Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system.
[en] Metabolite assays are required to characterise how metabolism changes between genotypes during development and in response to environmental perturbations. They provide a springboard to identify important regulatory sites and investigate the underlying mechanisms. Due to their small size, Arabidopsis seeds pose a technical challenge for such measurements. A set of assays based on a novel enzymic cycling system between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase have been developed and optimised for use with growing Arabidopsis seeds. In combination with existing assays they provide a suite of high throughput, sensitive assays for the immediate precursors for starch (adenine diphosphate glucose) and lipid (acetyl coenzyme A, glycerol-3-phosphate) synthesis, as well as pyrophosphate, ATP, ADP and most of the glycolytic intermediates. A method is also presented to rapidly quench intact siliques, lyophilise them and then manually separate seeds for metabolite analysis. These techniques are used to investigate changes in overall seed metabolite levels during development and maturation, and in response to a stepwise decrease of the external oxygen concentration.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Gibon, Yves
Vigeolas, Hélène ; Université de Liège - ULiège > Département des sciences de la vie > Génétique
Tiessen, Axel
Geigenberger, Peter
Stitt, Mark
Language :
English
Title :
Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system.
Albert S., Delseney M., Martine D. (1997) BANYULS, a novel negative regulator of flavonoid synthesis in the Arabidopsis seed coat. Plant Cell 11:289-299.
Baumlein H., Misera S., Luersson H., Koelle K., Horstmann C., Wobus U., Müller A.J. (1994) The FUS3 gene of Arabidopsis is a regulator of gene expression during late embyogenesis. Plant J. 6:379-387.
Bergmeyer H.U. Methods of enzymatic analysis, VCH, Weinheim, Germany; 1987.
Boisson M., Gomord V., Audran C., Berger N., Dubrucq B., Granier F., Lerouge P., Faye L., Caboche M., Lepiniec L. (2001) Arabidopsis glucosidase I mutants reveal a critical role of N-glycan trimming in seed development. EMBO J. 20:1010-1019.
Browse J., Somerville C.R. (1994) Glycerolipids. Arabidopsis , (Ed. by by E. M. Meyerowitz and C. R. Somerville, Cold.). Spring Laboratory Press, New York; 881-912.
Caspar T. (1994) Genetic dissection of the biosynthesis, degradation and biological function of starch. Arabidopsis , (Meyerowitz E. M. and Somerville C. R., eds). Cold Spring Laboratory Press, New York, NY; 913-936.
Chapple C., Vogt T., Ellis B.E., Somerville C.R. (1992) An Arabidopsis mutant defective in the general phenylpropanoid pathway. Plant Cell 4:1413-1424.
Dancer J.E., Ap Rees T. (1989) Effects of 2,4-dinitrophenol and anoxia on the inorganic pyrophosphate content of the spadix of Arum maculatum and the root apices of Pisum sativum. Planta 178:421-424.
De Bruiyn S.M., Ooms J.J.J., Karssen C.M., Vreugendhil D. (1997) Effects of abscisic acid on reserve formation in developing Arabidopsis seeds. Acta Bot. 46:263-277.
Debeaujon I., Leon-Kloosterziel K.M., Koornneef M. (2000) Influence of the testa on seed dormancy, germination and longevity in Arabidopsis. Plant Physiol. 122:403-413.
Debeaujon I., Peeters A.J.M., Leon-Kloosterziel K.M., Koornneef M. (2001) The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat epithelium. Plant Cell 13:853-871.
Drew M.C. (1997) Oxygen deficiency and root metabolism: Injury and acclimation under hypoxia and anoxia. Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:223-250.
Farré E.M., Geigenberger P., Willmitzer L., Trethewey R.N. (2000) A possible role for pyrophosphate in the coordination of cytosolic and plastidial carbon metabolism within the potato tuber. Plant Physiol. 123:681-688.
Ferrandiz C., Pelaz S., Yanofsky M.F. (1999) Control of carpel and fruit development in Arabidopsis. Ann. Rev. Biochem. 68:321-354.
Focks N., Benning C. (1998) Wrinkled1, A novel low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism. Plant Physiol. 118:91-101.
Galili G., Tang G., Zhu X., Amir R., Levanony H., Shy G., Herman E.M. (2000) Plant seeds: An exciting model system for dissecting molecular and cellular regulation of metabolic processes. Israel J. Plant Sci. 48:181-187.
Galili G., Tang G., Zhu X., Gakiero B. (2001) Lysine catabolism in plants: A stress and development superregulated metabolic pathway. Curr. Opinion Plant Biol. 4:261-266.
Geigenberger P., Fernie A.R., Gibon Y., Christ M., Stitt M. (2000) Metabolic activity decreases as an adaptive response to low internal oxygen in growing potato tubers. Biol. Chem. 381:723-740.
Geigenberger P., Hajirezaei M., Geiger M., Deiting U., Stitt M. (1998) Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased ativities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers. Planta 205:428-437.
Geigenberger P., Lerchl J., Stitt M., Sonnewald U. (1996) Phloem-specific expression of pyrophosphatase inhibits long distance transport of carbohydrates and amino acids in tobacco plants. Plant Cell Environ. 19:43-55.
Geigenberger P., Merlo L., Reimholz R., Stitt M. (1994) When growing potato tubers are detached from their mother plant there is a rapid inhibition of starch synthesis, involving inhibition of ADP-glucose pyrophosphorylase. Planta 193:486-493.
Geigenberger P., Reimholz R., Geiger M., Merlo L., Canale V., Stitt M. (1997) Regulation of sucrose and starch metabolism in potato tubers in response to short-term water deficit. Planta 201:502-518.
Hajirezaei M., Sonnewald U., Viola R., Carlisle S., Dennis D.T., Stitt M. (1994) Transgenic potato plants with strongly decreased expression of pyrophosphate: Fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers. Planta 192:16-30.
Jellito T., Sonnewald U., Willmitzer L., Hajiirzaei M.R., Stitt M. (1992) Inorganic pyrophosphate contents and metabolites in leaves and tubers of potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol. Planta 188:238-244.
Kaul S., Koo H.L., Jenkins J., Rizzo M., Rooney T., Tallon L.J., Feldblyum T., Nierman W., Benito M.I., Lin X.Y., Town C.D., Venter J.C., Fraser C.M., Tabata S., Nakamura Y., Kaneko T., Sato S., Asamizu E., Kato T., Kotani H., Sasamoto S., Ecker J.R., Theologis A., Federspiel N.A., Palm C.J. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796-815.
Koornneef M., Hanhart C.J., Hilhorst H.W.M., Karssen C.M. (1989) In-Vivo inhibition of seed development and reserve protein accumulation in recombinants of abscisic acid biosynthesis and responsiveness mutants in Arabidopsis thaliana. Plant Physiology 90:463-469.
Kuang A., Crispi M., Musgrave M.E. (1998) Control of seed development in Arabidopsis by atmospheric oxygen. Plant Cell Environ. 21:71-78.
Lowry O.H., Passonneau J.V. A Flexible System of Enzymatic Analysis, Academic Press, New York, NY; 1972.
Meinke D.W. (1994) Seed development in Arabidopsis thaliana. Arabidopsis , (Meyerowitz, E.M. and Somerville, C.R. eds). Cold Spring Laboratory Press, New York, NY; 253-295.
Menegus F., Cattaruzza L., Chersi A., Fronza G. (1989) Differences in the anaerobic lactate-succinate production and in the changes of cell sap pH for plants with high and low resistance to anoxia. Plant Physiol. 90:29-32.
Merlo L., Geigenberger P., Hajiirezaei M.R., Stitt M. (1993) Changes of carbohydrates, metabolites and enzyme activities in potato tubers during development, and within a single tuber along a stolon-apex gradient. J. Plant Physiol. 142:392-402.
Nesi N., Debeaujon I., Clarisse J., Pelletier G., Caboche M., Lepeniec L. (2000) The TT8 gene encodes a basic helix-loop-helix domain enzyme required for expression of DFR and BAN genes in Arabidopsis siliques. Plant Cell 12:1863-1878.
Nisselbaum J.S., Green S. (1969) A simple ultramicro method for determination of pyridine nucleotides in tissues. Anal. Biochem. 27:212-217.
Ooms J.A.A., Leon-Kloosterziel K.M., Bartels D., Koorneef M., Karssen C.M. (1993) Acquisition of dessication tolerance and longevity in seeds of Arabidopsis thaliana: A comparative study using abscisic acid-insensitve abi3 mutants. Plant Physiol. 102:185-1191.
Parcy F., Valon C., Kohara A., Misera S., Giradaut J. (1997) The ABSCISIC ACID-INSENSITIVE3, FUSCA3 and LEAFY COTYLEDON1 loci act in concert to control multiple aspects of Arabidopsis seed development. Plant Cell 9:1265-1277.
Peroappuram C.S., Steinhauer L., Barton D.L., Taylor D.C., Brock C., Jitao Z. (2000) The plastidic phosphglucomutase from Arabidopsis. A reversible enzyme reaction with an important role in metabolic control. Plant Physiol. 122:1193-1999.
Poirier Y., Ventre G., Caldelari D. (1999) Increased flow of fatty acids towards beta-oxidation in developing seeds of Arabidopsis deficient in diacylglyceraol acyltransferase activity or synthesising medium-chain-length fatty acids. Plant Physiol. 121:1359-1366.
Porterfield D.M., Kuang A., Smith P.J.S., Crispi M.L., Musgrave M.E. (1999) Oxygen-depleted zones inside reproductive structures of Brassicaceae: Implications for oxygen control of seed development. Can. J. Bot. 77:1439-1446.
Quebedeaux B., Hardy R.W.F. (1975) Reproductive growth and dry matter production of Glycine max(L.) Merr. in response to oxygen concentration. Plant Physiol. 55:102-107.
Quebedeaux B., Hardy R.W.F. (1976) Oxygen concentration: Regulation of crop growth and productivity. CO2 Metabolism and Plant Productivity , (Burris, R.H. and Black, C.C. eds). University Park Press, Baltimore; 185-204.
Rawsthorne S., Kang F., Eastmond P.J. (1999) Carbon flux to fatty acids in plastids. Regulation of Primary metabolism in Plants , (Kruger, N.J., Hill, S.A. and Ratcliffe, R.G. eds). Kluwer Academic Press, Dordrecht; 137-158.
Raz V., Bergervoet J.H.W., Koornneef M. (2001) Sequential steps for developmental arrest ion Arabidopsis seeds. Development 128:243-252.
Ap Rees T., Fuller W.A., Wright B.W. (1977) Measurements of glycolytic intermediates during the onset of thermogenesis in the spadix of Arum maculatum. Biochem. Biophys. Acta 461:274-282.
Reiter W.-D. (1994) Glycerolipids. Arabidopsis , (Meyerowitz, E.M. and Somerville, C.R. eds). Cold Spring Laboratory Press, New York, NY; 955-988.
Roessner U., Luedemann A., Brust D., Fiehn O., Linke T., Willmitzer L., Fernie A.R. (2001) Metabolic profiling allows comprehensive phenotyping of genetically or environmentally modified plant systems. Plant Cell 13:11-29.
Saavedra-Lira E., Ramirez-Silva L., Perez-Montfort R. (1998) Expression and characterization of recombinant pyruvate phosphate dikinase from Entamoeba histolytica. Biochem. Biophys. Acta 1382:47-54.
Shirley B.W. (1998) Flavonoids in seeds and grains. Physiological function and the genetics of biosynthesis. Seed Sci. Res. 8:415-422.
Smyth D.R., Bowman J.L., Meyerowitz E.M. (1990) Early flower development in Arabidopsis. Plant Cell 2:755-767.
Stitt M. (1998) Pyrophosphate as an alternative energy donor in the cytosol of plant cells: An enigmatic alternative to ATP. Bot. Acta 111:167-175.
Stitt M., Lilley R.McC., Gerhardt R., Heldt H.W. (1989) Determination of metabolite levels in specific cells and subcellular compartments of plant leaves. Meth. Enzymol. 174:518-552.
Stitt M., Wirtz W., Heldt H.W. (1980) Metabolite levels during induction in the chloroplast and extra-chloroplast compartments of spinach protoplast. Biochim. Biophys. Acta 593:85-102.
Vicient C.M., Natacha B.-E., Delseney M. (2000) Changes in gene expression in the leafy cotyledon (lec1) and fusca3 (fus3) mutants of Arabidopsis thaliana L. J. Exp. Bot. 51:995-1003.
Walker A.R., Davison P.A., Bolognesi-Winfield A.C., James C.M., Svrinivasan N., Blundell T.L., Esch J., Marks M.D., Gray J. (1999) The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiuation and antrocyanin synthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell 11:1337-1349.
White J.S., White D.C. Source Book of Enzymes, CRC Press, Boca Raton, FL; 1997.
Wisman E., Hartmann U., Sagasser M., Baumann E., Palme K., Hahlbrock K., Saedler H., Weisshaar B. (1998) Knock-out mutants from a En-1 mutagenised Arabidopsis thaliana population generated phenylpropanoid biosynthsis phenotypes. Proc. Natl. Acad. Sci. USA 95:12432-12437.
