Non-mitochondrial ATP transport.

Exchange of organelle ATP with cytosolic ADP through the ADP/ATP carrier is a well-characterized feature of mitochondrial metabolism. Obligate intracellular bacteria, such as Rickettsia prowazekii, and higher-plant plastids possess another type of adenylate transporter, which exchanges bacterial or plastidic ADP for ATP from the eukaryotic (host cell) cytoplasm. The bacterial and plastidic transporters are similar but do not share significant sequence similarities with the mitochondrial carrier. Recent molecular and biochemical studies are providing deeper insight into the functional and evolutionary relationships between the bacterial and the plant transport proteins.

Functional characterisation and cell specificity of BvSUT1, the transporter that loads sucrose into the phloem of sugar beet (Beta vulgaris L.) source leaves.

Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the ß-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks.

Solute pores, ion channels, and metabolite transporters in the outer and inner envelope membranes of higher plant plastids.

All plant cells contain plastids. Various reactions are located exclusively within these unique organelles, requiring the controlled exchange of a wide range of solutes, ions, and metabolites. In recent years, several proteins involved in import and/or export of these compounds have been characterized using biochemical and electrophysiological approaches, and in addition have been identified at the molecular level. Several solute channels have been identified in the outer envelope membrane. These porin-like proteins in the outer envelope membrane were formerly thought to be quite unspecific, but have now been shown to exhibit significant substrate specificity and to be highly regulated. Therefore, the inter-envelope membrane space is not as freely accessible as previously thought. Transport proteins in the inner envelope membrane have been characterized in more detail. It has been proved unequivocally that a family of proteins (including triose phosphate-/phosphoenolpyruvate-, and glucose 6-phosphate-specific transporters) permit the exchange of inorganic phosphate and phosphorylated intermediates. A new type of plastidic 2-oxoglutarate/malate transporter has been identified and represents the first carrier with 12 putative transmembrane domains, to be located in the inner envelope membrane. The plastidic ATP/ADP transporter also contains 12 putative transmembrane domains and possesses striking structural similarity to ATP/ADP transporters found in intracellular, human pathogenic bacteria.

Glucose- and ADPGlc-dependent starch synthesis in isolated cauliflower-bud amyloplasts. Analysis of the interaction of various potential precursors.

Recently, we have demonstrated that isolated cauliflower-bud amyloplasts incorporate glucose 6-phosphate at high rates into newly synthesized starch (Neuhaus et al. (1993) Plant Physiol. 101, 573-578). Here we have analyzed the incorporation of radioactively labeled glucose and ADPglucose into newly synthesized starch. It could be shown that glucose incorporation into starch exhibits a typical substrate saturation behaviour and is linear with time for at least 40 min. The incorporation of glucose is strongly dependent upon the intactness of the plastids and upon the presence of both, ATP and 3-phosphoglyceric acid. Using 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) we showed that glucose is taken up into isolated cauliflower-bud amyloplasts as the free glucose molecule, rather than as glucose 6-phosphate. Glucose incorporation into newly synthesized starch is strongly inhibited in the presence of low concentrations of glucose 6-phosphate. The radioactively labeled glucose moiety of ADPglucose is also incorporated into starch. This incorporation can be saturated at increased concentrations of ADPglucose. ATP significantly inhibits the incorporation of the glucose moiety of ADPglucose into starch. This inhibition can be reinforced by the additional presence of glucose 6-phosphate. Glucose 6-phosphate-dependent starch synthesis is not strongly inhibited in the presence of glucose or ADPglucose indicating that glucose 6-phosphate is the precursor for starch synthesis in isolated cauliflower-bud amyloplasts.

Oxidation of Imported or Endogenous Carbohydrates by Isolated Chloroplasts from Green Pepper Fruits.

Recently, we demonstrated that intact chloroplasts isolated from green pepper (Capsicum annum L.) fruits use exogenous glucose-6-phosphate (Glc-6-P) as the most efficient precursor for starch biosynthesis (O. Batz, R. Scheibe, H.E. Neuhaus [1995] Planta 196: 50-57). Here we demonstrate that these chloroplasts transport this hexose phosphate in counter-exchange for orthophosphate. By measuring the release of 14CO2 from [1-14C]Glc-6-P, we show that isolated fruit chloroplasts also use exogenous Glc-6-P as a substrate for the oxidative pentose-phosphate pathway. The rate of decarboxylation appears to be linear with time and is significantly reduced in the presence of Triton X-100, indicating that the reaction is dependent on plastid integrity. Pyruvate has been identified as a positive effector for flux through the oxidative pentose-phosphate pathway. However, the highest rates of Glc-6-P-driven oxidative pentosephosphate pathway activity are achieved in the presence of nitrite, 2-oxoglutarate, and glutamine, indicating a strong interaction between nitrogen metabolism and this pathway. In addition, we show that carbohydrates liberated during starch mobilization are used as substrates for the oxidative pentose-phosphate pathway. Orthophosphate was found to act as an activator for the observed 14CO2 release from carbohydrates formerly bound as starch. In this context, we demonstrate that exogenous Glc-6-P competes with endogenous carbohydrates. A possible interaction between exogenous and endogenous carbohydrates is discussed with respect to altered levels of carbohydrates during fruit development.

Characterization of Glucose-6-Phosphate Incorporation into Starch by Isolated Intact Cauliflower-Bud Plastids.

Intact plastids from cauliflower (Brassica oleracea var Prince de Bretagne) buds were isolated according to the method described by Journet and Douce (E.P. Journet and R. Douce [1985] Plant Physiol 79: 458-467). Incubation of these plastids with various 14C-labeled compounds revealed that glucose-6-phosphate can act as a precursor for starch synthesis. However, significant rates (incorporation of 120 nmol glucose mg-1 protein h-1) could only be observed when both 3-phosphoglyceric acid and ATP were present as well. Starch synthesis in isolated plastids was strongly dependent upon the intactness of the organelle. The presence of a high-affinity ATP/ADP translocator with a Km for ATP of 12 [mu]M was demonstrated by uptake experiments with [14C]ATP. ADP inhibited both ATP uptake and effector-stimulated starch synthesis. Effector-stimulated glucose-6-phosphate-dependent starch synthesis was not significantly influenced by fructose-6-phosphate or 2-deoxyglucose-6-phosphate but was strongly inhibited by triose phosphate and inorganic phosphate. Starch synthesis was also inhibited by 4,4[prime]-diisothio-cyanostilbene-2,2[prime]-disulfonate, which is known to be a potent inhibitor of the chloroplast phosphate translocator. The data presented here support the view that starch biosynthesis in heterotrophic tissues is powered by increasing levels of cytosolic 3-phosphoglyceric acid and ATP when glucose-6-phosphate is available.

Induction of Hexose-Phosphate Translocator Activity in Spinach Chloroplasts.

Many environmental and experimental conditions lead to accumulation of carbohydrates in photosynthetic tissues. This situation is typically associated with major changes in the mRNA and protein complement of the cell, including metabolic repression of photosynthetic gene expression, which can be induced by feeding carbohydrates directly to leaves. In this study we examined the carbohydrate transport properties of chloroplasts isolated from spinach (Spinacia oleracea L.) leaves fed with glucose for several days. These chloroplasts contain large quantities of starch, can perform photosynthetic 3-phosphoglycerate reduction, and surprisingly also have the ability to perform starch synthesis from exogenous glucose-6-phosphate (Glc-6-P) both in the light and in darkness, similarly to heterotrophic plastids. Glucose-1-phosphate does not act as an exogenous precursor for starch synthesis. Light, ATP, and 3-phosphoglyceric acid stimulate Glc-6-P-dependent starch synthesis. Short-term uptake experiments indicate that a novel Glc-6-P-translocator capacity is present in the envelope membrane, exhibiting an apparent Km of 0.54 mM and a Vmax of 2.9 [mu]mol Glc-6-P mg-1 chlorophyll h-1. Similar results were obtained with chloroplasts isolated from glucose-fed potato leaves and from water-stressed spinach leaves. The generally held view that sugar phosphates transported by chloroplasts are confined to triose phosphates is not supported by these results. A physiological role for a Glc-6-P translocator in green plastids is presented with reference to the source/sink function of the leaf.

Characterisation of a concentrative type of adenosine transporter from Arabidopsis thaliana (ENT1,At).

Here we report on the isolation of an Arabidopsis thaliana cDNA that is able to complement a Saccharomyces cerevisiae mutant unable to synthesise adenine. This cDNA encodes a highly hydrophobic protein (ENT1,At) of 428 amino acids, showing high similarity to the human nucleoside transporter hENT1. Yeast cells expressing ENT1,At are able to grow on adenosine-containing media, adenosine import exhibited an apparent affinity (K(M)) of 3.6 microM, and led to accumulation of this nucleoside within the yeast cell. Transport is inhibited by various nucleosides. Typical inhibitors of ENT-type nucleoside transporters do not inhibit (3)H-adenosine import. The presence of protonophores abolished adenosine import, indicating that ENT1,At catalyse a proton-dependent adenosine transport. This is the first functional characterisation of a plant nucleoside transport protein.

Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants.

We have isolated an Arabidopsis thaliana cDNA encoding a highly hydrophobic membrane protein of 589 amino acids which contains 12 potential transmembrane helices and shows a high degree of similarity (43.5% identity, 66.2% similarity) to the ATP/ADP translocase of the Gram-negative bacterium Rickettsia prowazekii, an obligate intracellular parasite responsible for the epidemic typhus. This rickettsial translocator resides in the cytoplasmic membrane and allows the bacterium to exploit the host cytoplasmic ATP pool. We hypothesize that the A. thaliana homolog of the R. prowazekii ATP/ADP translocase is the functional eukaryotic equivalent and resides in the plastid inner envelope membrane where it functions as an ATP importer.

Identification of an Arabidopsis solute carrier critical for intracellular transport and inter-organ allocation of molybdate.

Plants represent an important source of molybdenum in the human diet. Recently, MOT1 has been identified as a transport protein responsible for molybdate import in Arabidopsis thaliana L.; however, the function of the homologous protein MOT2 has not been resolved. Interestingly, MOT2-GFP analysis indicated a vacuolar location of this carrier protein. By site directed mutagenesis at the N-terminal end of MOT2, we identified a di-leucine motif that is essential for driving the protein into the vacuolar membrane. Molybdate quantification in isolated vacuoles showed that this organelle serves as an important molybdate store in Arabidopsis cells. When grown on soil, leaves from mot2 T-DNA mutants contained more molybdate, whereas mot2 seeds contained significantly less molybdate than corresponding wild-type (Wt) tissues. Remarkably, MOT2 mRNA accumulates in senescing leaves and mot2 leaves from plants that had finished their life cycle had 15-fold higher molybdate levels than Wt leaves. Reintroduction of the endogenous MOT2 gene led to a Wt molybdate phenotype. Thus, mot2 mutants exhibit impaired inter-organ molybdate allocation. As total concentrations of the molybdenum cofactor (Moco) and its precursor MPT correlates with leaf molybdate levels, we present novel evidence for an adjustment of Moco biosynthesis in response to cellular MoO4²â» levels. We conclude that MOT2 is important for vacuolar molybdate export, an N-terminal di-leucine motif is critical for correct subcellular localisation of MOT2 and activity of this carrier is required for accumulation of molybdate in Arabidopsis seeds. MOT2 is a novel element in inter-organ translocation of an essential metal ion.

Starch degradation in chloroplasts isolated from C3 or CAM (crassulacean acid metabolism)-induced Mesembryanthemum crystallinum L.

C3 or crassulacean acid metabolism (CAM)-induced Mesembryanthemum crystallinum plants perform nocturnal starch degradation which is linear with time. To analyse the composition of metabolites released by isolated leaf chloroplasts during starch degradation we developed a protocol for the purification of starch-containing plastids. Isolated chloroplasts from C3 or CAM-induced M. crystallinum plants are also able to degrade starch. With respect to the endogenous starch content of isolated plastids the rate of starch degradation in intact leaves. The combined presence of Pi, ATP, and oxaloacetate is identified to be the most positive effector combination to induce starch mobilization. The metabolic flux through the oxidative pentose-phosphate pathway in chloroplasts isolated from CAM-induced M. crystallinum is less than 3.5% compared with other metabolic routes of starch degradation. Here we report that starch-degrading chloroplasts isolated from CAM-induced M. crystallinum plants use exogenously supplied oxaloacetate for the synthesis of malate. The main products of starch degradation exported into the incubation medium by these chloroplasts are glucose 6-phosphate, 3-phosphoglyceric acid, dihydroxyacetone phosphate and glucose. The identification of glucose 6-phosphate as an important metabolite released during starch degradation is in contrast to the observations made on all other types of plastids analysed so far, including chloroplasts isolated from M. crystallinum in the C3 state. Therefore, we analysed the transport properties of isolated chloroplasts from M. crystallinum. Surprisingly, both types of chloroplasts, isolated from either C3 or CAM-induced plants, are able to transport glucose 6-phosphate in counter exchange with endogenous Pi, indicating the presence of a glucose 6-phosphate translocator as recently demonstrated to occur in other types of plastids. The composition of metabolites released and the stimulatory effect of oxaloacetate on the rate of starch degradation are discussed with respect to the acidification observed for CAM leaves during the night.

Evidence for two types of phosphate translocators in sweet-pepper (Capsicum annum L.) fruit chromoplasts.

We have investigated whether there is evidence for the presence of different types of phosphate translocators in envelopes purified from pepper-fruit chromoplasts. A method was developed that allowed the purification of envelope membranes from isolated pepper-fruit chromoplasts. Proteoliposomes containing envelope-membrane proteins are able to import inorganic phosphate (P1) or glucose 6-phosphate (Glc6P). In both cases, the rate of import is strongly dependent upon preloading of proteoliposomes with either P1, dihydroxyacetone phosphate (DHAP) or Glc6P. This demonstrates the presence of a phosphate translocator activity catalysing a counter exchange of phosphorylated intermediates. Interestingly, a high external concentration of Glc6P does not strongly inhibit P1 uptake into proteoliposomes preloaded with DHAP, whereas external Glc6P strongly inhibits P1 uptake into proteoliposomes preloaded with Glc6P. This observation strongly indicates that two types of phosphate translocator are present in chromoplast envelopes from red-pepper fruits. These data are discussed with respect to the possible physiological function of two types of phosphate translocator in one type of plastid.

NONPHOTOSYNTHETIC METABOLISM IN PLASTIDS.

Nonphotosynthetic plastids are important sites for the biosynthesis of starch, fatty acids, and the assimilation of nitrogen into amino acids in a wide range of plant tissues. Unlike chloroplasts, all the metabolites for these processes have to be imported, or generated by oxidative metabolism within the organelle. The aim of this review is to summarize our present understanding of the anabolic pathways involved, the requirement for import of precursors from the cytosol, the provision of energy for biosynthesis, and the interaction between pathways that share common intermediates. We emphasize the temporal and developmental regulation of events, and the variation in mechanisms employed by different species that produce the same end products.

Purification of highly intact plastids from various heterotrophic plant tissues: analysis of enzymic equipment and precursor dependency for starch biosynthesis.

Starting with a protocol originally developed for the purification of intact plastids from cauliflower buds [Journet and Douce (1985) Plant Physiol. 79, 458-467] we have modified this method to obtain intact heterotrophic plastids from etiolated barley leaves (Hordeum vulgare) and pea (Pisum sativum) and maize (Zea mays) endosperm. Two subsequent centrifugation steps on Percoll gradients were performed, the first as an isopycnic, the second as zonal, centrifugation step in a swing-out rotor. Percoll density and centrifugation time were adjusted for the various tissues. The obtained plastid preparations are characterized by a low degree of contamination with other cellular components and an intactness of at least 90%. In isolated maize endosperm amyloplasts, starch synthesis is driven by exogenously applied hexose phosphates (glucose 6-phosphate and glucose 1-phosphate) rather than by dihydroxyacetone phosphate. The hexose-phosphate-dependent starch synthesis is strictly dependent upon the intactness of the plastids and is increased up to 9-fold when ATP and 3-phosphoglyceric acid are added to the incubation medium. The occurrence of fructose-1,6-bisphosphatase and malate dehydrogenases in some plastid types is discussed in relation to their possible role in starch synthesis.

Identification of the putative hexose-phosphate translocator of amyloplasts from cauliflower buds.

Starch synthesis in amyloplasts isolated from cauliflower buds is strongly inhibited by the addition of micromolar concentrations of 4,4'-di-isothiocyano-2,2'-stilbenedisulphonic acid (DIDS). Using [3H]DIDS it was possible to label specifically a 31.6 kDa membrane protein of the envelope fraction of isolated amyloplasts. The intensity of the radioactive label was decreased in the presence of glucose 6-phosphate or dihydroxyacetone phosphate, indicating that this protein might be the amyloplastic hexosephosphate translocator.

Analysis of carbohydrate transport across the envelope of isolated cauliflower-bud amyloplasts.

Using isolated amyloplasts from cauliflower buds, we have characterized the interaction and transport of various carbohydrates across the envelope membrane of a heterotrophic plastid. According to our results, glucose 6-phosphate (Glc6P) and glucose 1-phosphate (Glc1P) do not share the same transport protein for uptake into cauliflower-bud amyloplasts. Glc6P-dependent starch synthesis is strongly inhibited in the presence of dihydroxyacetone phosphate (DHAP) or 4,4'-di-isothiocyano-2,2'- stilbenedisulphonic acid (DIDS), whereas Glc1P-dependent starch synthesis is hardly affected by these compounds. Analysis of the Glc6P uptake into proteoliposomes reconstituted from the envelope proteins of cauliflower-bud amyloplasts indicate that Glc6P is taken up in a counter-exchange mode with Pi, DHAP or Glc6P, whereas Glc1P does not act as a counter-exchange substrate. Pi is a strong competitive inhibitor of Glc6P uptake (Ki 0.8 mM) into proteoliposomes, whereas Glc1P does not significantly inhibit Glc6P transport. Beside a hexose-phosphate translocator, these amyloplasts possess an envelope protein mediating the transport of glucose across the membrane. This translocator exhibits an apparent Km for glucose of 2.2 mM and is inhibited by low concentrations of phloretin, known to be a specific inhibitor of glucose-transport proteins. Maltose inhibits the uptake of glucose (Ki 2.3 mM), indicating that both carbohydrates share the same translocator.

Subcellular compartmentation of uridine nucleotides and nucleoside-5' -diphosphate kinase in leaves.

The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools.

The impact of decreased activity of starch-branching enzyme on photosynthetic starch synthesis in leaves of wrinkled-seeded peas.

The effect of a reduction of the activity of starch-branching enzyme (1,4-α-(D)-glucan, 1,4-α-(D)-glucan-6-glycosyl transferase; EC 2.4.1.18) on photosynthetic starch synthesis and photosynthate partitioning has been studied in leaves of pea (Pisum sativum L.). Leaves of wrinkled-seeded peas, recessive at the rugosus locus (rr), contained lower activity of branching enzyme than leaves of near-isogenic round-seeded peas, dominant at the rugosus locus (RR). Western blots showed that one isoform of the enzyme is absent from rr leaves, corresponding to the isoform that is absent from rr embryos. RR and rr leaves had identical rates of starch synthesis and photosynthesis at low irradiances. At high irradiances the rate of starch synthesis was decreased by up to 40% in rr relative to RR leaves. There was no corresponding increase of sucrose synthesis in rr leaves; instead, the rate of photosynthesis was decreased. This inhibition of photosynthesis was more marked at low than at high temperatures and was accompanied by increased oscillatory behaviour, rr leaves contained higher levels of ADP glucose and glycerate 3-phosphate than RR leaves in low and high light. The contribution of these results to our understanding of the distribution of control in the pathways of starch and sucrose synthesis is discussed.

Transport of Phosphoenolpyruvate by Chloroplasts from Mesembryanthemum crystallinum L. Exhibiting Crassulacean Acid Metabolism.

Chloroplasts from CAM-Mesembryanthemum crystallinum can transport phosphoenolpyruvate (PEP) across the envelope. The initial velocities of PEP uptake in the dark at 4 degrees C exhibited saturation kinetics with increasing external PEP concentration. PEP uptake had a V(max) of 6.46 (+/-0.05) micromoles per milligram chlorophyll per hour and an apparent K(mpep) of 0.148 (+/-0.004) millimolar. The uptake was competitively inhibited by Pi (apparent K(i) = 0.19 millimolar), by glycerate 3-phosphate (apparent K(i) = 0.13 millimolar), and by dihydroxyacetone phosphate, but malate and pyruvate were without effect. The chloroplasts were able to synthesize PEP when presented with pyruvate. PEP synthesis was light dependent. The prolonged synthesis and export of PEP from the chloroplasts required the presence of Pi or glycerate 3-phosphate in the external medium. It is suggested that the transport of pyruvate and PEP across the chloroplasts envelope is required during the gluconeogenic conversion of carbon from malate to storage carbohydrate in the light.

Transport Processes and Corresponding Changes in Metabolite Levels in Relation to Starch Synthesis in Barley (Hordeum vulgare L.) Etioplasts.

Intact etioplasts with an intactness of 85% and with a cytosolic and a mitochondrial contamination of less than 10% were isolated from 8-d-old dark-grown barley (Hordeum vulgare) leaves. These plastids contained starch equivalent to 21.5 mumol of glucose per mg protein. From various likely precursors applied to isolated etioplasts, only dihydroxyacetone phosphate (DHAP) had significant effects on metabolite levels and on the internal ATP/ADP ratio. The concentration dependence of DHAP uptake exhibited saturation characteristics with half saturation at 0.36 mm DHAP and a maximal velocity of 6.6 mumol mg(-1) of protein h(-1). The transport was significantly inhibited by inorganic phosphate, pyridoxal-5'-phosphate, and 4,4'-diisothiocyano-2,2'-stilbenedisulfonate. The rate of glucose-6-phosphate uptake was much lower and not saturable up to a concentration of 10 mm. Exogenously applied [(14)C]DHAP was incorporated into starch at a rate of 0.14 mumol of DHAP mg(-1) of protein h(-1). Enzyme activities required to convert DHAP into starch were found to be present in etioplasts. Furthermore, enzymes generating ATP from DHAP for ADPglucose synthesis were also detected. Finally, a scheme is presented suggesting DHAP uptake to serve both as carbon skeleton and as energy source for starch synthesis, mediated by a translocator with properties similar to those of the triose phosphate translocator from chloroplasts.