Characterisation and expression of the pathway from UDP-glucose to UDP-xylose in differentiating tobacco tissue.
The pathway from UDP-glucose to UDP-xylose has been characterised in differentiating tobacco tissue. A xylogenic suspension cell culture of tobacco has been used as a source for the purification of the enzymes responsible for the oxidation of UDP-glucose to UDP-glucuronic acid and its subsequent decarboxylation to UDP-xylose. Protein purification and transcriptional studies show that two possible candidates can contribute to the first reaction. Most of the enzyme activity in the cultured cells could be accounted for by a protein with an Mr of 43 kDa which had dual specificity for UDP-glucose and ethanol. The cognate cDNA, with similarity to alcohol dehydrogenases (NtADH2) was expressed in E. coli to confirm the dual specificity. A second UDP-glucose dehydrogenase, corresponding to the monospecific form, ubiquitous amongst plants and animals, could not be purified from the tobacco cell cultures. However, two cDNAs were cloned with high similarity to the family of UDP-glucose dehydrogenases. Transcripts of both types of dehydrogenase showed highest expression in tissues undergoing secondary wall synthesis. The UDP-glucuronate decarboxylase was purified as polypeptides of Mr 87 and 40 kDa. Peptide fingerprinting of the latter polypeptide identified it as a form of UDP-glucuronate decarboxylase and functionality was established by expressing the cognate cDNA in E. coli. Expression of 40 kDa polypeptide and its corresponding mRNA was also found to be highest in tissues associated with secondary wall formation.
Bindschedler
Laurence V
LV
School of Biological Sciences, Royal Holloway, University of London, Egham, TW20 0EX, UK.
Wheatley
Edward
E
Gay
Elaine
E
Cole
Jim
J
Cottage
Amanda
A
Bolwell
G Paul
GP
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014540:Uridine Diphosphate Xylose / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism
D018076:DNA, Complementary; D010940:Plant Proteins; D012333:RNA, Messenger; D014540:Uridine Diphosphate Xylose; D014994:Xylose; D014533:Uridine Diphosphate Glucose Dehydrogenase; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase; D014532:Uridine Diphosphate Glucose