RESUMEN

The architecture of plant metabolism includes substantial duplication of metabolite pools and enzyme catalyzed reactions in different subcellular compartments. This poses challenges for understanding the regulation of metabolism particularly in primary metabolism and amino acid biosynthesis. To explore the extent to which amino acids are made in single compartments and to gain insight into the metabolic precursors from which they derive, we used steady state (13) C labelling and analysed labelling in protein amino acids from plastid and cytosol. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major component of green tissues and its large and small subunits are synthesized from different pools of amino acids in the plastid and cytosol, respectively. Developing Brassica napus embryos were cultured in the presence of [U-(13) C]-sucrose, [U-(13) C]-glucose, [U-(13) C]-glutamine or [U-(13) C]-alanine to generate proteins. The large subunits (LSU) and small subunits (SSU) of Rubisco were isolated and the labelling in their constituent amino acids was analysed by gas chromatography-mass spectrometry. Amino acids including alanine, glycine and serine exhibited different (13) C enrichment in the LSU and SSU, demonstrating that these pools have different metabolic origins and are not isotopically equilibrated between the plastid and cytosol on the time scale of cellular growth. Potential extensions of this novel approach to other macromolecules, organelles and cell types of eukaryotes are discussed.

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RESUMEN

The biosynthesis of cell wall polymers involves enormous fluxes through central metabolism that are not fully delineated and whose regulation is poorly understood. We have established and validated a liquid chromatography tandem mass spectrometry method using multiple reaction monitoring mode to separate and quantify the levels of plant cell wall precursors. Target analytes were identified by their parent/daughter ions and retention times. The method allows the quantification of precursors at low picomole quantities with linear responses up to the nanomole quantity range. When applying the technique to Arabidopsis (Arabidopsis thaliana) T87 cell cultures, 16 hexose-phosphates (hexose-Ps) and nucleotide-sugars (NDP-sugars) involved in cell wall biosynthesis were separately quantified. Using hexose-P and NDP-sugar standards, we have shown that hot water extraction allows good recovery of the target metabolites (over 86%). This method is applicable to quantifying the levels of hexose-Ps and NDP-sugars in different plant tissues, such as Arabidopsis T87 cells in culture and fenugreek (Trigonella foenum-graecum) endosperm tissue, showing higher levels of galacto-mannan precursors in fenugreek endosperm. In Arabidopsis cells incubated with [U-(13)C(Fru)]sucrose, the method was used to track the labeling pattern in cell wall precursors. As the fragmentation of hexose-Ps and NDP-sugars results in high yields of [PO(3)](-)/or [H(2)PO(4)](-) ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure (13)C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments.

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A simple flow-switching device has been designed for use as a comprehensive two-dimensional gas chromatography modulator. The device is constructed from fused silica tubing, t-unions, and a solenoid valve. A series of experiments were conducted to determine the influence of primary flow, secondary flow, modulation time, and device dimensions on the performance of the modulator. The flow-switching device was found to produce pulses with widths near the theoretical minimum. High-performance was maintained over a wide range of modulation times. The flow-switching device did not introduce extra broadening along the primary retention axis. However, the modulator performance was optimal only over a narrow range of primary to secondary flow ratios. The ideal flow ratio is determined by the dimensions of the tubes that connect the t-unions. A simple flow resistance model has been developed that can predict the dimensions that will produce optimal results for a specified primary to secondary flow ratio. Thus, it is possible to construct a device that operates near the theoretical limit without numerous alterations. Under optimal conditions, the flow switching modulator generates peaks that are narrower than those produced by a diaphragm valve.