Selective Formation of Photosystem 1 under Illumination at Low Intensity

Analyses of chlorophylls a and b and P700 in the wheat leaves grown for 8 days under illumination with white light at different intensities suggested selective formation of photosystem 1 of the photosynthesis at low light intensities. This was confirmed for the two types of chloroplasts isolated from leaves grown at light intensities of 1.1 and 240 μ W/cm², respectively, by measuring their pigment compositions, activities of photosystems 1 and 2, and absorption and fluorescence spectra. The chloroplasts developed at the low intensity showed properties only of photosystem 1 while those developed at the high intensity showed properties of both photosystems 1 and 2. Only photosystem 1 particles were obtained by fractionation of low intensity chloroplasts by treatment with digitonin followed by centrifugation, while high intensity chloroplasts could be fractionated into photosystem-1 and photosystem-2 particles. When the leaves grown at low light intensity were illuminated with strong light, photosystem 2 was developed. The fluorescence emission spectrum of low intensity chloroplasts at 77°K showed two peaks at 685 and 734 nm, and the spectrum of high intensity chloroplasts showed three peaks at 685, 697 and 740 nm.     

On the role of ß-carotene in the reaction center chlorophyll a antennae of photosystem I

Absorption and low temperature fluorescence emission spectra were measured on chloroplast thylakoids and on purified reaction center chlorophyll a-protein complexes of photosystem I, CP-a₁. A clear association between the presence of ß-carotene and the occurrence of far red absorbing and emitting chlorophyll a components of the reaction center antennae of photosystem I was demonstrated. For this study chloroplasts and CP-a₁ were obtained from normal and carotenoid deficient plant material of various sources. The experimental material included 1) lyophilized pea chloroplasts extracted with petroleum ether, 2) the carotenoid deficient mutant C-6E of Scenedesmus obliquus and 3) wheat chloroplasts derived from normal and SAN-9789 treated plants. Removal of carotenoids, most likely principally ß-carotene, caused a loss of long wavelength absorbing chlorophylls in chloroplasts and purified CP-a₁, and the loss or diminution of the long wavelength peak seen in the low temperature fluorescence emission spectrum. This association between ß-carotene and special chlorophyll a forms may explain both the photoprotective and antenna functions ascribed to ß-carotene. In the absence of carotenoids in wheat and in the Scenedesmus mutant, the chlorophyll a antenna of photosystem I was extremely photosensitive. A triplet-triplet resonance energy transfer from chlorophyll a to ß-carotene and a singlet-singlet energy transfer from excited ß-carotene to chlorophyll would explain the photoprotective and antenna functions, respectively. The role of this association in determining some of the fluorescence properties of photosystem I is also discussed.     

Extraction and Reconstitution of Lyophilized Photosystem I Subchloroplast Fragments from Spinach Chloroplasts

Lyophilized photosystem I subchloroplast fragments prepared from spinach chloroplasts were extracted and reconstituted. Hexane extraction eliminated 26 % of the photosystem I activity without removing chlorophylls, and the reconstitution with β-carotene, an unkown lipid or chlorophyll a restored almost all the activity. Extraction with hexane–acetone (2:1, v/v) eliminated 81% of the activity with removal of 78% of the chlorophylls. In this case, reconstitution with chlorophyll a showed complete restoration of the activity, but β-carotene, the unknown lipid, and plastoquinone A caused a rather inhibitory effect. A possible explanation is that photosystem I reaction centers are closely surrounded by chlorophyll a; and β-carotene, the unknown lipid, and plastoquinone A function only through chlorophyll a.     

Formation of photosynthetic pigments and quinones and development of photosynthetic activity in barley etioplasts during greening in intermittent and continuous white light

The appearance and development of photosynthetic activity, and the accumulation of chlorophylls, carotenoids and quinones, was investigated in etiolated barley shoots (Hordeum vulgare L. cv. Villa) during greening in flash light, periodic light-dark cycles, and continuous white light. Greening and the development of photosynthetic activity was delayed in flash and periodic light compared to continuous white light. Photosystem II activity occurred after 6 light-dark cycles and increased continuously during greening. After 3 h greening in continuous white light, photosystem II activity appeared with a very high rate and decreased to that of a green leaf after 50 h greening. Parallel to the development of photosynthetic activity, light stimulated the biosynthesis of prenyllipids. Moreover, chlorophylls and those carotenoids and quinones that are contained in etioplasts in relatively small amounts, were particularly enhanced in their biosynthesis. Chlorophyll a was synthesized without a lag phase during greening in flash light, whereas a 2 h lag phase occurred in continuous white light. In all three modes of illumination the formation of chlorophyll a exceeded that of chlorophyll b. After 4 flashes and 2 light-dark cycles, chlorophyll b could be detected with a very high initial a/b ratio. Higher chlorophyll a/b ratios were reached after 200 flashes (a/b=10.9) and 50 light-dark cycles (a/b=6.6) than after 50 h continuous white light (a/b=3.3). The formation of carotenes, lutein, violaxanthin and neoxanthin was also enhanced by light. This was also confirmed for plast-ouinone-9. ?-tocopherol,α-tocoquinone and phylloquinone. A comparison of the carotenoid and quinone composition of the differentiating thylakoid membrane before and after onset of photosynthesis, reveals that the photosynthetic membrane is already equipped with photosynthetic pigments and quinones before the appearance of photosystem II activity. It is concluded that during development of the photo-synthetic apparatus the thylakoid membrane with its structural and functional constituents is formed first. In a second and slower process the water splitting enzyme system and enzymes of the Calvin cycle are activated.     

Photochemical Apparatus Organization in the Chloroplasts of Two Beta vulgaris Genotypes

The composition and structural organization of thylakoid membranes of a low chlorophyll mutant of Beta vulgaris was investigated using spectroscopic, kinetic and electrophoretic techniques. The data obtained were compared with those of a standard F1 hybrid of the same species. The mutant was depleted in chlorophyll b relative to the hybrid and it had a higher photosystem II/photosystem I reaction center (Q/P₇₀₀) ratio and a smaller functional chlorophyll antenna size. Analysis of thylakoid membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the mutant lacked a portion of the chlorophyll a/b light-harvesting complex but was enriched in the photosystem II reaction center chlorophyll protein complex. Comparison of functional antenna sizes and of photosystem stoichiometries determined electrophoretically were in good agreement with those determined spectroscopically. Both approaches indicated that about 30% of the total chlorophyll was associated with photosystem I and about 70% with photosystem II. A greater proportion of photosystem II_β was detected in the mutant. The results suggest that a higher photosystem II to photosystem I ratio in the sugar beet mutant has apparently compensated for the smaller photosystem II chlorophyll light-harvesting antenna in its chloroplasts. Moreover, a lack of chlorophyll a/b light-harvesting complex correlates with the abundance of photosystem II_β. It is proposed that a developmental relationship exists between the two types of photosystem II where photosystem II_β is a precursor form of photosystem II_α occurring prior to the addition of the chlorophyll a/b light-harvesting complex and grana formation.     

Chloroplast Reactions of Photosynthetic Mutants in Zea mays

Three seedling lethal mutants of Zea mays with impaired photosynthesis are described. These recessive mutants were selected on the basis of high chlorophyll fluorescence. They have normal chlorophyll pigmentation but are unable to fix CO₂ fully. Evidence is presented from fluorescence characteristics of isolated chloroplasts that both photosystem I and II mutants were isolated. Using conventional measures of photosynthetic electron transport, we suggest that the photosystem I mutant has limited ability to reduce NADP. The other two mutants are clearly blocked in photosystem II, one possibly lacking the primary electron acceptor.     

Photophosphorylation and Ultrastructural Development in Pinus nigra Chloroplasts, Grown under Different Spectral Composition of Light

Developed pine seedlings synthesize chlorophyll in darkness. Their photosystem II reducing capacity is very low. The development of chloroplast structure and of photophosphorylation ability has been studied in plastids isolated from Pinus nigra (var. austriaca) developed for 14 days under different spectral compositions of light as compared with chloroplasts isolated from seedlings grown under white light or in darkness. Chloroplast structure was studied by electron microscopy. Cyclic and non-cyclic photophosphorylation were studied under white light. The spectral bands which are efficient for the development of granal structures are different from those needed to make photosystem II functional: red light makes photosystem II functional but does not permit the formation of granal structure, and under yellow light the granal structure develops but photosystem II is not fully functional. Orange light alone fulfils both these purposes. The spectral band around 650 nm seems effective in making the photosystem II functional.     

Properties of light-harvesting chlorophyll a/b-protein, and photosystem I chlorophyll a-protein, purified from digitonin extracts of spinach chloroplasts by isoelectrofocusing

A procedure for purifying both light-harvesting chlorophylla/b-protein and photosystem I chlorophyll -protein from digitoninextracts of spinach chloroplasts is described. This procedureuses isoelectrofocusing on Ampholine at the last step and permitsisolating all of the chlorophyll-proteins from the same extractin a better yield and a highly pure state. The purified light-harvesting chlorophyll a/b-protein whichhas an isoelectric point (pi) of 4.35 (?0.1) and a single polypeptideof 24 kilodaltons (kD), shows slightly higher chlorophyll a/Aratio of 1.35 than the values reported for the preparationsobtained by anionic detergents. This chlorophyll-protein exhibitsa markedly high and sharp fluorescence band at 681 nm at 77?Kwhich is not found on the chloroplast emission spectrum. Photosystem I chlorophyll a-protein focuses on Ampholine intotwo bands with pi values of 4.75 (?0.1) and 4.80 (?0.1). Thesetwo fractions show the same absorption spectra (maximum at 678nm at room temperature) and emission spectra (maximum at 734nm at 77?K) and have the same constituent polypeptides: onelarge band at 55–64 kD and six minor bands (21.5, 20,19, 18, 16 and 15 kD). The polypeptide composition and the P-700to chlorophyll a ratio (1 to ca. 80) of this preparation arevery similar to those of the photosystem I reaction center preparationobtained from Swiss chard chloroplasts by Bengis and Nelson(8). (Received October 31, 1978; )     

Inhibition of photosynthetic electron transport and formation of inactive chlorophyll in winter stressed Pinus silvestris

Kinetics of fluorescence at room temperature, electron transport and photooxidation of P700 and cytochrome f have been studied in chloroplasts isolated from active and winter stressed Pinus silvestris. The winter stress induced block in the electron transport chain between the two photosystems is close to the site of plastoquinone, since winter stress and DCMU caused the same type of inhibition of the reoxidation of the primary electron acceptor Q of photosystem II. No winter inhibition of the electron transport between cytochrome f and P700 was observed. Time course studies of P700 photooxidation in chloroplasts of active and winter stressed pine have shown that the photosynthetic unit size must be about equal in the two types of chloroplasts. An apparent increase of the photosynthetic unit size was induced by winter stress, as revealed by the high chlorophyll/P700 ratio of winter stressed pine. The phenomenon is explained by the formation of photosynthetically inactive chlorophyll. Low-temperature fluorescence emission spectra were recorded when either chlorophyll a (433 nm) or chlorophyll b (477 nm) were preferentially excited. Winter stress induced the formation of a chlorophyll a fraction emitting at 673 nm. This chlorophyll is most likely derived from the chlorophyll a antennae of the two photosystems, and it probably contributes to the photosynthetically inactive pool of chlorophyll in winter stressed pine. The light harvesting chlorophyll a/b complex is relatively resistant to winter stress.     

Differences between Fragaria clones by fourth derivative room temperature spectroscopy of intact leaves

Clones of Fragaria were characterized by the attenuance (absorption) spectra of their photosynthetic apparatus. This method uses fourth derivative analysis of spectra of intact lamina of detached leaves at room temperature (20–25 C). Twenty-one different species and hybrids of Fragaria were examined. F. chiloensis accessions frequently have characteristic fourth derivative spectra distinct from that of other Fragaria species. These spectra are characterized by a strong band that appears to be photosystem I component Ca 693; and is correlated with a higher chlorophyll b content per unit area of leaf, and a lower chlorophyll a/b ratio.Abbreviations Chl chlorophyll - chiloensis Fragaria chiloensis - 4th deriv fourth derivative - PSI photosystem I - PSH photosystem II     

Stoichiometric determination of pheophytin in photosystem II of oxygenic photosynthesis

Pheophytin and chlorophyll extracted from oxygen-evolving photosystem II particles, chloroplast thylakoids and cyanobacterial cells were separated by column chromatography with DEAE-Toyopearl, and quantitatively determined by spectrophotometry. The molecular ratio of chlorophyll a+b to pheophytin a was about 100 in spinach photosystem II particles and about 140 in spinach thylakoids. Using flash spectrophotometry of P680 and measurement of flash-induced oxygen yield, the molecular ratio of the chlorophyll to the photochemical reaction center II was determined to be about 200 in the photosystem II particles. These findings suggest that the stoichiometry in photosystem II particles is one reaction center II and two pheophytin a molecules per about 200 chlorophyll molecules. The same stoichiometry for pheophytin to the reaction center II was obtained in the cyanobacteria, Anacystis nidulans and Synechocystis PCC 6714. A quantitative determination of pheophytin a and the electron donor P700 in stroma thylakoids from pokeweed suggests that photosystem I does not contain pheophytin.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Effects of pigment-deficient mutants on the accumulation of photosynthetic proteins in maize

We have monitored the accumulation of photosynthetic proteins in developing pigment-deficient mutants of Zea mays. The proteins examined are the CO₂-fixing enzymes, phoshoenolpyruvate carboxylase (E.C. 4.1.1.31) and ribulose-1,5-bisphosphate carboxylase (E.C.4.1.1.39), and three thylakoid membrane proteins, the light-harvesting chlorophyll a/b binding protein (LHCP) of photosystem II, the 65 kilodalton chlorophyll a binding protein of photosystem I and the alpha subunit polypeptide of coupling factor I. Using a sensitive protein-blot technique, we have compared the relative quantities of each protein in mutants and their normal siblings. Carboxylase accumulation was found to be independent of chlorophyll content, while the amounts of the thylakoid proteins increase at about the same time as chlorophyll in delayed-greening mutants. The relative quantity of LHCP is closely correlated with the relative quantity of chlorophyll at all stages of development in all mutants. Because pigment-deficient mutants are arrested at early stages in chloroplast development, these findings suggest that the processes of chloroplast development, chlorophyll synthesis and thylakoid protein accumulation are coordinated during leaf development but that carboxylase accumulation is controlled by different regulatory mechanisms. A white leaf mutant was found to contain low levels of LHCP mRNA, demonstrating that the accumulation of LHCP mRNA is not controlled exclusively by phytochrome.     

Nomenclature for membrane-bound light-harvesting complexes of cyanobacteria

Accessory chlorophyll-binding proteins (CBP) in cyanobacteria have six transmembrane helices and about 11 conserved His residues that might participate in chlorophyll binding. In various species of cyanobacteria, the CBP proteins bind different types of chlorophylls, including chlorophylls a, b, d and divinyl-chlorophyll a, b. The CBP proteins do not belong to the light-harvesting complexes (LHC) superfamily of plant and algae. The proposed new name of CBP for this class of proteins, which is a unique accessory light-harvesting superfamily in cyanobacteria, clarifies the confusion of names of prochlorophytes chlorophyll binding protein (Pcb), PSII-like light-harvesting proteins and iron-stress-induced protein A (IsiA). The CBP complexes are a member of a larger family that includes the chlorophyll a-binding proteins CP43 and CP47 that function as core antennas of photosystem II.     

Comparative ultrastructural properties of pallisade tissue and spongy tissue chloroplasts from normal and mutant leaves of Pisum sativum L.*

Abstract. The ultrastructure of chloroplasts from palisade and spongy tissue was studied in order to analyse the adaptation of chloroplasts to the light gradient within the bifacial leaves of pea. Chloroplasts of two nuclear gene mutants of Pisum sativum (chlorotica-29 and chlorophyll b-less 130A), grown under normal light conditions, were compared with the wild type (WT) garden-pea cv. ‘Dippes Gelbe Viktoria’. The differentiation of the thylakoid membrane system of plastids from normal pea leaves exhibited nearly the same degree of grana formation in palisade and in spongy tissue. Using morphometrical measurements, only a slight increase in grana stacking capacity was found in chloroplasts of spongy tissue. In contrast, chloroplasts of mutant leaves differed in grana development in palisade and spongy tissue, respectively. Their thylakoid systems appeared to be disorganized and not developed as much as in chloroplasts from normal pea leaves. Grana contained fewer lamellae per granum, the number of grana per chloroplast section was reduced and the length of appressed thylakoid regions was decreased. Nevertheless, chloroplasts of the mutants were always differentiated into grana and stroma thylakoids. The structural changes observed and the reduction of the total chlorophyll content correlated with alterations in the polypeptide composition of thylakoid membrane preparations from mutant chloroplasts. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), polypeptide bands with a relative molecular mass of 27 and 26 kilodalton (kD) were markedly reduced in mutant chloroplasts. These two polypeptides represented the major apoproteins of the light harvesting chlorophyll a/b complex from photosystem II (LHC-II) as inferred from a comparison with the electrophoretic mobility of polypeptides isolated from the LHC-II.     

Membrane structure and function. II. Alterations in the photo-induced absorption changes after treatment of isolated chloroplasts with large pulses of the ruby laser

Treatment of isolated chloroplasts with high-energy pulses of the ruby laser causes graded structural changes in the chloroplast membranes and is here correlated with the biochemical changes produced. The laser treatment caused decreases in the photoinducible absorption changes of cytochromes b₅₅₉, b₅₆₃, and P₅₂₀ (the carotenoid shift), but smaller decreases in cytochrome f. The decreases correlated with the quantum efficiency alterations produced by the laser treatment. Ferricyanide photoreduction and O₂ evolution was only slightly affected by the laser treatment. The slow phase of the dark recovery kinetics of P₅₂₀ was increased maximally by the lowest laser input energies and NADP⁺ photoreduction induced by carbonylcyanide-P-trifluoromethoxyphenylhydrazone (FCCP) was decreased maximally by the lowest energies, suggesting that uncoupling of the chloroplasts was the most sensitive parameter. This was corroborated by our previous observation (5) that chloroplast membrane bound surface particles (coupling factor) was the ultrastructural change most sensitive to the laser pulses. Electron flow from photosystem II to photosystem I was not altered by the laser treatment. The laser treatments did not cause a detectable decrease in total chlorophyll in the chloroplasts, however, approximately 10% of the total chlorophyll was present in the solution phase after the treatment, whereas no detectable cytochromes were present in the solution phase.     

Chlorophyll composition of Photosystems IIα, IIβ and I in tobacco chloroplasts

The antenna composition of the Photosystems II_α, II_β and I was studied in tobacco chloroplasts. Absorbance spectra, recorded at 4 K, were analyzed for the wild type and the mutants Su/su and Su/su var. Aurea, containing higher concentrations of the photosystems. With chloroplasts of Su/su we measured the action spectra of the three photosystems from 625 to 690 nm. Above 675 nm absorption by Photosystem I dominated. This sytem had a maximum at 678 nm and a shoulder at 660 nm. Of the long-wavelength chlorophyll a forms, absorbing at 690, 697 and 705 nm at 4 K, which are generally assigned to Photosystem I, the 697 nm form occurred in an amount of four molecules per reaction center of Photosystem I in each type of chloroplast. The Photosystem II_α spectrum was characterized by maxima at 650 and 672 nm, showing clearly the participation of the chlorophyll a and b containing light-harvesting complex. In the mutants the light-harvesting complex has a chlorophyll a to chlorophyll b ratio of more than 1; the amount of the 672 nm chlorophyll a was normal, whereas the amount of chlorophyll b was markedly decreased in the mutants relative to the wild type. The Photosystem II_β spectrum mainly consisted of a band at 683 nm.     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715–740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50-4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the lightharvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (F_v) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm.From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emittting at 685 nm appears to be directly modulated by the trapping state of the reaction center.     

The Photoxidative Alteration of Chlorophylls in Methyl Linoleate and Prooxidant Activity of Their Decomposition Products

Methyl linoleate containing chlorophylls and/or pheophytins was exposed to light in the presence of oxygen. The photooxidative reaction of both chlorophylls a and b was first-order, and the reaction rate for chlorophyll a was higher than that for chlorophyll b. On the other hand, pheophytins a and b hardly decomposed even after irradiation for 24 hr, and retained a green or a brownish-green color. In qualitative analysis of the photooxidation products of chlorophylls a and b, no pheophytins or pheophorbides were detected, while green and polar red pigments were observed on a thin layer chromatogram near the spot of chlorophyll and the origin, respectively. These photooxidation compounds also had prooxidant effects as well as did chlorophyll.