Studies on the evolution of thioredoxins: Properties of thioredoxinsf andm in a phototrophically and heterotrophically grown green alga

Thioredoxins are proteins that serve as redox carriers in plants, animals, and bacteria and are reduced with either NADPH or reduced ferredoxin (Buchanan, 1984; Holmgren, 1985). The ferredoxin-linked thioredoxin system of oxygenic photosynthetic tissues seems to have evolved after the heterotrophic NADP-linked thioredoxin system (Crawford, et al, 1984). Determination of the relatedness of the thioredoxins from these two systems may provide clues to the evolution of the oxygenic photosynthetic system. To this end, we investigated the thioredoxins in Chlamydomonas reinhardii, a green alga that can grow both phototrophically and heterotrophically. We observed the presence of chloroplast thioredoxins ~ and ~ under both culture conditions. The heterotrophic NADP-linked thioredoxin system was not detected in abundance under either heterotrophic or phototrophic culture. Fractionation of the phototrophic cells revealed at least two m-type and two ~-type thioredoxins. The Chlamydomonas m thioredoxins were similar to their counterparts from higher plants. They reacted equally to antibodies raised against corn thioredoxin m and appeared as single bands of approximately 12 kDa on SDS polyacrylamide gel electrophoresis. In contrast, the thioredoxins of Chlamydomonas were quite different from the thioredoxin ~ in higher plants. Unlike higher plant proteins, the Chlamydomonas ~ thioredoxins neither adhered to DEAE-cellulose columns nor showed cross reactivity with antibodies to the thioredoxin ~ of corn or spinach when tested in an enzyme-linked immunosorption assay (ELISA) or by Western blot. The Chlamydomonas thioredoxin ~ activity was separated into two peaks by fast protein liquid chromatography (FPLC) on a Mono S column. These findings suggest that the ~ thioredoxins of oxygenic photosynthetic systems are conserved. The unique