Characterisation and expression of the pathway from UDP-glucose to UDP-xylose in differentiating tobacco tissue

被引:38
作者
Bindschedler, LV
Wheatley, E
Gay, E
Cole, J
Cottage, A
Bolwell, GP [1 ]
机构
[1] Univ London, Royal Holloway, Sch Biol Sci, Egham TW20 0EX, Surrey, England
[2] Adv Technol Cambridge Ltd, Cambridge CB4 0WA, England
关键词
hemicellulose; UDP-glucose dehydrogenase; UDP-glucuronate decarboxylase; UDP-xylose; tobacco; xylan; xylogenesis;
D O I
10.1007/s11103-004-7795-7
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 M-r 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 M-r 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.
引用
收藏
页码:285 / 301
页数:17
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