sucrose-6-phosphate and trehalose-6-phosphate

Phosphorylated carbohydrates are central metabolites involved in key plant metabolic pathways, such as glycolysis and central carbon metabolism. Such pathways influence plant growth, development, and stress responses to environmental changes, and ultimately, reflect the plant's energy status. The high polarity of these metabolites, the variety of isomeric structures (e.g., glucose-1-phosphate (G1P)/fructose-6-phosphate (F6P)/mannose-6-phosphate (M6P)/G6P, sucrose-6-phosphate (S6P)/T6P), and rapid metabolic turnover makes their analysis particularly challenging. In this chapter, we describe the use of a set of known phosphorylated carbohydrates to develop and validate a hydrophilic interaction chromatography (HILIC) triple quadrupole (QqQ) tandem mass spectrometry (MS/MS) method in the highly sensitive and selective multiple reaction monitoring (MRM) mode for the target analysis of G1P, F6P, M6P, G6P, S6P, T6P, and the sugar nucleotide uridine 5-diphospho-glucose (UDPG). We present detailed information regarding HILIC column chemistry and practical considerations when coupling it with a QqQ-MS system.

Topics: Carbohydrate Metabolism; Glucosephosphates; Hydrophobic and Hydrophilic Interactions; Sucrose; Sugar Phosphates; Tandem Mass Spectrometry; Trehalose

Trehalose-6-phosphate (T6P) is an important signaling metabolite involved in plant growth control that inhibits the sucrose nonfermenting-1-related protein kinase 1 (SnRK1), a key regulator of energy and carbon metabolism in plants. The quantification of T6P in plant tissues is fundamental to improve our understanding of sugar signaling and the links between plant growth and development in response to stress conditions. However, the almost undetectable levels of T6P together with the complex plant matrix and the presence of T6P isomers such as sucrose-6-phosphate (S6P), makes the detection of this metabolite challenging. This work describes the development and validation of a hydrophilic interaction chromatography (HILIC) method for the on-line coupling with negative ion electrospray (ESI) triple quadrupole tandem mass spectrometry (MS/MS) in the highly sensitive and selective multiple reaction monitoring (MRM) mode for the target analysis of metabolic intermediates of the biosynthesis of trehalose, including glucose-6-phosphate (G6P), uridine 5-diphospho-glucose (UDPG), T6P (and its isomer S6P). Enhanced signal in the MRM mode and improved chromatographic separation for each compound were obtained using piperidine and methylphosphonic acid as additives in the HILIC mobile phase. The optimized HILIC-ESI-QqQ-MS/MS method increases the range of sensitive analytical methodologies for the quantification of key low-abundant metabolites, and was applied to quantify the fluctuations of S6P, T6P and G6P in Medicago truncatula plants in response to environmental stress. The levels of S6P, T6P, and G6P in M. truncatula plant tissues (roots and leaves) exposed to a water deficit and recovery treatment, ranged from 30 to 150pmolg

Topics: Biosynthetic Pathways; Chromatography, Liquid; Hydrophobic and Hydrophilic Interactions; Limit of Detection; Medicago truncatula; Metabolome; Plant Leaves; Reference Standards; Solutions; Sucrose; Sugar Phosphates; Tandem Mass Spectrometry; Trehalose

Trehalose 6-phosphate (Tre6P) is an essential signal metabolite in plants, linking growth and development to carbon metabolism. The sucrose-Tre6P nexus model postulates that Tre6P acts as both a signal and negative feedback regulator of sucrose levels. To test this model, short-term metabolic responses to induced increases in Tre6P levels were investigated in Arabidopsis thaliana plants expressing the Escherichia coli Tre6P synthase gene (otsA) under the control of an ethanol-inducible promoter. Increased Tre6P levels led to a transient decrease in sucrose content, post-translational activation of nitrate reductase and phosphoenolpyruvate carboxylase, and increased levels of organic and amino acids. Radio-isotope ((14)CO2) and stable isotope ((13)CO2) labelling experiments showed no change in the rates of photoassimilate export in plants with elevated Tre6P, but increased labelling of organic acids. We conclude that high Tre6P levels decrease sucrose levels by stimulating nitrate assimilation and anaplerotic synthesis of organic acids, thereby diverting photoassimilates away from sucrose to generate carbon skeletons and fixed nitrogen for amino acid synthesis. These results are consistent with the sucrose-Tre6P nexus model, and implicate Tre6P in coordinating carbon and nitrogen metabolism in plants.

Topics: Amino Acids; Arabidopsis; Carbon; Escherichia coli; Gene Expression; Glucosyltransferases; Nitrate Reductase; Nitrogen; Phosphoenolpyruvate Carboxylase; Phosphorylation; Plants, Genetically Modified; Protein Processing, Post-Translational; Sucrose; Sugar Phosphates; Trehalose; Ubiquitination