Biosynthesis of UDP‐glucuronic acid in developing soybean embryos: possible role of UDP‐sugar pyrophosphorylase

Abstract

During soybean [Glycine max (L.) Merrill] embryo development, cell wall polysaccharides (CWPs) derived from UDP‐glucuronic acid (UDP‐GlcA) (uronic acids, arabinose, xylose) exhibited a linear increase during the period of 25–45 days after flowering (daf). At embryo maturity, CWPs derived from UDP‐GlcA accounted for 39% of total CWPs. To ascertain the relative importance of the nucleotide sugar oxidation (NSO) and the myo‐inositol oxidation (MIO) pathways to UDP‐GlcA biosynthesis, UDP‐glucose (UDP‐Glc) dehydrogenase (UDP‐Glc DH, EC 1.1.1.22) and UDP‐glucuronic acid pyrophosphorylase (UDP‐GlcA PPase, EC 2.7.7.44) activities, respectively, were measured in desalted extracts of developing embryos. UDP‐Glc DH and UDP‐GlcA PPase activities, expressed on a per seed basis, increased 3.5‐ and 3.9‐fold, respectively, during the period of 25–45 daf. However, UDP‐GlcA PPase activity was 35–50‐fold greater than UDP‐Glc DH activity. The soybean UDP‐sugar pyrophosphorylase gene (USP1), a homolog of pea USP, and a candidate gene for UDP‐GlcA PPase, was cloned and the recombinant enzyme characterized. Recombinant soybean USP1 (71 kDa) exhibited high activity with glucuronic acid 1‐phosphate (GlcA‐1‐P), glucose 1‐phosphate (Glc‐1‐P) and galactose 1‐phosphate (Gal‐1‐P), but low activity with mannose 1‐phosphate (Man‐1‐P), N‐acetylglucosamine 1‐phosphate and Glc‐6‐P. Determination of kinetic constants indicated that USP1 has a higher affinity for GlcA‐1‐P (Km= 0.14 ± 0.02 mM) than for Glc‐1‐P (Km= 0.23 ± 0.02 mM). Semiquantitative RT‐PCR was used to measure transcript levels of the UDP‐glucose DH (UGD) and USP gene families in developing soybean embryos. Transcript levels, normalized to the 18S rRNA controls, were greater for UGD than USP throughout embryo development. The possibility that USP serves as UDP‐GlcA PPase, the terminal enzyme of the MIO pathway, is discussed.