Radioisotope ratios discriminate between competing pathways of cell wall polysaccharide and RNA biosynthesis in living plant cells

• Published in: The Plant journal : for cell and molecular biology 2007-10, Vol.52 (2), p.252-262
• Main Authors: Sharples, Sandra C, Fry, Stephen C
• Format: Article
• Language: English
• Subjects: Arabidopsis; Arabidopsis - cytology; Biological and medical sciences; biomass; Botany; Carbon - metabolism; Carbon Radioisotopes; Cell kinetics; Cell physiology; Cell Survival; Cell Wall - chemistry; Cells, Cultured; Cellular biology; Fundamental and applied biological sciences. Psychology; Isotope Labeling; Molecular biology; myo-inositol; Phosphatidylinositols - chemistry; Phosphatidylinositols - metabolism; plant cell walls; Plant physiology and development; polysaccharide biosynthesis; Polysaccharides - biosynthesis; Polysaccharides - chemistry; Ribonucleic acid; ribose biosynthesis; RNA; RNA, Messenger - metabolism; RNA, Plant - biosynthesis; Sucrose - chemistry; Sucrose - metabolism; sugar-nucleotides; Tritium - metabolism
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313x.2007.03225.x

Cell wall polysaccharides are synthesized from sugar-nucleotides, e.g. uridine 5'-diphosphoglucose (UDP-Glc), but the metabolic pathways that produce sugar-nucleotides in plants remain controversial. To help distinguish between potentially 'competing' pathways, we have developed a novel dual-radiolabelling strategy that generates a remarkably wide range of ³H:¹⁴C ratios among the various proposed precursors. Arabidopsis cell cultures were fed traces of d-[1-³H]galactose and a ¹⁴C-labelled hexose (e.g. d-[U-¹⁴C]fructose) in the presence of an approximately 10⁴-fold excess of non-radioactive carbon source. Six interconvertible 'core intermediates', galactose 1-phosphate [leftright arrow] UDP-galactose [leftright arrow] UDP-glucose [leftright arrow] glucose 1-phosphate [leftright arrow] glucose 6-phosphate [leftright arrow] fructose 6-phosphate, showed a large decrease in ³H:¹⁴C ratio along this pathway from left to right. The isotope ratio of a polysaccharide-bound sugar residue indicates from which of the six core intermediates its sugar-nucleotide donor substrate stemmed. Polymer-bound galacturonate, xylose, arabinose and apiose residues (all produced via UDP-glucuronate) stemmed from UDP-glucose, not glucose 6-phosphate; therefore, UDP-glucuronate arose predominantly by the action of UDP-glucose dehydrogenase rather than through the postulated competing pathway leading from glucose 6-phosphate via myo-inositol. The data also indicate that UDP-galacturonate was not formed by a hypothetical UDP-galactose dehydrogenase. Polymer-bound mannose and fucose residues stemmed from fructose 6-phosphate, not glucose 1-phosphate; therefore GDP-mannose (guanosine 5'-diphosphomannose) arose predominantly by a pathway involving phosphomannose isomerase (via mannose phosphates) rather than through a postulated competing pathway involving GDP-glucose epimerization. Curiously, the ribose residues of RNA did not stem directly from hexose 6-phosphates, but predominantly from UDP-glucose; an alternative to the textbook pentose-phosphate pathway therefore predominates in plants.