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

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 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.

The Molecular Basis of Shoot Responses of Maize Seedlings to Trichoderma harzianum T22 Inoculation of the Root: A Proteomic Approach

“…Regulation of these enzymes is important in understanding the partitioning of carbon into hemicellulose away from starch, Suc, and cellulose, which are irreversible processes. Biochemical evidence suggests that the timing of expression of UDP-Glc dehydrogenase and of UDP-glucuronate decarboxylase may control the flux of carbon into hemicellulose in differentiating vascular tissues (Harper and Bar-Peled, 2002;Bindschedler et al, 2005). Both UDP-Glc dehydrogenase and UDP-GlcUA decarboxylase were upregulated, indicating that this biochemical pathway leading to cell wall synthesis is increased in leaves of Trichoderma-inoculated plants.…”

Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis

“…Also a xylogenic Nicotiana tabacum cell-suspension culture is reported to contain a dual-specificity ADH/UDPGDH enzyme in addition to a mammalian-type UDPGDH enzyme (Bindschedler et al 2005). The Phaseolus enzyme had a relatively high K m for UDP-Glc (5.5 mM; Robertson et al 1996); most plant UDPGDHs have been reported to have K m values of 0.02-0.3 mM, e.g.…”

Cell Wall Polysaccharide Synthesis

“…The enzyme is well characterized from numerous organisms and crystal structures are available. In plants, the gene encoding this activity was first described in soybean (460), and soon after, as genome sequence became available, numerous UDPGDH gene isoforms that share high amino acid sequence similarity to each other were identified in Arabidopsis (461), poplar (462), tobacco (463), maize (464), and Dunaliella (465). In Arabidopsis, four UDPGDH isozymes are present and, likewise, multiple UDPGDH isoforms are found in the genome of every plant species that has been sequenced.…”

“…Recently, the tobacco ADH homologous genes, cloned and expressed in bacteria, had activity on both ethanol and UDPGlc. But again, the UDPGDH assay was determined by spectrophotometer assays (463). Whether recombinant ADH catalyzed the formation, UDP-GlcA was not explicitly confirmed.…”

“…In the second stage, the NADH-bound enzyme reduces the UDP-4ketopentose to UDP-Xyl resulting in the release of NAD-bound enzyme (403). Multiple distinct UXS isoforms encoding this enzyme activity were reported in Arabidopsis (431), rice (474), barley (475), and tobacco (463). Uxs isoforms are very specific enzymes and act only on UDP-GlcA.…”