Linear dichroism of microalgae, developing thylakoids and isolated pigment-protein complexes in stretched poly(vinyl alcohol) films at 77 K

A variety of unicellular algae, thylakoids from higher plants in different stages of maturity and isolated pigment-protein complexes were oriented in stretched polyvinyl alcohol films. Low temperature linear dichroism (LD) spectra of Chlorella pyrenoidosa and higher plant thylakoids in the films were very similar to those obtained after orientation of similar samples using magnetic or electric fields. Positive LD bands corresponding to Chl a (670) and (682) and negative bands due to Chl a (658) and Chl b(648) were resolved in spectra of the light harvesting Chl a/b protein. Chl b (648) and Chl a (658) and (670) were not seen in the LD spectrum of thylakoids from plants grown in intermittent light, the Chl b-less mutant of barley, Euglena gracilis or the cyanobacteria, Phormidium luridum and Anacystis nidulans, but did appear upon chloroplast maturation in Romaine lettuce and during the greening of etiolated and intermittent light plants. The highly oriented long wavelength Chl a (682) in the light-harvesting complex may represent residual PS II whose peak dichroism is centered at 681 nm. The PS I preparation had a Chl a/b ratio of approx. 6 and the LD spectrum was positive with a maximum at 690-694 nm and a band of lower amplitude at 652 nm. The minor LD band was not observed in PS I preparations from organisms that lack chl b such as the cyanobacteria, intermittent light plants and the Chl b-less mutant of barley. We suggest that the 652 nm band is due to Chl b molecules associated with the antenna of PS I and are distinct from those on the light harvesting complex whose orientation is different. We also conclude that all the Chl a forms are oriented and that the long geometric axes of the pigment-protein complexes, as deduced from the configuration they assume in the stretched films, are axes that normally lie parallel to the plane of the native thylakoid.     

Action spectra and functional antenna sizes of Photosystems I and II in relation to the thylakoid membrane organization and pigment composition

The functional organization of competent photosynthetic units in developing thylakoids from intermittent-light grown pea as well as in the unstacked, stacked and phosphorylated stacked thylakoids from its mature chloroplasts was characterized by polarographic measurements of action spectra, reaction centre contents and optical cross-sections for PS I-mediated O2 uptake and PS II-mediated O2 evolution. The minimum antenna sizes of 60 and 37 chlorophyll a molecules for PS I and PS II, respectively, were determined in developing thylakoids with a ratio of Chl a/Chl b>50. In mature chloroplasts, the embedded light-harvesting chlorophyll a/b-binding (LHC) protein complexes increased the PS I and PS II effective antenna sizes by 3–6 times depending on the thylakoid membrane organization. In unstacked thylakoids, a randomization of PS I, PS II and LHC II led to the most uniform spectral distribution of light harvesting between the two photosystems but caused the maximal difference of their antenna sizes to be 370 and 100 Chls for the competent PS I and PS II units, respectively. Following the Mg2+-induced stacking of thylakoids, opposite complementary changes of the action spectra, antenna sizes and Chl a/Chl b ratios indicated a redistribution of a LHC II pool of 100 Chl ( a + b) molecules from PS I to PS II. Unlike to the stroma-exposed PS II in unstacked thylakoids, the granal PS II units of 200 Chls demonstrated an additional 2-fold increase of the effective antenna size due to energy transfer within PS II dimers under strong background illumination, which closed >90% of reaction centres. Protein phosphorylation of the stacked thylakoids induced a significant inactivation of the O2-evolving PS II centres but did not cause complementary changes of the action spectra and antenna sizes of the competent PS I and PS II. In this case, light harvesting parameters of the O2-evolving PS II units were nearly unaffected, whereas the obvious relative increase of the PS I activity at 650 nm and its decrease at >700 nm both in the action spectrum and optical cross-section measurements might suggest a substitution of PS I units in the O2-reducing fraction by another distinct fraction of -type which in turn is not the same to PS I units in unstacked thylakoids.     

The coleoptile chloroplast: Distinct distribution of xanthophyll cycle pigments,and enrichment in Photosystem II

Recent studies have shown that coleoptile chloroplasts operate the xanthophyll cycle, and that their zeaxanthin concentration co-varies with their sensitivity to blue light. The present study characterized the distribution of photosynthetic pigments in thylakoid pigment–protein complexes from dark-adapted and light-treated coleoptile and mesophyll chloroplasts, the low temperature fluorescence emission spectra, and the rates of PS I and PS II electron transport in both types of chloroplasts from 5-day-old corn seedlings. Pigments were extracted from isolated PS I holocomplex, LHC IIb trimeric and LHC II monomeric complexes and analyzed by HPLC. Chlorophyll distribution in coleoptile thylakoids showed 31% of the total collected Chl in PS I and 65% in the light harvesting complexes of PS II. In mesophyll thylakoids, the values were 44% and 54%, respectively. Mesophyll and coleoptile PS I holocomplexes differed in their Chl t a/Chl t b ratios (8.1 and 6.1, respectively) and -carotene content. In contrast, mesophyll and coleoptile LHC IIb trimers and LHC II monomers had similar Chl t a/Chl t b ratios and -carotene content. The three analyzed pigment–protein complexes from dark-adapted coleoptile chloroplasts contained zeaxanthin, whereas there was no detectable zeaxanthin in the complexes from dark-adapted mesophyll chloroplasts. In both chloroplast types, zeaxanthin and antheraxanthin increased markedly in the three pigment–protein complexes upon illumination, while violaxanthin decreased. In mesophyll thylakoids, zeaxanthin distribution as a percentage of the xanthophyll cycle pool was: LHC II monomers > LHC IIb trimers > PS I holocomplex, and in coleoptile thylakoids, it was: LHC IIb trimers > LHC II monomers = PS I holocomplex. Low temperature (77 K) fluorescence emission spectra showed that the 686 nm emission of coleoptile chloroplasts was approximately 50% larger than that of mesophyll chloroplasts when normalized at 734 nm. The pigment and fluorescence analysis data suggest that there is relatively more PS II per PS I and more LHC I per CC I in coleoptile chloroplasts than in mesophyll chloroplasts. Measurements of t in vitro uncoupled photosynthetic electron transport showed approximately 60% higher rates of electron flow through PS II in coleoptile chloroplasts than in mesophyll chloroplasts. Electron transport rates through PS I were similar in both chloroplast types. Thus, when compared to mesophyll chloroplasts, coleoptile chloroplasts have a distinct PS I pigment composition, a distinct chlorophyll distribution between PS I and PS II, a distinct zeaxanthin percentage distribution among thylakoid pigment–protein complexes, a higher PS II-related fluorescence emission, and higher PS II electron transport capacity. These characteristics may be associated with a sensory transducing role of coleoptile chloroplasts.     

Orientation of pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. II. Linear dichroism spectra of isolated pigment-protein complexes oriented in polyacrylamide gels at 300 and 100 K

The absorption and linear dichroism (LD) spectra (380–780 nm) of isolated light-harvesting complex (LHC), Photosystem I (PS I), Photosystem II (PS II), as well as intact thylakoids have been determined at 300 and 100 K. The samples were oriented in squeezed polyacrylamide gel. The low-temperature spectra of LHC and PS I present LD signals which are characteristic enough to be recognized in the LD spectrum of thylakoids. Tentative assignments of the various features of the LD spectra to the major photosynthetic pigments are discussed. A shoulder in the low-temperature absorption spectra is observed at about 673 nm in all the systems under investigation. The absence of an associated LD signal suggests that this ubiquitous chlorophyll (Chl) a form is non-dichroic. Furthermore, in the three isolated chlorophyll-protein complexes described in this study the sign of the LD signal indicates that both the Q_y transition of the Chl a and the carotenoid molecules are preferentially oriented parallel to the largest dimension(s) of the particles.     

Linear dichroism of microalgae,developing thylakoids and isolated pigment-protein complexes in stretched poly (vinyl alcohol) films at 77 K

A variety of unicellular algae, thylakoids from higher plants in different stages of maturity and isolated pigment-protein complexes were oriented in stretched polyvinyl alcohol films. Low temperature linear dichroism (LD) spectra of Chlorella pyrenoidosa and higher plant thylakoids in the films were very similar to those obtained after orientation of similar samples using magnetic or electric fields.Positive LD bands corresponding to Chl a (670) and (682) and negative bands due to Chl a (658) and Chl b (648) were resolved in spectra of the light harvesting Chl a/b protein. Chl b (648) and Chl a (658) and (670) were not seen in the LD spectrum of thylakoids from plants grown in intermittent light, the Chl b-less mutant of barley, Euglena gracilis or the cyanobacteria, Phormidium luridum and Anacystis nidulans, but did appear upon chloroplast maturation in Romaine lettuce and during the greening of etiolated and intermittent light plants. The highly oriented long wavelength Chl a (682) in the light-harvesting complex may represent residual PS II whose peak dichroism is centered at 681 nm. The PS I preparation had a Chl $\text{a}\text{b}$ ratio of approx. 6 and the LD spectrum was positive with a maximum at 690–694 nm and a band of lower amplitude at 652 nm. The minor LD band was not observed in PS I preparations from organisms that lack Chl b such as the cyanobacteria, intermittent light plants and the Chl b-less mutant of barley. We suggest that the 652 nm band is due to Chl b molecules associated with the antenna of PS I and are distinct from those on the light harvesting complex whose orientation is different. We also conclude that all the Chl a forms are oriented and that the long geometric axes of the pigment-protein complexes, as deduced from the configuration they assume in the stretched films, are axes that normally lie parallel to the plane of the native thylakoid.     

A new type of subchloroplast fragments isolated from pea chloroplasts in the presence of digitonin

Heavy fragments were isolated from pea chloroplasts using digitonin treatment and differential centrifugation. The particles were characterized by a significantly lowered chlorophyll a/b ratio, contents of photosystem I (PS I) proteins and ATPase, as well as of amount of P700. The content of photosystem II (PS II) proteins decreased insignificantly, whereas that of proteins of the light-harvesting complex II did not change. The absorption and low-temperature fluorescence spectra were indicative of a decreased content of PS I. Electron microscopy of ultrathin sections of heavy fragment preparations identified them as grana with reduced content of thylakoids. The diameter of these particles was practically the same as within chloroplasts. Comparison of various characteristics of the fragments and chloroplasts from which the fragments were isolated allowed us to define a high degree of preservation of marginal regions in thylakoids present in the heavy fragment particles. Analysis of the results shows that the procedure of fragmentation produces grana with high extent of thylakoid integrity. The phenomenon of reduction of the thylakoid content in grana, occurring as our heavy fragments, is considered in the frame of our previous hypothesis concerning the peculiarities of grana organization in the transversal direction.     

Carotenoid distribution and deepoxidation in thylakoid pigment-protein complexes from cotton leaves and bundle-sheath cells of maize

In response to excess light, the xanthophyll violaxanthin (V) is deepoxidized to zeaxanthin (Z) via antheraxanthin (A) and the degree of this deepoxidation is strongly correlated with dissipation of excess energy and photoprotection in PS II. However, little is known about the site of V deepoxidation and the localization of Z within the thylakoid membranes. To gain insight into this problem, thylakoids were isolated from cotton leaves and bundle-sheath strands of maize, the pigment protein-complexes separated on Deriphat gels, electroeluted, and the pigments analyzed by HPLC. In cotton thylakoids, 30% of the xanthophyll cycle pigments were associated with the PS I holocomplex, including the PS I light-harvesting complexes and PS I core complex proteins (CC I), and about 50% with the PS II light-harvesting complexes (LHC II). The Chl was evenly distributed between PS I and PS II. Less than 2% of the neoxanthin, about 18% of the lutein, and as much as 76% of the -carotene of the thylakoids were associated with PS I. Exposure of pre-darkened cotton leaves to a high photon flux density for 20 min prior to thylakoid isolation caused about one-half of the V to be converted to Z. The distribution of Z among the pigment-protein complexes was found to be similar to that of V. The distribution of the other carotenoids was unaffected by the light treatment. Similarly, in field-grown maize leaves and in the bundle-sheath strands isolated from them, about 40% of the V present at dawn had been converted to Z at solar noon. Light treatment of isolated bundle-sheath strands which initially contained little Z caused a similar degree of conversion of V to Z. As in cotton thylakoids, about 30% the V+A+Z pool in bundle-sheath thylakoids from maize was associated with the PS I holocomplex and the CC I bands and 46% with the LHC II bands, regardless of the extent of deepoxidation. These results demonstrate that Z is present in PS I as well as in PS II and that deepoxidation evidently takes place within the pigment-protein complexes of both photosystems.Abbreviations A antheraxanthin - CC I, CC II Core or reaction center complex of PS I, PS II - CP Chl protein - EPS epoxidation state - F_m Chl fluorescence at closed PS II reaction centers - IEF isoelectric focussing gels - LHC I, LHC II light-harvesting complex of PS I, PS II - OE oxygen evolving polypeptide - PFD photon flux density - PS I^* PS I holocomplex - V violaxanthin - Z zeaxanthin - antibody against C.I.W.-D.P.B. Publication No. 1127.     

High Yield Non-detergent Isolation of Photosystem I-Light-harvesting Chlorophyll II Membranes from Spinach Thylakoids: IMPLICATIONS FOR THE ORGANIZATION OF THE PS I ANTENNAE IN HIGHER PLANTS*

Styrene-maleic acid copolymer was used to effect a non-detergent partial solubilization of thylakoids from spinach. A high density membrane fraction, which was not solubilized by the copolymer, was isolated and was highly enriched in the Photosystem (PS) I-light-harvesting chlorophyll (LHC) II supercomplex and depleted of PS II, the cytochrome b₆/f complex, and ATP synthase. The LHC II associated with the supercomplex appeared to be energetically coupled to PS I based on 77 K fluorescence, P₇₀₀ photooxidation, and PS I electron transport light saturation experiments. The chlorophyll (Chl) a/b ratio of the PS I-LHC II membranes was 3.2 ± 0.9, indicating that on average, three LHC II trimers may associate with each PS I. The implication of these findings within the context of higher plant PS I antenna organization is discussed.     

Changes in lipid composition of Photosystem 1 particles from thylakoids phosphorylated under reductive or anaerobic conditions

Changes in lipid composition of Photosystem 1 (PS 1) particles isolated from thylakoids phosphorylated under reductive or anaerobic conditions have been studied. Under reductive conditions, there was an increase in monogalactosyldiacylglycerol containing highly saturated fatty acids and phosphatidylglycerol containing transhexadecenoic fatty acid. Under anaerobic conditions, the amount of all lipid classes was increased. As we have shown earlier (S. V. Manuilskaya, O. I. Volovik, A. I. Mikhno, A. I. Polischuk and S. M. Kochubey (1990) Photosynthetica 24: 419–423) these changes were due to a co-migration of some lipid species and light-harvesting chlorophyll a/b complex LHC II from PS 2 to PS 1. These data allow us to conclude that LHC II consists of the lipoproteins containing specific lipids. Different composition of lipids co-migrating with LHC II under various conditions of phosphorylation might be caused by the variety of LHC II subpopulations transferred under each reductive condition.Abbreviations PS 1 Photosystem 1 - PS 2 Photosystem 2 - LHC II light-harvesting chlorophyll a/b protein complex II - Chl chlorophyll - MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol - PG phosphatidylglycerol - SQDG sulfoquinovosyldiacylglycerol     

Reversed-phase HPLC determination of chlorophyll a' and phylloquinone in Photosystem I of oxygenic photosynthetic organisms. Universal existence of one chlorophyll a' molecule in Photosystem I.

Chlorophyll (Chl) a', the C132-epimer of Chl a, is a constituent of the primary electron donor (P700) of Photosystem (PS) I of a thermophilic cyanobacterium Synechococcus (Thermosynechococcus) elongatus, as was recently demonstrated by X-ray crystallography. To determine whether PS I of oxygenic photosynthetic organisms universally contains one molecule of Chl a', pigment compositions of thylakoid membranes and PS I complexes isolated from the cyanobacteria T. elongatus and Synechocystis sp. PCC 6803, the green alga Chlamydomonas reinhardtii, and the green plant spinach, were examined by simultaneous detection of phylloquinone (the secondary electron acceptor of PS I) and Chl a' by reversed-phase HPLC. The results were compared with the Chl a/P700 ratio determined spectrophotometrically. The Chl a'/PS I ratios of thylakoid membranes and PS I were about 1 for all the organisms examined, and one Chl a' molecule was found in PS I even after most of the peripheral subunits were removed. Chl a' showed a characteristic extraction behaviour significantly different from the bulk Chl a in acetone/methanol extraction upon varying the mixing ratio. These findings confirm that a single Chl a' molecule in P700 is the universal feature of PS I of the Chl a-based oxygenic photosynthetic organisms.     

Organization of the pigment molecules in the chlorophyll a/b/c containing alga Mantoniella squamata (Prasinophyceae) studied by means of absorption, circular and linear dichroism spectroscopy

In order to obtain information on the organization of the pigment molecules in chlorophyll (Chl) a/b/c-containing organisms, we have carried out circular dichroism (CD), linear dichroism (LD) and absorption spectroscopic measurements on intact cells, isolated thylakoids and purified light-harvesting complexes (LHCs) of the prasinophycean alga Mantoniella squamata. The CD spectra of the intact cells and isolated thylakoids were predominated by the excitonic bands of the Chl a/b/c LHC. However, some anomalous bands indicated the existence of chiral macrodomains, which could be correlated with the multilayered membrane system in the intact cells. In the red, the thylakoid membranes and the LHC exhibited a well-discernible CD band originating from Chl c, but otherwise the CD spectra were similar to that of non-aggregated LHC II, the main Chl a/b LHC in higher plants. In the Soret region, however, an unusually intense (+) 441 nm band was observed, which was accompanied by negative bands between 465 and 510 nm. It is proposed that these bands originate from intense excitonic interactions between Chl a and carotenoid molecules. LD measurements revealed that the Q(Y) dipoles of Chl a in Mantoniella thylakoids are preferentially oriented in the plane of the membrane, with orientation angles tilting out more at shorter than at longer wavelengths (9 degrees at 677 nm, 20 degrees at 670 nm and 26 degrees at 662 nm); the Q(Y) dipole of Chl c was found to be oriented at 29 degrees with respect to the membrane plane. These data and the LD spectrum of the LHC, apart from the presence of Chl c, suggest an orientation pattern of dipoles similar to those of higher plant thylakoids and LHC II. However, the tendency of the Q(Y) dipoles of Chl b to lie preferentially in the plane of the membrane (23 degrees at 653 nm and 30 degrees at 646 nm) is markedly different from the orientation pattern in higher plant membranes and LHC II. Hence, our CD and LD data show that the molecular organization of the Chl a/b/c LHC, despite evident similarities, differs significantly from that of LHC II.     

The stoichiometry and antenna size of the two photosystems in marine green algae, Bryopsis maxima and Ulva pertusa, in relation to the light environment of their natural habitat

The stoichiometry and antenna sizes of the two photosystems in two marine green algae, Bryopsis maxima and Ulva pertusa, were investigated to examine whether the photosynthetic apparatus of the algae can be related to the light environment of their natural habitat. Bryopsis maxima and Ulva pertusa had chlorophyll (Chl) a/b ratios of 1.5 and 1.8, respectively, indicating large levels of Chl b, which absorbs blue-green light, relative to Chl a. The level of photosystem (PS) II was equivalent to that of PS I in Bryopsis maxima but lower than that of PS I in Ulva pertusa. Analysis of Q(A) photoreduction and P-700 photo-oxidation with green light revealed that >50% of PS II centres are non-functional in electron transport. Thus, the ratio of the functional PS II to PS I is only 0.46 in Bryopsis maxima and 0.35 in Ulva pertusa. Light-response curves of electron transport also provided evidence that PS I had a larger light-harvesting capacity than did the functional PS II. Thus, there was a large imbalance in the light absorption between the two photosystems, with PS I showing a larger total light-harvesting capacity than PS II. Furthermore, as judged from the measurements of low temperature fluorescence spectra, the light energy absorbed by Chl b was efficiently transferred to PS I in both algae. Based on the above results, it is hypothesized that marine green algae require a higher ATP:NADPH ratio than do terrestrial plants to grow and survive under a coastal environment.     

Energy and electron transfer in photosystem II of a chlorophyll b-containing Synechocystis sp. PCC 6803 mutant

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm. Time-resolved fluorescence decay measurements upon Chl excitation showed a significant reduction in the amplitude of the 60-70 ps component of fluorescence decay in open Chl b-containing PS II centers. This may indicate slower energy transfer from the PS II core antenna to the reaction center pigments or slower energy trapping. Chl b and pheophytin b were present in isolated PS II reaction centers. Pheophytin b can be reversibly photoreduced, as evidenced from the absorption bleaching at approximately 440 and 650 nm upon illumination in the presence of dithionite. Upon excitation at 685 nm, transient absorption measurements using PS II particles showed some bleaching at 650 nm together with a major decrease in absorption around 678 nm. The 650 nm bleaching that developed within approximately 10 ps after the flash and then remained virtually unchanged for up to 1 ns was attributed to formation of reduced pheophytin b and oxidized Chl b in some PS II reaction centers. Chl b-containing PS II had a lower rate of charge recombination of Q(A)(-) with the donor side and a significantly decreased yield of delayed luminescence in the presence of DCMU. Taken together, the data suggest that Chl b and pheophytin b participate in electron-transfer reactions in PS II reaction centers of Chl b-containing mutant of Synechocystis without significant impairment of PS II function.     

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20°C (nonhardened rye, RNH) or 5°C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.

Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20°C indicated that RNH and RH thylakoids contained the same proportions of fast (α) and slow (β) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for α components increased and the rate constant for β components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5°C.

     

A photosystem II-phycobilisome preparation from the red alga, Porphyridium cruentum: oxygen evolution, ultrastructure, and polypeptide resolution

The photosystem II-phycobilisome preparation, isolated by lauryldimethyl amine oxide treatment, had a greatly reduced chlorophyll content, with an average ratio of 90 chlorophyll a/phycobilisome as compared to approximately 1200 Chl/phycobilisome in unfractionated thylakoids. P700 was not detected in the particles. By electron microscopy the preparations were relatively homogeneous and were generally devoid of chloroplast membranes. In negatively stained preparations phycobilisome particles were seen often in clusters of two and three, probably due to retention of hydrophobic thylakoid fragments. The preparation was deficient in photosystem I chlorophyll complexes, but enriched in polypeptides of 85 to 92, approximately 43, and approximately 26 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 43- and 26-kDa polypeptides are attributable to the PS II core and the oxygen-evolving complex, respectively.     

Antenna chlorophyll a complexes in mutant and developing barley

The chlorophyll a antenna of photosystems I and II were each isolated after detergent treatment by gel electrophoresis or sucrose gradient centrifugation from a b-less mutant of barley grown in daylight and from wildtype barley developed in intermittent light. We identified each fraction by both its electrophoretic position and PS I activity (P700 content) in the case of the mutant, and by both PS I and PS II activity (DCIP reduction from DPC) in the light-limited plants. The proportion of Chl a in each photosystem was estimated from the amount in each gel or sucrose gradient band, and from addition of the areas under the absorption spectra (650–710 nm) of each fraction to match the spectrum of the solubilized thylakoids. The latter method was possible because the spectrum (77 K) of each fraction was unique; in the mutant about 70% of chlorophyll is associated with PS I and 30% with PS II. In the light-limited plants, the reverse is true with nearly 70% associated with PS II. RESOL analyses of both absorption and fluorescence emission spectra of all isolated fractions indicated an abnormal arrangement of antenna chlorophyll molecules in the light-limited, developing membranes even though their reaction centers are fully functional.Abbreviations DCIP dichlorophenolindophenol - DOC deoxycholate - DPC diphenylcarbazide - DL daylight - ImL intermittent light - LHC light-harvesting Chl a/b protein complex - PAGE polyacrylamide gel electrophoresis DPB-CIW No. 778     

The detection,isolation and characterization of a light-harvesting complex which is specifically associated with Photosystem I

10% of the chlorophyll associated with a ‘native’ Photosystem (PS) I complex (110 chlorophylls/P-700) is chlorophyll (Chl) b. The Chl b is associated with a specific PS I antenna complex which we designate as LHC-I (i.e., a light-harvesting complex serving PS I). When the native PS I complex is degraded to the core complex by LHC-I extraction, there is a parallel loss of Chl b, fluorescence at 735 nm, together with 647 and 686 nm circular dichroism spectral properties, as well as a group of polypeptides of 24-19 kDa. In this paper we present a method by which the LHC-I complex can be dissociated from the native PS I. The isolated LHC-I contains significant amounts of Chl b (Chl $\text{a}\text{b ? 3.7}$). The long-wavelength fluorescence at 730 nm and circular dichroism signal at 686 nm observed in native PS I are maintained in this isolated complex. This isolated fraction also contains the low molecular weight polypeptides lost in the preparation of PS I core complex. We conclude that we have isolated the PS I antenna in an intact state and discuss its in vivo function.     

Chromatic Regulation of Photosystem Composition in the Photosynthetic System of Red and Blue-Green Algae

A chromatic adaptation in the photosynthetic quantum yield forthe light mainly absorbed by chlorophyll a (Chl a light) firstfound by Yocum (1951) was studied with one red and three blue-greenalgal strains. When the cells were grown under a weak Chl alight, the quantum yield in all the strains increased. Comparisonof photosystem (PS) compositions, including phycobilin (PBP)and Chl a antennae, reaction centers I and II, in the cellsgrown under the light mainly absorbed by PBP and Chl a revealedthat changes in quantum yield could be attributed to changesin the ratio of PS I/II; PS I/II becomes larger than 1 underPBP light but decreases to 1 in most cases under Chl a light.The change in the PS I/II ratio is due solely to the changesin the PS I population in the cell; PS II remains constant.These results are similar to the intensity-dependent responsein PS composition. A common hypothesis for both the chromatic and intensity-inducedregulation of PS composition was proposed based on the ideaof balance between the electron flow from H₂O to NADP drivenby PS I and II and the cyclic one driven by PS I. (Received May 16, 1985; Accepted September 4, 1985)     

On the long-wavelength spectral forms of chlorophyll a in Photosystem I: Spectroscopic and immunological investigations on a greening mutant of the green alga Scenedesmus obliquus

The origin of the long-wavelength chlorophyll (Chl) absorption (_peak > 680 nm) and fluorescence emission (_peak > 685 nm) has been investigated on Scenedesmus mutants (C-2A-series, lacking the ability to synthesize chlorophyll in the dark) grown at 0.3 (LL), 10 (ML) and 240 µE s^–1 m^–2(HL). LL cells are arrested in an early greening state; consequently, Chl availability determines the phenotype. LL thylakoids are totally lacking long-wavelength Chl; nonetheless, PS I and PS II are fully functional. Gel electrophoresis and Western blots indicate that four out of seven resolved LHC polypeptides seem to require a high Chl availability for assembly of functional chlorophyll-protein complexes. The PS I core-complex of ML and HL thylakoids contains long-wavelength chlorophylls, but in the PS I core-complex of LL thylakoids these pigments are lacking. We conclude that long-wavelength pigments are only present in the PS I core in the case of high Chl availability. The following hypothesis is discussed: Chl availability determines not only the LHC polypeptide pattern, but also the number of bound Chl molecules per individual pigment-protein complex. Chl-binding at non-obligatory, peripheral sites of the pigment-protein complex results in long-wavelength Chl. In the case of low Chl availability, these sites are not occupied and, therefore, the long-wavelength Chl is absent.     

Low temperature development of winter rye leaves alters the detergent solubilization of thylakoid membranes

Thylakoids isolated from leaves of winter rye (Secale cereale L. cv Puma) grown at either 20 or 5°C were extracted with the nonionic detergents Triton X-100 and octyl glucoside. Less total chlorophyll was extracted from 5°C thylakoids by these detergents under all conditions, including pretreatment with cations. Thylakoids from either 20 or 5°C leaves were solubilized in 0.7% Triton X-100 and centrifuged on sucrose gradients to purify the light harvesting complex (LHCII). Greater yields of LHCII were obtained by cation precipitation of particles derived from 20°C thylakoids than from 5°C thylakoids. When 20 and 5°C thylakoids were phosphorylated and completely solubilized in sodium dodecyl sulfate, no differences were observed in the ³²Pi-labeling characteristics of the membrane polypeptides. However, when phosphorylated thylakoids were extracted with octyl glucoside, extraction of LHCII associated with the 5°C thylakoids was markedly reduced in comparison with the extraction of LHCII from 20°C membranes. Since 20 and 5°C thylakoids exhibited significant differences in the Chl content and Chl a/b ratios of membrane fractions produced after solubilization with either Triton X-100 or octyl glucoside, and since few differences between the proteins of the two membranes could be observed following complete denaturation in sodium dodecyl sulfate, we conclude that the integral structure of the thylakoid membrane is affected during rye leaf development at low temperature.