Low-temperature Fluorescence Emission Spectra Research Articles

Disruption of Chlorophyll Organization and Function in Powdery Mildew-Diseased Cucumber Leaves and Its Control by the HyperparasiteAmpelomyces quisqualis

The hyperparasite Ampelomyces quisqualis is a potential biological control agent for powdery mildew (PM) disease. Cucumber plants were exposed to PM with and without subsequent A. quisqualis treatment. These plants were examined in comparison to healthy uninfected plants. Disease symptoms, including chlorosis and necrosis, were most prominent on the plants exposed only to PM. Electron micrographs of leaf sections of these diseased plants indicated marked deterioration in the morphological organization of chloroplast membranes. In comparison, chloroplasts of A. quisqualis-treated plants seemed undamaged and like those of uninfected plants. Low-temperature fluorescence emission spectra of diseased leaf tissue showed a dramatic increase in the far-red :red emission ratio in comparison with uninfected and A. quisqualis-treated leaves. Room-temperature fluorescence transients revealed reduced variable fluorescence in the diseased plants. Both sets of fluorescence data indicate a disease-correlated increase in levels of uncoupled chlorophyll. Fluorescence methods, thus, are proposed as ideal means for assessing hyperparasite effectiveness in controlling PM.

Chlorophyll Fluorescence Properties of Chloroplast Membranes Isolated from Jasmonic Acid-Treated Barley Seedlings

Summary Effects of in vivo jasmonic acid OA)-treatments on the photosynthetic apparatus of barley chloroplasts were studied by chlorophyll fluorescence measurements. Low temperature (77K) fluorescence emission spectra indicate that the peak at 735 nm originating from photosystem I (PSI) is considerably reduced and the F735/F695 ratio is markedly lower in chloroplasts isolated from JA-treated plants than in control chloroplasts. This effect is most pronounced at an exogenous JA concentration of 10 -4 M Concomitant with this the F695/F685 ratio is enhanced in JA-treated chloroplasts, the effect being also concentration dependent. It is demonstrated that chlorophyll a fluorescence levels (F o , F m ) are also increased to 160% and 123%, respectively, as a result of JA (10 -4 M) treatment. However, the variable fluorescence (F v = F m - F o ) remains almost constant under the same conditions. The fluorescence ratio F v /F m exhibits a certain decrease in chloroplasts isolated from JA-treated plants. These data are discussed in terms of possible involvement of JA-induced alteration at the level of supramolecular organization of the thylakoid membranes.

Changes Induced by Ultraviolet-B Radiation in Vegetative Growth, Foliar Characteristics and Photosynthetic Activities in Vigna unguiculata

The effect of short-term ultraviolet-B (UV-B) radiation on growth and foliar characteristics was studied in Vigna unguiculata (L.) Walp. Daily UV-B exposure was varied from 0.9 to 4.5 kJ m-2 d-1 (15-75 min at 1 J m-2 s-1). Low doses of UV-B irradiation (1.8 kJ m-2 s-1) produced varying responses on growth and leaf morphology. Inhibition of growth and shoot length is attributed to the destruction of endogenous auxin levels by UV-B. Chl b content decreased more than Chl a. Room temperature absorption spectra of carotenes and xanthophyll extracts of treated chloroplasts showed an increased synthesis of carotene. Xanthophylls of UV-B treated seedlings exhibited marked spectral changes which confirm that the light harvesting chlorophyll protein assembly is affected under short term UV-B treatment. Inhibition by 50% of photosynthetic CO2 uptake was observed in seedlings after 3 days of treatment at 1.8 kJ m-2 s-1, which was accounted for by changes in primary carboxylase as well as at the primary photochemical reactions. Low temperature fluorescence emission spectra of chloroplasts reveal that strong UV-B radiation, besides acting directly at the photosystem level, induces a change in the stoichiometry of PS I to PS II.

Identification of four universal protochlorophyllide forms in dark-grown leaves by analyses of the 77 K fluorescence emission spectra

Low-temperature (77 K) fluorescence emission spectra of dark-grown leaves from 12 different plant species were measured, corrected and analysed at different stages of development. The second and fourth derivative of the spectra indicated the peak positions of possible spectral bands, which were used for resolution of the fluorescence spectra into gaussian components. Based on the intensities of the gaussian components, statistical analyses were performed to determine a possible connection between the main peaks and their respective long-wavelength vibrational sublevels. Four universal protochloro-phyll(ide) forms were distinguished, with the main emission bands (half-bandwidths parentheses) at 633 (14.5), 645 (14.3), 657 (10.5) and 670 (20.1) nm respectively. Their vibrational sublevels were found at 693 (20.8), 710 (17.3), 726 (17.7) and 740 (21.0) nm respectively. The spectral characteristics of the different protochlorophyll(ide) forms are discussed in relation to their possible molecular structures and interactions with the surrounding membrane components.

PH dependent chlorophyll fluorescence quenching in spinach thylakoids from light treated or dark adapted leaves

The pH dependence of maximum chlorophyll fluorescence yield (Fm) was examined in spinach thylakoids in the presence of nigericin to dissipate the transthylakoid pH gradient. 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) was present to eliminate photochemical quenching. Thylakoids were prepared from dark adapted leaves ('dark' thylakoids) or preilluminated leaves ('light' thylakoids). In the latter there had been approximately 50% conversion of the xanthophyll violaxanthin to zeaxanthin, while no conversion had occurred in the former. In the presence of a reductant such as ascorbate, antimycin A sensitive quenching was observed (half maximal quenching at 5 μM), whose pH dependence differed between the two types of thylakoid. Preillumination of leaves resulted in more quenching at pH values where very little quenching was observed in 'dark' thylakoids (pH 5-7.6). This was similar to activation of high-energy-state quenching (qE) observed previously (Rees D, Young A, Noctor G, Britton G and Horton P (1989) FEBS Lett 256: 85-90). Thylakoids isolated from preilluminated DTT treated leaves, that contained no zeaxanthin, behaved like dark thylakoids. A second form of quenching was observed in the presence of ferricyanide, that could be reversed by the addition of ascorbate. This was not antimycin A sensitive and showed the same pH dependence in both types of thylakoid. The former type of quenching, but not the latter, showed similar low temperature fluorescence emission spectra to qE, and was considered to occur by the same mechanism.

THE FORMATION OF A SHORT-WAVELENGTH CHLOROPHYLLIDE FORM AT PARTIAL PHOTOTRANSFORMATION OF PROTOCHLOROPHYLLIDE IN ETIOPLAST INNER MEMBRANES

Abstract— Partial phototransformation caused the formation and successive accumulation of spectral forms of chlorophyllide in etioplast inner membrane fractions from wheat (Triticum aestivum L. cv. Walde), Low-temperature fluorescence emission and circular dichroism spectra showed the formation of two chlorophyllide forms with emission maxima at 683 and 694 nm, respectively. The light dependent accumulation of the two forms differed in prolamellar body (PLB) and prothylakoid (PT) fractions. The 694 nm form was preferentially found in PLB fractions which before irradiation were characterized by a 657 nm emitting protochlorophyllide form and a regular PLB structure. The 683 nm form accumulated to a higher extent in PT fractions which before irradiation contained mainly shortwavelength protochlorophyllide forms. The results indicate that at least two photoactive protochlorophyllide forms must be considered. The major part of protochlorophyllide, with a fluoresence emission maximum at 657 nm, is transformed to the 694 nm emitting chlorophyllide first at high light doses. A minor part, revealed only after Gaussian resolution, has a fluorescence emission maximum at 645 nm and was transformed by low light doses to chlorophyllide emitting at 683 nm.

Restoration of both an oligomeric form of the light-harvesting antenna CP II and a fluorescence state II-state I transition by Δ3- trans-hexadecenoic acid-containing phosphatidylglycerol, in cells of a mutant of Chlamydomonas reinhardtii

To define the role of the Δ 3- trans-hexadecenoic acid (16:1- trans) in the thylakoid membrane, restoration experiments were carried out using a Photosystem II (PS II)-lacking but low-fluorescent mutant, mf 2, of Chlamydomonas reinhardtii. This mutant is unable to synthesize 16:1- trans, lacks an oligomeric form of the main light-harvesting chlorophyll (Chl) a + b antenna, CP II, and shows an impaired regulation of the excitation energy distribution. Whole cells were incubated for 39 h in the presence of liposomes of 16:1- trans-containing phosphatidylglycerol (PG-16:1- trans), in the light at 25°C. Then lipids and Chl-protein complexes were analyzed and low-temperature fluorescence emission spectra, both in state I and in state II (oxidized and reduced plastoquinone pool), were measured. The results indicated: (1) a relatively important content of 16:1- trans specifically incorporated in the chloroplast phosphatidylglycerol (PG); (2) an appreciable amount of CP II oligomeric form; (3) the occurrence of a clear state II-state I transition, as shown by a ratio of the CP II fluorescence at 682 nm to the photosystem I fluorescence at 712 nm, which was 3.8-times higher in state I than in state II. These restorations were not observed when cells of mf 2 were incubated in the presence of palmitate-containing PG, of oleate-containing phosphatidylcholine or of PG-16:1- trans + cycloheximide. It is concluded that: (1) the oligomeric form of CP II is essential for a good excitation energy transfer towards the PS II region and, consequently, to a good state II-state I transition in the distribution of excitation energy; (2) PG-16:1- trans probably plays an essential role in stabilizing neo-formed CP II oligomers during the assembly of new Chl-protein complexes in the chloroplast.

Light-Dependent Assembly of the Components of Photosystem II Core Complex CPa in Mutant C-6D of Scenedesmus obliquus

Cells of pigment mutant C-6D of the unicellular green alga Scenedesmus obliquus synthesize only chlorophyll a and carotenoid precursors during heterotrophic growth in the dark. These cells have photosystem I-activity but no photosystem II-activity and lack the core-complex CPa of photosystem II. Upon transfer to light, the cells immediately form carotenoids and an active photosystem II (Humbeck et aI., 1989). In this report we demonstrate that the apoproteins of CPa are already present in the dark. Upon transfer to light, pigment-protein complex CPa is quickly assembled. The low temperature fluorescence excitation and emission spectra do not change from the first assembly gf CPa until further development of photosystem II. Illumination with monochromatic light reveals that light of the blue as well as of dhe red region of the spectrum induces the formation of CPa. The mode of pigment-protein complex CPa assembly under illumination in mutant C-6D is discussed.

Chlorophyll—protein complexes related to photosystem I in Chlamydomonas reinhardtii

A study of the photosystem I (PS I)-related chlorophyll (Chl)—protein complexes of the unicellular green alga Chlamydomonas reinhardtii was performed, using the wild type and six photosynthesis mutants. Three complexes have been previously described in the literature: CP Ia, undissociated PS I complex; CP 0, Chl a + b peripheral antenna of PS I; CP I, Chl a complex corresponding to the PS I reaction center and its core antenna. Another Chl a complex, CP 0a, has been more recently described by us. The electrophoretic behaviors of these complexes and of their polypeptide subunits have been studied using different solubilization mixtures and lithium dodecylsulfate polyacrylamide gels. It was determined that the non-denatured complex CP 0a shows an apparent relative molecular mass ( M r in the 80 000 range and that two polypeptides of M r = 50 000 and 38 000 resulted from its dissociation. Low temperature fluorescence emission spectra of cells of the wild type and of three mutants were measured both in oxidized and reduced plastoquinone pool conditions. It appeared obvious that, in a CP 0a-lacking mutant, the excitation energy transfer from the main light-harvesting antenna towards PS I was impaired and inefficient. This is interpreted as indicating that CP 0a plays the essential role of a connecting antenna between the peripheral antenna CP 0 and the core antenna (CP I) of PS I.

Chlorophyll precursors in the plasma membrane of a cyanobacterium, Anacystis nidulans: Characterization of protochlorophyllide and chlorophyllide by spectrophotometry, spectrofluorimetry, solvent partition, and high performance liquid chromatography

Plasma membranes were isolated and separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation of crude membranes prepared by French pressure cell extrusion of lysozyme-treated Anacystis nidulans. Two distinct populations of chlorophyll-free plasma membrane vesicles were obtained exhibiting buoyant densities of 1.087 and 1.100 g/cm3 as opposed to a uniform density of 1.192 g/cm3 for thylakoid membranes. Plasma and thylakoid membranes were characteristically different also with respect to fatty acid and protein composition, cytochrome oxidase activity, and pigment content as analyzed by spectrophotometry, spectrofluorimetry, and high performance liquid chromatography. Apart from carotenoids, chlorophyll a was the only major photosynthetic pigment detected in thylakoid membranes while plasma membranes contained virtually no chlorophyll a but (besides large amounts of carotenoids) protochlorophyllide a and chlorophyllide a as revealed by solvent partition (between n-hexane and acetone or methanol), room and low temperature fluorescence emission and excitation spectra, and analytical separation and identification by high performance liquid chromatography and comparison with authentic standards. The protochlorophyllide in the plasma membrane could be transformed into chlorophyllide in the dark in vitro by incubating the membrane preparation with NADPH; NADP+ effected the reverse transition.

Spectral Properties and Composition of Reaction Center and Ancillary Polypeptide Complexes of Photosystem II Deficient Mutants of Synechocystis 6803

The polypeptide composition and spectral properties of three photosystem II (PSII) deficient mutants of the cyanobacterium Synechocystis 6803 have been determined. The levels of the 43 and 47 kilodalton chlorophyll-binding proteins and the reaction center component D2 are affected differently in each mutant; the 33 kD polypeptide of the oxygen-evolving complex is found at wild-type levels in all three. The 43 and 47 kilodalton proteins are implicated as important elements in the assembly and/or stability of the PSII reaction center, although the loss of one of these polypeptides does not lead to the loss of all PSII proteins. Low temperature fluorescence emission spectra of wild-type cells reveal chlorophyll-attributable peaks at 687 (PSII), 696 (PSII), and 725 (photosystem I) nanometers. All three mutants retain the 725 nanometer fluorescence but lack the 696 nanometer peak. This suggests that the latter fluorescence arises from PSII reaction center chlorophyll or results from interactions among functional PSII components in vivo. Cells that contain the 43 kilodalton and lack the 47 kilodalton protein, retain the 687 fluorescence; furthermore, in as much as this fluorescence is absent from cells without the 43 kilodalton protein, the 687 nanometer peak is judged to emanate from the 43 kilodalton chlorophyll-protein. A new peak, probably previously obscured, is revealed at 691 nanometers in cells that retain the 47 kilodalton protein but lack the 43 kilodalton polypeptide, suggesting that emission near 691 nanometers can be attributed to the 47 kilodalton polypeptide. Membrane-bound phycobilisomes are retained in these cells as is coupled-energy transfer between phycocyanin and allophycocyanin. Energy transfer to photosystem I by way of phycocyanin excitation proceeds as in wild-type cells despite the absence of certain PSII components.

Chloroplast biogenesis at cold-hardening temperatures. Kinetics of trans-?3-hexadecenoic acid accumulation and the assembly of LHCII

Etiolated seedlings developed at cold-hardening temperatures (5°C) exhibited etioplasts with considerable vesiculation of internal membranes compared to etioplasts developed at 20°C regardless of the osmotic concentration employed during sample preparation. This vesiculation disappeared during exposure to continuous light at 5°C. This transformation of 5°C and 20°C etioplasts to chloroplasts under continuous light at 5° and 20°C respectively proceeded normally with the initial development of non-appressed lamellae and the subsequent appearance of granal stacks. However, chloroplasts developed at 5°C exhibited fewer lamellae per granum than chloroplasts developed at 20°C.Although the polypeptide complements of etioplasts and chloroplasts developed at 5° or 20°C were not significantly different, monomeric light harvesting complex (LHCII3) was assembled into oligomeric light harvesting complex (LHCII1) during chloroplast biogenesis at 20°C (oligomer:monomer =1.8) whereas monomeric LHCII predominated at 5°C (oligomer:monomer =0.3). Low temperature fluorescence emission spectra of isolated thylakoids indicated that both the F685/F735 and F695/F735 were significantly higher after greening at 5°C than at 20°C. In addition, chloroplast biogenesis at 5°C was associated with a low ratio of trans-Δ3-hexadecenoic acid (0.5) in phosphatidylglycerol whereas at 20°C biogenesis was associated with a high ratio (1.6). Comparative kinetics indicated that the maximization of the trans-Δ3-hexadecenoic acid level precedes the assembly of monomeric LHCII into oligomeric LHCII during biogenesis at 20°C. It is suggested that low developmental temperatures modulate the assembly of LHCII by reducing the trans-Δ3-hexadecenoic acid content of phosphatidylglycerol such that monomeric or some intermediate form of LHCII predominates.

Temperature dependence of antennae chlorophyll fluorescence kinetics in photosystem I reaction centre protein

CPl, the isolated reaction centre (RC) chlorophyll(chl)-protein of plant photosystem I(PSI) containing P700 and ca. 40 antenna Chl has been isolated using sodium dodecyl sulphate and gel electrophoresis. It retained the triplet e.s.r. polarisation pattern characteristic of active charge separation and recombination. Low-temperature and time-resolved fluorescence emission spectra showed that at least tow discrete antenna chl forms were present, and excitation energy transfer between them and P700 was studied by measuring chl sub-nanosecond fluorescence decay kinetics over a range of temperatures and emission wavelengths, using ca. 100 ps Ar-ion laser excitation pulses and single-photon detection, resulting in ca. 10 ps time resolution. The two forms are F720, emitting at 720 nm (low-energy sites within the antenna) and F690, emitting at 690–695 nm. The latter form was only observed at short times ( 1 ns) components were of small amplitude and have no significance for energy transfer. The lifetime of the shortest resolved component varied in a complex way with temperature between 30 and 150 ps, also dependent on emission wavelength. At T > 200 K the lifetime was 40 ± 10 ps, independent of wavelength, dependence with a distinct minimum at 690–695 nm. A model is presented for energy transfer between the discrete chl antenna forms which accounts for the change of the observed lifetimes with temperature. In this model F690 forms a core antenna close to the RC and can transfer energy to P700 even at 10 K. Endothermic energy transfer out of F720, which is inhibited by low temperatures, gives rise to the observed temperature dependence of the F690 and F720 fluorescence lifetimes.

Properties of the water-soluble chlorophyll-synthetic linear macromolecular complexes

The properties of the water-soluble chlorophyll (Chl) a or b-synthetic linear macromolecular (poly(vinylpyrrolidone)(PVP), poly(ethylene glycol)(PEG) or poly(vinyl alcohol)(PVA)) complexes were investigated. The low-temperature (77 K) fluorescence emission spectra suggested that the Chl a or b-PVP and Chl b-PVA complexes had the similar form of Chl a or b to that of Chl a or b in ethyl ether or Triton X-100, probably a monomeric form, while the Chl a or b-PEG and Chl a-PVA complexes contained the several undefined forms of Chl a or b. The Chl a-PVP complex was eluted at the same volume of the corresponding PVP applied independently to a column of Sephadex G-100, but the Chl a-PEG and Chl a-PVA complexes showed the elution patterns similar to that of Chl a-bovine serum albumin (BSA) complex. The Chls in the synthetic linear macromolecular complexes were stable against oxidative stress of photobleaching, but they were easily hydrolyzed by chlorophyllase, as were the Chls in both the BSA complexes and the isolated light-harvesting Chl a b- protein complex (LHC). A possible localization of Chls within linear macromolecular complexes was suggested so that the tetrapyrrole ring and phytol group of Chl would be surrounded by a linear macromolecular (PVP) chain or a number of linear macromolecular (PEG or PVA) chains, whereas the hydrophyllic edge of porphyrin ring, adjacent to the phytol group, would be exposed to the environment of a water medium.

Chlorophyll Fluorescence in Leaves and Cells of Light-Grown Maize ( Zea mays) during Development

A comparison of chlorophyll a fluorescence in segments chosen from different parts of developing maize ( Zea mays L. cv. Golden Bantam) leaf show differences in the relative variable fluorescence intensities of the induction time course at room temperature (Kautsky curves). The low temperature fluorescence emission spectra of intact leaf samples and diluted powder samples which were prepared from different sections of the leaf were measured. Older leaf segments (tip) showed more variable fluorescence and less relative fluorescence of light harvesting chlorophyll a/b-protein complex and of Photosystem II chlorophyll a-protein complex at 77K compared to young leaf segments (base). These results show (1) that the functions of mesophyll and bundle sheath chloroplasts differ during development of the maize leaf (2) that the amount of chlorophyll protein complexes present varies along the maize leaf from base to tip.

Characterization of New Strains of Photosynthesis Mutants of <italic>Chlamydomonas Reinhardtii</italic> IV Impaired Excitation Energy Transfer in Three Low Fluorescent Mutants

Three new mutants of Chlamydomonas reinhardtii were characterized, which showed maximum Chl fluorescence yields close to the initial minimum level and clearly lower than the wild type maximum yield. Two of these mutants, mf 1 and mf 2, also showed anomalies characteristic of mutants lacking functional PS II: absence of PS II-related photochemical activity and of the variable component of fluorescence, deficiencies of high potential Cyt b-559, of the Chl-protein complexes CP III and CP IV and of five PS II-related proteins. The third mutant, mf 3, was able to perform photosynthesis and did not show any of the above deficiencies. Low-temperature fluorescence emission spectra indicated clearly that, whatever the oxido-reduction state of the plastoquinone pool, the emission of the main light-harvesting antenna at 682 nm was weaker and the emission of the PS I core antenna at 712–716 nm was more important in mf 1 and mf 2 than in a “classical” PS II-deficient mutant. In mf 3, unlike the wild type, the PS II-related emissions at 686 and 696 nm were relatively less important than that at 712–716 nm. Low-temperature fluorescence excitation spectra showed that, in the three mutants, the light captured by the main Chl a+b antenna, CP II, was the most efficient in inducing PS I fluorescence. These results indicate that, in the three mutants, the energy captured by the main light-harvesting antenna is always preferentially transferred towards PS I. They will be discussed in terms of membrane defects leading to an impaired regulation mechanism for the distribution of the excitation energy between the two photosystems.

Excitation-energy redistribution in the cryptomonad alga Cryptomonas ovata

We have studied the redistribution of excitation energy in the cryptomonad alga Cryptomonas ovata. Low-temperature fluorescence emission spectra from cells preilluminated with light 1 and light 2 show that preferential excitation of Photosystem II (PS II) leads to decreased fluorescence emission from chlorophyll (Chl) a associated with PS II relative to the emission following the preferential excitation of Photosystem I (PS I). The fluorescence change is indicative of a light-state transition by the cells. However, comparision of measurements of the kinetics of P-700 photooxidation by cells fixed with glutaraldehyde following illumination with light 1 or light 2 shows that the relative activity of PS I is lower in cells fixed in light 2. This is in contrast to the expectation for cells in State 2. Excitation spectra for the fluorescence emission from PS II Chl a show that preferential excitation of PS II leads to a decreased probability for energy transfer from phycoerythrin and Chl c 2 to PS II when compared to cells in which PS I is preferentially excited. This result is in accordance with recent picosecond time-resolved fluorescence studies (Bruce, D., Biggins, J., Charbonneau, S. and Thewalt, M. (1987) in Progress in Photosynthesis Research (Biggins, J., ed.), Vol. II, pp. 777–780, Martinus Nijhoff, Dordrecht) and we, therefore, suggest that C. ovata does not undergo a classical light-state transition. However, preferential excitation of PS II or PS I appears to cause pigment-protein conformational changes which change the probability for energy transfer from phycoerythrin to PS II, and we suggest that this may be a mechanism for photoprotection of PS II. Studies of the kinetics of excitation-energy redistribution, and of the effects of electron-transport inhibitors and uncouplers of photophosphorylation indicate that the mechanism for excitation-energy redistribution in C. ovata and phycobilisome-containing organisms may be similar.

Chlorophyll-protein complexes of barley photosystem I.

Photosystem I (PSI) preparations with a chlorophyll a/b ratio of 6.0 were isolated from barley thylakoids using two different methods. The high-molecular-mass complex (CP1a) which is resolved by non-denaturing gel electrophoresis had the same properties as a PSI preparation (PSI-200) isolated by Triton X-100 solubilisation of thylakoids followed by sucrose gradient ultracentrifugation. This material had a chlorophyll:P700 ratio of 208:1 and was composed of three different chlorophyll-protein complexes which could be separated from each other by solubilising the PSI preparation in dodecyl maltoside followed by sucrose gradient ultracentrifugation. Approximately half of the chlorophyll, including all the chlorophyll b, was located in two antenna complexes designated LHCI-680 and LHCI-730, which were identified by their characteristic low-temperature fluorescence emission spectra. The rest of the chlorophyll a was associated with the PSI reaction centre, P700 Chla-P1, which fluoresced at 720 nm. Each chlorophyll-protein complex had a unique polypeptide composition and characteristic circular dichroic and absorption spectra. The use of dodecyl maltoside instead of dodecyl sulphate resulted in a less denatured form of LHCI-680, which fluoresced at 690 nm at 77 K. One of the sucrose gradient fractions contained a complex consisting of only LHCI-730 and P700 Chla-P1 which fluoresced at 731 nm, indicating that LHCI-730 is structurally associated with P700 Chla-P1 and quenches its fluorescence. Approximately three-quarters of the light-harvesting antenna chlorophyll was in LHCI-730, but only about one-quarter of the normal complement of LHCI-730 was required to quench the reaction centre. By reducing the amount of Triton relative to the chlorophyll concentration, a PSI preparation (chlorophyll a/b ratio of 3.5) with a chlorophyll:P700 ratio of 300:1 was isolated. It contained no photosystem II, but a significant amount of LHCII which was functionally connected to the PSI reaction centre. Reconstitution studies demonstrated that excitation energy transfer from LHCII to PSI requires the presence of LHCI-680, and we propose that, in PSI, the following linear excitation energy transfer sequence occurs: LHCII----LHCI-680----LHCI-730----P700 Chla-P1.

Spectroscopic properties of chloroplast grana membranes and of the core of Photosystem II

An oxygen-evolving Photosystem II core complex essentially free of the light-harvesting chlorophyll a b protein complex, containing 45 chlorophylls per reaction center was isolated from spinach chloroplasts. Its structural integrity was established by studying its photochemistry and spectral properties. The absorption spectrum measured at 4 K revealed the presence of at least five spectrally distinct chlorophyll a species. The same bands, but in different proportions, were observed in a Photosystem II grana preparation used as starting material for the preparation of the core complex. The relative contributions of these components to the overall absorption were calculated by deconvoluting this spectrum into Gaussian bands. The core complex was enriched in a long-wave band located at 683 nm, which presumably reflects the presence of 8–10 pigment molecules that are closely associated with the reaction center. Low temperature fluorescence emission spectra showed the characteristic Photosystem II emission bands located at 685 nm (F 685) and at 695 nm (F 685). The two states giving rise to these emissions are in thermal equilibrium down to 70 K. It is suggested that F 685 arises from a chlorophyll a species absorbing at 676 nm and that F 695 is the result of fluorescence from the photoactive pheophytin a absorbing around 683 nm.

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

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.