Light-harvesting Chlorophyll Ab-protein Complex Research Articles

Accumulation of the NON-YELLOW COLORING 1 protein of the chlorophyll cycle requires chlorophyll b in Arabidopsis thaliana.

Chlorophyll a and chlorophyll b are interconverted in the chlorophyll cycle. The initial step in the conversion of chlorophyll b to chlorophyll a is catalyzed by the chlorophyll b reductases NON-YELLOW COLORING 1 (NYC1) and NYC1-like (NOL), which convert chlorophyll b to 7-hydroxymethyl chlorophyll a. This step is also the first stage in the degradation of the light-harvesting chlorophyll a/b protein complex (LHC). In this study, we examined the effect of chlorophyll b on the level of NYC1. NYC1 mRNA and NYC1 protein were in low abundance in green leaves, but their levels increased in response to dark-induced senescence. When the level of chlorophyll b was enhanced by the introduction of a truncated chlorophyllide a oxygenase gene and the leaves were incubated in the dark, the amount of NYC1 was greatly increased compared with that of the wild type; however, the amount of NYC1 mRNA was the same as in the wild type. In contrast, NYC1 did not accumulate in the mutant without chlorophyll b, even though the NYC1 mRNA level was high after incubation in the dark. Quantification of the LHC protein showed no strong correlation between the levels of NYC1 and LHC proteins. However, the level of chlorophyll fluorescence of the dark adapted plant (Fo ) was closely related to the accumulation of NYC1, suggesting that the NYC1 level is related to the energetically uncoupled LHC. These results and previous reports on the degradation of chlorophyllide a oxygenase suggest that the a feedforward and feedback network is included in chlorophyll cycle.

A light-harvesting siphonax-anthin-chlorophylla/b-protein complex of marine green alga,Bryopsis corticulans

A light-harvesting chlorophyll a/b-protein complex (LHCP) was isolated directly from thylakoid membranes of marine green alga,Bryopsis corticulans, by two consecutive runs of liquid chromatography. The trimeric form of the light-harvesting complex has been obtained by sucrose gradient ultracentrifugation. The result of SDS-PAGE shows that the light-harvesting complex is composed of at least five apoproteins in which a protein with apparent molecular weight of about 31 kD was never found in the major light-harvesting complex (LHC II) from higher plants. The isolatedBryopsis corticulans light-harvesting complex contains a specific carotenoid, siphonaxanthin, as well as chlorophyll (Chl)a, Chlb, neoxanthin and violaxanthin. Siphonaxanthin which is present in the light-harvesting siphonaxanthin-chlorophylla/b-protein complex ofBryopsis corticulans is responsible for enhanced absorption in the blue-green region (530 nm). Efficient energy transfer from both siphonaxanthin and Chlb to Chla inBryopsis corticulans LHCP, which has similar absorption and fluorescence emission spectra to those of the lutein-chlorophylla/b-protein of higher plants, proved that molecular arrangement of the light-harvesting pigments was highly ordered in theBryopsis corticulans LHCP. The siphonaxanthin-chlorophyll a/b-proteins allow enhanced absorption of blue-green light, the predominant light available in deep ocean waters or shaded subtidal marine habitats.

Calcium-induced accumulation of apoproteins of the light-harvesting chlorophyll [formula omitted] complex in cucumber cotyledons in the dark

A small amount of the apoproteins of the light-harvesting chlorophyll ab-protein complex of photosystem II accumulated in cucumber cotyledons during illumination with intermittent light. However, when these cotyledons were incubated with calcium in the dark after intermittent light, accumulation of the apoproteins occurred. In order to determine whether calcium affected the rate of synthesis or breakdown of the apoproteins, pulse-chase experiments were carried out. Without calcium, the apoproteins were actively synthesized during dark incubation after intermittent illumination, but they were broken down during the subsequent chase period. In the presence of calcium, however, the rate of synthesis of the apoproteins was reduced, but they, nevertheless, accumulated during the subsequent chase period as their breakdown stopped. in contrast, the apoproteins of CP43 and CP47 (Photosystem II core CPs) completely disappeared with calcium treatment. These results indicate that calcium stabilizes the apoproteins of the light-harvesting chlorophyll ab-protein complex of photosystem II and destabilizes the CP43 and CP47 apoproteins.

Spectroscopic analysis of chlorophyll photobleaching in spinach thylakoids, grana and light-harvesting chlorophyll a/b protein complex

The absorption spectra of the chlorophyll photobleaching in the wavelength range 600–750 nm of thylakoids, the BBY grana preparation and purified light-harvesting chlorophyll a/b protein complex (LHCP) are presented. These spectra are described as a linear combination of gaussian components over the wavelength range 635–750 nm and the photobleaching of each component is evaluated. We find that all the spectral species of chlorophyll examined are bleached and, for thylakoids and BBY grana preparations, that the bleaching is greatest for those spectral components that peak at longer wavelengths. On the other hand, photobleaching of the LHCPII spectral components is more homogeneous.

Acclimation by the Thylakoid Membranes to Growth Irradiance and the Partitioning of Nitrogen Between Soluble and Thylakoid Proteins

Three characteristics of shade plants are reviewed. Firstly, they have relatively more chlorophyll b and the associated light-harvesting chlorophyll a/b-protein complex (LHC). Two currently accepted reasons for this are not supported by quantitative analysis. Instead, the reduced protein cost of complexing chlorophyll in LHC and the turnover of the 32 kDa herbicide binding protein are considered. Secondly, shade plants have low electron transport capacities per unit of chlorophyll. This is primarily related to a reduction in the amount of electron transport components such as the cytochrome f complex and the ATPase. The nitrogen cost of the thylakoid membranes per unit of light absorbed is thereby reduced, but the irradiance range over which light is used with high efficiency is also reduced. Thirdly, shade plants have less RuP2 carboxylase and other soluble proteins for a given amount of chlorophyll. However, while the ratio of RuP2 carboxylase protein to thylakoid protein declined, the ratio of the RuP2 carboxylase activity to electron transport activity increased. For several species, the relationship between the rate of CO2 assimilation and leaf nitrogen content depends on the irradiance during growth.

The influence of permanent light and of intermittent light on the reconstitution of the light-harvesting system in regreeningEuglena gracilis

The regreening of etiolatedEuglena gracilis under intermittent light of 2 minutes light/98 minutes darkness was re-examined. In contrast to other investigations theEuglena culture medium contained no organic carbon compound. Therefore the known catabolic repression of thylakoid differentiation,i.e., of chlorophyll-protein complex assembly inEuglena gracilis was avoided. Under these conditions the light-harvesting chlorophyll a/b protein complex (LHCP) was assembled and inserted into the thylakoids. This was demonstrated by (i) the presence of chlorophyll b, (ii) the presence of the LHCP apoproteins and (iii) the proof of thylakoid stacking. These results, which are in contrast to earlier observations, are discussed with regard to the different levels of regulation of chloroplast differentiation inEuglena gracilis and in higher plants.

Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts.

Phycobiliproteins obtained by dissociation of phycobilisomes were reassociated in vitro with intact thylakoids or isolated photosystems I and II preparations obtained from cyanophytes (prokaryotes) or green algae (eukaryotes) to form bound phycobilisome complexes. Energy transfer from Fremyella diplosiphon phycobiliproteins to chlorophyll a of reaction centers I and II was measured in: complexes containing intact thylakoids of the cyanophytes F. diplosiphon or Anacystis nidulans and the eukaryotic algae Euglena gracilis and mutants of Chlamydomonas reinhardtii; complexes containing isolated photosystem II particles of A. nidulans or C. reinhardtii; and complexes containing reaction center I of F. diplosiphon or C. reinhardtii. Energy transfer from phycoerythrin to chlorophyll a of photosystem II could be demonstrated in complexes containing phycobilisomes bound to cyanophyte thylakoids or isolated photosystem II particles of A. nidulans or C. reinhardtii. Bound phycobilisomes did not transfer energy to photosystem II within green algae thylakoids containing altered forms of light-harvesting chlorophyll a/b-protein complex (LHC) II antenna, reduced amounts of LHC II, or chlorophyll b, or chlorophyll b-less mutants, nor to chlorophyll a of photosystem I of intact thylakoids or isolated reaction centers. We conclude that phycobilisomes can form a specific and functional association with photosystem II particles of both cyanophytes and eukaryotic thylakoids. This interaction appears to be hindered by the presence of LHC II antenna in the eukaryotic thylakoids.

Modification of the Photosystem II light-harvesting chlorophyll [formula omitted] protein complex in maize during chill-induced photoinhibition

The changes in the functional and molecular organization of the light-harvesting chlorophyll ab protein complex (LHC II), associated with Photosystem II (PS II), are examined when maize leaves are exposed to high light at 5°C for 6 h. Chlorophyll-fluorescence kinetic analyses of thylakoids isolated from mesophyll cells of stressed and control leaves demonstrate that the stress produces a reduction in energy transfer from LHC II to PS II. The polypeptide complements of thylakoids isolated from stressed leaves showed the accumulation of a 31 kDa polypeptide; no other changes in thylakoid polypeptides were observed between control and stress leaves. The 31 kDa polypeptide was a component of purified LHC II from stressed leaves and was immunologically related to the LHC II polypeptides. Differences in fluorescence emission spectra at 77 K of purified LHC II from control and stressed leaves suggested that the stress induces a perturbation of the environment of the chlorophyll species. The appearance of the 31 kDa polypeptide correlated with a modification of LHC II and its functional association with PS II. It is suggested that this polypeptide is a precursor of LHC II polypeptides and is inserted into the membrane prior to processing.

Thylakoid-protein phosphorylation during the life cycle of Scenedesmus obliquus in synchronous culture

The phosphorylation of thylakoid proteins, which comprise apoproteins of the light-harvesting chlorophyll a/b-protein complex (LHCP), was investigated in vivo and in vitro during the development of Scenedesmus obliquus in synchronous cultures. The in-vitro and in-vivo protein phosphorylation exhibited a maximum activity in cells with maximum photosynthetic capacity (8th hour) and miximum activity in cells with minimum photosynthetic capacity (16th hour). The major phosphorylated polypeptides in vivo were the 24/25-kDa and 28-30-kDa apoprotein of the LHCP, a protein of about 32 kDa, and some smaller polypeptides within the range 10 to 20 kDa. In vitro, the main phosphoproteins were the 28-30-kDa apoprotein and the protein characterized by an apparent molecular weight of 32 kDa. Pulse-chase experiments in vivo established that the latter had the fastest radioactivity turnover of the thylakoidal phosphoproteins.

Monoclonal antibodies specific for the light-harvesting chlorophyll a/b-protein complex (LHC) : Detection of conserved antigenic determinants in LHC from different species

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-protein complex (LHC) of pea and characterised using the enzyme linked immunosorbent assay (with purified LHC or intact thylokoids) or immunoblotting using chloroplast proteins transferred from SDS-PAGE gels. Several clones showed strong binding to the two major polypeptides of pea LHC, even after trypsin or proteinase K treatment. The two antibodies with the most efficient binding to pea LHC were shown to cross-react with polypeptides from green algae and higher plants; indicating sequential similarities and the presence of several closely related polypeptides between phylogenetically distant species.

Evidence for the role of the light-harvesting chlorophyll [formula omitted] protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II α and PS II β centres. On excitation at 550 nm the major contribution was from PS II β centres, that from PS II α centres was only minimal. Mg 2+ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II α component was observed. Thylakoids from a chlorophyll- b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II β centres being the major contributors; the PS II α contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II α and PS II β centres. Mg 2+ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II α and PS II β to the fluorescence induction kinetics. PS II α characteristics are produced by LHCP association with PS II, whereas PS II β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.

Three-dimensional structure of the light-harvesting chlorophyll a/b–protein complex

Photosynthesis in the chloroplasts of green plants is carried out by a highly organized system of membranes. Chlorophyll–protein complexes in the photosynthetic membrane perform specialized functions which result in the biochemical fixation of solar energy. The light-harvesting chlorophyll a/b protein complex (LHC) has a key role in this process 1,2. A striking property of the LHC that distinguishes it from other chlorophyll–protein complexes and makes it suitable for structural studies by Fourier methods is its tendency to form crystalline arrays in vitro3. I report here the three-dimensional structure of pea LHC determined at 16 A resolution by electron microscopy of two-dimensional crystals and image analysis. A three-dimensional map shows that the LHC is a highly asymmetric transmembrane protein con posed of three structurally equivalent monomers. The large surface area exposed on one side of the trimeric complex suggests a functional role in membrane interaction. The ability of LHC isolated from widely different plant species to form similar crystalline arrays4–6 suggests that the structure reported here is of general significance.

Die Organisation der Pigmente im lichtsammelnden Chlorophyll a/b-Protein-Komplex (LHC) aus Vicia faba II. Die Übertragung der Anregungsenergie im isolierten und mit Lipiden rekombinierten LHC

Summary The light-harvesting chlorophyll a/b-protein complex (LHC) was extracted from Vicia faba using Triton X-100. This complex was reconstituted with various lipids to form LHC — proteoliposomes. Four different fractions of liposomes are found. Two of them can be regarded as LHC — proteoliposomes. Higher concentrations of lipids act as detergents which is indicated by degradation of the long wavelength spectral forms of chlorophyll a in absorption, circular dichroic and fluorescence spectra and disturbance of energy transfer from chlorophyll b to chlorophyll a. The highest aggregated proteoliposome fraction shows the same perfect energy transfer as in dialysed or cation-precipitated LHC.

Die Organisation der Pigmente im Lichtsammelnden Chlorophyll a/b-Protein-Komplex (LHC) aus Vicia faba I. Das Spektralformensystem der Chlorophylle im LHC

Summary The light-harvesting chlorophyll a/b-protein complex (LHC) from Vicia faba was isolated according to a modification of the method described by Shutilova et al. (1979) using Triton X-100 and followed by a removal of most of the detergent molecules. Continuing the investigations of the structure and function of chlorophyll forms in the LHC a method for shaping the profile curve to fit the optical spectra is proposed. All the spectra could best be fitted with synthetic functions consisting of Gaussian and Lorentzian portions in a fixed ratio for all the spectral forms of each pigment. In conformity with French et al. (1972) only four components of chlorophyll a in the longwavelength region are detectable in the spectra of LHC in different states of aggregation. Applying the exciton theory the numbers of individual pigment molecules and the molecular weight of an intact LHC monomer are estimated.

The action of lipases on chloroplast membranes. III. The effect of lipid hydrolysis on chlorophyll-protein complexes in thylakoid membranes

Bean thylakoid membranes treated with various lipolytic enzymes (bean galactolipase, phospholipases A2, C, D) showed marked changes in their acyl lipid composition. As a consequence of acyl lipids hydrolysis, destruction of some chlorophyll a-protein complexes (CP1a, CP1, CPa) or monomerization of the oligomeric of light harvesting chlorophyll a/b protein complex (LHCP) was observed. It is concluded that galactolipids and phosphatidylcholine are responsible for the stability of CP1a, CP1 and CPa, respectively. Phosphatidylglycerol and to some extent monogalactosyldiacylglycerol are essential for the stabilization of oligomeric structures of light harvesting chlorophyll a/b protein complex.

Thylakoid protein kinase activity and associated control of excitation energy distribution during chloroplast biogenesis in wheat

The activity of thylakoid protein kinase and the regulation of excitation energy distribution between photosystems I and II was examined during chloroplast biogenesis in light-grown Triticum aestivum (wheat) leaves. The specific activity of the thylakoid protein kinase decreased some six-fold during development from the young plastids at the base of the 7-d-old leaf to the mature chloroplasts at the leaf tip. Appreciable activity was also detected in plastids isolated from etiolated leaves. In mature chloroplasts the majority of phosphate was incorporated into the Mr=26,000 apo-proteins of the light-harvesting chlorophyll a/b-protein complex (LHCP). However, at early stages of chloroplast development and in the etioplast, the phosphate was predominantly incorporated into a polypeptide of Mr=9,000 dalton. Immature thylakoids, isolated from the base of the leaf, had relatively low concentrations of LHCP and could perform a State 1-State 2 transition, as demonstrated by ATP-induced quenching of photosystem II fluorescence. Analyses of photosystem I and photosystem II fluorescence-induction curves from intact leaf tissue demonstrated that this transition occurs in vivo at early stages of leaf development and, therefore, may play an important role in regulating energy transduction during chloroplast biogenesis.

Organization of Chlorophyll a in the Light-Harvesting Chlorophyll a/b Protein Complex as Shown by Circular Dichroism

The development of thylakoid stacking, accumulation of the light-harvesting chlorophyll a/b protein complex (LHCP), and the changes of circular dichroism (CD) which reflect the organization of chlorophyll molecules in greening thylakoids of bean Phaseolus vulgaris cv Red Kidney leaves were investigated.Chloroplasts formed under intermittent light contained large double sheets of membrane with extensive appression in addition to separate lamellae. Thylakoids of such chloroplasts were devoid of LHCP and exhibited a relatively small CD in the chlorophyll absorption region. Upon continuous illumination, the rearrangement of membranes to characteristic grana and the accumulation of the LHCP was accompanied by the gradual appearance of the very intense CD signal with peaks at 682 to 684 (+) and 665 to 672 nanometers (-). The magnitude of differential absorption was approximately 100 times larger than that of the chlorophyll a in solution. This suggests a superhelical liquid crystal-like organization for LHCP, a texture which can be altered by changes of the electric field in the photosynthetic membranes.

Thylakoid protein phosphorylation during State 1—State 2 transitions in osmotically shocked pea chloroplasts

In osmotically shocked pea chloroplasts illuminated with modulated blue-green light (light 2), phosphorylation of the light-harvesting chlorophyll a b- protein complex (LHCP) accompanies the slow decrease in modulated fluorescence that indicates adaptation to light absorbed predominantly by Photosystem II (State 2). On subsequent additional illumination with continuous far-red light (absorbed predominantly by Photosystem I; light 1) both effects are reversed: modulated chlorophyll fluorescence emission increases (indicating adaptation towards State 1) and LHCP is dephosphorylated. Net phosphorylation and dephosphorylation of LHCP induced by light 2 and excess light 1, respectively, occur on the same time scale as the ATP-dependent chlorophyll fluorescence changes indicative of State 2 and State 1 transitions. The phosphatase inhibitor NaF (10 mM), stimulates the effect of blue-green light on fluorescence and prevents the effect of far-red light. These results provide a demonstration that light of different wavelengths can control excitation energy distribution between the two photosystems via the plastoquinol-activated LHCP phosphorylation mechanism suggested previously (Allen, J.F., Bennett, J., Steinback, K.E. and Arntzen, C.J. (1981) Nature 291, 25–29; and Horton, P. and Black, M.T. (1980) FEBS Lett. 119, 141–144).

The reconstitution of a Photosystem II protein complex, P-700-chlorophyll a-protein complex and light-harvesting chlorophyll [formula omitted

A Photosystem II reaction centre protein complex was extracted from spinach chloroplasts using digitonin. This complex showed (i) high rates of dichloroindophenol and ferricyanide reduction in the presence of suitable donors, (ii) low-temperature fluorescence at 685 nm with a variable shoulder at 695 nm which increased as the complex aggregated due to depletion of digitonin and (iii) four major polypeptides of 47, 39, 31 and 6 kDa on dissociating polyacrylamide gels. The Photosystem II protein complex, together woth the P-700-chlorophyll a protein complex and light-harvesting chlorophyll a b- protein complex (LHCP) also isolated using digitonin, were reconstituted with lipids from spinach chloroplasts to form proteoliposomes. The low-temperature (77 K) fluorescence properties of the various proteoliposomes were analysed. The F 685 F 695 ratios of the Photosystem II reaction centre protein complex-liposomes decreased as the lipid to protein ratios were increased. The F 681 F 697 ratios of LHCP-liposomes were found to behave similarly. Light excitation of chlorophyll b at 475 nm stimulated emission from both the Photosystem II protein complex ( F 685 and F 695) and the P-700-chlorophyll a-protein complex ( F 735) when LHCP was reconstituted with either of these complexes, demonstrating energy transfer between LHCP and PS I or II complexes in liposomes. No evidence was found for energy transfer from the PS II complex to the P-700-chlorophyll a-protein complex reconstituted in the same proteoliposome preparation. Proteoliposome preparations containing all three chlorophyll-protein complexes showed fluorescence emission at 685, 700 and 735 nm.

Chloroplast protein phosphorylation and chlorophyll fluorescence quenching. Activation by tetramethyl- p-hydroquinone, an electron donor to plastoquinone

When tetramethyl- p-benzoquinone (TMQ) is reduced to tetramethyl- p-hydroquinone (TMQH 2) by NaBH 4, TMQH 2 will act as an electron donor in isolated chloroplasts. The resulting electron transport is highly sensitive to inhibition by 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB), and the site of donation is inferred to be plastoquinone, in agreement with previous findings. In contrast, when TMQ is added to chloroplasts with ascorbate as reductant, the resulting electron transport is relatively insensitive to DBMIB, and so plastoquinone is assumed not to be involved. In darkness, TMQH 2 activates the chloroplast protein kinase that phosphorylates the light-harvesting chlorophyll a b- protein complex (LHCP), while TMQ with ascorbate does not. TMQH 2 also activates ATP-dependent chlorophyll fluorescence quenching to a much greater extent than does TMQ with ascorbate. These findings are explained by the recent proposal that phosphorylation of LHCP is activated by reduced plastoquinone. They are therefore evidence for plastoquinone-regulated protein phosphorylation as a mechanism for self-adjustment of distribution of excitation between the two light reactions of photosynthesis.