High-throughput colorimetric method for the parallel assay of glyoxylic acid and ammonium in a single extract

Abstract

Glyoxylate and ammonia are signature metabolites of a major plant metabolic pathway, the C 2 oxidative carbon cycle, also known as photorespiration. This complex salvage pathway recycles most of the carbon that is lost from the Calvin cycle in the form of phosphoglycolate as a consequence of the oxygenation reaction of RuBP carboxylase-oxygenase (RuBisCO) 2 and constitutes a significant metabolic flux in photosynthesizing leaves of C 3 plants [1]. Mutations in enzymes of this pathway usually are lethal, underlining the importance of this salvage pathway [2][3][4]. Although all enzymes and some of the metabolite transporters involved in this highly compartmentalized pathway have been identified [5][6][7][8][9], information about the majority of the transport proteins and the processes regulating the pathway is still missing [1,4], possibly because mutations in the corresponding genes cause subtle metabolic phenotypes. Thus, a high-throughput method to quantify metabolites known to accumulate in photorespiratory mutants, glyoxylate and ammonium, is desirable. Here we present improved versions of two colorimetric methods for the quantification of ammonia and glyoxylate that make these procedures suitable for high-throughput and sensitive quantification of these signature metabolites in plant tissues.

New Connections across Pathways and Cellular Processes: Industrialized Mutant Screening Reveals Novel Associations between Diverse Phenotypes in Arabidopsis

“…1W). The high Gly and Ser contents are indicative of a defect in the photorespiratory pathway (Bräutigam et al, 2007). The reduction in F v /F m after high light treatment indicates an increase in photoinhibition of PSII (Takahashi et al, 2007).…”

ArabidopsisPhosphoglycerate Dehydrogenase1 of the Phosphoserine Pathway Is Essential for Development and Required for Ammonium Assimilation and Tryptophan Biosynthesis

“…Amino acid concentrations were quantified by precolumn online derivatization with orthophthaldehyde in combination with fluorescence detection (Lindroth and Mopper, 1979). The ammonium concentration was determined using a protocol described by Bräutigam et al (2007). For the analysis of glucosinolates, ;100 mg (fresh weight) plant tissue was lyophilized overnight, homogenized with a tissue lyzer, and extracted with 1 mL 80% methanol (v/v) containing benzyl-glucosinolate as an internal standard.…”

Point Mutation of a Plastidic Invertase Inhibits Development of the Photosynthetic Apparatus and Enhances Nitrate Assimilation in Sugar-treated Arabidopsis Seedlings

“…Levels of sucrose and hexose were determined according to Tamoi et al (36). The ammonium content of the seedlings was assayed by the method of Bräutigam et al (37). Glutamic acid and glutamine levels were determined with a L-glutamic acid measuring kit (Roche Applied Science) according to the manufacturer's instructions with some modifications.…”