Soybean galactinol synthase forms fagopyritol B1 but not galactopinitols: substrate feeding of isolated embryos and heterologous expression

Abstract

Soybean (Glycine max (L.) Merrill) seeds accumulate sucrose, raffinose, stachyose and lesser amounts of galactopinitol A, galactopinitol B and fagopyritol B1 in axis and cotyledon tissues as part of the seed maturation process. Somatic embryos appear to be deficient in D-pinitol and galactopinitols, indicating a lack of synthesis by embryo tissues in vitro. Isolated immature soybean zygotic embryos were fed myo-inositol, D-pinitol, D-chiro-inositol and sucrose, individually and in combination, to evaluate the role of substrate availability on galactosyl cyclitol accumulation during precocious maturation. Feeding myo-inositol transiently doubled galactinol accumulation with little effect on other soluble carbohydrates. Feeding D-pinitol increased free D-pinitol 8-fold, galactopinitol A 4.5-fold and galactopinitol B 4.2-fold. Stachyose concentration was 2-fold higher in cotyledons after feeding D-pinitol than after feeding D-chiro-inositol. Feeding D-chiro-inositol increased fagopyritol B1 17-fold in the axis and 7-fold in the cotyledons, but did not increase other soluble carbohydrates. Feeding D-pinitol and D-chiro-inositol together reduced uptake of D-chiro-inositol and steady-state accumulation of galactinol and galactopinitols by 50%, compared to feeding D-pinitol alone. Increasing sucrose concentration from 0 to 200 mM had no effect. Recombinant soybean galactinol synthase, heterologously expressed in Escherichia coli, catalysed the synthesis of fagopyritol B1 and galactinol, but not galactopinitols. These results were consistent with the following interpretations: D-pinitol and D-chiro-inositol were transported from maternal tissues and not synthesized in the embryo, D-chiro-inositol uptake into embryos may be reduced by D-pinitol, fagopyritol B1 was synthesized by galactinol synthase while galactopinitols were not, and fagopyritol B1 and galactopinitols accumulated in response to the supply of free D-chiro-inositol and D-pinitol to embryos.