Radioisotope ratios discriminate between competing pathways of cell wall polysaccharide and RNA biosynthesis in living plant cells.
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-(3)H]galactose and a 14C-labelled hexose (e.g. D-[U-14C]fructose) in the presence of an approximately 10(4)-fold excess of non-radioactive carbon source. Six interconvertible 'core intermediates', galactose 1-phosphate <--> UDP-galactose <--> UDP-glucose <--> glucose 1-phosphate <--> glucose 6-phosphate <--> 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.
Sharples
Sandra C
SC
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, Edinburgh, UK.
Fry
Stephen C
SC
D017360:Arabidopsis / Q000166:cytology*; D002244:Carbon / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002470:Cell Survival; D002473:Cell Wall / Q000737:chemistry*; D002478:Cells, Cultured; D007553:Isotope Labeling; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D018749:RNA, Plant / Q000096:biosynthesis*; D013395:Sucrose / Q000737:chemistry / Q000378:metabolism; D014316:Tritium / Q000378:metabolism*
D002250:Carbon Radioisotopes; D010716:Phosphatidylinositols; D011134:Polysaccharides; D012333:RNA, Messenger; D018749:RNA, Plant; D014316:Tritium; D013395:Sucrose; D002244:Carbon