Plant Physiology

Lovatt, Carol J.; Cheng, Anne H.

doi: 10.1104/pp.75.3.511pmid: 16663656

Abstract Lovatt et al. (1979 Plant Physiol 64: 562-569) have previously demonstrated that end-product inhibition functions as a mechanism regulating the activity of the orotic acid pathway in intact cells of roots excised from 2-day-old squash plants (Cucurbita pepo L. cv Early Prolific Straightneck). Uridine (0.5 millimolar final concentration) or one of its metabolites inhibited the incorporation of NaH14CO3, but not [14C]carbamylaspartate or [14C]orotic acid, into uridine nucleotides (ΣUMP). Thus, regulation of de novo pyrimidine biosynthesis was demonstrated to occur at one or both of the first two reactions of the orotic acid pathway, those catalyzed by carbamylphosphate synthetase (CPSase) and aspartate carbamyltransferase (ACTase). The results of the present study provide evidence that ACTase alone is the site of feedback control by added uridine or one of its metabolites. Evidence demonstrating regulation of the orotic acid pathway by end-product inhibition at ACTase, but not at CPSase, includes the following observations: (a) addition of uridine (0.5 millimolar final concentration) inhibited the incorporation of NaH14CO3 into ΣUMP by 80% but did not inhibit the incorporation of NaH14CO3 into arginine; (b) inhibition of the orotate pathway by added uridine was not reversed by supplying exogenous ornithine (5 millimolar final concentration), while the incorporation of NaH14CO3 into arginine was stimulated more than 15-fold when both uridine and ornithine were added; (c) incorporation of NaH14CO3 into arginine increased, with or without added ornithine when the de novo pyrimidine pathway was inhibited by added uridine; and (d) in assays employing cell-free extracts prepared from 2-day-old squash roots, the activity of ACTase, but not CPSase, was inhibited by added pyrimidine nucleotides. 1 Supported by the Citrus Research Center and Agricultural Experiment Station of the University of California, Riverside, California. This content is only available as a PDF. © 1984 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Beudeker, Rob F.; Tabita, F. Robert

doi: 10.1104/pp.75.3.516pmid: 16663657

Abstract The utilization of nitrate and ammonia as nitrogen sources had different effects on the metabolism of glycolate in Cholorella sorokiniana. During photolithotrophic growth with nitrate as nitrogen source, glycolate was metabolized via the glycine-serine pathway. Ammonia, produced as a result of glycolate metabolism, was reassimilated by glutamine synthetase. Two isoforms of this enzyme were present at different relative abundance in C. sorokiniana wild type and in a mutant with an increased capacity for the metabolism of glycolate (strain OR). During photolithotrophic growth in the presence of ammonia as sole nitrogen source, several lines of evidence indicated that glycolate was metabolized to malate, pyruvate, tricarboxylic acid cycle intermediates and related amino acids in C. sorokiniana wild-type cells. Malate synthase was induced and glycine decarboxylase and serine-glyoxylate aminotransferase were repressed in cells grown with ammonia. An inverse correlation was observed between aminating NADPH-glutamate dehydrogenase and the in vivo glycine decarboxylation rate. 2 Present address: R. F. Beudeker, Gist-Brocades, Section FOG, 2600 MA Delft, The Netherlands. 1 Supported by Dow Chemical Company. A preliminary account of these investigations was presented at the Third International Symposium on Microbial Growth of C1 Compounds, held in Minneapolis from September 6-10, 1983. This content is only available as a PDF. © 1984 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Valenti, Vincenzo; Stanghellini, Maria A.; Pupillo, Paolo

doi: 10.1104/pp.75.3.521pmid: 16663658

Abstract Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP+ to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP+. The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves. Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K m of NADP+ being 2.5-fold higher for pI 4.3 form. Free ATP (K m = 0.64 millimolar) and ADP (K m = 1.13 millimolar) act as competitive inhibitors with respect to NADP+ in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition. 1 Supported by grants of the Consiglio Nazionale delle Ricerche (Rome) for years 1980 and 1981. This content is only available as a PDF. © 1984 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Ta, Trung Chanh; Joy, Kenneth W.; Ireland, Robert J.

doi: 10.1104/pp.75.3.527pmid: 16663659

Abstract The fate of nitrogen originating from the amide group of asparagine in young pea leaves (Pisum sativum) has been studied by supplying [15N-amide]asparagine and its metabolic product, 2-hydroxysuccinamate (HSA) via the transpiration stream. Amide nitrogen from asparagine accumulated predominantly in the amide group of glutamine and HSA, and to a lesser extent in glutamate and a range of other amino acids. Treatment with 5-diazo,4-oxo-L-norvaline (DONV) a deamidase inhibitor, caused a decrease in transfer of label to glutamine-amide. Virtually no 15N was detected in HSA of leaves supplied with asparagine and the transaminase inhibitor aminooxyacetate. When [15N]HSA was supplied to pea leaves, most of the label was also found in the amide group of glutamine and this transfer was blocked by the addition of methionine sulfoximine, which caused a large increase in NH3 accumulation. DONV was not specific for asparaginase, and inhibited the deamidation of HSA, causing a decrease in transfer of 15N into glutamine-amide, NH3, and other amino acids. It is concluded from these results that use of the amide group of asparagine as a nitrogen source for young pea leaves involves deamidation of both asparagine and its transamination product HSA (possibly also oxosuccinamate). The amide group, released as ammonia, is then reassimilated via the glutamine synthetase/glutamate synthase system. 1 Supported by grants from Natural Sciences and Engineering Research Council of Canada to K. W. J. and R. J. I. This content is only available as a PDF. © 1984 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Stabenau, Helmut; Winkler, U.; Säftel, W.

doi: 10.1104/pp.75.3.531pmid: 16663660

Abstract The algae Mougeotia and Eremosphaera were used for isolation of microbodies with the characteristics of leaf peroxisomes and unspecialized peroxisomes, respectively. In both types of organelles, the following enzymes of the β-oxidation pathway were determined: acyl-CoA oxido-reductase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase. There are indications that the peroxisomal oxidoreductase of both algae is a H2O2-forming oxidase rather than a dehydrogenase. The enzymes enoyl-CoA hydratase and acyl-CoA oxidoreductase are located also in the mitochondria from Eremosphaera but not from Mougeotia. The mitochondrial acyl-CoA oxidizing enzyme was found to be a dehydrogenase. The specific activities of acyl-CoA oxidase and enoyl-CoA hydratase are lower than in spinach leaf peroxisomes. However, the activity of 3-hydroxyacyl-CoA dehydrogenase in the peroxisomes of both algae is almost 2-fold higher. The capability for degradation of fatty acids is a common feature of all different types of peroxisomes from algae. 1 Present address: Universität Oldenburg, Fachbereich Biologie, Postfach 2503, D-2900 Oldenburg, West Germany. This content is only available as a PDF. © 1984 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Quader, Hartmut

doi: 10.1104/pp.75.3.534pmid: 16663661

Abstract The effect of tunicamycin (TM) on the development of the cell wall in Oocystis solitaria has been investigated. It was found that 10 micromolar TM completely stops the assembly of new microfibrils as observed at the ultrastructural level. During cell wall formation, freeze fracture replicas of the E-face of the plasma membrane reveal two major substructures: the terminal complexes (TC), paired and unpaired, and the microfibril imprints extending from unpaired TCs. In cells treated for 3 hours or longer with TM, the TCs are no longer visible, whereas microfibril imprints are still present. Because of the reported highly selective mode of action of TM, our results implicate a role for lipid-intermediates in cellulose synthesis in O. solitaria. It is assumed that TM prevents the formation of a glycoprotein which probably is a fundamental part of the TCs and may act as a primer for the assembly of the microfibrils. 2 Present address: Zellenlehre, Universität Heidelberg, Im Neuenheimer Feld 230, D-6900 Heidelberg, Federal Republic of Germany. 1 Supported by the Deutsche Forschungsgemeinschaft. This content is only available as a PDF. © 1984 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Chueca, Ana; Lázaro, Juan José; Gorgé, Julio López

doi: 10.1104/pp.75.3.539pmid: 16663662

Abstract Etiolated spinach (Spinacia oleracea L. var Winter Giant) seedlings show a residual photosynthetic fructose-1,6-bisphosphatase activity, which sharply rises under illumination. This increase in activity is due to a light-induced de novo synthesis, as it has been demonstrated by enzyme labeling experiments with 2H2O and [35S]methionine. The rise of bisphosphatase activity under illumination is strongly inhibited by cycloheximide, but not by the 70S ribosome inhibitor lincocin, which shows the nuclear origin of this chloroplastic enzyme. 1 Supported by a grant from Comisión Asesora de Investigación Científica y Técnica of Spain. This content is only available as a PDF. © 1984 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Gerhardt, Richard; Heldt, Hans W.

doi: 10.1104/pp.75.3.542pmid: 16663663

Abstract This paper describes a technique for measuring the in vivo metabolite levels in the chloroplast stroma, the cytosol, and the vacuole of spinach (Spinacia oleracea U.S.A. hybrid 424) leaves. Spinach leaves were freeze stopped and the frozen tissue was ground and lyophilized. The dry material was homogenized by sonication in a mixture of carbon tetrachloride and heptane, and fractionated by density gradient centrifugation. Measurements of the activity of marker enzymes in various subcellular compartments show the chloroplastic material mainly appearing in the lightest fractions and the cytosolic material in the middle of the gradient, whereas most of the vacuolar material is found in the heaviest fraction. Using the measured distributions of metabolites and of marker enzymes in each fraction of the gradient, the subcellular distribution of the metabolite can be calculated. As a first application, the new fractionation technique was used to investigate the subcellular contents of malate and sucrose in spinach leaves. The results show striking diurnal changes of sucrose and malate, with both substances primarily located in the vacuolar compartment. About three times more malate is present at the end of the day than at the end of the night. The sucrose content in the vacuole falls from a maximum of 45 millimolars at the end of the day to an almost undetectable value of approximately 1 millimolar at the end of the night. 1 Supported by the Deutsche Forschungsgemeinschaft. This content is only available as a PDF. © 1984 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Stitt, Mark; Herzog, Bernd; Heldt, Hans W.

doi: 10.1104/pp.75.3.548pmid: 16663664

Abstract A mechanism is proposed for a feed-forward control of photosynthetic sucrose synthesis, which allows withdrawal of carbon from the chloroplast for sucrose synthesis to be coordinated with the rate of carbon fixation. (a) Decreasing the rate of photosynthesis of spinach (Spinacia oleracea, U.S. hybrid 424) leaf discs by limiting light intensities or CO2 concentrations leads to a 2-to 4-fold increase in fructose 2,6-bisphosphate. (b) This increase can be accounted for by lower concentrations of metabolites which inhibit the synthesis of fructose 2,6-bisphosphate, such as dihydroxyacetone phosphate and 3-phosphoglycerate. (c) Thus, as photosynthesis decreases, lower levels of dihydroxyacetone phosphate should inhibit the cytosolic fructose bisphosphatase via simultaneously lowering the concentration of the substrate fructose 1,6-bisphosphate, and raising the concentration of the inhibitor fructose 2,6-bisphosphate. 1 Supported by the Deutsche Forschungsgemeinschaft. This content is only available as a PDF. © 1984 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Stitt, Mark; Kürzel, Birgit; Heldt, Hans W.

doi: 10.1104/pp.75.3.554pmid: 16663665

Abstract The role of fructose 2,6 bisphosphate in partitioning of photosynthate between sucrose and starch has been studied in spinach (Spinacia oleracea U.S. hybrid 424). Spinach leaf material was pretreated to alter the sucrose content, so that the rate of starch synthesis could be varied. The level of fructose 2,6-bisphosphate and other metabolites was then related to the accumulation of sucrose and the rate of starch synthesis. The results show that fructose 2,6-bisphosphate is involved in a sequence of events which provide a fine control of sucrose synthesis so that more photosynthate is diverted into starch in conditions when sucrose has accumulated to high levels in the leaf tissue. (a) As sucrose levels in the leaf rise, there is an accumulation of triose phosphates and hexose phosphates, implying an inhibition of sucrose phosphate synthase and cytosolic fructose 1,6-bisphosphatase. (b) In these conditions, fructose 2,6-bisphosphate increases. (c) The increased fructose 2,6-bisphosphate can be accounted for by the increased fructose 6-phosphate in the leaf. (d) Fructose 2,6-bisphosphate inhibits the cytosolic fructose 1,6-bisphosphatase so more photosynthate is retained in the chloroplast, and converted to starch. 1 Supported by the Deutsche Forschungsgemeinschaft. This content is only available as a PDF. © 1984 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)