Alginate is a major cell wall polymer of brown algae. The precursor for the polymer is GDP-mannuronic acid, which is believed to be derived from a four-electron oxidation of GDPmannose through the enzyme GDP-mannose dehydrogenase (GMD). So far no eukaryotic GMD has been biochemically characterized. We have identified a candidate gene in the Ectocarpus siliculosus genome and expressed it as a recombinant protein in Escherichia coli. The GMD from Ectocarpus differs strongly from related enzymes in bacteria and is as distant to the bacterial proteins as it is to the group of UDP-glucose dehydrogenases. It lacks the C-terminal ϳ120 amino acid domain present in bacterial GMDs, which is believed to be involved in catalysis. The GMD from brown algae is highly active at alkaline pH and contains a catalytic Cys residue, sensitive to heavy metals. The product GDP-mannuronic acid was analyzed by HPLC and mass spectroscopy. The K m for GDP-mannose was 95 M, and 86 M for NAD ؉ . No substrate other than GDP-mannose was oxidized by the enzyme. In gel filtration experiments the enzyme behaved as a dimer. The Ectocarpus GMD is stimulated by salts even at low molar concentrations as a possible adaptation to marine life. It is rapidly inactivated at temperatures above 30°C.

Galactose toxicity (Gal-Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP-galactose (UDP-Gal) and UDP-glucose is catalyzed by the enzymes galactokinase, UDP-sugar pyrophosphorylase (USP) and UDPgalactose 4-epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose-mediated toxicity in plants. In this study, we investigated the role of galactose-containing metabolites like galactose-1-phosphate (Gal-1P) and UDP-Gal in Gal-Tox. Recently published data from mouse models suggest that a reduction of the Gal-1P level via an mRNA-based therapy helps to overcome Gal-Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal-1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L À1 Gal). Nucleotide sugar analysis via high-performance liquid chromatography-mass spectrometry revealed highly elevated UDP-Gal levels in roots of seedlings grown on 1.5 mmol L À1 galactose versus 1.5 mmol L À1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal-feeding conditions, indicating that glycoproteins are a major target for elevated UDP-Gal levels in plants.