Cation control of fluorescence emission,light scatter,and membrane stacking in pigment mutants of Chlamydomonas reinhardi

The effect of monovalent and divalent cations on thylakoid membrane stacking, light scatter, and fluorescence yield were examined in broken-cell preparations of the wild type of Chlamydomonas reinhardi and mutants lacking various pigment-protein complexes. Membrane stacking as determined by electron microscopy and light scatter at 540 nm shows an approximate linear proportionality. In a mutant lacking photosystem II reaction centers, stimulation of light scatter and of fluorescence yield show different kinetics at a given cation concentration and show different titration curves as a function of cation concentration. Control of membrane stacking and fluorescence yield is attributed, respectively, to two locations differing in their anionic charge density. In a mutant lacking chlorophyll-protein complex I, the cation effect on the fluorescence yield is reversed giving rise to a decrease in fluorescence intensity upon cation addition instead of the usual increase. We conclude that the site of energy spillover between the two photosystems is located exterior to chlorophyll-protein complex I but not at the junction of chlorophyll-protein complex I and the rest of the light-harvesting antenna.     

Light-independent synthesis of LHC IIb polypeptides and assembly of the major pigmented complexes during the initial stages of Pinus palustris seedling development

Pinus palustris has a greatly reduced need for light to initiate chloroplast development in comparison to angiosperms. Light is not required for chlorophyll synthesis in dark-grown Pinus palustris seedlings. However, embryos do not contain chlorophyll, and synthesis is limited to seedlings having cotyledon lengths between about 0.5 cm and 2.0 cm. The final amount of chlorophyll accumulated by dark-grown seedlings is about one fifth of that in light-grown seedlingsat the same stage. The major light-harvesting chlorophyll a/b-polypeptides of Photosystem II (LHC IIb) are absent in the embryos but begin to accumulate in seedlings of 0.5 cm cotyledon length, irrespective of the light conditions. Although dark-grown seedlings accumulate most of the pigmented complexes seen in light-grown seedlings, there are differences in the subunit structure of some of them. These findings suggest that the majority of the components of the photosynthetic membrane do not require light for induction of synthesis or assembly into complexes, but that the final forms seen in light-grown seedlings may require light.Abbreviations ALA 5-amino levulinic acid - glucoside -D-glucopyranoside - LHC light-harvesting complex - lhc genes encoding LHCs - PS photosystem     

Euploidy in Ricinus: EUPLOIDY EFFECTS ON PHOTOSYNTHETIC ACTIVITY AND CONTENT OF CHLOROPHYLL-PROTEINS

The effects of nuclear genome duplication on the chlorophyll-protein content and photochemical activity of chloroplasts, and photosynthetic rates in leaf tissue, have been evaluated in haploid, diploid, and tetraploid individuals of the castor bean, Ricinus communis L. Analysis of this euploid series revealed that both photosystem II (2,6-dichlorophenolindophenol reduction) and photosystem I oxygen uptake (N,N,N′,N′-tetramethyl-p-phenylenediamine to methyl viologen) decrease in plastids isolated from cells with increasingly larger nuclear complement sizes. Photosynthetic O₂-evolution and ¹⁴CO₂-fixation rates in leaf tissue from haploid, diploid, and tetraploid individuals were also found to decrease with the increase in size of the nuclear genome. Six chlorophyll-protein complexes, in addition to a zone of detergent complexed free pigment, were resolved from sodium dodecyl sulfate-solubilized thylakoid membranes from cells of all three ploidy levels. In addition to the P700-chlorophyll a-protein complex and the light-harvesting chlorophyll a/b-protein complex, four minor complexes were revealed, two containing only chlorophyll a and two containing both chlorophyll a and b. The relative distribution of chlorophyll among the resolved chlorophyll-protein complexes and free pigment was found to be similar for all three ploidy levels.     

Events surrounding the early development of Euglena chloroplasts. 15. Origin of plastid thylakoid polypeptides in wild-type and mutant cells

Techniques are described for the isolation of plastid thylakoid membranes from light-grown and dark-grown cells of Euglena gracilis var. bacillaris, and from mutants affecting plastid development. These membranes, which have minimal contamination with other cell fractions, are localized in sucrose gradients by using the thylakoid membrane sulfolipid as a specific marker. The plastid thylakoid membrane polypeptides isolated from these membranes were separated on SDS polyacrylamide gels and yielded patterns containing 30–40 polypeptides. Light-grown strain Z gave patterns identical with bacillaris. Since the plastid thylakoid polypeptide patterns obtained from dark-grown wild-type cells and from a bleached mutant W₃BUL in which plastid DNA is undetectable are identical, it appears that the proplastid thylakoid polypeptides of wild-type cannot be coded in plastid DNA and are probably coded in nuclear DNA. The plastid thylakoid polypeptide patterns obtained from various dark-grown mutants are identical to those obtained from dark-grown wild-type cells. Light-grown mutants, making large but abnormal chloroplasts, show a correlation between the amount of chlorophyll formed and the amount of a plastid thylakoid polypeptide thought to be associated with one of the pigment-protein light-harvesting complexes. Treatment with SAN 9789 (4-chloro-5-(methyl-amino)-2-(α,α,α,-trifluoro-m-tolyl)-3-(2H(pyridazinone) known to block carotenoid synthesis at the level of phytoene, causes a progressive loss of all plastid thylakoid polypeptides during growth in darkness and results in the establishment of a new, lower steady-state level of sulfolipid. At least ten of the plastid thylakoid polypeptides become labeled when isolated chloroplasts are supplied with radioactive amino acids; of these six are undectable in W₃BUL and are, therefore, candidates for coding by plastid DNA.     

Development of Photosystem I and Photosystem II Activities in Leaves of Light-grown Maize (Zea mays)

To compare chloroplast development in a normally grown plant with etiochloroplast development, green maize plants (Zea mays), grown under a diurnal light regime (16-hour day) were harvested 7 days after sowing and chloroplast biogenesis within the leaf tissue was examined. Determination of total chlorophyll content, ratio of chlorophyll a to chlorophyll b, and O₂-evolving capacity were made for intact leaf tissue. Plastids at different stages of development were isolated and the electron-transporting capacities of photosystem I and photosystem II measured. Light saturation curves were produced for O₂-evolving capacity of intact leaf tissue and for photosystem I and photosystem II activities of isolated plastids. Structural studies were also made on the developing plastids. The results indicate that the light-harvesting apparatus becomes increasingly efficient during plastid development due to an increase in the photosynthetic unit size. Photosystem I development is completed before that of photosystem II. Increases in O₂-evolving capacity during plastid development can be correlated with increased thylakoid fusion. The pattern of photosynthetic membrane development in the light-grown maize plastids is similar to that found in greening etiochloroplasts.     

Specificity of the chlorophyll-to-protein binding in the chlorophyll-protein complexes of the thylakoid

We tried to establish whether the chlorophyll-protein complexes of the thylakoid, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, originate from real entities existing in vivo, or are mere artifacts of the sodium dodecyl sulfate solubilization procedure. Making use of the finding that etiolated leaves exposed to periodic light form selectively the chlorophyll-protein complexes CPI and CPa, while after transfer to continuous light they form in addition the light-harvesting complexes (J. H. Argyroudi-Akoyunoglou, Z. Feleki, and G. Akoyunoglou, 1971, Biochem. Biophys. Res. Commun.45, 606–614; J. H. Argyroudi-Akoyunoglou and G. Akoyunoglou 1979, FEBS Lett.104, 78–84) we tried to see whether the latter complexes contain newly formed chlorophyll. We labeled the chlorophyll a formed in periodic light with δ-[¹⁴C]aminolevulinic acid, and determined the specific radioactivity of chlorophyll in the complexes formed before or after transfer to continuous light. We found that the light-harvesting complexes contain primarily newly formed and nonradioactive chlorophyll. The results suggest that (i) the chlorophyll a of CPI and CPa formed in periodic light does not exchange with that of the light-harvesting complexes formed after transfer to continuous light. (ii) The light-harvesting complexes formed after transfer to continuous light contain primarily newly formed chlorophylls a and b. (iii) The binding of chlorophyll to protein in the complexes is specific and not an artifact of the sodium dodecyl sulfate action. (iv) As the thylakoid membrane grows and differentiates, the chlorophyll synthesized binds on the apoproteins of the complexes in a stepwise manner.     

Photosynthetic apparatus of pea thylakoid membranes : response to growth light intensity

We investigated the effect of growth light intensity on the photosynthetic apparatus of pea (Pisum sativum) thylakoid membranes. Plants were grown either in a growth chamber at light intensities that ranged from 8 to 1050 microeinsteins per square meter per second, or outside under natural sunlight. In thylakoid membranes we determined: the amounts of active and inactive photosystem II, photosystem I, cytochrome b/f, and high potential cytochrome b₅₅₉, the rate of uncoupled electron transport, and the ratio of chlorophyll a to b. In leaves we determined: the amounts of the photosynthetic components per leaf area, the fresh weight per leaf area, the rate of electron transport, and the light compensation point. To minimize factors other than growth light intensity that may alter the photosynthetic apparatus, we focused on peas grown above the light compensation point (20-40 microeinsteins per square meter per second), and harvested only the unshaded leaves at the top of the plant. The maximum difference in the concentrations of the photosynthetic components was about 30% in thylakoids isolated from plants grown over a 10-fold range in light intensity, 100 to 1050 microeinsteins per square meter per second. Plants grown under natural sunlight were virtually indistinguishable from plants grown in growth chambers at the higher light intensities. On a leaf area basis, over the same growth light regime, the maximum difference in the concentration of the photosynthetic components was also about 30%. For peas grown at 1050 microeinsteins per square meter per second we found the concentrations of active photosystem II, photosystem I, and cytochrome b/f were about 2.1 millimoles per mol chlorophyll. There were an additional 20 to 33% of photosystem II complexes that were inactive. Over 90% of the heme-containing cytochrome f detected in the thylakoid membranes was active in linear electron transport. Based on these data, we do not find convincing evidence that the stoichiometries of the electron transport components in the thylakoid membrane, the size of the light-harvesting system serving the reaction centers, or the concentration of the photosynthetic components per leaf area, are regulated in response to different growth light intensities. The concept that emerges from this work is of a relatively fixed photosynthetic apparatus in thylakoid membranes of peas grown above the light compensation point.     

Light-Harvesting Chlorophyll a/b Protein : Membrane Insertion, Proteolytic Processing, Assembly into LHC II, and Localization to Appressed Membranes Occurs in Chloroplast Lysates

The apoprotein of the light-harvesting chlorophyll a/b protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem II. LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll-protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process.     

Chemical Cross-linking of Neighboring Thylakoid Membrane Polypeptides

Cross-linking between protein components of whole spinach (Spinacia oleracea var. Nobel) thylakoids and of photosystem I- and II-enriched thylakoid fractions has been produced by reaction with the bifunctional imidoester dimethyl-3,3′-dithiobispropionimidate dihydrochloride as well as by the oxidation of intrinsic sulfydryl groups with an orthophenanthrolinecupric ion complex. The mixture of membrane proteins and their cross-linked products has been analyzed by two-dimensional sodium dodecyl sulfate electrophoresis, with a reductive cleavage step of the cross-linkages before the second dimension. Cross-linked aggregates up to a molecular weight of about 130 kilodaltons (kD) were analyzed, and it was inferred that the polypeptides appearing together in the same aggregates were neighbors within the membrane.

In thylakoids as well as in isolated photosystem fractions, oligomers were formed by cross-linking polypeptides of the 60 to 90 kD range, among them the polypeptides of the chlorophyll-protein complex I. Polypeptides of 46, 19, and 12 kD were cross-linked to these complexes. Polypeptides of 25 and 22 kD, which are related to the chlorophyll-protein complex II, were cross-linked in thylakoids as well as in photosystem II fractions, suggesting that in the membrane these molecules are close together. In photosystem II fractions an oligomer having a molecular weight of about 60 kD was formed by cross-linking several polypeptides of different molecular weights: 40, 25, and 22 kD.

Our cross-linking experiments show that protein interactions in the thylakoid membrane occurred mainly among the polypeptides of the two chlorophyll-protein complexes, thus suggesting an oligomeric nature of these apoproteins.

     

Loss of Albino3 leads to the specific depletion of the light-harvesting system

The chloroplast Albino3 (Alb3) protein is a chloroplast homolog of the mitochondrial Oxa1p and YidC proteins of Escherichia coli, which are essential components for integrating membrane proteins. In vitro studies in vascular plants have revealed that Alb3 is required for the integration of the light-harvesting complex protein into the thylakoid membrane. Here, we show that the gene affected in the ac29 mutant of Chlamydomonas reinhardtii is Alb3.1. The availability of the ac29 mutant has allowed us to examine the function of Alb3.1 in vivo. The loss of Alb3.1 has two major effects. First, the amount of light-harvesting complex from photosystem II (LHCII) and photosystem I (LHCI) is reduced >10-fold, and total chlorophyll represents only 30% of wild-type levels. Second, the amount of photosystem II is diminished 2-fold in light-grown cells and nearly 10-fold in dark-grown cells. The accumulation of photosystem I, the cytochrome b(6)f complex, and ATP synthase is not affected in the ac29 mutant. Mild solubilization of thylakoid membranes reveals that Alb3 forms two distinct complexes, a lower molecular mass complex of a size similar to LHC and a high molecular mass complex. A homolog of Alb3.1, Alb3.2, is present in Chlamydomonas, with 37% sequence identity and 57% sequence similarity. Based on the phenotype of ac29, these two genes appear to have mostly nonredundant functions.     

Effect of Light Quality on the Composition, Function, and Structure of Photosynthetic Thylakoid Membranes of Asplenium australasicum (Sm.) Hook

The effect of light quality on the composition, function and structure of the thylakoid membranes, as well as on the photosynthetic rates of intact fronds from Asplenium australasicum, a shade plant, grown in blue, white, or red light of equal intensity (50 microeinsteins per square meter per second) was investigated. When compared with those isolated from plants grown in white and blue light, thylakoids from plants grown in red light have higher chlorophyll a/chlorophyll b ratios and lower amounts of light-harvesting chlorophyll a/b-protein complexes than those grown in blue light. On a chlorophyll basis, there were higher levels of PSII reaction centers, cytochrome f and coupling factor activity in thylakoids from red light-grown ferns, but lower levels of PSI reaction centers and plastoquinone. The red light-grown ferns had a higher PSII/PSI reaction center ratio of 4.1 compared to 2.1 in blue light-grown ferns, and a larger apparent PSI unit size and a lower PSII unit size. The CO₂ assimilation rates in fronds from red light-grown ferns were lower on a unit area or fresh weight basis, but higher on a chlorophyll basis, reflecting the higher levels of electron carriers and electron transport in the thylakoids.

The structure of thylakoids isolated from plants grown under the three light treatments was similar, with no significant differences in the number of thylakoids per granal stack or the ratio of appressed membrane length/nonappressed membrane length. The large freeze-fracture particles had the same size in the red-, blue-, and white-grown ferns, but there were some differences in their density. Light quality is an important factor in the regulation of the composition and function of thylakoid membranes, but the effects depend upon the plant species.

     

Compensatory Alterations in the Photochemical Apparatus of a Photoregulatory, Chlorophyll b-Deficient Mutant of Maize

Characterization of the functional organization of the photochemical apparatus in the light sensitive chlorophyll b-deficient oil yellow-yellow green (OY-YG) mutant of maize (Zea mays) is presented. Spectrophotometric and kinetic analysis revealed substantially lower amounts of the light harvesting complex of photosystem II (LHCII-peripheral) in high light-grown OY-YG thylakoids. However, accumulation of a tightly bound LHCII appears unaffected by the lesion. Changes in photosystem (PS) stoichiometry include lower amounts of PSII with characteristic fast kinetics (PSII_α) and a substantial accumulation of PSII centers with characteristic slow kinetics (PSII_β) in the thylakoid membrane of the OY-YG mutant. Thus, PSII_β is the dominant photosystem in the mutant chloroplasts. In contrast to wild type, roughly 80% of the mutant PSII_β centers are functionally coupled to the plastoquinone pool and are probably localized in the appressed regions of the thylakoid membrane. These centers, designated PSII_β-Q_B-reducing (Q_B being the secondary electron quinone acceptor of PSII), are clearly distinct from the typical PSII_β-Q_B-nonreducing centers found in the stroma lamellae of wild-type chloroplasts. It is concluded that the observed changes in the stoichiometry of electron-transport complexes reflect the existence of a regulatory mechanism for the adjustment of photosystem stoichiometry in chloroplasts designed to correct any imbalance in light absorption by the two photosystems.     

Light stress photodynamics of chlorophyll-binding proteins in Arabidopsis thaliana thylakoid membranes revealed by high-resolution mass spectrometric studies

In higher plants the light energy is captured by the photosynthetic pigments that are bound to photosystem I and II and their light-harvesting complex (LHC) subunits. In this study, we examined the photodynamic changes within chlorophyll-protein complexes in the thylakoid membrane of Arabidopsis thaliana leaves adapted to low light and subsequently exposed to light stress. Chlorophyll-protein complexes were isolated using sucrose density gradient centrifugation and blue-native polyacrylamid gel electrophoresis (BN-PAGE). Proteome analysis was performed using SDS-PAGE, HPLC and high resolution mass spectrometry. We identified several rarely expressed and stress-induced chlorophyll-binding proteins, showed changes in localization of early light-induced protein family and LHC protein family members between different photosynthetic complexes and assembled/disassembled subcomplexes under light stress conditions and discuss their role in a variety of light stress-related processes.     

Characterization of early events leading to the formation of chlorophyll-proteins of PSII in Euglena gracilis

Development of chlorophyll-proteins in photosystem II was studied with Euglena gracilis Z. during dark-light transition. Upon illumination of the dark-grown cells, protochlorophyllide was photoconverted to chlorophyll(ide) a with a low efficiency (14%). After a lag time of 1-2 h, chlorophylls, apoproteins of antenna chlorophyll-protein complex CP 43/47 and of light-harvesting chlorophyll-protein complex (LHCII) accumulated in the thylakoid membrane in a coordinated fashion. There was, however, a significant difference in the stability between the newly formed LHCII and CP 43/47 judging from non-denaturing lithium dodecyl sulfate-polyacrylamide gel electrophoresis. The possibility that efficiencies of incorporation and stabilization of chlorophylls in the apoproteins differ among the chlorophyll-proteins in the early stage of greening of Euglena is discussed.     

Chlorophyll-Protein Complexes from the Red-Tide Dinoflagellate, Gonyaulax polyedra Stein : Isolation, Characterization, and the Effect of Growth Irradiance on Chlorophyll Distribution

A comparision of high (330 microeinsteins per meter squared per second) and low (80 microeinsteins per meter squared per second) light grown Gonyaulax polyedra indicated a change in the distribution of chlorophyll a, chlorophyll c₂, and peridinin among detergent-soluble chlorophyll-protein complexes. Thylakoid fractions were prepared by sonication and centrifugation. Chlorophyll-protein complexes were solubilized from the membranes with sodium dodecyl sulfate and resolved by Deriphat electrophoresis. Low light cells yielded five distinct chlorophyll-protein complexes (I to V), while only four (I′ to IV′) were evident in preparations of high light cells. Both high molecular weight complexes I and I′ were dominated by chlorophyll a absorption and associated with minor amounts of chlorophyll c. Both complexes II and II′ were chlorophyll a-chlorophyll c₂-protein complexes devoid of peridinin and unique to dinoflagellates. The chlorophyll a:c₂ molar ratio of both complexes was 1:3, indicating significant chlorophyll c enrichment over thylakoid membrane chlorophyll a:c ratios of 1.8 to 2:1. Low light complex III differed from all other high or low light complexes in that it possessed peridinin and had a chlorophyll a:c₂ ratio of 1:1. Low light complexes IV and V and high light complexes III′ and IV′ were spectrally similar, had high chlorophyll a:c₂ ratios (4:1), and were associated with peridinin. The effects of growth irradiance on the composition of chlorophyll-protein complexes in Gonyaulax polyedra differed from those described for other chlorophyll c-containing plant species.     

Calcium-induced formation of chlorophyll b and light-harvesting chlorophyll complex in cucumber cotyledons in the dark

Dark-grown cucumber seedlings were exposed to intermittent light (2 min light and 98 min dark) and then cotyledons were incubated with 50 mM CaCl₂ in the dark. Chlorophyll (Chl) a was selectively accumulated under intermittent light and Chl b was accumulated during the subsequent dark incubation with CaCl₂. The change in chlorophyll-protein complexes during Chl b accumulation induced by CaCl₂ in the dark was investigated by SDS-polyacrylamide gel electrophoresis. Chlorophyll-protein complex I and free chlorophyll were major chlorophyll-containing bands of the cotyledons intermittently illuminated 10 times. When these cotyledons were incubated with CaCl₂ in the dark, the light-harvesting Chl complex was formed. When the number of intermittent illumination periods was extended to 55, small amounts of Chl b and light-harvesting Chl complex were recognized at the end of intermittent light treatment, and these two pigments were further increased during the subsequent incubation of the cotyledons with CaCl₂ in the dark compared to water controls.     

Modulations of the thylakoid system in snow xanthophycean alga cultured in the dark for two months: comparison between microspectrofluorimetric responses and morphological aspects

Summary. The response of the plastid was studied, with a special emphasis on thylakoid structure and function, in a snow filamentous xanthophycean alga (Xanthonema sp.) incubated in darkness for two months. Microspectrofluorimetric analyses were performed on single living cells to study the variations in the assembly of the chlorophyll-protein complexes of photosystem II, in comparison with cells grown in light. In parallel, changes in micro- and submicroscopic plastid morphology and in photosynthetic pigment content were monitored. Throughout the experiment, the lamellar architecture of thylakoids in the alga was relatively well preserved, whereas photosystem II underwent disassembly and degradation triggered by prolonged darkness. Conversely, the light-harvesting complex of photosystem II proved to be relatively stable for long periods in darkness. Moreover, a role of the peripheral antennae in determining thylakoid arrangement in xanthophycean algae is implied. Although the responses observed in Xanthonema sp. can be considered in terms of acclimation to darkness, the progressive destabilisation of the light-harvesting complex of photosystem II testifies to incipient ageing of the cells after 35 days. Correspondence and reprints: Department of Natural and Cultural Resources, University of Ferrara, Corso Ercole I d’Este 32, 44100 Ferrara, Italy.     

The role of chlorophyll-protein complexes in the function and structure of chloroplast thylakoids

Summary The photosynthetic pigments of chloroplast thylakoid membranes are complexed with specific intrinsic polypeptides which are included in three supramolecular complexes, photosystem I complex, photosystem II complex and the light-harvesting complex. There is a marked lateral heterogeneity in the distribution of these complexes along the membrane with photosystem II complex and its associated light-harvesting complex being located mainly in the stacked membranes of the grana partitions, while photosystem I complex is found mainly in unstacked thylakoids together with ATP synthetase. In contrast, the intermediate electron transport complex, the cylochrome b-f complex, is rather uniformly distributed in these two membrane regions. The consequences of this lateral heterogeneity in the location of the thylakoid complexes are considered in relation to the function and structure of chloroplasts of higher plants.