Abstract
UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis.
Highlights
The biosynthesis of hemicelluloses and pectins in higher plants is mainly regulated by the cytosolic enzyme UDP-glucose dehydrogenase (UGDH) (EC 1.1.1.22), that catalyzes the sugar interconversion involving the fourelectron, NAD+-linked, oxidation of UDP-glucose to UDP-glucuronate (Amino et al, 1985; Gibeaut, 2000)
UDP-GlcA is the precursor of many sugar nucleotides, including UDP-galacturonic acid (UDP-GalA), UDP-xylose (UDP-Xyl), UDP-arabinose (UDP-Ara) and UDP-apiose (UDP-Api), which are the substrates for the polymer synthases involved in the formation of pectins and hemicelluloses (Gibeaut and Carpita, 1994)
A developmentally regulated and often transient expression pattern for UGDH transcripts was observed by Seitz et al (2000), suggesting that the isoforms are expressed in cells only when sugar nucleotides derived from the UDP-GlcA are needed for the synthesis of new cell wall polymers
Summary
The biosynthesis of hemicelluloses and pectins in higher plants is mainly regulated by the cytosolic enzyme UDP-glucose dehydrogenase (UGDH) (EC 1.1.1.22), that catalyzes the sugar interconversion involving the fourelectron, NAD+-linked, oxidation of UDP-glucose to UDP-glucuronate (Amino et al, 1985; Gibeaut, 2000). A developmentally regulated and often transient expression pattern for UGDH transcripts was observed by Seitz et al (2000), suggesting that the isoforms are expressed in cells only when sugar nucleotides derived from the UDP-GlcA are needed for the synthesis of new cell wall polymers. Studies on enzymes participating in the sugar nucleotide interconversion showed that UGDH is often the least active enzyme of the pathway, and is present in low amounts, leading to the conclusion that this enzyme might be rate-limiting for the synthesis of cell wall precursors, since there is a demand for UDP-glucuronate in all plant tissues and organs at all stages of development (Dalessandro and Northcote, 1977a,b; Robertson et al, 1995; Gibeaut, 2000). We isolated and identified three UGDH protein isoforms from the cambial region of 3- and 22-year-old trees, using 2-DE gels and LC-MS/MS
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