Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry    

Abstract

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 NDPsugar 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 ] 2 and/or [H 2 PO 4 ] 2 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.Plant cell walls are the most abundant renewable resources (Pauly and Keegstra, 2008a). Much of the current biotechnological research on plant cell wall synthesis involves manipulating these biosynthetic processes to obtain higher concentrations of starches or oil, which show much promise in biofuel production, or to alter cell wall composition for easier breakdown. A detailed knowledge of these processes is essential to understanding and utilizing plant cell wall materials as well as for progress in understanding plant growth and structural development (Pauly and Keegstra, 2008b). However, research into cell wall biosynthesis has been hindered by our limited understanding of the metabolic processes that produce cell walls and particularly their regulation. Progress in this area is limited by the difficulty of differentiating among the compounds involved and of analyzing the fluxes through the biochemical network of wall biosynthesis. Many of the metabolic steps involve isomeric sugars, including hexose-Ps and nucleotidesugars (NDP-sugars) that serve as direct precursors to plant cell wall biosynthesis. Separate quantification of these sugars has been difficult to achieve.Much of the current research on identifying and differentiating among different metabolic pathways involves the use of chromato...

Relationships of Leaf Net Photosynthesis, Stomatal Conductance, and Mesophyll Conductance to Primary Metabolism: A Multispecies Meta-Analysis Approach

“…3; Supplemental Data Set S2). Interestingly, these sugars and the hydroxybenzoic acid derivatives could be mostly related with the synthesis and maintenance of the cell wall structure and its pectin matrix (Schnitzler et al, 1992;Alonso et al, 2010;Chen et al, 2013;Franková and Fry, 2013). Cell wall cannot be considered as a fixed element in cell metabolism.…”

Engineering Mammalian Mucin-type O-Glycosylation in Plants

“…The resulting PCR product was then N-terminally fused to YFP by cloning into the pC2300u vector containing YFP (34), yielding pC2300u-35SPro-PpSP-MUC1-3.5TR-YFP(His) (MUC1-YFP). Two additional target peptides, under control of the ubiquitin promoter and terminator system, were created as follows: 1) pC2300D-UbiPro-NtSP-INF␣2B-GM-UbiTerm (INF␣2B) encoding interferon ␣2B (GenBank TM accession number AY255838.1), INF␣2B, with an N-terminal signal peptide NtSP and C-terminal glyco-module ((SP) 10 ), T7 (MASMTGGQQMG), and His 6 tag; 2) pC2300D-UbiProNtSP-MUC16 -1.2TR-UbiTerm (MUC16) encoding MUC16 -1.2TR (33) (GenBank TM accession number AF414442.2) with an N-terminal signal peptide NtSP and C-terminal T7, and His 6 tag was codon optimized for expression in Arabidopsis and synthesized by GenScript. Fragments encoding each mucin type peptide were subcloned into the HindIII site of pCAMBIA2300.…”

“…5A). Co-expression of a construct encoding the full secreted INF␣2B cytokine tagged with T7 and an Arabinogalactan Protein type (SP) 10 glycomodule (INF␣2B-T7-AGP) with the T2-2A-CytoEpi construct resulted in a mobility shift of a minor fraction of the protein (ϳ30 to ϳ31 kDa) and reactivity with VVA (Fig. 5B).…”

Rapid Kinetic Labeling of Arabidopsis Cell Suspension Cultures: Implications for Models of Lipid Export from Plastids

“…Although tobacco (Nicotiana tabacum) Bright Yellow 2 (BY2) cells have been the most widely used cell culture system, the abundant information and molecular and genetic tools available for Arabidopsis (Arabidopsis thaliana) have increased interest in T-87 cells as a model for molecular and biochemical investigations (e.g. Alonso et al, 2010). In addition, a high-throughput procedure for the testing of large numbers of transgenes in a 96-well format has been described (Ogawa et al, 2008).…”