Abstract
A variety of metabolites are routinely assayed after perchloric acid extraction of plant tissues. A common technique uses coupled enzyme assays that produce or consume pyridine nucleotides together with spectrophotometric detection at 340 nm. Because of the presence of pigments in plant tissues, the high absorbance of such extracts usually limits the amount of extract that can be assayed spectrophotometrically. Here, we show that after batch adsorption with AG50WX8 (H+ form), the absorbance of potato root perchloric acid extracts at 340 nm is significantly reduced. This clean up procedure does not interfere with the assay or the recovery of anionic metabolites such as hexose phosphates. It therefore facilitates spectrophotometric assays of metabolites in plant extracts with high absorbance.
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References
Bergmeyer HU, Michal G (1974a) D-glucose-1-phosphate. In: Bergmeyer HU (ed) Methods of enzymatic analysis, 2nd edn. Academic Press, Weinheim, pp 1233–1237
Bergmeyer HU, Michal G (1974b) D-glucose-6-phosphate and D-fructose-6-phosphate. In: Bergmeyer HU (ed) Methods of enzymatic analysis, 2nd edn. Academic Press, Weinheim, pp 1238–1242
Bräutigam A, Gagneul D, Weber AP (2007) High-throughput colorimetric method for the parallel assay of glyoxylic acid and ammonium in a single extract. Anal Biochem 362:151–153. doi:10.1016/j.ab.2006.12.033
Brown CR (2005) Antioxidants in potato. Am J Potato Res 82:163–172
Canvin DT, Beevers H (1961) Sucrose synthesis from acetate in the germinating castor bean: kinetics and pathway. J Biol Chem 236:988–995
Cheremishinoff NP, Moressi AC (1978) Carbon adsorption applications. In: Cheremishinoff NP, Moressi AC (eds) Carbon adsorption handbook. Ann Arbor Science, Ann Arbor, pp 1–53
Geigenberger P, Stamme C, Tjaden J, Schulz A, Quick PW, Betsche T, Kersting HJ, Neuhaus HE (2001) Tuber physiology and properties of starch from tubers of transgenic potato plants with altered plastidic adenylate transporter activity. Plant Physiol 125:1667–1678. doi:10.1104/pp.125.4.1667
Haake V, Geiger M, Walch-Liu P, Engels C, Zrenner R, Stitt M (1999) Changes in aldolase activity in wild-type potato plants are important for acclimation to growth irradiance and carbon dioxide concentration, because plastid aldolase exerts control over the ambient rate of photosynthesis across a range of growth conditions. Plant J 17:479–489. doi:10.1046/j.1365-313X.1999.00391.x
Hanson AD, Rathinasabapathi B, Rivoal J, Burnet M, Dillon MO, Gage DA (1994) Osmoprotective compounds in the Plumbaginaceae: a natural experiment in metabolic engineering of stress tolerance. Proc Natl Acad Sci USA 91:306–310. doi:10.1073/pnas.91.1.306
Hausler RE, Fischer KL, Flugge UI (2000) Determination of low-abundant metabolites in plant extracts by NAD(P)H fluorescence with a microtiter plate reader. Anal Biochem 281:1–8. doi:10.1006/abio.2000.4556
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x
Rivoal J, Hanson AD (1993) Evidence for a large and sustained glycolytic flux to lactate in anoxic roots of some members of the halophytic genus limonium. Plant Physiol 101:553–560
Rivoal J, Hanson AD (1994) Metabolic control of anaerobic glycolysis. Overexpression of lactate dehydrogenase in transgenic tomato roots supports the Davies-Roberts hypothesis and points to a critical role for lactate secretion. Plant Physiol 106:1179–1185
Rivoal J, Ricard B, Pradet A (1989) Glycolytic and fermentative enzyme induction during anaerobiosis in rice seedlings. Plant Physiol Biochem 27:43–52
Saglio PH, Pradet A (1980) Soluble sugars, respiration, and energy charge during aging of excised maize root tips. Plant Physiol 66:516–519. doi:10.1104/pp.66.3.516
Stitt M, Gerhardt R, Kurzel B, Heldt HW (1983) A role for fructose 2, 6-bisphosphate in the regulation of sucrose synthesis in spinach leaves. Plant Physiol 72:1139–1141. doi:10.1104/pp.72.4.1139
Vroh Bi I, Harvengt L, Chandelier A, Mergeai G, Du Jardin P (1996) Improved RAPD amplification of recalcitrant plant DNA by the use of activated charcoal during DNA extraction. Plant Breed 115:205–206. doi:10.1111/j.1439-0523.1996.tb00905.x
Acknowledgments
Russet Burbank seed potatoes used for the generation of transformed roots were kindly provided by Les Semences Élites du Québec, Les Buissons, QC, Canada. S. D. was supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. This work was supported by a discovery grant from the Natural Sciences and Engineering Research Council of Canada to J. R.
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Communicated by M. Stobiecki.
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Dorion, S., Rivoal, J. A rapid ion exchange procedure that facilitates spectrophotometric assays of phosphorylated metabolites in potato extracts. Acta Physiol Plant 31, 855–859 (2009). https://doi.org/10.1007/s11738-009-0292-5
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DOI: https://doi.org/10.1007/s11738-009-0292-5