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Radioisotope ratios discriminate between competing pathways of cell wall polysaccharide and RNA biosynthesis in living plant cells
Author(s) -
Sharples Sandra C.,
Fry Stephen C.
Publication year - 2007
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2007.03225.x
Subject(s) - biochemistry , galactokinase , galactose , glucuronate , sugar phosphates , mannose , nucleotide sugar , biology , dehydrogenase , arabinose , fructose , pentose phosphate pathway , isomerase , polysaccharide , fructokinase , nucleotide , carbohydrate , sugar , xylose , phosphate , biosynthesis , enzyme , glycolysis , escherichia coli , fermentation , gene
Summary 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 3 H: 14 C ratios among the various proposed precursors. Arabidopsis cell cultures were fed traces of d ‐[1‐ 3 H]galactose and a 14 C‐labelled hexose (e.g. d ‐[U‐ 14 C]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 3 H: 14 C 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.