Plant Cell. 2004 Dec;16(12):3304-25. Epub 2004 Nov 17.

A Robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness.

Gibon Y, Blaesing OE, Hannemann J, Carillo P, Höhne M, Hendriks JH, Palacios N, Cross J, Selbig J, Stitt M.

Source

Max Planck Institute of Molecular Plant Physiology, Science Park Golm, 14476 Golm-Potsdam, Germany. gibon@mpimp-golm.mpg.de

Abstract

A platform has been developed to measure the activity of 23 enzymes that are involved in central carbon and nitrogen metabolism in Arabidopsis thaliana. Activities are assayed in optimized stopped assays and the product then determined using a suite of enzyme cycling assays. The platform requires inexpensive equipment, is organized in a modular manner to optimize logistics, calculates results automatically, combines high sensitivity with throughput, can be robotized, and has a throughput of three to four activities in 100 samples per person/day. Several of the assays, including those for sucrose phosphate synthase, ADP glucose pyrophosphorylase (AGPase), ferredoxin-dependent glutamate synthase, glycerokinase, and shikimate dehydrogenase, provide large advantages over previous approaches. This platform was used to analyze the diurnal changes of enzyme activities in wild-type Columbia-0 (Col-0) and the starchless plastid phosphoglucomutase (pgm) mutant, and in Col-0 during a prolongation of the night. The changes of enzyme activities were compared with the changes of transcript levels determined with the Affymetrix ATH1 array. Changes of transcript levels typically led to strongly damped changes of enzyme activity. There was no relation between the amplitudes of the diurnal changes of transcript and enzyme activity. The largest diurnal changes in activity were found for AGPase and nitrate reductase. Examination of the data and comparison with the literature indicated that these are mainly because of posttranslational regulation. The changes of enzyme activity are also strongly delayed, with the delay varying from enzyme to enzyme. It is proposed that enzyme activities provide a quasi-stable integration of regulation at several levels and provide useful data for the characterization and diagnosis of different physiological states. As an illustration, a decision tree constructed using data from Col-0 during diurnal changes and a prolonged dark treatment was used to show that, irrespective of the time of harvest during the diurnal cycle, the pgm mutant resembles a wild-type plant that has been exposed to a 3 d prolongation of the night.

PMID:: 15548738; [PubMed - indexed for MEDLINE]
PMCID:: PMC535875

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Figure 1.

Principles of Stopped Assays for Enzyme Activities That Use a Cycling Assay to Determine the Product of the Reaction.
(A) and (B) Determinations based on the glycerol-3P-cycling assay.
(C) and (D) Determinations based on the NADP-cycling assay.
(E) and (F) Determinations based on the NAD-cycling assay.
(A), (C), and (E) represent the stopped step leading to glycerol-3P or dihydroxyacetone-P, NADPH, or NAD(H), respectively, directly or via coupled reactions. The enzyme measured is in bold. For clarity, products that are not measured have been omitted as well as the cosubstrates required for the coupling reactions.
(B), (D), and (F) represent the principles of the cycling reactions for the determination of glycerol-3P or dihydroxyacetone-P, NADP(H), or NAD(H), respectively.
AGPase, glucose-1-phosphate adenyltransferase; Ald, aldolase; AlaAT, Ala aminotransferase; AspAT, Asp aminotransferase; CS, citrate synthase; FBPase, fructose-1,6-bisphosphatase; FruK, fructokinase; Fum, fumarase; G3PDH, glycerol-3P dehydrogenase; G3POX, glycerol-3P oxidase; G6PDH, glucose-6P dehydrogenase; GlcK, glucokinase; GAP DH, glyceraldehyde-3P dehydrogenase; GK, glycerokinase; GLDH, glutamate dehydrogenase; ICDH, isocitrate dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; PEPC, phosphoenolpyruvate carboxylase; PFP, pyrophosphate-dependent phosphofructokinase; PGI, phosphoglucose isomerase; PGK, phosphoglycerate kinase; PK, pyruvate kinase; ShikDH, shikimate dehydrogenase; SPS, sucrose-phosphate synthase; TPI, triose-P isomerase; UMPK, UMP kinase. Abbreviations for chemicals are as follows: 2OG, 2-oxoglutarate; 3PGA, 3-phosphoglycerate; 6PG, 6-phosphogluconate; ADPG, adenine-diphosphoglucose; BPGA, bisphosphoglycerate; DAP, dihydroxyacetone-P; F6P, fructose-6P; FBP, fructose-1,6-bisP; Fum, fumarate; G, glycerol; G3P, glycerol-3P; GAP, glyceraldehyde-3P; i-Cit, isocitrate; Mal, malate; MTT, methylthiazolyldiphenyl-tetrazolium bromide; OxA, oxaloacetate; PEP, phosphoenol pyruvate; Pyr, pyruvate; Shik, shikimate.

A Robot-Based Platform to Measure Multiple Enzyme Activities in Arabidopsis Using a Set of Cycling Assays: Comparison of Changes of Enzyme Activities and Transcript Levels during Diurnal Cycles and in Prolonged Darkness

Plant Cell. Plant Cell;16(12):3304-3325.