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[W]

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.

Regulation of Leaf Starch Degradation by Abscisic Acid Is Important for Osmotic Stress Tolerance in Plants

“…This nearly linear pattern of starch biosynthesis and degradation is retained under changing photoperiods or if plants are subject to a sudden early or late dusk, as long as the total circadian rhythm remains at 24 h (Sulpice et al, 2014;Graf et al, 2010). It is indeed observed that plants degrade starch faster in long days than in short days, demonstrating that plants somehow anticipate the length on the following night (Gibon et al, 2004;Lu et al, 2005). Such a tight regulation of starch degradation rates prevents carbon starvation or nonproductive carbon sequestration, thereby supporting continued growth during the night (Stitt and Zeeman, 2012).…”

Changes in the Phosphoproteome and Metabolome Link Early Signaling Events to Rearrangement of Photosynthesis and Central Metabolism in Salinity and Oxidative Stress Response in Arabidopsis

“…The concentrations of the products of these stopped reactions, such as glycerol-3-P, were then determined using cycling assays. The individual stopped assays and the subsequent cycling assays were described previously (Gibon et al, 2004). Accordingly, the cytosolic enzyme has maximum activity at pH 6.7, whereas the isoform is inactive.…”

“…The supernatant was the raw extract and used for the assays. Enzyme activities were assayed using the method as described before (Gibon et al, 2004). Reactions were started with the addition of a substrate or cofactor and incubated at 30°C for 20 min.…”

SlARF4, an Auxin Response Factor Involved in the Control of Sugar Metabolism during Tomato Fruit Development

“…Absorbances were read at 340 nm in MP96 readers (SAFAS). ADP-Glc pyrophosphorylase and NADP-glyceraldehyde-3-P dehydrogenase were assayed as described in Gibon et al (2004).…”