Progress in manipulating ascorbic acid biosynthesis and accumulation in plants

Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide that was first identified in 1978 as a quantitatively minor component of suspension-cultured sycamore cell walls. Subsequent studies have shown that RG-II is present in the primary walls of angiosperms, gymnosperms, lycophytes, and pteridophytes and that its glycosyl sequence is conserved in all vascular plants examined to date. This is remarkable because RG-II is composed of at least 12 different glycosyl residues linked together by more than 20 different glycosidic linkages. However, only a few of the genes and proteins required for RG-II biosynthesis have been identified. The demonstration that RG-II exists in primary walls as a dimer that is covalently cross-linked by a borate diester was a major advance in our understanding of the structure and function of this pectic polysaccharide. Dimer formation results in the cross-linking of the two homogalacturonan chains upon which the RG-II molecules are constructed and is required for the formation of a three-dimensional pectic network in muro. This network contributes to the mechanical properties of the primary wall and is required for normal plant growth and development. Indeed, changes in wall properties that result from decreased borate cross-linking of pectin may lead to many of the symptoms associated with boron deficiency in plants.