Inter- and intracellular distribution of amino acids and other metabolites in maize (Zea mays L.) leaves

In illuminated maize (Zea mays L.) leaves, the distribution of triose phosphates, 3-phosphoglycerate, malate and various amino acids between the chloroplastic and the extrachloroplastic compartments of mesophyll and bundle-sheath cells, and the total vacuolar fraction of the leaves, was determined by a combination of previously published methods, for separating mesophyll from bundle-sheath material, and for nonaqueous subcellular fractionation. The results show that the triose phosphate/3-phosphoglycerate ratio in the extrachloroplastic fraction of the mesophyll cells is about 20-fold higher than in the bundle-sheath cells, which is in accordance with a triose phosphate/phosphoglycerate shuttle postulated previously. Whereas the vacuolar compartment was shown to contain most of the cellular malate, amino acids were found to be almost absent from this compartment. The amino-acid pattern in the extrachloroplastic fraction of the bundle-sheath cells largely resembled the pattern in whole leaves. These results show that for future studies the analysis of amino-acid contents in whole maize leaves can be used as a measure for the amino-acid levels in the cytosol of bundle-sheath cells.Abbreviations BS bundle sheath - Chl chlorophyll - Man -mannosidase - ME malic enzyme - MDH malate dehydrogenase - MS mesophyll - PEPCase phosphoenolpyruvate carboxylase - PGA 3-phosphoglycerate - trioseP triose phosphate This work was supported by the Bundesminister für Forschung und Technologie.     

Changes in activity of some enzymes involved in glucose utilization and formation in developing rat liver

1. The activities of some enzymes involved in both the utilization of glucose (pyruvate kinase, ATP citrate lyase, NADP-specific malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-specific isocitrate dehydrogenase, all present in the supernatant fraction of liver homogenates) and the formation of glucose by gluconeogenesis (glucose 6-phosphatase in the whole homogenate and fructose 1,6-diphosphatase, phosphopyruvate carboxylase, NAD-specific malate dehydrogenase and fumarase in the supernatant fraction) have been determined in rat liver around birth and in the postnatal period until the end of weaning. 2. The activities of those enzymes involved in the conversion of glucose into lipid are low during the neonatal period and increase with weaning. NADP-specific malate dehydrogenase first appears and develops at the beginning of the weaning period. 3. The marked increase in cytoplasmic phosphopyruvate carboxylase activity at birth is probably the major factor initiating gluconeogenesis at that time. 4. The results are discussed against the known changes in dietary supplies and the known metabolic patterns during the period of development.     

Lipid peroxidation and the degradation of cytochrome P-450 heme

The enzyme content and functional capacities of mesophyll chloroplasts from Atriplex spongiosa and maize have been investigated. Accompanying evidence from graded sequential blending of leaves confirmed that mesophyll cells contain all of the leaf pyruvate, P_i dikinase, and PEP carboxylase activities and a major part of the adenylate kinase and pyrophosphatase. 3-Phosphoglycerate kinase, NADP glyceraldehyde-3-P-dehydrogenase, and triose-P isomerase activities were about equally distributed between mesophyll and bundle sheath cells but other Calvin cycle enzymes were very largely or solely located in bundle sheath cells. In A. spongiosa extracts of predominantly mesophyll origin the proportion of the released pyruvate, P_i dikinase, adenylate kinase, pyrophosphatase, 3-phosphoglycerate kinase, and NADP glyceraldehyde-3-P dehydrogenase retained in pelleted chloroplasts was similar but varied between 30 and 80% in different preparations. The proportion of these enzymes and NADP malate dehydrogenase recovered in maize chloroplast preparations varied between 15 and 35%. Washed chloroplasts retained most of the activity of these enzymes but ribulose diphosphate carboxylase and other Calvin cycle enzyme activities were undetectable. Among the evidence for the integrity of these chloroplasts was their capacity for light-dependent conversion of pyruvate to phosphoenolpyruvate and O₂ evolution when 3-phosphoglycerate or oxaloacetate were added. These results support our previous conclusions about the function of mesophyll chloroplasts in C₄-pathway photosynthesis and clearly demonstrate that they lack Calvin cycle activity.     

Metabolic Activities in Extracts of Mesophyll and Bundle Sheath Cells of Panicum miliaceum (L.) in Relation to the C(4) Dicarboxylic Acid Pathway of Photosynthesis

The activities of certain enzymes related to the carbon assimilation pathway in whole leaves, mesophyll cell extracts, and bundle sheath extracts of the C₄ plant Panicum miliaceum have been measured and compared on a chlorophyll basis. Enzymes of the C₄ dicarboxylic acid pathway—phosphoenolpyruvate carboxylase and NADP-malic dehydrogenase—were localized in mesophyll cells. Carbonic anhydrase was also localized in mesophyll cell extracts. Ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, and ribulose diphosphate carboxylase—enzymes of the reductive pentose phosphate pathway—were predominantly localized in bundle sheath extracts. High activities of aspartate and alanine transaminases and glyceraldehyde-3-P dehydrogenase were found about equally distributed between the photosynthetic cell types. P. miliaceum had low malic enzyme activity in both mesophyll and bundle sheath extracts.     

Iron deficiency and the structure and physiology of maize chloroplasts

The ultrastructure of mesophyll chloroplasts of maize (Zea mays L.) was more severely affected by iron deficiency that induced mild chlorosis than was the ultrastructure of bundle sheath plastids. Ferredoxin and ribulose diphosphate carboxylase levels were severely decreased by iron deficiency. Malic enzyme was less affected, and phosphoenolpyruvate carboxylase activity remained high even under severe iron deficiency. Iron deficient leaves fixed carbon into malic and aspartic acids but the rate of entrance of carbon into the sugar phosphates and sucrose was greatly reduced compared to the control. Chlorophyll a/b ratios ranged from low values of less than 2 in severely iron deficient leaves to high values exceeding 4 in leaves showing little iron deficiency.     

The argentia mutation delays normal development of photosynthetic cell-types in Zea mays

Photosynthesis in C4-type grasses such as maize involves the interaction of two cell types (bundle sheath (BS) and mesophyll (M)) which both contain cell-specific photosynthetic enzymes. Malate dehydrogenase, phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase are located in the M cells and malic enzyme and ribulose bisphosphate carboxylase are found in the BS cells. We have studied photosynthetic development in leaves of the temperature-sensitive greening mutant argentia (ar). We have determined that with the exception of malate dehydrogenase, levels of C4 enzymes are lower in ar leaves than in normal leaves. Malate dehydrogenase accumulates identically in both. Using in situ immunolocalization techniques with normal and ar leaves, we have observed a developmental pattern of C4 protein accumulation. In normal leaves protein was detected first in cells surrounding the median vein, then in cells surrounding other major veins, and finally in cells surrounding minor veins. In ar leaves, C4 enzymes accumulate in the correct cell type and in this same order but their appearance is delayed. Furthermore, BS cell development is delayed with respect to M cell development. The observed pattern of photosynthetic development reflects an earlier manifested pattern of vascular development yet the timing of vascular differentiation in ar mutants appears to be normal.     

Role of phosphoenolpyruvate carboxylation in Acetobacter xylinum

Glucose-grown cells of Acetobacter xylinum oxidized acetate only when the reaction mixture was supplemented with catalytic quantities of glucose or intermediates of the citrate cycle. Extracts, prepared by sonic treatment, catalyzed the formation of oxalacetate when incubated with phosphoenolpyruvate (PEP) and bicarbonate. Oxalacetate was not formed in the presence of pyruvate plus adenosine triphosphate. The ability to promote carboxylation of PEP was lower in succinate-grown cells than in glucose-grown cells. PEP carboxylase, partially purified from extracts by ammonium sulfate fractionation, catalyzed the stoichiometric formation of oxalacetate and inorganic phosphate from PEP and bicarbonate. The enzyme was not affected by acetyl-coenzyme A or inorganic phosphate. It was inhibited by adenosine diphosphate in a manner competitive with PEP (K(1) = 1.3 mm) and by dicarboxylic acids of the citrate cycle; of these, succinate was the most potent inhibitor. It is suggested that the physiological role of PEP carboxylase in A. xylinum is to affect the net formation of C(4) acids from C(3) precursors, which are essential for the maintainance of the citrate cycle during growth on glucose. The relationship of PEP carboxylase to other enzyme systems metabolizing PEP and oxalacetate in A. xylinum is discussed.     

Correlations between photosynthetic enzymes,CO2-fixation and plastid structure in an albino mutant of Zea mays L.

Summary An albino seedling of Zea mays L. was investigated for its potential for CO₂-assimilation. In the mesophyll the number, dimensions and fine structure of chloroplasts are drastically reduced but to a lesser extent in the bundle sheath. Chlorophyll concentration is zero and carotenoid concentration almost zero. Albinism also exerts a strong influence on the stroma of bundle sheath chloroplasts; ribulose-1.5-biphosphate carboxylase (EC 4.1.1.39) activity and glyceraldehyde-3-phosphate dehydrogenase (NADP) (EC 1.2.1.13) activity is not detectable. The C₄-enzymes phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (decarboxylating) (EC 1.1.1.40) and the non-photosynthetic linked enzymes malate dehydrogenase (NAD) (EC 1.1.1.37), aspartate-2-oxoglutarate aminotransferase (EC 1.1.1.37), aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.1.) and glyceraldehyde-3-phosphate dehydrogenase (NAD) (EC 1.2.1.1.) are present in the albino seedling with activities comparable to those in etiolated maize seedlings. The potential for CO₂ fixation of the albino seedlings exceeds that of comparable dark seedlings considerably. The results are discussed with regard to enzyme localization of the C₄ pathway of photosynthesis.Abbreviations Aspartate aminotransferase L-aspartate-2-oxoglutarate aminotransferase-EC 2.6.1.1. - GAPDH (NAD) glyceraldehyde-3-phosphate dehydrogenase (NAD dep.)-EC 1.2.1.12 - GAPDH (NADP) glyceraldehyde-3-phosphate dehydrogenase (NADP dep.)-EC 1.2.1.13 - malic enzyme malate dehydrogenase (NADP dep., decarboxylating)-EC 1.1.1.40 - MDH malate dehydrogenase (NAD dep.)-1.1.1.37 - PEP carboxylase phosphoenolpyruvate carboxylase-EC 4.1.1.31 - RuDP carboxylase ribulose-1.5-biphosphate carboxylase-EC 4.1.1.39     

The C4-dicarboxylic acid pathway of photosynthesis. Identification of intermediates and products and quantitative evidence for the route of carbon flow

1. When leaves with the C(4)-dicarboxylic acid pathway of photosynthesis are exposed to (14)CO(2) the major labelled compounds formed, in order of labelling, are dicarboxylic acids, 3-phosphoglycerate, bexose phosphates and sucrose. During the present studies several quantitatively minor intermediates were identified and their labelling behaviour is described. 2. The pattern of labelling of dihydroxyacetone phosphate, fructose 1,6-diphosphate and ribulose di- and mono-phosphates during radiotracer pulse-chase experiments was consistent with their operation as intermediates in the pathway of carbon dioxide fixation. 3. Serine, glycine, alanine and glutamate had labelling patterns typical of products secondary to the main flow of carbon. 4. The mechanism of the transfer of label from C-4 of dicarboxylic acids to C-1 of 3-phosphoglycerate was also examined. Evidence consistent with pyruvate being derived from C-1, C-2 and C-3 of oxaloacetate, and for a relationship between ribulose 1,5-diphosphate and the acceptor for the C-4 carboxyl group, was obtained. 5. Evidence is provided that, under steady-state conditions, essentially all the label incorporated from (14)CO(2) into C-1 of 3 phosphoglycerate enters via C-4 of the dicarboxylic acids. These and other studies indicated that the route via dicarboxylic acids is essentially the sole route for entry of carbon into 3-phosphoglycerate.     

In situ immunofluorescent localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in mesophyll of C4 dicotyledonous plants

The location of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) in the leaf mesophyll of some dicotyledonous C4 plants was confirmed by immunofluorescent labelling. The anti-RuBPCO immune serum was obtained by inoculating a rabbit with commercially obtained RuBPCO. Specificity of these antibodies was tested by immunodiffusion, immunoelectrophoresis, and Western blotting. Fresh hand-cuts of leaves from dicotyledonous C4 plants, Amaranthus caudatus, A. dubius, Gomphrena globosa, and Portulaca oleracea, were incubated with the conjugated anti-RuBPCO immune serum and then with a commercial FITC-anti-rabbit IgG conjugate. Nerium oleander was used a control C3 plant pattern and Zea mays as a C4 plant pattern. The immunofluorescent label was distributed in both mesophyll and bundle sheath in all the C4 plants tested. It is an unequivocal proof that in the C4 dicotyledonous plants the RuBPCO is not only located in the chloroplasts of the bundle sheath cells but also in the chloroplasts of the mesophyll cells. In these plants therefore, the C4 pathway cannot exclusively be viewed as an intercellular level concentration mechanism. In the mesophyll cytoplasm, phosphoenolpyruvate carboxylase traps CO2, while in the mesophyll chloroplasts, RuBPCO operates with atmospheric CO2 and CO2 from the C4 decarboxylation step at an intracellular level, which could mean a significant energetic economy. The CO2 from photorespiration could be saved and reincorporated. Location of RuBPCO in the mesophyll and/or bundle sheath chloroplasts is a matter of inter- and intracellular compartmentation which makes another variation of C4 photosynthetic pathway possible. This revised version was published online in September 2006 with corrections to the Cover Date.     

Changes in photosynthetic carbon flow in transgenic rice plants that express C4-type phosphoenolpyruvate carboxykinase from Urochloa panicoides

A cDNA encoding phosphoenolpyruvate carboxykinase (PCK) of Urochloa panicoides (a PCK-type C4 plant) was expressed in rice (Oryza sativa cv Tsukinohikari) plants under the control of the promoter of a maize (Zea mays) gene for phosphoenolpyruvate carboxylase or pyruvate, orthophosphate dikinase with the transit peptide of the small subunit of Rubisco. Crude extracts prepared from the green leaves of transgenic plants had high PCK activity and the newly expressed PCK was localized in chloroplasts. In labeling experiments with (14)CO(2) up to 20% of the radioactivity was incorporated into 4C compounds (malate, oxaloacetate, and aspartate) in excised leaves of transgenic plants, as compared with about 1% in excised leaves of control plants. There was a positive correlation between PCK activity and the extent of labeling of 4C compounds. When L-[4-(14)C]malate was fed to excised leaves the extent of incorporation of radioactivity into sucrose was 3-fold greater in transgenic plants than in control plants and the level of radiolabeled aspartate was significantly lower in transgenic plants. These results indicate that the ectopic expression of PCK in rice chloroplasts was able partially to change the carbon flow in mesophyll cells into a C4-like photosynthetic pathway. Such a strategy appears to provide a possible method for enhancing the photosynthetic capacity of C3 plants.     

Photosynthetic Metabolism of Aspartate in Mesophyll and Bundle Sheath Cells Isolated from Digitaria sanguinalis (L.) Scop., a NADP-Malic Enzyme C(4) Plant

Mesophyll cells and bundle sheath strands isolated from leaves of the C(4) plant Digitaria sanguinalis (L.) Scop. are capable of utilizing aspartate as a Hill oxidant. The resulting O(2) evolution upon illumination depends on the presence of 2-oxoglutarate, is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and is stimulated by methylamine. The rate of aspartate-dependent O(2) evolution with mesophyll cells was similar to those with phosphoenolpyruvate + CO(2) or with oxalacetate. Amino-oxyacetate, an inhibitor of aspartate aminotransferase, inhibited the aspartate-dependent O(2) evolution. Aspartate aminotransferase and NADP(+) -malate dehydrogenase are located in the mesophyll chloroplasts. These data suggest that aspartate is converted to oxalacetate via aspartate aminotransferase in the chloroplasts of mesophyll cells and that oxalacetate is subsequently reduced to malate, which is coupled to the photochemical evolution of O(2). This suggestion is further verified by the inhibition of phosphoenolpyruvate-dependent (14)CO(2) fixation by aspartate + 2-oxoglutarate, which presumably acts as oxalacetate and competes with phosphoenolpyruvate + CO(2) for NADPH. dl-Glyceraldehyde inhibited aspartate-dependent O(2) evolution in the bundle sheath strands but not in the mesophyll cells. The data indicate that aspartate may be converted to malate in both mesophyll and bundle sheath cells. In NADP(+) -malic enzyme species, aspartate may exist as a C(4)-dicarboxylic acid reservoir which can contribute to the C(4) cycle through its conversion to malate.     

Differential Localization of Fraction I Protein between Chloroplast Types

The soluble proteins of C₃ and C₄ mesophyll chloroplasts and C₄ bundle sheath extracts have been analyzed by gel electrophoresis for fraction I protein. Gel scans of soluble protein from C₄ bundle sheath extracts and C₃ mesophyll chloroplasts showed typical fraction I protein peaks that could be identified by ribulose diphosphate carboxylase activity. No such peak was observed for C₄ mesophyll chloroplasts, which also lacked both large and small subunits of ribulose diphosphate carboxylase on sodium dodecyl sulfate gels. The absence of fraction I protein in these chloroplasts was reflected in the soluble protein to chlorophyll ratios, which were roughly 3-fold lower than the ratio obtained for C₃ chloroplasts. The carboxylating enzyme in C₄ mesophyll cells, phosphoenolpyruvate carboxylase, was found to be a major protein in the cytoplasm of C₄ mesophyll protoplasts, and had higher mobility than fraction I protein.     

The effect of dietary and hormonal conditions on the activities of glycolytic enzymes in rat epididymal adipose tissue

1. Measurements were made of the activities of nine glycolytic enzymes in epididymal adipose tissues obtained from rats that had undergone one of the following treatments: starvation; starvation followed by re-feeding with bread or high-fat diet; feeding with fat without preliminary starvation; alloxan-diabetes; alloxan-diabetes followed by insulin therapy. 2. In general, the activities of the glycolytic enzymes of adipose tissue, unlike those of liver, were not greatly affected by the above treatments. 3. The ;key' glycolytic enzymes, phosphofructokinase and pyruvate kinase, were generally no more adaptive in response to physiological factors than other glycolytic enzymes such as glucose phosphate isomerase, fructose diphosphate aldolase, triose phosphate isomerase, glycerol 3-phosphate dehydrogenase, phosphoglycerate kinase and lactate dehydrogenase. 4. Adiposetissue pyruvate kinase did not respond to feeding with fat in a manner similar to the liver enzyme. 5. Glyceraldehyde phosphate dehydrogenase had a behaviour pattern unlike the other eight glycolytic enzymes studied in that its activity was depressed by feeding with fat and was not restored to normal by re-feeding with a high-fat diet after starvation. These results are discussed in relation to the requirements of adipose tissue for glycerol phosphate in the esterification of fatty acids. 6. A statistical analysis of the results permitted the writing of linear equations describing the relationships between the activities of eight of the enzymes studied. 7. Evidence is presented for the existence of two constant-proportion groups amongst the enzymes studied, namely (i) glucose phosphate isomerase, phosphoglycerate kinase and lactate dehydrogenase, and (ii) triose phosphate isomerase, fructose diphosphate aldolase and pyruvate kinase. 8. Mechanisms for maintaining the observed relationships between the activities of the enzymes in the tissue are discussed.     

Variation with age in the photosynthetic carbon fixation pattern by leaves of Amaranthus paniculatus and Oryza sativa: Change in the primary carboxylation but no shift from C4 or C3 pathway

Abstract The pattern of photosynthetic carbon fixation by leaves of Amaranthus paniculatus L. (a C₄ plant) and Oryza sativa L. (a C₃ plant) varied with age. Younger leaves of A. paniculatus incorporated ¹⁴CO₂ into malate and aspartate while senescent leaves fixed predominantly into phosphoglycerate (PGA) and sugar phosphates. Only developing leaves of O. sativa formed malate/aspartate whereas mature and senescent leaves produced PGA/sugar phosphates as the initial labelled products. Correspondingly the ratio of phosphoenolpyruvate/ribulose bisphosphate (RuBP) carboxylase activities was higher in younger leaves of A. paniculatus and developing leaves of O. sativa than in older leaves. However, pulse chase experiments revealed that the main donors of carbon to end products, irrespective of leaf stage, were C₄ acids and PGA in A. paniculatus and O. sativa respectively. The results suggest that although an apparent change from initial β-carboxylation to RuBP carboxylation occurs during leaf ontogeny in both the plants, the overall leaf photosynthesis remains C₄ or C₃. The high rate of ¹⁴CO₂ incorporation into PGA/sugar phosphates by senescent leaves of A. paniculatus is suggested to be partly due to the increased intercellular spaces in their mesophyll, allowing greater access of CO₂ directly to RuBP carboxylase in the bundle sheath.     

Subcellular Volumes and Metabolite Concentrations in Potato (Solanum tuberosum cv. Désirée) Leaves1

Cellular and subcellular volumes in mature leaves of potato (Solanum tuberosum cv. Désirée) were determined stereologically from light and electron micrographs. Leaves of ten-week-old plants with a total leaf volume of 623 μL per mg chlorophyll (Chl) were found to be composed of 12 % epidermis, 68 % mesophyll, 5 % vascular tissue, 3 % apoplast and 16 % gas space. In the epidermal cells 97 % of the volume was occupied by the vacuole. The mesophyll cells consisted (as expressed per mg Chl) of 323 μL (76 %) vacuole, 35 μL (8 %) chloroplast stroma, 22 μL (5 %) cytosol plus nucleus and peroxisomes, and 4μL, (1 %) mitochondria. A comparison of these values with subcellular volumes previously determined for spinach and barley leaves, shows that the relative sizes of the subcellular volumes are strikingly similar. Subcellular concentrations of carbohydrates, of the phosphorylated intermediates of carbohydrate metabolism, of malate, and of amino acids have been evaluated from measurements of the corresponding subcellular metabolite contents determined using the non-aqueous fractionation technique. Malate, glucose and fructose were found to accumulate in the vacuole, whereas the concentration of sucrose and amino acids in the cytosol were much higher than in the vacuole. The amino acid concentration in the chloroplast stroma is similar to that in the cytosol. Phosphorylated intermediates of carbohydrate metabolism are confined to the chloroplast stroma and the cytosol, confirming the validity of the fractionation method. Whereas triose phosphates and fructose-l,6-bisphosphate are concentrated in the stroma, the concentrations of hexose monophosphates were highest in the cytosol. Since the subcellular metabolite distribution in potato leaves reported here is very similar to that previously described for spinach and barley leaves, we conclude that it may be characteristic for mesophyll cells in general.     

Properties of an inducible C 4 -dicarboxylic acid transport system in Bacillus subtilis

The transport of the tricarboxylic acid cycle C(4)-dicarboxylic acids was studied in both the wild-type strain and tricarboxylic acid cycle mutants of Bacillus subtilis. Active transport of malate, fumarate, and succinate was found to be inducible by these dicarboxylic acids or by precursors to them, whereas glucose or closely related metabolites catabolite-repressed their uptake. l-Malate was found to be the best dicarboxylic acid transport inducer in succinic dehydrogenase, fumarase, and malic dehydrogenase mutants. Succinate and fumarate are accumulated over 100-fold in succinic dehydrogenase and fumarase mutants, respectively, whereas mutants lacking malate dehydrogenase were unable to accumulate significant quantities of the C(4)-dicarboxylic acids. The stereospecificity of this transport system was studied from a comparison of the rates of competitive inhibition of both succinate uptake and efflux in a succinate dehydrogenase mutant by utilizing thirty dicarboxylic acid analogues. The system was specific for the C(4)-dicarboxylic acids of the tricarboxylic acid cycle, neither citrate nor alpha-ketoglutarate were effective competitive inhibitors. Of a wide variety of metabolic inhibitors tested, inhibiors of oxidative phosphorylation and of the formation of proton gradients were the most potent inhibitors of transport. From the kinetics of dicarboxylic acid transport (K(m) approximately 10(-4) M for succinate or fumarate in succinic acid dehydrogenase and fumarase mutants) and from the competitive inhibition studies, it was concluded that an inducible dicarboxylic acid transport system mediates the entry of malate, fumarate, or succinate into B. subtilis. Mutants devoid of alpha-ketoglutarate dehydrogenase were shown to accumulate both alpha-ketoglutarate and glutamate, and these metabolites subsequently inhibited the transport of all the C(4)-dicarboxylic acids, suggesting a regulatory role.     

Generation and maintenance of concentration gradients between the mesophyll and bundle sheath in maize leaves

(1) Experiments have been carried out to test the proposal that intercellular transport of carbon occurs by diffusion during photosynthesis in C-4 plants. (2) The intercellular distribution of metabolites has been compared in different conditions. A partial separation of the mesophyll and bundle sheath was obtained by homogenisation in liquid N₂, followed by filtration through nylon nets with differing aperture. (3) Concentration gradients between the bundle sheath and mesophyll were found for 3-phosphoglycerate, triose phosphates, malate and pyruvate during photosynthesis. These gradients are shown to be large enough to allow rapid intercellular transport by diffusion. They disappear when photosynthesis is prevented by removal of light or CO₂. (4) The concentration gradients for triose phosphates and 3-phosphoglycerate are due to the differing capacity of the bundle sheath and mesophyll to reduce 3-phosphoglycerate. (5) The distribution of carbon between the malate/pyruvate and 3-phosphoglycerate/triose phosphate shuttles is flexible, and may be controlled by phosphoenolpyruvate carboxylase. (6) The maintenance of these large concentration gradients has consequences for the regulation of sucrose synthesis and the Calvin cycle.     

Pyridine and Adenine Nucleotide Status, and Pool Sizes of a Range of Metabolites in Chloroplasts, Mitochondria and the Cytosol/Vacuole of Avena Mesophyll Protoplasts during Dark/Light Transition: Effect of Pyridoxal Phosphate

Leaf mesophyll protoplasts of Avena sativa L. underwent dark/lighttransition in the absence or presence of pyridoxal phosphate(PLP), a specific inhibitor of the phosphate translocator ofchloroplasts. By combining rapid fractionation of protoplastswith enzymatic cycling, the contents of metabolites (adeninenucleotides, pyridine nucleotides, triose phosphates, 3-phosphoglycerate,inorganic phosphate, aspartate, malate, oxaloacetate, glutamate,2-oxoglutarate and citrate) associated with the chloroplasts,mitochondria and cytosol/vacuole were determined. Fluctuationsof metabolite pools occurred in all compartments on illumination.Mitochondria showed immediate inhibition of their tricarboxylicacid cycle activities, as indicated by a transiently increasedNADH/NAD⁺ ratio and elevated malate contents within 60 s ofillumination. During this period large transient increases intriose phosphates took place in all fractions. Incubation of intact protoplasts with PLP severely affectedthe compartmented pool sizes, producing the typical patternof inhibition of the chloroplast phosphate translocator. Themitochondrial pool sizes of the metabolites responded to light,if at all, differently than did the controls. ¹ Dedicated to Prof. Hubert Ziegler on the occasion of his 60thbirthday. (Received July 23, 1984; Accepted October 19, 1984)     

Adaptation responses in C4 photosynthesis of maize under salinity

The effect of salinity on C(4) photosynthesis was examined in leaves of maize, a NADP-malic enzyme (NADP-ME) type C(4) species. Potted plants with the fourth leaf blade fully developed were treated with 3% NaCl solution for 5d. Under salt treatment, the activities of pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent malate dehydrogenase (NADP-MDH) and NAD-dependent malate dehydrogenase (NAD-MDH), which are derived mainly from mesophyll cells, increased, whereas those of NADP-ME and ribulose-1,5-bisphosphate carboxylase, which are derived mainly from bundle sheath cells (BSCs), decreased. Immunocytochemical studies by electron microscopy revealed that PPDK protein increased, while the content of ribulose-1,5-bisphosphate carboxylase/oxygenase protein decreased under salinity. In salt-treated plants, the photosynthetic metabolites malate, pyruvate and starch decreased by 40, 89 and 81%, respectively. Gas-exchange analysis revealed that the net photosynthetic rate, the transpiration rate, stomatal conductance (g(s)) and the intercellular CO(2) concentration decreased strongly in salt-treated plants. The carbon isotope ratio (δ(13)C) in these plants was significantly lower than that in control. These findings suggest that the decrease in photosynthetic metabolites under salinity was induced by a reduction in gas-exchange. Moreover, in addition to the decrease in g(s), the decrease in enzyme activities in BSCs was responsible for the decline of C(4) photosynthesis. The increase of PPDK, PEPCase, NADP-MDH, and NAD-MDH activities and the decrease of NADP-ME activity are interpreted as adaptation responses to salinity.