On sodium-dodecyl-sulfate polyacrylamide gels, purified glutathione reductase (GR; EC 1.6.4.2) from the leaves of two- to three-week-old pea (Pisum sativum L. cv. Birte) seedlings was represented by a single band with an apparent molecular weight of 55 kilodaltons. This polypeptide was resolved to multiple isoforms by two-dimensional electrophoresis. Fractionation of protoplasts and purification of subcellular organelles has shown that enzyme activity is associated with the chloroplasts, mitochondria and cytosol (in this order, approx. 77%, 3%, and 20% of the total activity). Distinct multiple isoforms of the enzyme, which differed in isoelectric point and were compartment-specific, were resolved from purified mitochondria and chloroplasts. The latency of the glutathione reductase activity which co-purified on Percoll gradients with the mitochondrial marker enzyme, cytochrome-c oxidase (EC 1.9.3.1.), indicated that this enzyme was within the mitochondrion. The mitochondrial glutathione reductase activity was strongly dependent on NADPH and not NADH.
Measurements of metabolic fluxes in whole embryos and isolated plastids have revealed major changes in the pathways of carbon utilization during cotyledon filling by oilseed rape (Brassica napus L.) embryos. In the early cotyledon stage (stage A), embryos used sucrose (Suc) predominantly for starch synthesis. Plastids isolated from these embryos imported glucose-6-phosphate (Glc-6-P) and partitioned it to starch and fatty acids synthesis and to the oxidative pentose phosphate pathway in the ratio of 2:1:1 on a hexose basis. Of the substrates tested, Glc-6-P gave the highest rates of fatty acid synthesis by the plastids and pyruvate was used weakly. By the midto late-cotyledon stage (stage C), oil accumulation by the embryos was rapid, as was their utilization of Suc for oil synthesis in vitro. Plastids from C-stage embryos differed markedly from those of stage-A embryos: (a) pyruvate uptake and utilization for fatty acid synthesis increased by respectively 18-and 25-fold; (b) Glc-6-P partitioning was predominantly to the oxidative pentose phosphate pathway (respective ratios of 1:1:3); and (c) the rate of plastidial fatty acid synthesis more than doubled. This increased rate of fatty synthesis was dependent upon the increase in pyruvate uptake and was mediated through the induction of a saturable transporter activity.
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