N.K. Boardman

Fractionation of the photochemical systems of photosynthesis I. Chlorophyll contents and photochemical activities of particles isolated from spinach chloroplasts

Spinach chloroplasts were fragmented by incubation with digitonin and the resulting chlorophyll-containing particles separated by differential centrifugation. Particles pelleted by forces of 1000 × g and 10 000 × g had a lower ratio of chlorophyll a/chlorophyll b (chl a/chl b) than the chloroplasts, whereas the smaller particles which either sedimented at 50 000–144 000 × g or remained in the 144 000 × g supernatant had higher ratios than the chloroplasts. Chl a/chl b ratios were usually determined spectrophotometrically in 80% acetone, but essentially the same ratios were obtained after transfer of the pigments to ether or after separation of the chlorophylls by thin-layer chromatography. The small particles were inactive in the Hill reaction, either with ferricyanide, trichlorophenolindophenol or NADP+ as oxidant, but they photoreduced NADP+ if provided with the electron-donor couple, sodium ascorbate and dichlorophenolindophenol, and both ferredoxin and NADP reductase. The 1000 × g and 10 000 × g fractions showed Hill activity, but the rate of reduction of NADP+ in the presence of ferredoxin and NADP reductase was lower than the rates of reduction of ferricyanide and trichlorophenolindophenol. The particles with the high chl a/chl b ratios appear to be representative of System 1 or the “long wavelength” system of photosynthesis, whereas the 1000 × g and 10 000 × g fractions appear to be enriched in particles representative of System 2. Chloroplasts were also fragmented by Triton X-100 and Nonidet P-40; in contrast to the digitonin treatment, the particles so obtained were photochemically inactive.

Fractionation of the photochemical systems of photosynthesis. II. Cytochrome and carotenoid contents of particles isolated from spinach chloroplasts

Abstract 1. Spinach chloroplasts were fragmented by incubation with digitonin, and the resulting particles examined for their cytochrome and carotenoid contents. Cytochrome difference spectra were recorded both at 20°C and 77°K, usually without prior extraction of the chlorophylls. 2. It is concluded that at least three cytochromes are localized in the chloroplast; cytochromes f and b 6 with α bands at 554 and 563 mμ respectively, and another cytochrome with an α band at about 559 mμ at 20°C and 557 mμ at 77°K. The cytochromes are present in the approximate molar ratio of 1:2:2, respectively. It is assumed tentatively that cytochrome 559 (chloroplasts) is a b -type cytochrome. The chloroplast contains 430 molecules of chlorophyll per molecule of cytochrome f and 118 molecules of chlorophyll per molecule of cytochrome b (cytochrome b 6 plus cytochrome 559). 3. The results presented for the particles prepared by digitonin treatment of chloroplasts show that cytochromes f and b 6 are associated with Photosystem 1 and cytochrome 559 with Photosystem 2. Cytochromes f and b 6 are partly solubilized from the Photosystem 1 particle by the digitonin, and the method of preparation of the particles was modified to minimize this loss. Cytochrome 559 appears to be tightly bound to the Photosystem 2 particle. 4. The four major carotenoids, β-carotene, lutein, violaxanthin and neoxanthin were found to be present in both types of particles, but in varying proportions. The particles representative of Photosystem 1 were enriched in β-carotene.

Composition of the photosystems and chloroplast structure in extreme shade plants

Abstract Chloroplasts were isolated from leaves of three species of tropical rainforest plants, Alocasia macrorrhiza, Cordyline rubra and Lomandra longifolia; these species are representative of extreme “shade” plants. It was found that shade plant chloroplasts contained 4–5 times more chlorophyll than spinach chloroplasts. Their chlorophyll a/chlorophyll b ratio was 2.3 compared with 2.8 for spinach. Electron micrographs of leaf sections showed that the shade plant chloroplasts contained very large grana stacks. The total length of partitions relative to the total length of stroma lamellae was much higher in Alocasia than in spinach chloroplasts. Freeze-etching of isolated chloroplasts revealed both the small and large particles found in spinach chloroplasts. Despite their increased chlorophyll content, low chlorophyll a/chlorophyll b ratio, and large grana, the shade plant chloroplasts were fragmented with digitonin to yield small fragments (D-144) highly enriched in Photosystem I, and large fragments (D-10) enriched in Photosystem II. The degree of fragmentation of the shade plant chloroplasts was remarkably similar to that of spinach chloroplasts, except that the subchloroplast fragments from the shade plants had lower chlorophyll a/chlorophyll b ratios than the corresponding fragments from spinach. The D-10 fragments from the shade plants had chlorophyll a/chlorophyll b ratios of 1.78-2.00 and the D-144 fragments ratios of 3.54–4.07. We conclude that Photosystems I and II of the shade plants have lower proportions of chlorophyll a to chlorophyll b than the corresponding photosystems of spinach. The lower chlorophyll a/chlorophyll b ratio of shade plant chloroplasts is not due to a significant increase in the ratio of Photosystem II to Photosystem I in these chloroplasts. The extent of grana formation in higher plant chloroplasts appears to be related to the total chlorophyll content of the chloroplast. Grana formation may simply be an means of achieving a higher density of light-harvesting assemblies and hence a more efficient collection of light quanta.

Studies on a barley mutant lacking chlorophyl b. I. Photochemical activity of isolated chloroplasts

Abstract 1. 1. Chloroplasts were isolated from the leaves of a barley mutant which lacked chlorophyll b and compared with the chloroplasts from the normal strain which had the normal content of chlorophyll a and chlorophyll b. 2. 2. Hill-reaction measurements were made with either 2,3′,6-trichlorophenol-indophenol, ferricyanide or NADP+ as oxidant. At a light intensity of 40 000 lux, the mutant chloroplasts were more active per mg of total chlorophyll than the normal chloroplasts, but only slightly more active per mg of chlorophyll a. Normal chloroplasts were saturated at a light intensity of about 30 000 lux whereas the mutant chloroplasts required an intensity of nearly 60 000 lux for saturation. At low light intensities ( 3. 3. Microscopic examination under phase contrast showed no significant differences in the appearance of isolated chloroplasts from the normal and mutant plants. The lack of chlorophyll b in the mutant was confirmed by absorption and spectrofluorimetric measurements at the temperature of liquid nitrogen. The spectrofluorimetric method was capable of detecting one molecule of chlorophyll b in the presence of 1000 molecules of chlorophyll a. 4. 4. The results are discussed in relation to the function of chlorophyll b in photosynthesis.

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.

The stacking of chloroplast thylakoids: Quantitative analysis of the balance of forces between thylakoid membranes of chloroplasts, and the role of divalent cations

Abstract An analysis is made of the van der Waals dispersion attractive forces and electrostatic repulsive forces between the grana thylakoid membranes of chloroplasts. These forces are determined for negatively charged surfaces with a pK a value of 4.7 for a bulk pH of 7.0 with a range of mono- and divalent cation concentrations and intermembrane spacing in the range 10 to 80 A. For equilibrium under dark conditions, it is concluded that either there is extensive electrostatic binding of divalent cations (Mg 2+ ) to the negatively charged membrane groups (phospholipid, sulfolipid, and protein carboxyl), or a redistribution of these groups between stacked and unstacked regions must be invoked.

Photooxidation of cytochromes in leaves and chloroplasts at liquid-nitrogen temperature

Abstract 1. 1. Light minus dark difference spectra of leaves and chloroplasts at liquidnitrogen temperatures were recorded in a Cary spectrophotometer. The spectra showed minima at 547 nm and 557 nm, but no band was observed at 552 nm, the position of the α-band of cytochrome f at low temperatures. Similar spectra were obtained with spinach and normal peas and with a pea mutant deficient in chlorophyll. It is concluded that cytochrome b -559, but not cytochrome f is photooxidized at liquid-nitrogen temperatures. Light of wavelength 650 nm was much more effective than 710-nm light, indicating that the oxidation of cytochrome b -559 at low temperatures is driven by light absorbed by Photosystem II. 2. 2. The redox potential of the cytochrome which is photooxidized at low temperature was determined as 0.35 V at pH 7.2. 3. 3. Chloroplasts were illuminated at room temperature, frozen rapidly to the temperature of liquid nitrogen, and light minus dark difference spectra were recorded. The spectrum of untreated chloroplasts showed only one small band at 547 nm. Chloroplasts treated with 3(3,4-dichlorophenyl)-1,1-dimethylurea gave sharp bands at 548 nm and 552 nm due to cytochrome f , but no band at 557 nm. 3(3,4-Dichlorophenyl)-1,1-dimethylurea inhibited the low temperature photooxidation of cytochrome b -559 provided the chloroplasts were illuminated at room temperature before freezing. Cytochrome b -559 was photooxidized to some extent at room temperature if the chloroplasts were treated with carbonyl cyanide m -chlorophenylhydrazone. 4. 4. The results are difficult to reconcile with an electron transport scheme in which cytochrome b -559 is located on the main coupled pathway between the photoacts of Photosystems I and II. We suggest that cytochrome b -559 is on a side pathway connected to the reaction centre of Photosystem II.

Studies on a protochlorophyll-protein complex. I. Purification and molecular-weight determination.

Abstract An active protochlorophyll-protein complex was isolated from dark-grown bean leaves and purified by ammonium sulphate fractionation, treatment with calcium phosphate gel, density-gradient centrifugation and density-gradient electrophoresis. The purification was followed by spectrophotometric and ultracentrifugal analyses and it was concluded that the active protochlorophyll was associated with a protein boundary with a sedimentation coefficient of 18 S. However, this boundary was associated with at least two components which could be partly resolved by zone electrophoresis in a density gradient. It appears that the protochlorophyll-containing protein was contaminated with a colourless protein which sediments at the same rate. Evidence was also obtained from zone electrophoresis for the separation of an active and an inactive form of protochlorophyll. The molecular weight of the protochlorophyll-protein complex was calculated as 600 000 ± 50 000 from measurements of sedimentation coefficient (18.0 S), diffusion coefficient (2.70 × 10 −7 cm 2 /sec8, and partial specific volume (0.73 ml/g). Electron microscopy indicated an approximately spherical molecule. In the most purified preparations, there was an average of one protochlorophyll molecule per protein molecule.

The stacking of chloroplast thylakoids: Effects of cation screening and binding, studied by the digitonin method

Abstract The differential action of digitonin on stacked and unstacked chloroplast thylakoids was used to investigate the molecular interactions between thylakoid membranes. The yield of the heavy fraction which is obtained from chloroplasts after digitonin incubation and differential centrifugation was taken as a measure of the degree or tightness of membrane appression. The effects of various mono-, di-, and trivalent cations on the yield of the heavy fraction were studied, and the results interpreted in terms either of electrostatic screening or ion binding to the thylakoid membrane surface: Although there was some degree of cation specificity in the degree of thylakoid appression indicative of cation binding, the nonspecific screening effect was much more important in determining the overall balance of forces. It is postulated that stacking occurs in regions of low net surface charge density, with a possible segregation of excess negative charges into nonstacked regions.

Photochemical systems in mesophyll and bundle sheath chloroplasts of C4 plants

Abstract 1. The agranal bundle sheath chloroplasts of Sorghum bicolor possess very low Photosystem II activity compared with the grana-containing mesophyll chloroplasts. 2. Sorghum mesophyll chloroplasts have a chlorophyll (chl) and carotenoid composition similar to that of spinach chloroplasts. In contrast, the sorghum bundle sheath chloroplasts have a higher chl a chl b ratio; they are enriched in β-carotene and contain relatively less xanthophylls as compared to sorghum mesophyll or spinach chloroplasts. 3. Sorghum mesophyll chloroplasts with 1 cytochrome f, 2 cytochrome b6 and 2 cytochrome b-559 per 430 chlorophylls have a cytochrome composition similar to spinach chloroplasts. Sorghum bundle sheath chloroplasts contain cytochrome f and cytochrome b6 in the same molar ratios as for the mesophyll chloroplasts, but cytochrome b-559 is barely detectable. 4. The chl/P700 ratios of mesophyll chloroplasts of S. bicolor and mesophyll and bundle sheath chloroplasts of Atriplex spongiosa are similar to that of spinach chloroplasts suggesting that these chloroplasts possess an identical photosynthetic unit size to that of spinach. The agranal bundle sheath chloroplasts of S. bicolor possess a photosynthetic unit which contains only about half as many chlorophyll molecules per P700 as found in the grana-containing chloroplasts. 5. The similarity of the composition of the bundle sheath chloroplasts of S. bicolor with that of the Photosystem I subchloroplast fragments, together with their smaller photosynthetic unit and low Photosystem II activities suggests that these chloroplasts are highly deficient in the pigment assemblies of Photosystem II.