Localization of galactinol,raffinose, and stachyose synthesis in Cucurbita pepo leaves

The biochemical pathway of stachyose synthesis was localized by immunocytochemical and ¹⁴C-labeling techniques in mature Cucurbita pepo L. leaves. Galactinol synthase (GaS; EC 2.4.1.123), the first unique enzyme in this pathway, was immunolocalized within the intermediary cells of minor veins in conventionally fixed and cryo-fixed, resin-embedded sections using polyclonal anti-GaS antibodies and protein A-gold. Intermediary cells are specialized companion cells with extensive symplastic connections to the bundle sheath. Gold particles were not seen over the non-specialized companion cells of larger veins or over intermediary cells in young leaves prior to the sink-source transition. In another approach to localization, radiolabel was measured in isolated mesophyll tissue and whole tissue of leaves that were lyophilized following a 90-s exposure to ¹⁴CO₂. Mesophyll, obtained by abrasion of the leaf surface, contained labeled sucrose, galactinol, raffinose and stachyose. However, the latter three labeled compounds constituted a smaller proportion of the neutral fraction than in whole-tissue samples, which also contained minor veins. We conclude that synthesis of galactinol, raffinose, and stachyose occurs in both mesophyll and intermediary cells, predominantly the latter.Abbreviations GaS galactinol synthase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresisWe thank John Pierce, Phillip Kerr, and Brace Schweiger for the gift of anti-GaS antibody and M.K. Kandasamy for helpful discussions. This research was supported by National Science Foundation grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.