump. This might result in elevated CO2/O-2, ratios in the mesophyll, concomitant with a more favoured carboxylation/oxygenation ratio of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).

The cDNA of the NADP-dependent malic enzyme gene Me2 from the C3 plant Flaveria pringlei was used for expression in Escherichia coli and Solanum tuberosum. A chimeric GST-Me2 gene complemented a malic enzyme deficient E. coli mutant. Two potato lines were transformed with Me2-cDNA constructs, one line already overexpressing the phosphoenolpyruvate carboxylase gene (ppc) from Corynebacterium glutamicum. Both genes were under the control of the constitutive 35S CaMV promoter. Increased levels of malic enzyme (ME) were found in chloroplasts of transformants. Western blot analysis indicated that the ME transit sequence was cleaved. Expression of both genes led to a significantly reduced electron requirement for apparent CO2 assimilation (e/A) at higher temperature. At low temperatures (15°C) 11 electrons per CO2 assimilated (e/A) were measured in controls, single transformants (ppc or Me2) and double transformants (ppc and Me2). However, when leaf temperature was raised to 36°C electron requirement of the double transformants (15 e/A) was 65% of controls or single transformants (23 e/A). Thus, the temperature dependent increase in electron requirement was reduced in the double transformants suggesting a suppression in the oxygenation reaction of Rubisco and with it presumably in the rate of photorespiratory CO2 release which is more marked at high light and high temperatures.

Plastids contain various transport proteins that mediate the exchange of metabolites between the plastids and the cytosol. These metabolite translocators reside in the inner envelope membrane and are involved in the translocation of photoassimilates in both photosynthetic and heterotrophic tissues. The characteristics of three of these translocators, all functioning as antiporters, are described in more detail. First, the chloroplast triose phosphate/phosphate translocator (cTPT) that exports the fixed carbon in form of triose phosphates and 3-phosphoglycerate from the chloroplasts in exchange for inorganic phosphate is described. Secondly, a phosphoenolpyruvate/phosphate translocator that is present in both photosynthetic and non-green tissues is considered. The main purpose of this transporter is presumably to supply the plastids with phosphoenolpyruvate as a substrate for the shikimate pathway, Finally, a dicarboxylate translocator that imports carbon skeletons into chloroplasts in exchange with malate for ammonia assimilation is discussed. This nitrogen source is used for the formation of amino acids that are subsequently exported.

We have purified a plastidic phosphate transport protein from maize endosperm membranes and cloned and sequenced the corresponding cDNAs from maize endosperm, maize roots, cauliflower buds, tobacco leaves, and Arabidopsis leaves. All of these cDNAs exhibit high homology to each other but only approximately 30% identity to the known chloroplast triose phosphate/phosphate translocators. The corresponding genes are expressed in both photosynthetically active tissues and in nongreen tissues, although transcripts were more abundant in nongreen tissues. Expression of the coding region in transformed yeast cells and subsequent transport measurements of the purified recombinant translocator showed that the protein mediates transport of inorganic phosphate in exchange with C3 compounds phosphorylated at C-atom 2, particularly phosphoenolpyruvate, which is required inside the plastids for the synthesis of, for example, aromatic amino acids. This plastidic phosphate transporter is thus different in structure and function from the known triose phosphate/phosphate translocator. We propose that plastids contain various phosphate translocators with overlapping substrate specificities to ensure an efficient supply of plastids with a single substrate, even in the presence of other phosphorylated metabolites.

Solanum tuberosum was transformed with a chimeric gene consisting of the constitutive 35S CaMV promoter, a potato ribulose bisphosphate carboxylase (Rubisco) small subunit transit sequence (rbcS), and the phosphoenolpyruvate synthetase gene (ppsA) from E. coli. Transgenic plants were regenerated producing (phosphoenolpyruvate-) PEP-synthetase in amounts as much as 0.1% of total soluble protein. Western blot analysis indicated that most of the protein is located in the chloroplasts and that the transit sequence is cleaved off. Electron microscopy of leaves revealed that PEP-synthetase specific immunogold labeling was most pronounced over the chloroplast matrix occurring in between the thylakoid stacks. PEP-synthetase activity was detected in isolated chloroplasts of transgenic plants. Chloroplast morphology and starch production in leaves were affected.