Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.     

Nuclear pea mutants deficient in chlorophyll b and the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II

Eight chlorophyll b deficient nuclear mutants of pea (Pisum sativum L.) have been characterized by low temperature fluorescence emission spectra of their leaves and by the ultrastructure, photochemical activities and polypeptide compositions of the thylakoid membranes. The room temperature fluorescence induction kinetics of leaves and isolated thylakoids have also been recorded. In addition, the effects of Mg²⁺ on the fluorescence kinetics of the membranes have been investigated. The mutants are all deficient in the major polypeptide of the light-harvesting chlorophyll a/b protein of photosystem II. The low temperature fluorescence emission spectra of aurea-5106, xantha-5371 and –5820 show little or no fluorescence around 730 nm (photosystem I fluorescence), but possess maxima at 685 and 695 nm (photosystem II fluorescence). These three mutants have low photosystem II activities, but significant photosystem I activities. The long-wavelength fluorescence maximum is reduced for three other mutants. The Mg²⁺ effect on the variable component of the room temperature fluorescence (685 nm) induction kinetics is reduced in all mutants, and completely absent in aurea-5106 and xantha-5820. The thylakoid membranes of these 2 mutants are appressed pairwise in 2-disc grana of large diameter. Chlorotica-1-206A and–130A have significant long-wavelength maxima in the fluorescence spectra and show the largest Mg²⁺ enhancement of the variable part of the fluorescence kinetics. These two mutants have rather normally structured chloroplast membranes, though the stroma regions are reduced. The four remaining mutants are in several respects of an intermediate type.Abbreviations Chl chlorophyll - CPI Chi-protein complex I, F_o, F_v - F_m parameters of room temperature chlorophyll fluorescence induction kinetics - F685, F695 and F-1 components of low temperature chlorophyll emission with maximum at 685, 695 and ca 735 nm, respectively - PSI photosystem I - PSII photosystem II - LHCI and LHCII light-harvesting chlorophyll a/b complexes associated with PSI and PSII, respectively - SDS sodium dodecyl sulfate     

Isolation of two different subpopulations of the light-harvesting chlorophyll a/b complex of photosystem II (LHCII)

Isolated LHCII from spinach has been solubilized and fractionated by non-denaturing isoelectric focusing to yield two subpopulations with different polypeptide but equal chlorophyll composition. One LHCII subpopulation contains only a 27 kDa polypeptide while the other contains the 27 and 25 kDa polypeptides in about equal amounts. The polypeptide patterns of the two subpopulations closely correspond to those suggested previously for the inner LHCII and peripheral LHCII, respectively.     

Evidence for the role of the light-harvesting chlorophyll 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 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. Mg²⁺ 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 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. Mg²⁺ 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.     

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     

Assembly of the precursor and processed light-harvesting chlorophyll a/b protein of Lemna into the light-harvesting complex II of barley etiochloroplasts

When the in vitro synthesized precursor of a light-harvesting chlorophyll a/b binding protein (LHCP) from Lemna gibba is imported into barley etiochloroplasts, it is processed to a single form. Both the processed form and the precursor are found in the thylakoid membranes, assembled into the light-harvesting complex of photosystem II. Neither form can be detected in the stromal fraction. The relative amounts of precursor and processed forms observed in the thylakoids are dependent on the developmental stage of the plastids used for uptake. The precursor as well as the processed form can also be detected in thylakoids of greening maize plastids used in similar uptake experiments. This detection of a precursor in the thylakoids, which has not been previously reported, could be a result of using rapidly developing plastids and/or using an heterologous system. Our results demonstrate that the extent of processing of LHCP precursor is not a prerequisite for its inclusion in the complex. They are also consistent with the possibility that the processing step can occur after insertion of the protein into the thylakoid membrane.     

The structure of membrane crystals of the light-harvesting chlorophyll a/b protein complex

Membrane crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts were investigated using electron microscopy and image analysis. The membrane crystals formed upon precipitation of the detergent-solubilized complex with mono- and divalent cations in the presence of small amounts of Triton X-100. The crystalline fraction contained two polypeptides of 25,000 and 27,000 mol wt. Freeze-dried and freeze-etched specimens showed a periodic honeycomb structure on the surface of membrane crystals. Double replicas of freeze-fractured sheets showed a hexagonal lattice of particles on both fracture faces. Image analysis of negatively stained membrane crystals suggested that they had threefold rather than sixfold symmetry in projection. A projection map at 20-A resolution revealed two triangular structural units of opposite handedness per crystallographic unit cell. The structural units appeared to be inserted bidirectionally into the membrane, alternating in orientation perpendicular to the membrane plane.     

Three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts

Two forms of three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea have been obtained. Crystals of one form grew as hexagonal plates measuring up to 150 micron across and 2 to 3 micron in thickness. Electron diffraction patterns of thin hexagonal plates showed sharp reflections to a resolution of 3.7 A on a hexagonal reciprocal lattice. The unit cell in projection (a = 127.0 A) and the symmetry of the diffraction pattern (6 mm) suggested that the hexagonal plates were highly ordered stacks of two-dimensional crystals suitable for structure analysis by electron microscopy and image processing. Crystals of a second form grew as dark green octahedra measuring roughly 0.5 mm across. Low-resolution X-ray diffraction patterns suggested a large cubic unit cell (a = 390 A). SDS/polyacrylamide gel electrophoresis of single octahedral crystals showed the same polypeptide composition as the starting solution, one major band at 24,000 apparent molecular weight and two satellite bands of 23,000 and 23,500 apparent molecular weight.     

Characterization of different isoforms of the light-harvesting chlorophyll a/b complexes of photosystem II in bamboo

The major light-harvesting chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb) play important roles in energy balance of thylakoid membrane. They harvest solar energy, transfer the energy to the reaction center under normal light condition and dissipate excess excitation energy under strong light condition. Many bamboo species could grow very fast even under extremely changing light conditions. In order to explain whether LHCIIb in bamboo contributes to this specific characteristic, the spectroscopic features, the capacity of forming homotrimers and structural stabilities of different isoforms (Lhcb1-3) were investigated. The apoproteins of the three isoforms of LHCIIb in bamboo are overexpressed in vitro and successfully refolded with thylakoid pigments. The sequences of Lhcb1 and Lhcb2 are similar and they are capable of forming homotrimer, while Lhcb3 lacks 10 residues in the N terminus and can not form the homotrimeric structure. The pigment stoichiometries, spectroscopic characteristics, thermo- and photostabilities of different reconstituted Lhcbs reveal that Lhcb3 differs strongly from Lhcb1 and Lhcb2. Lhcb3 possesses the lowest Qy transition energy and the highest thermostability. Lhcb2 is the most stable monomer under strong illumination among all the isoforms. These results suggest that in spite of small differences, different Lhcb isoforms in bamboo possess similar characteristics as those in other higher plants.     

Evidence for the role of the light-harvesting chlorophyll a/b 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 and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II centres was only minimal. Mg²⁺ 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 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 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 and PS II_β centres. Mg²⁺ 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 and PS II_β to the fluorescence induction kinetics. PS II 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.     

Polyadenylated mRNA for the light-harvesting chlorophyll a/b protein

In thylakoid membranes isolated from green plants of parsley, pea, and barley, the light-harvesting chlorophyll a/b protein complex (LHCP, mol. weight: 25,000), is a major constituent. Poly(A)RNA isolated from these species was translated in a wheat germ, cell-free system. The in vitro translation products were treated with antibodies raised against the LHCP. This treatment resulted in the precipitation of a precursor protein (mol. weight: 29,000). Poly(A)RNA was also prepared from a cell culture ofPetroselinum that does not develop chloroplasts upon illumination. This poly(A)RNA is capable of stimulating amino acid incorporation in the in vitro translation system, however, it does not direct the synthesis of LHCP.     

Analysis of heat-induced disassembly process of three different monomeric forms of the major light-harvesting chlorophyll a/b complex of photosystem II

The temperature-dependent disassembly process of three monomeric isoforms, namely Lhcb1, Lhcb2, and Lhcb3, of the major light-harvesting chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb) were characterized by observing the changes of absorption spectra, circular dichroism (CD), and dissociation processes of the bound pigments to the in vitro reconstituted complexes subjected to high temperatures. Our results suggest that the three isoforms of LHCIIb undergo conformational rearrangements, structural changes, and dissociations of the bound pigments when the ambient temperature increases from 20 to 90°C. The conformation of the complexes changed sensitively to the changing temperatures because the absorption peaks in the Soret region (436 and 471?nm) and the Qy region (650-660 and 680?nm) decreased immediately upon elevating the ambient temperatures. Analyzing temperature-dependent denaturing and pigment dissociation process, we can divide the disassembly process into three stages: The first stage, appeared from 20°C to around 50-60°C, was characterized by the diminishment of the absorption around 650-660 and 680?nm, accompanied by the blue-shift of the peak at 471?nm and disappearance of the absorbance at 436?nm, which is related to changes in the transition energy of the Chl b cluster, and the red-most Chl a cluster in the LHCIIb. The second stage, beginning at about 50-60°C, was signified by the diminishment of the CD signal between (+)483?nm and (-)490?nm, which implied the disturbance of dipole-dipole interaction of pigments, and the onset of the pigment dissociation. The last stage, beginning at about 70-80°C, indicates the complete dissociation of the pigments from the complex. The physiological aspects of the three stages in the denaturing process are also discussed.     

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.     

Structural stability and properties of three isoforms of the major light-harvesting chlorophyll a/b complexes of photosystem II

Three isoforms of the major light-harvesting chlorophyll (Chl) a/b complexs of photosystem II (LHCIIb) in the pea, namely, Lhcb1, Lhcb2, and Lhcb3, were obtained by overexpression of apoprotein in Escherichia coli and by successfully refolding these isoforms with thylakoid pigments in vitro. The sequences of the protein, pigment stoichiometries, spectroscopic characteristics, thermo- and photostabilities of different isoforms were analysed. Comparison of their spectroscopic properties and structural stabilities revealed that Lhcb3 differed strongly from Lhcb1 and Lhcb2 in both respects. It showed the lowest Qy transition energy, with its reddest absorption about 2 nm red-shifted, and the highest photostability under strong illuminations. Among the three isoforms, Lhcb 2 showed lowest thermal stability regarding energy transfer from Chl b to Chl a in the complexes, which implies that the main function of Lhcb 2 under high temperature stress is not the energy transfer.     

Determination of relative chlorophyll binding affinities in the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex (LHCIIb) of photosystem II can be reconstituted in vitro from its recombinant apoprotein in the presence of a mixture of carotenoids and chlorophylls a and b. By varying the chlorophyll a/b ratio in the reconstitution mixture, the relative amounts of chlorophyll a and chlorophyll b bound to LHCIIb can be changed. We have analyzed the chlorophyll stoichiometry in recombinant wild type and mutant LHCIIb reconstituted at different chlorophyll a/b ratios in order to assess relative affinities of the chlorophyll-binding sites. This approach reveals five sites that exclusively bind chlorophyll b. Another site exhibits a slight preference of chlorophyll b over chlorophyll a. The remaining six sites are filled preferentially with chlorophyll a but also tolerate chlorophyll b when this is offered at a large excess. Three of these chlorophyll a-affine sites could be assigned to distinct positions defined by the three-dimensional LHCIIb structure. Exclusive chlorophyll b sites complemented by chlorophyll a sites that are selective only to a certain extent are consistent with the observation that chlorophyll b but not chlorophyll a is essential for reconstituting stable LHCIIb. These data offer an explanation why a rather constant chlorophyll a/b ratio is observed in native LHCIIb despite the apparent promiscuity of some binding sites.     

Structural stability and properties of three isoforms of the major light-harvesting chlorophyll a/b complexes of photosystem II

Three isoforms of the major light-harvesting chlorophyll (Chl) a/b complexs of photosystem II (LHCIIb) in the pea, namely, Lhcb1, Lhcb2, and Lhcb3, were obtained by overexpression of apoprotein in Escherichia coli and by successfully refolding these isoforms with thylakoid pigments in vitro. The sequences of the protein, pigment stoichiometries, spectroscopic characteristics, thermo- and photostabilities of different isoforms were analysed. Comparison of their spectroscopic properties and structural stabilities revealed that Lhcb3 differed strongly from Lhcb1 and Lhcb2 in both respects. It showed the lowest Qy transition energy, with its reddest absorption about 2 nm red-shifted, and the highest photostability under strong illuminations. Among the three isoforms, Lhcb 2 showed lowest thermal stability regarding energy transfer from Chl b to Chl a in the complexes, which implies that the main function of Lhcb 2 under high temperature stress is not the energy transfer.     

Crystallization of the light-harvesting chlorophyll a/b complex within thylakoid membranes

We have found that treatment of the photosynthetic membranes of green plants, or thylakoids, with the nonionic detergent Triton X-114 at a 10:1 ratio has three effects: (a) photosystem I and coupling factor are solubilized, so that the membranes retain only photosystem II (PS II) and its associated light-harvesting apparatus (LHC-II); (b) LHC-II is crystallized, and so is removed from its normal association with PS II; and (c) LHC-II crystallization causes a characteristic red shift in the 77 degrees K fluorescence from LHC-II. Treatment of thylakoids with the same detergent at a 20:1 ratio results in an equivalent loss of photosystem I and coupling factor, with LHC-II and PS II being retained by the membranes. However, no LHC-II crystals are formed, nor is there a shift in fluorescence. Thus, isolation of a membrane protein is not required for its crystallization, but the conditions of detergent treatment are critical. Membranes with crystallized LHC-II retain tetrameric particles on their surface but have no recognizable stromal fracture face. We have proposed a model to explain these results: LHC-II is normally found within the stromal half of the membrane bilayer and is reoriented during the crystallization process. This reorientation causes the specific fluorescence changes associated with crystallization. Tetrameric particles, which are not changed in any way by the crystallization process, do not consist of LHC-II complexes. PS II appears to be the only other major complex retained by these membranes, which suggests that the tetramers consist of PS II.     

Cytokinins modulate the expression of genes encoding the protein of the light-harvesting chlorophyll a/b complex

Summary Tobacco cell suspension cultures responded to cytokinins (for instance kinetin) by full chloroplast differentiation. The hormone had the effect of stimulating the appearance of a few prominent plastid proteins. Synthesis of the light-harvesting chlorophyl a/b-binding protein (LHCP) in response to kinetin was noteworthy (Axelos M. et al.: Plant Sci Lett 33:201–212, 1984).Poly(A)⁺RNAs were prepared from cells grown in the presence of or without added kinetin. Poly(A)⁺RNA recovery and translation activity were not quantitatively altered by the hormone treatment. In vitro translation of polyadenylated mRNA into precursor polypeptides of LHCP (pLHCP) was quantified by immunoprecipitation and SDS-PAGE fractionation of pLHCP immunoprecipitates: pLHCP-mRNA translating activity was found to be stimulated in parallel to mature LHCP accumulation by kinetin-induced cells.Dot-blot and northern-blot hybridizations of poly(A)⁺RNA were carried out, using as a probe a pea LHCP-cDNA clone (Broglie R. et al.: Proc Natl Acad Sci USA 78: 7304–7308, 1981). A ten-fold increase of the level of pLHCP-encoding sequences was observed in poly(A)⁺RNA prepared from 9-d kinetin-stimulated cells, compared to control cells. Oligo(dT)-cellulose-excluded RNA fractions exhibited very low hybridization levels, in the same ratios as those obtained with poly(A)⁺RNA.Thus, the expression of LHCP-gene activity, in response to kinetin addition to tobacco cell suspension cultures, is regulated by the level of pLHCP-encoding mRNA rather than by translational or post-translational controls. re]19850218 rv]19850605 ac]19850613