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

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Abstract

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.

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Abbreviations

ADH:

alcohol dehydrogenase

M r :

apparent molecular mass

Nt:

Nicotiana tabacum

UDPGlc:

UDP-glucose

UDPGIcA:

UDP-glucuronic acid

UDPGlcADCX:

UDP-glucuronate decarboxylase

UDP:

glucose dehydrogenase

UDP:

uridine diphosphate

UDPXyl:

UDP-xylose

UXS:

UDP-xylose synthase

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Author notes

  1. Edward Wheatley

    Present address: National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK

Authors and Affiliations

  1. School of Biological Sciences, Royal Holloway, University of London, Egham, TW20 0EX, UK

    Laurence V. Bindschedler, Edward Wheatley, Elaine Gay & G. Paul Bolwell

  2. Advanced Technologies (Cambridge) Ltd., 210 Cambridge Science Park, Milton Road, Cambridge, CB4 0WA, UK

    Jim Cole & Amanda Cottage

Authors
  1. Laurence V. Bindschedler
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  2. Edward Wheatley
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  3. Elaine Gay
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  4. Jim Cole
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  5. Amanda Cottage
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  6. G. Paul Bolwell
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Correspondence to G. Paul Bolwell.

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Bindschedler, L.V., Wheatley, E., Gay, E. et al. Characterisation and expression of the pathway from UDP-glucose to UDP-xylose in differentiating tobacco tissue. Plant Mol Biol 57, 285–301 (2005). https://doi.org/10.1007/s11103-004-7795-7

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