Site of Synthesis of NADPH

Abstract

The site of synthesis of the plastid membrane-located enzyme, protochlorophyllide reductase, has been determined. Plastid ribosome-deficient and normal rye (Secale cereake L., cv Rheidol) plants were grown in darkness at 33°C and 22°C, respectively. Extracts from these plants were analyzed for the levels of different ribosomal RNAs and cytochrome f and the activity of a number of enzymes with well-established sites of synthesis. The results confirmed that the higher temperature had induced a specific inhibition of protein synthesis in the plastids. The activity and level of protochlorophyllide reductase was unaffected by growth at the higher temperature, suggesting it to be a cytoplasmically synthesized enzyme.NADPH-Pchlide oxidoreductase is an enzyme of the Chl biosynthetic pathway and is responsible for the light-dependent reduction of Pchilde in higher plants. The enzyme is present at high activity in etiolated tissue (13) and has recently been purified and shown to be one of the major proteins of the etioplast internal membrane system (2, 21). Light exerts an interesting regulatory effect on the enzyme in that illumination ofpreviously dark-grown plants results in a decrease in both the activity (19) and amount (2) of the enzyme. To characterize this light effect, it is obviously essential to gain information about the synthesis of the enzyme, and in this paper the site of synthesis of the enzyme is reported.In an attempt to determine whether the enzyme is synthesized in the cytoplasm or within the plastid, it was decided to make use of the observation that a deficiency of plastid ribosomes can be induced in certain higher plants by growth at high temperatures, a phenomenon first described and extensively investigated by Feierabend (11,12). Rye was chosen for study because in this species an elevated growth temperature sufficient to bring about an almost total lack of plastid ribosomes has little effect on cytoplasmic or mitochondrial protein synthesis (12). An alternative approach to answer the question might have been the analysis of plastid ribosome-deficient mutants (5); however, in view of the findings of Bradbeer et at. (7) that certain cytoplasmically synthesized plastid enzymes can still occur, but in greatly reduced amounts, in plastid ribosome-free mutants, it was felt that measurement of Pchilde reductase activity in plants with a heatinduced plastid ribosome deficiency would yield more reliable data regarding the site of synthesis of the enzyme. A previous study of the enzymes of Chl synthesis failed to detect a significant level of Pchilde reductase, measured as photoconvertible Pchilde, in such high-temperature-grown plants (10), but in the present work use was made of a much more sensitive assay of enzymic activity (13). A growth temperature of 33°C was adopted here to 'Supported by the Science and Engineering Research Council.

The light‐dependent control of chloroplast development in barley (Hordeum vulgare L)

“…From these results it has been concluded that the NADPH-protochlorophyllide oxidoreductase is synthesized on 80s ribosomes. Similar results have been obtained with other cereal plants [24]. They are consistent with our finding of a polyadenylated mRNA that codes for a high molecular weight precursor of the NADPH-protochlorophyllide oxidoreductase [lo].…”

Terpenoid Metabolism in Plastids

“…On the other hand, Cyt c oxidase activity exhibited a comparable level in extracts made from seedlings grown at 25 and 340C (Table I). The inhibition of RuBP carboxylase is readily explained by the fact that the synthesis of the large subunit coded by the plastid genome is prevented under the experimental conditions used, whereas that affecting NADPglyceraldehyde phosphate dehydrogenase could be tentatively correlated to the control exercised by the plastid on cytoplasmically synthetized polypeptides (5), as noted also in Secale (16, 19).…”

“…Presently, concerning enzymes involved in the biogenesis of plastid terpenoids, the results obtained for phytoene synthetase are to be compared with those obtained for NADPH:Pchlide oxidoreductase (2,19), which is synthesized as a high mol wt precursor in the cytoplasm. Obviously, phytoene synthetase belongs to the family of plastidic stroma and membrane proteins made on 80S ribosomes and ultimately processed by posttranslational mechanisms (1 1,14,26).…”