Photoreactions of Cytochrome b-559 and cyclic electron flow in photosystem II of intact chloroplasts.

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO2 showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.

Cytochrome b-563 redox changes in intact CO-fixing spinach chloroplasts and in developing pea chloroplasts.

Intact spinach chloroplasts, capable of high rates of photochemical oxygen evolution with CO2 as electron acceptor (120-350 mumol O2 mg chlorophyll-1 h-1) were examined for cytochrome redox changes. The response of the cytochromes in intact chloroplasts to oxidants and reductants appears to be governed by the permeability of the chloroplast envelope. The low potential cytochromes (b-559LP and b-563) were more slowly reduced at 25 degrees C by dithionite than is the case with broken chloroplasts. At 0 degrees C, the reduction of the low potential cytochromes in intactchloroplasts was extremely slow. The chloroplast envelope is impermeable to ferricyanide, slowly permeable to ascorbate and rapidly permeable to reduced dichlorophenolindophenol. Light-induced redox changes of cytochrome b-563 in intact chloroplasts were examined both at 0 degrees and 25 degrees C. A red/far-red antagonism on the redox changes of cytochrome b-563 was observed at 0 degrees C under anaerobic conditions. 3-(3,4-dichlorophenyl)-1, 1-dimethlyurea (DCMU) inhibited the photoreduction of cytochrome b-563 in red light following far-red illumination. The photooxidation of cytochrome b-563 under anaerobic conditions was not influenced by DCMU or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The photoreduction of cytochrome b-563 under aerobic conditions was much less efficient than its photooxidation under anaerobic conditions. Developing pea chloroplasts showed much greater light-induced redox changes of cytochrome b-563 than did intact spinach chloroplasts. Our data are consistent with the view that cytochrome b-563 functions on a cyclic pathway around Photosystem I, but it appears that cyclic flow is sensitive to the relative poising of the redox levels of cytochrome b-563 and the components of the non-cylic pathway.

Effects of pH and Oxygen on Photosynthetic Reactions of Intact Chloroplasts.

Oxygen inhibition of photosynthesis was studied with intact spinach (Spinacia oleracea L.) chloroplasts which exhibited very high rates of photosynthetic CO(2) reduction and were insensitive to additions of photosynthetic intermediates when CO(2) was available at saturating concentrations. Photosynthetic rates were measured polarographically as O(2) evolution, and the extent of the reduction of substrate was estimated from the amount of O(2) evolved. With CO(2) as substrate, inhibition of photosynthesis by O(2) was dependent on pH. At pH values above 8, rates of O(2) evolution were strongly inhibited by O(2) and only a fraction of the added bicarbonate was reduced before O(2) evolution ceased. The extent of O(2) evolution declined with increasing O(2) concentration and decreasing initial bicarbonate concentration. At pH 7.2, the initial photosynthetic rate was inhibited about 30% at high O(2) levels, but the extent of O(2) evolution was unaffected and most of the added bicarbonate was reduced. Photosynthetic O(2) evolution with 3-phosphoglycerate as substrate was similarly dependent on pH and O(2) concentration. In contrast, there was little effect of O(2) and pH on oxaloacetate-dependent oxygen evolution. Acid-base shift experiments with osmotically shocked chloroplasts showed that ATP formation was not affected by O(2). The results are discussed in terms of a balance between photosynthetic O(2) evolution and O(2) consumption by the ribulose diphosphate oxygenase reaction.

Light-dependent absorption and selective scattering changes at 518 nm in chloroplast thylakoid membranes.

The light-induced absorbance change at 518 nm of isolated chloroplasts consists of a rapid phase, and a slow phase which is complete in about 20 sec. The slow component of the 518 nm absorbance change correlates with the light-induced change in 90 degrees light scattering at 518 nm. Both show a similar time course, similar pH dependence with a maximum at pH 6.0, and similar sensitivity to inhibitors and to treatment of the chloroplasts with a low concentration of glutaraldehyde. Their light minus dark difference spectra are similar with maxima at about 520 nm. It is concluded that they are manifestations of the same phenomenon, and the slow absorbance increase at 518 nm is due to enhanced scattering. It is proposed that the light-induced changes in scattering at 518 nm reflect alterations in selective dispersion, due to proton uptake and conformational changes in the chloroplast thylakoid membrane.

Properties of Protochlorophyllide and Chlorophyll(ide) Holochromes from Etiolated and Greening Leaves.

Protochlorophyllide and chlorophyll(ide) holochromes (Pchl-H and Chl-H) were extracted from dark-grown and greening seedlings with saponin and partly purified by ammonium sulfate fractionation. Sephadex gel filtration in the presence of saponin showed that the photoactive saponin Pchl-H from dark-grown leaves of bean (Phaseolus vulgaris L. cv. Redlands Pioneer) or pea (Pisum sativum L. cv. Greenfeast) has an apparent molecular weight of about 170,000, compared with 51,000 to 75,000 for the saponin Pchl-H from barley (Hordeum vulgare L. cv. Svalöfs Bonus). Photoconversion of saponin Pchl-H from dark-grown barley seedlings yields Chl-H with an absorption maximum at 678 nm, and with no change in apparent molecular weight. Above 0 C, a spectral shift from 678 to 672 nm follows, and a change in apparent molecular weight from about 63,000 to 29,000 is observed.Saponin Chl-H extracted from barley leaves illuminated for 15 minutes has an absorption maximum at 670 nm and an apparent molecular weight greater than 100,000. This chlorophyll holochrome has photosystem I activity and it is eluted together with the cytochromes. Saponin holochrome extracted from barley leaves returned to darkness after a light period, contains chlorophyll(ide) and protochlorophyllide complexes. Gel chromatography yields a complete separation of Chl-H (apparent molecular weight > 100,000) and photoactive Pchl-H (63,000).It is proposed that Chl-H dissociates into a chlorophyll(ide) a carrier protein complex and a photoenzyme, before the incorporation of chlorophyll into the lamellar membrane.Spectrofluorimetry on partially photoconverted preparations of saponin holochrome from barley, bean, and pea gave no indication for resonance energy transfer from protochlorophyllide to chlorophyllide. The saponin holochromes gave high polarization values, in contrast with bean holochrome extracted without the aid of detergents and bean leaves.

Lipid biosynthesis by isolated plastids from greening pea, Pisum sativum.

Isolated etioplasts from 8-day-old dark-grown pea seedlings incorporated [1-(14)C]acetate into lipid at a relatively low rate. Plastids from seedlings that had been illuminated for at least 2 hr showed an enhanced incorporation provided the plastids were illuminated during incubation with the labeled acetate. Dark incubation or the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased the acetate-incorporating activity of the developing chloroplasts to the level observed with etioplasts. Light had a marked effect on the type of fatty acid into which acetate was incorporated by the developing chloroplasts. Unsaturated fatty acids (mostly oleic acid) accounted for 60-80% of the incorporated label if the plastids were illuminated, but in the dark or in the presence of DCMU the unsaturated acids accounted for only 0-15% of the label incorporated into lipid. The effect of ATP on incorporation was dependent on the maturity of the chloroplasts; mature pea chloroplasts were inhibited by ATP, whereas in developing plastids there was a slight stimulation by ATP. Inhibition of acetate incorporation into lipid by DCMU appears to be due to inhibition of noncyclic phosphorylation. Incorporation was restored by reduced 2,3,5,6-tetramethylphenylenediamine, which restored phosphorylation, but not by reduced N,N,N',N'-tetramethylphenylenediamine.

Development of Photochemical Activity and the Appearance of the High Potential Form of Cytochrome b-559 in Greening Barley Seedlings.

The development of photochemical activity during the greening of dark-grown barley seedlings (Hordeum vulgare L. cv. Svalöfs Bonus) was studied in relation to the formation of the high potential form of cytochrome b-559 (cytochrome b-559(HP)). Photosynthetic oxygen evolution from leaves was detected at 30 minutes of illumination. The rate of oxygen evolution per gram fresh weight of leaf was as high at 2 to 2.5 hours of greening as at 24 hours or in fully greened leaves. On a chlorophyll basis, the photosynthetic rate at 90 minutes of greening was 80-fold greater than the rate at 45 hours. It is concluded that the majority of photosynthetic units are functional at an early stage of greening, and that chlorophyll synthesis during greening serves to increase the size of the units.Plastids showed substantial photochemical oxygen evolution after a seedling greening time of 1 hour. However, a comparison of the relative activity of leaves and plastids at 2 hours and 24 hours of greening suggests that there was some inactivation of greening plastids during isolation. Appreciable photosystem I activity was observed as early as 15 minutes of greening.The synthesis of cytochrome b-559(HP) during greening does not correlate with the onset of oxygen evolution. Cytochrome b-559(HP) was absent from etioplasts and in most preparations of 2-hour plastids. The average amount of cytochrome b-559(HP) at 2 hours of greening was well below the level needed to provide 1 molecule of the carrier for each functional photosynthetic chain. The results suggest that cytochrome b-559(HP) is not essential for oxygen evolution. Cytochrome f, cytochrome b(6), and the low potential form of cytochrome b-559 were present in the etioplast. There was little increase in the levels of these cytochromes during 24 hours of greening.

Lipid Composition of Pea and Bean Leaves during Chloroplast Development.

The changes in composition of the complex lipids were followed during the greening of dark-grown pea (Pisum sativum) and bean (Phaseolus vulgaris) seedlings. No significant changes in glycerolipid concentrations in the leaves were observed during the early stages of greening (0-8 hour for peas and 0-12 hour for beans). On further greening, there was an increase in the proportion of galactolipids and a decrease in the phospholipids. The fatty acid composition of the galactolipids remained constant during 24 hours of greening, but there was a slight increase in alpha-linolenic acid at 72 hours in the bean. The percentage of alpha-linolenic acid in the phospholipids and in sulfolipid showed a marked increase between 24 and 72 hours in the bean. Trans-delta(3)-hexadecenoic acid was the major fatty acid of phosphatidyl glycerol in bean leaves at 72 hours, but it was barely detectable at 24 hours. The lipid composition of greening leaves is discussed in relation to the fine structure and photochemical activity of the developing plastids.