Accumulation of Chlorophyll a/b-Binding Polypeptides in Chlamydomonas reinhardtii y-1 in the Light or Dark at 38 degrees C : Evidence for Proteolytic Control

The kinetics of accumulation of light harvesting chlorophyll (Chl) a/b-binding polypeptides (LHCPs) in thylakoid membranes were analyzed during greening of Chlamydomonas reinhardtii y-1 at 38°C. Initial accumulation of LHCPs in thylakoid membranes was linear; LHCP precursors or polypeptides in transit within the chloroplast stroma were not detected. The rate of accumulation in the light was at least five-fold greater than that in the dark. The relatively small amount of LHCPs that accumulated in the dark was integrated properly in the membrane, as judged by the pattern of cleavage in vitro by exogenous proteases, and did not turn over at a significant rate in vivo. The kinetic data suggested that in y-1 cells either translation of LHCP mRNA was inhibited in the dark or newly synthesized polypeptides were degraded concurrently with transport into the chloroplast unless rescued by Chl. LHCPs accumulated in cells of the Chl b-deficient strain pg-113 at the same rate in the dark or the light at 38°C, an indication that light did not affect translation of LHCP mRNA. Membrane-associated LHCPs in pg-113 cells were completely degraded, in contrast to those in y-1 cells, by exogenous proteases, which suggested that pg-113 cells are deficient in a proteolytic activity. A peptidase was recovered from y-1 cells in a membrane fraction with a buoyant density slightly less than that of thylakoid membranes. Although a role for this activity in degradation of LHCPs has not been established, the specific activity of this peptidase in pg-113 cells was only 10 to 15% of the level in y-1 cells.     

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.     

Organization and Function of Chlorophyll in Membranes of Cyanobacteria during Iron Starvation

Cells of Anacystis nidulans strain R2 and of Synechococcus cedrorum were grown in an iron-deficient medium. Iron starvation induced several pronounced effects without influencing the viability of these cells. The phycocyanin and chlorophyll contents of these cells were depressed, and the absorption maxima of membrane-bound chlorophyll was blue-shifted by 5 nanometers. Cells showed a dramatic increase in original and in maximal chlorophyll fluorescence when monitored at room temperature. Low temperature chlorophyll fluorescence revealed a loss in fluorescence at 696 and 716 nanometers; much of the remaining fluorescence emission was at 686 nanometers. These changes suggest an alteration of membrane composition and structure. This was documented by an electrophoretic analysis of iron-deficient membranes. The prominent findings were: (a) large chlorophyll-protein complexes were not observed in iron-deficient membranes, although the chlorophyll-binding proteins were present; (b) the staining of acrylamide gels with 3,3′,5,5′-tetramethylbenzidine plus peroxide indicated that iron deficiency led to a decrease in the quantity of cytochromes. These results support a structural model of the relation between fluorescence and chlorophyll organization in Anacystis. In addition, they suggest a method for studying cytochrome and chlorophyll protein assembly in these membranes.     

Reorganization of the Photosystem II Unit in Developing Thylakoids of Higher Plants after Transfer to Darkness : Changes in Chlorophyll b, Light-Harvesting Chlorophyll Protein Content, and Grana Stacking

A light-dependent reversible grana stacking-unstacking process, paralleled by a reorganization of thylakoid components, has been noticed in greening etiolated bean (Phaseolus vulgaris, var. red kidney) leaves upon transfer to darkness. The reorganization, based on biochemical and biophysical criteria, involves mainly the photosystem II (PSII) unit components: upon transfer to darkness, the light-harvesting chlorophyll protein (LHCP), its 25 kilodalton polypeptide and chlorophyll b are decreased, while the CPa and its 42 kilodalton polypeptide are increased and new PSII units of smaller size are formed. This reorganization of components occurs only in thylakoids still in the process of development and not in those present in steady state conditions.

It is proposed that this process does not reflect the turnover of the LHCP component per se, but a regulatory process operating during development, by which the ratio of light-harvesting to PSII reaction center components, determined by the environmental conditions, controls the photosynthetic rate.

     

The Light-harvesting Chlorophyll a/b-Protein Complex of Chlamydomonas reinhardii

The molecular organization of chlorophyll in Chlamydomonas reinhardii has been shown to be essentially similar to that in higher plants. Some 50% of the chlorophyll in Chlamydomonas reinhardii chloroplast membranes has been shown to be located in a chlorophyll a/b-protein complex. The complex was isolated in a homogeneous form by hydroxylapatite chromatography of sodium dodecyl sulfate extracts of the chloroplast membranes. Its absorption spectrum exhibits two maxima in the red region at 670 and 652 nm due to the presence of equimolar quantities of chlorophylls a and b in the complex. Preparations of the chlorophyll-protein also contain some of each of the carotenoids observed in the intact chloroplast membrane, but not in the same proportions. The native complex (S value = 2.3S) exhibits a molecular weight of 28,000 ± 2,000 on calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, on the basis of its amino acid composition and other data a more probable molecular weight of about 35,000 was calculated. Each 35,000 dalton unit contains three chlorophyll a and three chlorophyll b molecules, and on the average one carotenoid molecule conjugated with probably a single polypeptide of 29,000 daltons. Comparison of spectral and biochemical characteristics demonstrates that this algal chlorophyll-protein is homologous to the previously described major light-harvesting chlorophyll a/b-protein of higher plants. It is anticipated that the Chlamydomonas complex functions solely in a light-harvesting capacity in analogy to the function determined for the higher plant component.     

Chlorophyll fluorescence changes at high temperatures induced by linear heating of greening barley leaves

A relative decrease of the high temperature part (above 60°C) of the chlorophyll fluorescence temperature curve during 3 h to 10 h greening period of barley (Hordeum vulgare L.) leaves was found to be concomitant to a decrease of Chl alb ratio and to a gradual increase of LHCP/core ratio found by electrophoresis and the ratio of granal to total length of thylakoid membranes. It is suggested that the high temperature part of the fluorescence temperature curve depends inversely on the relative amount of LHC II in thylakoid membranes.Abbreviations Chl a(b) chlorophyll a(b) - CPa chlorophyll a protein complex of PS II - CP1 P700 chlorophyll a protein complex of PS I - FP free pigments - FTC fluorescence temperature curve - F(T30) fluorescence intensity at 30°C - LHC II light harvesting complex II - LHCP light harvesting chlorophyll protein - LHCP3 (LHCPm) monomeric form of LHC II - LHCPo oligomeric form of LHC II complex - M1 first maximum of FTC - M2 second maximum (region) of FTC - PAA polyacrylamide - PAR photosynthetically active radiation - PS I(II) Photosystem I(II) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Chlorophyll a in bilayer membranes. I. The thermal phase diagram with distearoylphosphatidylcholine

Several groups have introduced chlorophyll a into artificial bilayer membranes in an attempt to develop a model system for studying the behavior of chlorophyll in the photosynthetic membrane. In order to investigate the organization of chlorophyll in these model systems, mixed bilayer systems containing chlorophyll a and distearoylphosphatidylcholine under conditions of excess water have been studied by differential thermal analysis. The resulting data suggest a phase diagram for this system consisting of a double eutectic with formation of a thermodynamic compound of defined stoichiometry between chlorophyll a and phospholipid at temperatures below the liquidus. The phase diagram may be simulated to obtain thermodynamic parameters characteristic of the compound phase. It is apparent that the organization and intermolecular interactions of chlorophyll in a bilayer membrane can very widely depending on the temperature and composition of the system. In particular, phase separation can occur within the membrane over certain temperature ranges, resulting in an inhomogeneous system. Thus in interpreting the physical and spectroscopic properties of chlorophyll a in bilayer membranes, it is essential to consider the phase state of the membrane and the organization and environment of the chlorophyll in the particular phase.     

Chloroplast Import of Light-Harvesting Chlorophyll a/b-Proteins with Different Amino Termini and Transit Peptides

We have previously isolated and sequenced two genes encoding light-harvesting chlorophyll a/b-proteins (LHCP) from Lemna gibba. One of these, AB30, encodes a protein that is highly homologous to LHCP sequences reported from other species, but the second, AB19, encodes a protein that has a transit peptide and first 12 amino-terminal residues of the mature protein that are substantially different. Despite these differences, we can demonstrate that AB19 encoded protein synthesized in vitro can be imported into isolated chloroplasts, and we provide evidence that at least some of the imported molecules are assembled into the light-harvesting complex of photosystem II. Thus, our results are consistent with the possibility that there are two functional forms of LHCP.     

Membrane phosphorylation leads to the partial detachment of the chlorophyll a/b protein from Photosystem II

The hypothesis that the chlorophyll fluorescence decline due to membrane phosphorylation is caused principally by the detachment and removal of LHCP from the LHCP-PS II matrix is examined. It is demonstrated that when membranes are phosphorylated in the dark (a) the fluorescence decline is greater when excited by light enriched in wavelengths absorbed mainly by LHCP (475 nm) than when excited by light absorbed to a large extent also by the PS II complex (435 nm), (b) titration with different artificial quenchers of chlorophyll fluorescence is unchanged after the phosphorylation-induced fluorescence decline, and (c) the F_v/F_m ratio does not change after the phosphorylation-induced fluorescence decline. These data indicate that it is indeed principally LHCP that interacts with the quencher (PS I presumably). This interaction involves a small fraction of the total PS II-coupled LHCP, which becomes functionally detached from the LHCP-PS II matrix.     

The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein

The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein (LHCP) is investigated in wild-type barley (Hordeum vulgare L.) and in the chlorophyll b-less mutant chlorina f2. In dark-grown plants a short red light pulse triggers the appearance of mRNA activity for the LHCP. While the accumulation of this mRNA is controlled by phytochrome (Apel (1979) Eur. J. Biochem. 97, 183–188), the red light treatment is not sufficient to induce the appearance of the LHCP within the membrane. Thus, at least one of the subsequent steps in the biosynthetic pathway leading to the assembly of the LHCP is controlled by light. The red light-induced mRNA is taken up into the polysomes during the subsequent dark period and is translated in vitro in a cell-free protein synthesizing system. However, an accumulation of the freshly synthesized polypeptide within the plant is not observed. The apparent instability of the polypeptide might be explained by the deficiency of chlorophyll in the red light-treated plants. In the chlorophyll b-less barley mutant chlorina f2 an accumulation of the freshly synthesized apoprotein of the LHCP can be observed in the light. Thus, chlorophyll a formation seems to be a light-dependent step which is required for the stabilization of the LHCP.Abbreviations mRNA messenger RNA - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecylsulfate - LHCP light-harvesting chlorophyll a/b protein     

Stage-Specific State I-State II Transitions during the Cell Cycle of Euglena gracilis

In synchronized Euglena gracilis (light-dark regime of 14:10 hours) the successive formation of the photosynthetic apparatus during cell ontogeny is correlated with large changes in photosynthetic efficiency (P Brandt, B von Kessel 1983 Plant Physiol 72: 616-619; B Kohnke, P Brandt 1984 Biochim Biophys Acta 766: 156-160). This observation led us to investigate the functional association of the chlorophyll a/b light-harvesting protein complex (LHCP) with photosystem I or II, because changes in energy flow to photosystem I or II and in energy transfer between the two photosystems can be a reason for these alterations. As criterion for the association of the LHCP with photosystem I or II, state transitions were determined after 15 minutes preillumination using wave-lengths of 725 or 620 nanometers. The state transitions were determined from measurements of fluorescence induction at room temperature, and fluorescence kinetics at 77 K. According to the obtained data (a) mobile LHCP is present only between the 6th and the 10th hour of the light-time of the cell cycle and (b) this functional relation of the LHCP to photosystem I only at this stage of Euglena chloroplast development is not accompanied by a decrease in stacking. A model for the organization of the newly inserted LHCP within the photosynthetic apparatus of E. gracilis is discussed.     

Light-Harvesting Chlorophyll a/b Complexes: Interdependent Pigment Synthesis and Protein Assembly

The biogenetic interdependence of light-harvesting chlorophyll (Chl) a/b proteins (LHCPs) and antenna pigments has been analyzed for two nuclear mutants of Chlamydomonas that have low levels of Chl b, neoxanthin, and loroxanthin. In mutant PA2.1, the apoprotein precursors (pLHCP II) of the major light-harvesting complex LHC II were synthesized at approximately wild-type rates, processed to their mature size, and rapidly degraded. Because the bulk of labile LHCP II in PA2.1 was soluble, a thylakoid integration factor apparently is defective in this strain. Chl a, Chl b, neoxanthin, and loroxanthin synthesis and accumulation were coordinately reduced in PA2.1, indicating that LHCP II play important regulatory or substrate roles in de novo synthesis of these pigments. Mutant GE2.27 is impaired principally in Chl b synthesis but nonetheless accumulated wild-type levels of all LHCPs. Topology studies of the GE2.27 LHCP II demonstrated that their insertion into thylakoids was incomplete even though they were not structurally altered. Thus, Chl b formation mediates conformational changes of LHCP II after thylakoid integration is initiated. GE2.27 also exhibited very low rates of neoxanthin synthesis and was unable to accumulate loroxanthin. Revertant GE2.27 strains with varying capacities for Chl b formation provided additional evidence that neoxanthin synthesis and accumulation are coupled with the final steps of LHCP II integration into thylakoids. We propose that biogenesis of LHC includes interdependent pigment synthesis/assembly events that occur during LHCP integration into the thylakoid membrane and that defects in these events account for the pleiotropic characteristics of many Chl b-deficient mutants.     

Isolation and Characterization of a New Minor Chlorophyll a/b-Protein Complex (CP24) from Spinach

We have identified a new minor chlorophyll a/b-protein complex in the thylakoid membranes of spinach (Spinacia oleracea L.), which migrates as a green band below CPII on mildly denaturing polyacrylamide gels. This complex, designated CP24, was isolated from octyl glucoside/sodium dodecyl sulfate solubilized spinach grana membrane fractions by preparative gel electrophoresis and has been characterized as to its spectral properties and polypeptide composition. CP24 has a room temperature absorption maximum at 668 nanometers, a chlorophyll a/b ratio between 0.8 and 1.2, and contains three or four polypeptides between 20 and 23 kilodaltons. CP24 was also identified in grana membrane preparations from peas (Pisum sativum) and barley (Hordeum vulgare). We postulate that CP24 functions as a linker component in photosystem II, acting to orient the photosystem II light harvesting components to ensure efficient energy transfer to the reaction center.     

Accumulation of Chlorophyll, Chloroplastic Proteins, and Thylakoid Membranes during Reversion of Chromoplasts to Chloroplasts in Citrus sinensis Epicarp

In vitro culture of pericarp segments from fruit of Citrus sinensis (L.) Osbeck cv Valencia was used to determine the temporal sequence in development of chloroplasts from chromoplasts during regreening of the epicarp. Regreening of chromoplasts closely resembled greening of etioplasts, except that regreening proceeded much more slowly. Chlorophyll, the light-harvesting chlorophyll a/b binding protein of photosystem II, the chlorophyll a binding protein of reaction center P-700 of photosystem I, thylakoid membranes, and adenosine triphosphate synthetase were all detected at very low levels in degreened epicarp. All of these increased in parallel during regreening of the epicarp. Ribulose 1,5-bisphosphate carboxylase (RuBPCase) levels were high in degreened epicarp and declined for the first 10 days of culture before reaccumulating in the regreening segments. Light was necessary for the accumulation of all of the chloroplastic components. A lack of exogenous nitrogen did not prevent the accumulation of any chloroplastic component except Ru-BPCase, although accumulation of the other components was reduced. Sucrose at 150 millimolar in media lacking nitrogen markedly inhibited the accumulation of chlorophyll and light-harvesting chlorophyll a/b-protein.     

Presence of the Photoactive Reaction-Center Chlorophyll of PSI (P700) in Dark-Grown Cells of a Chlorophyll-Deficient Mutant of Chlorella kessleri

Compositions of pigments and polypeptides of pale green membranesthat had been isolated from dark-grown cells of a chlorophyll-deficientmutant of Chlorella kessleri were investigated. They containedChl a in a level corresponding to about 1% of that present inthe thylakoid membranes isolated from autotrophically grownwild-type cells and a trace amount of chlorophyllide a, butneither Chl b nor carotenoids. The polypeptide profile of themutant membranes was similar to that of membranes isolated fromwild-type cells that were grown in the dark. Neither the chlorophyll-bindingsubunits of PSI nor the apoproteins of LHCP were detected bySDS-PAGE and immunoblot analysis. However, the light-minus-darkdifference spectrum of the mutant membranes revealed the presenceof the reaction-center chlorophyll of PSI (P700) at a molarratio of 190 chlorophyll (Chl a plus Chlide a) per P700. P700was more stable than Chl a and Chlide a in the light so thatprolonged illumination led to a decline in the Chl/P700 ratioto 24. The initial rate of P700 photooxidation in the mutantmembranes was comparable to that in CP1 isolated from the dark-grownwild-type cells. Under illumination with strong light, the initialrate was decreased in parallel to the decrease in Chl/P700 ratio.The results suggest that most of Chi present in the mutant membranescan transfer excitation energy to P700. (Received March 13, 1998; Accepted August 7, 1998)     

Chlorophyll-Protein Complexes from Euglena gracilis and Mutants Deficient in Chlorophyll b: II. Polypeptide Composition

Chlorophyll-protein complexes (CPs) obtained from thylakoids of Euglena gracilis Klebs var bacillaris Cori contain the following polypeptides (listed in parentheses in order of prominence after Coomassie R-250 staining of polyacrylamide gels): CP Ia (66, 18, 22, 22.5, 27.5, 21, 28, 24, 25.5, and 26 kilodaltons [kD]); CP I (66 kD); CPx (41 kD); LHCP₂ (an oligomer of LHCP) (26.5, 28, and 26 kD); CPy (27 and 19 kD); CPa (54 kD); and LHCP (26.5, 28, and 26 kD). Mutants of bacillaris low in chlorophyll b (Gr₁BSL, G₁BU, and O₄BSL; Chl a/b [mol/mol] = 50-100) which lack CP Ia, LHCP₂, and LHCP also lack or are deficient in polypeptides associated with these complexes in wild-type cells. Mutants G₁ and O₄, which also lack CPy, lack the CPy-associated polypeptides found in wild-type and Gr₁. Using an antiserum which was elicited by and reacts strongly and selectively with the SDS-treated major polypeptide (26.5 kD) of the LHCP complexes of wild-type, this polypeptide is undetectable in the mutants (0.25% of the level in wild-type on a cell basis); the antiserum does not react with the SDS-treated 28 kD polypeptide of the Euglena LHCP complexes and cross-reacts only very weakly with components in SDS-treated cells of Chlamydomonas reinhardtii Dangeard and chloroplasts of Spinacia oleracea L. cv Winter Bloomsdale. Rates of photosynthesis of the wild-type and mutant cells of Euglena are approximately equal on a cell basis when measured at light saturation, consistent with the selective loss of major antenna components but not CP I or CPa from the mutants.