Characterization of prolamellar bodies, from dark‐grown seedlings of Scots pine, containing light‐ and NADPH‐dependent protochlorophyllide oxidoreductase

Abstract

Cotyledons of conifers have a light‐independent pathway for chlorophyll biosynthesis. To investigate whether the prolamellar body of Scots pine (Pinus sylveslris L.) is similar to the better known prolamellar body of wheat, etioplast membrane fractions were isolated from cotyledons of dark‐grown Scots pine. Dark‐grown cotyledons contained both chlorophyll and protochlorophyllide, 158 and 10 nmol (g fresh weight)’respectively, and had a chlorophyll a to b ratio of 4.2. The content of glyco‐ and phospholipids was 7.1 μmol (g fresh weight)1. About 40 mol % of these lipids were the specific plastid lipids – monogalactosyl diacylglycerol. digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol in the relative amounts 50, 35 and 7 mol %. The mol ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was 1.7. Low temperature fluorescence emission spectra of intact cotyledons and homogenate showed maxima at 633, 657, 686, 696 nm and a broad peak at 725–735 nm. The maxima at 633 and 657 nm represented different forms of protochlorophyllide and the other emission maxima represented chlorophyll protein complexes. The 657 nm form of protochlorophyllide was phototransformable both in vivo and in the isolated membranes. The phototransformable protochlorophyllide was substantially enriched in the prolamellar body fraction. The specific activity of light dependent protochlorophyllide oxidoreductase in the prolamellar body fraction was found to be 2 nmol chlorophyllide formed [(mg protein)−1 min−1]. The molecular weight of the enzyme polypeptide was determined as 38 000 dalton with sodium dodecylsulphate‐polyacrylamide gel electrophoresis.

Immunodetection and photostability of NADPH-protochlorophyllide oxidoreductase in Pinus pinea L.

“…Overall our findings are consistent with the identification by Selstam and Widell (1986) of the dominant protein band in extracts of prolamellar bodies of Pinus sylvestris and Pinus jeffreyi as the light-dependent NADPH-Pchlide oxidoreductase [EC 1.6.99.1]. The immunological cross-reactivity detected by immunoblotting described in this paper confirms the similarity between a 38 kDa protein in pine and the light-dependent NADPH-Pchlide oxidoreductase of the same molecular weight in barley and demonstrates a conservation of the antigenic sites of the oxidoreductase protein in the development of the flowering plants.…”

“…As in angiosperms, the accumulation of Pchlide in gymnosperms is correlated with the accumulation of the light-dependent NADPH-Pchlide oxidoreductase (Selstam and Widell 1986). The enzyme has the same apparent location, molecular weight and relative activity in pine and wheat (Selstam et al 1987).…”

“…This compares with the two immunoreactive bands seen in etiolated oat, wheat and rye and greened oats and barley , Meyer et al 1983. Selstam et al (1986Selstam et al ( , 1987 have demonstrated that the major protein constituent of prolamellar bodies in Pinus sylvestris and Pinus jeffreyi has a molecular weight of 38 kDa. They have also shown that this fraction catalyses the light-dependent reduction of Pchlide to Chlide.…”

Homologues of the green algal gidA gene and the liverwort frxC gene are present on the chloroplast genomes of conifers

“…Evidently, conifer seedlings grown in the dark are capable of fight-independent reduction of Pchfide. However, the Pchlide reduction activity in plastid extracts from dark-grown spruce [ 17] and pine [36] seedlings has been found to be NADPH-and light-dependent, just as in darkgrown angiosperms, and the dominating polypeptide in prolamellar bodies from pine appeared similar to the NADPH-protochlorophyllide oxidoreductase of barley and wheat [ 36]. This would indicate that either a mechanism, which is disrupted upon extraction, eliminates or reduces the photon requirement of the reaction in vivo, or that an alternative enzymatic process for Pchlide reduction exists, which is not detected in vitro.…”

Chlorophyll Biosynthesis.

“…Crystalline prolamellar bodies and extensive grana are also found in chloroplasts of cotyledons of Norway spruce and other conifers developed in the dark (von Wettstein, 1958;Selstam and Widell, 1986). The light-independent protochlorophyllide reductase and the two light-dependent protochlorophyllide reductases expressed in the dark in developing conifer cotyledons make this differentiation of plastid structures understandable.…”