Immuno-gold localization of cytochrome f,light-harvesting complex,ATP synthase and ribulose 1,5-bisphosphate carboxylase/oxygenase

The proteins ribulose 1,5-bisphosphate carboxylase/oxygenase, ATP synthase, light-harvesting chlorophyll a/b protein, and cytochrome f, have been localized in mesophyll chloroplasts of barley (Hordeum vulgare L.) by electron microscopy of immunogold-labelled sections. The light-harvesting chlorophyll a/b protein and cytochrome f are shown to be present in the grana, both within the stacks and at the margins, and in the stromal membranes. Although the absolute amount of labelling for these proteins is greater in the grana than in the stromal membranes, when expressed as label/membrane length the partitioning appears approximately equal between appressed and non-appressed membranes for both the light-harvesting chlorophyll a/b protein and cytochrome f. ATP synthase is restricted to the non-appressed thylakoid membranes, and ribulose 1,5-bisphosphate carboxylase/oxygenase is uniformly distributed through the stromal contents.Abbreviations CF₁ ATP synthase - LHCPII light-harvesting chlorophyll a/b protein - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

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     

The light-harvesting chlorophyll a/b protein acts as a torque aligning chloroplasts in a magnetic field

Displacement of particles from the purified light-harvesting chlorophyll a/b protein aggregate (LHC) was studied in magnetic fields of various strengths (0 to 1.6 T) by polarized fluorescence measurements. Macromolecular aggregates of LHC have a considerable magnetic susceptibility which enables the particles to rotate and align with their nematic axes parallel with H. As LHC is embedded in a transmembrane direction thylakoids should align perpendicular to H, the mode of alignment experimentally observed in thylakoids. The value of the magnetic susceptibility could be estimated by relating it to the integral susceptibility of the chlorophyll molecules in LHC. The fitting of this value with the field strength dependency of the fluorescence polarization ratio (FP) revealed a relationship between the LHC content of various photosynthetic membranes and their capacity for alignment, which suggested that LHC might be the torque ordering chloroplasts in a magnetic field.Abbreviations LHC light-harvesting chlorophyll a/b protein - FP fluorescence polarization ratio, Iz/Iy     

Cadmium-induced changes in the composition and structure of the light-harvesting chlorophyll a/b protein complex II in radish cotyledons

Five-day-old etiolated radish ( Raphanux salivux L. cv. Saxa) seedlings exposed to white continuous light in the presence of Cd²⁺ (0.2 mM) showed characteristic changes in their light-harvesting chlorophyll a/b protein complex II after 48 h of greening. The content of its oligomeric supramolecular form was greatly diminished with a concomitant increase in the level of the monomer. The isolation of highly purified light-harvesting chlorophyll a/b protein complex II from control and Cd²⁺ treated radish cotyledons and a detailed analysis of its structure and composition revealed that first of all, Cd²⁺ altered the content of the specific phosphatidylglycerol fatty acid - trans -Δ³-hexadecenoic acid, widely accepted as a component responsible for the oligomerization of this chlorophyll-protein complex. This fatty acid in the thylakoid membrane phosphatidylglycerol pool seems to be very sensitive to different environmental stresses lowering its content, which indicates the vital significance of this component for the supramolecular organization and proper functioning of the light-harvesting chlorophyll a/b protein complex II.     

Chlorophyll-proteins from maize seedlings grown under intermittent light conditions

We studied the organization of the antenna system of maize (Zea mays L.) seedlings grown under intermittent light conditions for 11 d. These plants had a higher chlorophyll-a/b ratio, a higher ratio of carotenoids to chlorophyll and a lower ratio of chlorophyll to protein than plants grown in continuous light. We found all chlorophyll-protein complexes of maize to be present. However, the minor chlorophyll a/b-proteins CP29 and CP26, and to a greater extent CP24 and the major light-harvesting complex II were reduced relative to the photosystem (PS) II core-complex. Also the chlorophyll a/b-antennae of PSI were reduced relative to the reaction-centre polypeptides. When isolated by flatbed isoelectrofocussing, the chlorophyll-a/b complexes of PSII showed a higher chlorophyll-a/b ratio and a lower ratio of chlorophyll to protein than the same complexes from continuous light; additionally, they bound more carotenoids per protein than the latter. Thus the altered organization of the photosynthetic apparatus of plants from intermittent light is caused by two different factors: (i) the altered stoichiometry of chlorophyll-binding proteins and (ii) a different ratio of pigment to protein within individual chlorophyll-proteins.Abbreviations Chl chlorophyll - CL continuous light - F fraction - HPLC high-performance liquid chromatography - IEF isoelectrofocussing - IL intermittent light - LHCII light-harvesting complex II - PAGE polyacrylamide-gel electrophoresis - Phe pheophytin - SDS sodium dodecyl sulfate This work was supported by the grant no. 4.7240.90 from the Italian Ministry of Agriculture and Forestry. We thank Drs. R. Barbato (Dipartimento di Biologia, Padua, Italy) and Olivier Vallon (Institut de Biologie Physico-Chimique, Paris, France) for their gifts of antibodies, Drs. R. Barbato and P. Dainese (Dipartimento di Biologia, Padua, Italy) for fruitful discussion and Prof. G. Gennari (Dipartimento di Chimica fisica, Padua, Italy) for his assistance in recording the excitation spectra. J.M. was supported by a Stipendium from the Deutsche Forschungsgemeinschaft, which is gratefully acknowledged.     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Thylakoids from pea seedlings grown under intermittent light: biochemical and flash-spectrophotometric properties.

Thylakoid membranes were isolated from pea seedlings grown under intermittent light (2-min light/118-min dark cycles). These preparations differed from controls (thylakoids from plants grown under 16-h light/8-h dark cycles) in the following respects: 15 times smaller chlorophyll/protein ratio, 10 times greater chlorophyll a/b ratio, absence of light-harvesting chlorophyll a/b binding proteins, and 2-3-fold greater ratio of photosystem II over photosystem I. In addition we found the following: (1) Electrogenic electron transfer around cytochrome b6/f under flashing light was greatly enhanced, probably as a consequence of the greater photosystem II/photosystem I ratio. (2) The rate of proton uptake from the medium at the acceptor side of photosystem II was enhanced, probably by unshielding of the quinone binding domain. (3) The N,N'-dicyclohexylcarbodiimide sensitivity of the proton-pumping activity of photosystem II was absent, which was consistent with the attribution of a N,N'-dicyclohexylcarbodiimide-induced protonic short circuit to chlorophyll a/b binding proteins. (4) The sensitivity of oxygen evolution under continuous light to variations of pH or the concentration of Ca2+ was altered. Chlorophyll a/b binding proteins serve as light-harvesting antennas. We found in addition that they modulated the activity of water oxidation and, in particular, the proteolytic reactions around photosystem II.     

Synthesis and turnover of the light-harvesting chlorophylla/b-protein inLemna gibba grown with intermittent red light: possible translational control

Lemna gibba L. G-3 plants grown heterotrophically in the dark with intermittent red light (2 min every 8 h) contain a substantial amount of translatable mRNA encoding the light-harvesting chlorophyll (Chl)a/b-protein. However, very little [³⁵S]methionine is incorporated into the apoproteins during a 1-h labeling period in the dark in these plants compared to plants grown in continuous white light. The Chla/b-protein mRNA is found to be associated with functioning polysomes in plants grown in the dark with intermittent red illumination (R plants). The small amounts of the apoproteins which are synthesized by these plants are found in the membrane fraction; neither the mature apoproteins nor their precursor(s) can be detected immunologically in the soluble fraction. The protein does not accumulate in these plants. Pulse-chase experiments with the R plants demonstrate that the newly synthesized apoproteins have a half-life of about 10 h in the dark. This turnover is not sufficient to explain the observed 20-fold difference in [³⁵S]methionine incorporation into the apoprotein between white-light-grown and R plants. We therefore suggest that the synthesis of the Chla/b-apoproteins can be regulated by a light-dependent step at the level of translation, and that this regulation occurs after the initiation of translation.Abbreviations Chl chlorophyll - W Lemna plants grown in continuous white light - R plants grown heterotrophically in the dark with intermittent red light (2 min/8 h)     

Effect of light quality on chloroplast-membrane organization and function in pea

The effect of light quality during plant growth of chloroplast membrane organization and function in peas (Pisum sativum L. cv. Alaska) was investigated. In plants grown under photosystem (PS) I-enriched (far-red enriched) illumination both the PSII/PSI stoichiometry and the electrontransport capacity ratios were high, about 1.9. In plants grown under PSII-enriched (far-red depleted) illumination both the PSII/PSI stoichiometry and the electron-transport capacity ratios were significantly lower, about 1.3. In agreement, steady-state electron-transport measurements under synchronous illumination of PSII and PSI demonstrated an excess of PSII in plants grown under far-red-enriched light. Sodium dodecylsulfate polyacrylamide gel electrophoretic analysis of chlorophyll-containing complexes showed greater relative amounts of the PSII reaction center chlorophyll-protein complex in plants grown under farred-enriched light. Additional changes were observed in the ratio of light-harvesting chlorophyll a/b protein to PSII reaction center chlorophyll-protein under the two different light-quality regimes. The results demonstrate the dynamic nature of chloroplast structure and support the notion that light quality is an important factor in the regulation of chloroplast membrane organization and-function.Abbreviations and symbols Chl chlorophyll - CPa PSII reaction center chlorophyll protein complex - CPI PSI chlorophyll protein complex - FR-D light depleted in far-red sensitizing primarily PSII - FR-E light enriched in far-red sensitizing primarily PSI - LHCP PSII light-harvesting chlorophyll a/b protein complex - P ₇₀₀ primary electron donor of PSI - PSI, PSII photosystems I and II, respectively - Q primary electron acceptor of PSII     

Functional and structural organization of chlorophyll in the developing photosynthetic membranes of Euglena gracilis Z. IV. Light-harvesting properties of system II photosynthetic units and thylakoid ultrastructure during greening under intermittent light

Dark-grown, non-dividing Euglena gracilis Z cells were exposed for 100 h to intermittent light (15 s every 15 min darkness) and were then transferred to continuous light. During chloroplast differentiation, the development of light harvesting and trapping properties of Photosystem II was analyzed mainly with fluorescence induction measurements in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and was associated with observations on ultrastructural organisation of developing thylakoids using thin section and freeze-fracture methods. Results showed that: (a) the synthesis of chlorophyll b and probably that of the light-harvesting chlorophyll a/b-protein complex was more reduced by intermittent light than the formation of active system II reaction centers; (b) the size of the overall photosynthetic units, i.e. the number of chlorophyll molecules per O2 molecule evolved under a regime of repetitive saturating short flashes were reduced by 2-3 compared to those developed under continuous light; (c) the lack of chlorophyll induced by intermittent light affected more specifically the size of light-harvesting antennae of system II units, the optical cross-section of which was reduced by 3-4; (d) energy transfers did not occur between these small system II units in spite of high concentrations of PS II reaction centers and of a high trapping efficiency of the absorbed energy; (e) thylakoids developed under intermittent light were not stacked; (f) particles on exoplasmic fracture faces were significantly smaller than those developed under continuous light; (g) rapid synthesis of chlorophyll (Chl a and Chl b) upon exposure to continuous light of cells first greened under intermittent light are concomittant with rapid recovery of light-harvesting properties and structural characteristics of thylakoids developed under continuous light. These structural and functional observations are consistent with the hypothesis that system II units are organized in the photosynthetic membrane as individual and discrete entities, the morphological expression of which correspond to exoplasmic fracture face particles. They also support the model whereby energy transfers between physically connected system II units could occur across the partition between exoplasmic fracture face particles brought into contact in stacked regions.     

Preferential accumulation of apoproteins of the light-harvesting chlorophyll a/b-protein complex in greening barley leaves treated with 5-aminolevulinic acid

In order to study the coordinate accumulation of chlorophyll (Chl) and apoproteins of Chl-protein complexes (CPs) during chloroplast development, we examined changes in the accumulation of the apoproteins in barley (Hordeum vulgare L.) leaves when the rate of Chl synthesis was altered by feeding 5-aminolevulinic acid (ALA), a precursor of Chl biosynthesis. Pretreatment with ALA increased the accumulation of Chl a and Chl b 1.5- and 2.3-fold, respectively, after 12 cycles of intermittent light (2 min light followed by 28 min darkness). Apoproteins of the light-harvesting Chl a/b-protein complex of photosystem II (LHCII) were increased 2.4-fold with ALA treatment. However, apoproteins of the P700-Chl a-protein complex (CP1) and the 43-kDa apoprotein of a Chl a-protein complex of photosystem II (CPa) were not increased by ALA application. With respect to CPs themselves, LHCII was increased when Chl synthesis was raised by ALA feeding, whereas CP1 exhibited no remarkable increase. These results indicate that LHCII serves a role in maintaining the stoichiometry of Chl to apoproteins by acting as a temporary pool for Chl molecules.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll - CP chlorophyll-protein complex - CPa chlorophyll a-protein complex of PSII - CP1 P700-chlorophyll a-protein complex - LDS lithium dodecyl sulfate - LHCII light-harvesting chlorophyll a/b-protein complex of PSII This work was supported by the Grants-in-Aid for Scientific Research (04304004) from the Ministry of Education, Science and Culture, Japan.     

Differential expression of the genes for ribulose-1,5-bisphosphate carboxylase and light-harvesting chlorophyll a/b protein in the developing barley leaf

The expression of genes in particular for light-harvesting chlorophyll a/b protein (LHCP) and ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been studied in the developing barley leaf. This has been done by analysis of the occurrence of both proteins within the different regions (1 to 6, beginning from the base) of the primary 7-day-old leaf. It has been found that LHCP already appears in the base of the leaf, whereas RuBPCase is primarily expressed in the apical expanding part of the leaf. The distribution of the mRNAs for both proteins within this gradient is in accordance with that of the proteins themselves, indicating that gene expression is not regulated at the level of translation in both cases. The poly(A) mRNA for LHCP occurs mainly in the basic sections 2 and 3, whereas that for RuBPCase is found throughout the leaf but primarily in the apical sections of the leaf.Abbreviations LHCP light-harvesting chlorophyll a/b protein - RuBPCase ribulose-1,5-bisphosphate carboxylase - TCA trichloroacetic acid     

Energy transfer and pigment composition in three chlorophyll b-containing light-harvesting complexes isolated from Mantoniella squamata (Prasinophyceae), Chlorella fusca (Chlorophyceae) and Sinapis alba

Light-harvesting Chl a/b protein complexes were isolated from the higher plant Sinapis alba, the green alga Chlorella fusca, and the prasinophycean alga Mantoniella squamata by mild gel electrophoresis. The energy transfer from chlorophyll b and the accessory xanthophyll was measured by means of fluoresence spectroscopy at 77 K. The pigment composition of the isolated antenna complexes was determined by high performance liquid chromatography in order to calculate the number of light absorbing molecules per chlorophyll a in the different light-harvesting complexes. These results were complemented by the quantitation of the pigments in total thylakoids as well as in the different electrophoretic fractions. On the basis of these data the in vivo ratios of xanthophylls per chlorophyll a could be estimated. The results show that the light-harvesting complexes from Chlorella and from Sinapis exhibit identical ratios of total xanthophylls per chlorophyll a. By contrast, in the prasinophycean alga Mantoniella, the light-harvesting complex markedly differs from the other chlorophyll b containing proteins. It contains, in addition to neoxanthin and violaxanthin, high amounts of prasinoxanthin and its epoxide, which contribute significantly to light absorption. The concentration of chlorophyll b in the complex is very much higher in the antenna of Mantoniella than in those of Chlorella and Sinapis. Furthermore, it must be emphasized that in addition to chlorophyll b, a third chlorophyll species acts in the energy transfer to chlorophyll a. This chlorophyll c-like pigment is found to be present in a concentration which improves very efficiently the absorption in blue light. In light of these results it can be concluded that the absorption cross section in Mantoniella is higher not only because of an enhanced number of light-harvesting particles in the membrane, but also because of a higher ratio of accessory pigments to chlorophyll a.Abbreviations Chl Chlorophyll - FP Free Pigments - HPLC High Performance Liquid Chromatography - LHC Light-harvesting Chlorophyll protein complex - PAGE Polyacrylamide Gel Electrophoresis - PS Photosystem     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.Abbreviations kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b-binding protein - M_r relative molecular mass - PSII photosystem II - SDS sodium dodecyl sulphate     

Energization and ultrastructural pattern of thylakoids formed under periodic illumination followed by continuous light

Bean leaves grown under periodic illumination (56 cycles of 2 min light and 98 min darkness) were subsequently exposed to continuous illumination, and in connection with granum formation and accumulation of the light-harvesting pigment-protein complex thermoluminescence and light-induced shrinkage of thylakoid membranes were studied. Juvenile chloroplasts with large double sheets of thylakoids obtained under periodic light exhibited low temperature spectra of polarized fluorescence yielding fluorescence polarization (FP) values < 1 at 695 nm, characteristic for pheophytin emission. In the course of maturation under continuous light when normal grana appeared and the chlorophyll a/b light-harvesting photosystem II complex was incorporated into the membrane, at 695 nm the relative intensity of fluorescence dropped and FP changed to a value of > 1, suggesting an overlap between the emission of pheophytin and that of the chlorophyll a/b light-harvesting photosystem II complex. Thermoluminescence glow curves recorded with juvenile thylakoids displayed a relatively high proportion of emission at low temperatures (around -10°C) while with mature chloroplasts, more thermoluminescence originated from energetically deeper traps (discharged around 28°C). This means that during thylakoid development the capacity of the membrane to stabilize the separated charges increases, which might be favourable for the ultimate conservation of energy. The more extensive energization of mature thylakoids was also indicated by a light-induced decrease in the thickness of the membranes upon illumination; a change which could not be detected in juvenile thylakoids.Abbreviations EDTA ethylenediamine tetraacetic acid - Hepes 4-(2-hydroxy ethyl)-1-piperazine ethane sulfonic acid Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledons

Cotyledons were excised from imbibed watermelon seeds, grown for 4 days in darkness on water or 10 M benzyladenine (BA) and then tested for the presence of the light-harvesting chlorophyll a/b protein (LHCP) and its mRNA. LHCP was assayed immunologically by western blotting of SDS gels: the protein was present in plastids, but it was not recovered with the thylakoid fraction. Antibodies directed against LHCP precipitated a 32 kDa polypeptide from translation products of poly(A) RNA of cotyledons only if these had been grown on BA. Taken together the data suggest that in absence of light cytokinins are necessary for the maintenance of a detectable level of LHCP-mRNA as well as for synthesis of the protein.     

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     

High light fluence impairs light-harvesting Chl a/b complex apoprotein syntheses and/or membrane uptake in the CD3 mutant of wheat

The CD3 mutant of wheat is a chlorophyll(Chlo-deficient mutant the phenotype of which depends upon the accumulation of the light-harvesting Chl a/b protein complex in leaves in response to the intensity of illumination. In the present studies, the rates of synthesis and/or uptake, and degradation of the light-harvesting Chl apoprotein in chloroplasts of wild-type wheat ( Triticum aestivum L. selection ND 496) and CD3 wheat leaf segments were examined in response to two different intensities of illumination. We were interested particularly in the 21. 23 kDa proteins of the light-harvesting Chl a/b complex of photosystem I (LHCI) and the 25. 27. 29 kDa proteins of the light-harvesting Chl a/b complex of photosystem II (LHCII). The accumulation of [³⁵S]-Met into the light-harvesting Chl protein of CD3 wheat chloroplasts was impaired by a high but not by a low light fluence. The levels of radiolabel in the supernatant fractions of leaf tissue homogenates from the wild-type and CD3 wheats were not significantly different over time, suggesting that the cellular uptake of [³⁵S]-Met was not limiting in the mutant. The high fluence did not enhance the degradation of light-harvesting Chl protein from CD3 wheat thylakoids. Our data indicate an impairment in the light-harvesting Chl protein synthesis/membrane uptake system in CD3 wheat leaves under high fluence. A recovery in levels of the inner LHCPII, but not of LHCPI, was observed in the Chl-deficient wheat mutant after a prolonged (4 days) exposure to high fluence. Under low fluence, LHCP was added to both photosystem II (PSH) and photosystem I (PSI) but only that added to PSI remained in thylakoids after seedlings were switched to high fluence.     

Overexpression of chlorophyllide a oxygenase (CAO) enlarges the antenna size of photosystem II in Arabidopsis thaliana

The light-harvesting efficiency of a photosystem is thought to be largely dependent on its photosynthetic antenna size. It has been suggested that antenna size is controlled by the biosynthesis of chlorophyll b. To verify this hypothesis, we overexpressed the enzyme for chlorophyll b biosynthesis, chlorophyllide a oxygenase (CAO), in Arabidopsis thaliana by transforming the plant with cDNA for CAO under the control of the 35S cauliflower mosaic virus promoter. In the early de-etiolation phase, when the intrinsic CAO expression is very low, the chlorophyll a: b ratio was drastically decreased from 28 to 7.3, indicating that enhancement of chlorophyll b biosynthesis had been successfully achieved. We made the following observations in full-green rosette leaves of transgenic plants. (1) The chlorophyll a : b ratio was reduced from 2.85 to 2.65. (2) The ratio of the peripheral light-harvesting complexes (LHCII) to the core antenna complex (CPa) resolved with the green-gel system increased by 20%. (3) The ratio of the light-harvesting complex II apoproteins (LHCP) to 47-kDa chlorophyll a protein (CP47), which was estimated by the results of immunoblotting, increased by 40%. These results indicated that the antenna size increased by at least 10-20% in transgenic plants, suggesting that chlorophyll b biosynthesis controls antenna size. To the best of our knowledge, this is the first report on enlargement of the antenna size by genetic manipulations.