Abstract
An analytical workflow was developed for the absolute quantification of uridine diphosphate (UDP)-sugars in plant material in order to compare their metabolism both in wild-type Arabidopsis thaliana and mutated plants (ugd2,3) possessing genetic alterations within the UDP-glucose dehydrogenase genes involved in UDP-sugar metabolism. UDP-sugars were extracted from fresh plant material by chloroform-methanol-water extraction and further purified by solid-phase extraction with a porous graphitic carbon adsorbent with extraction efficiencies between 80 ± 5 % and 90 ± 5 %. Quantitative determination of the UDP-sugars was accomplished through HPLC separation with a porous graphitic carbon column (HypercarbTM) which was interfaced to electrospray ionization Orbitrap mass spectrometry. The problem of instable retention times due to redox processes on the stationary phase were circumvented by grounding of the column effluent and incorporation of a column regeneration procedure using acetonitrile-water containing 0.10 % trifluoroacetic acid. The method was calibrated using external calibration and UDP as internal standard. Calibration functions were approximated by first- or second-order regression analysis for concentrations spanning three orders of magnitude. Upon injecting sample volumes of 2.65 μL, the limits of detection for the UDP-sugars were in the 70 nmol L−1 range. Six different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-arabinose, UDP-xylose, UDP-glucuronic acid, and UDP-galacturonic acid were found in concentrations of 0.4 to 38 μg/g plant material. Data evaluation by analysis of variance (ANOVA) revealed statistically significant differences in UDP-sugar concentrations between wild-type and mutant plants, which were found to conclusively mirror the impaired metabolic pathways in the mutant plants. Figureᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-7746-3) contains supplementary material, which is available to authorized users.
Highlights
As nucleotide sugars in plants are responsible for the conversion of sun energy into usable plant biomass, research in this area is essential for a better understanding of the enzymatic pathways necessary for nucleotide sugar production [1]
We believe that oxidation due to the oxidative potential applied to the ESI source makes the stationary phase more hydrophilic which entails increased retention of the highly hydrophilic uridine diphosphate (UDP)-sugars
Since the oxidative potential applied to the ESI source is known to have an influence on the redox state of the porous graphitic carbon (PGC) stationary phase, electrical grounding of the column effluent was performed in order to stabilize retention times
Summary
As nucleotide sugars in plants are responsible for the conversion of sun energy into usable plant biomass, research in this area is essential for a better understanding of the enzymatic pathways necessary for nucleotide sugar production [1]. A polysaccharide comprising a linear chain of several hundred to over ten thousand β(1→4) linked D-glucose units, is an important structural component of the primary cell walls of green plants. Cellulosic biomass is the biggest contributor to the production of biomass on the planet. It is synthesized at the plasma membrane using UDP-Glc as glycosyl donor, which represents the most prominent nucleotide sugar in plants and which is provided by either photosynthesis or from the cleavage of sucrose by sucrose synthase. The intercellular levels of nucleotide sugars as well as their corresponding glycosyltransferases have been shown to be of utmost importance to cell wall formation and characteristics
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