Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

A collection of 17 monoclonal antibodies elicited against the light-harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC-II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b.     

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.     

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.     

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.     

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     

A major light-harvesting polypeptide of photosystem II functions in thermal dissipation

Under high-light conditions, photoprotective mechanisms minimize the damaging effects of excess light. A primary photoprotective mechanism is thermal dissipation of excess excitation energy within the light-harvesting complex of photosystem II (LHCII). Although roles for both carotenoids and specific polypeptides in thermal dissipation have been reported, neither the site nor the mechanism of this process has been defined precisely. Here, we describe the physiological and molecular characteristics of the Chlamydomonas reinhardtii npq5 mutant, a strain that exhibits little thermal dissipation. This strain is normal for state transition, high light-induced violaxanthin deepoxidation, and low light growth, but it is more sensitive to photoinhibition than the wild type. Furthermore, both pigment data and measurements of photosynthesis suggest that the photosystem II antenna in the npq5 mutant has one-third fewer light-harvesting trimers than do wild-type cells. The npq5 mutant is null for a gene designated Lhcbm1, which encodes a light-harvesting polypeptide present in the trimers of the photosystem II antennae. Based on sequence data, the Lhcbm1 gene is 1 of 10 genes that encode the major LHCII polypeptides in Chlamydomonas. Amino acid alignments demonstrate that these predicted polypeptides display a high degree of sequence identity but maintain specific differences in their N-terminal regions. Both physiological and molecular characterization of the npq5 mutant suggest that most thermal dissipation within LHCII of Chlamydomonas is dependent on the peripherally associated trimeric LHC polypeptides.     

An amino-proximal hydrophobic domain in the major light-harvesting chlorophyll a/b-protein is essential for membrane integration and protein stability

The major light-harvesting chlorophyll a/b-protein (LHCP) of higher plant chloroplasts is a nuclearencoded, integral thylakoid membrane protein that binds photosynthetic pigments and occurs in situ in an oligomeric form. We have previously examined structural and functional domains of the mature apoprotein by use of mutant LHCPs and in vitro assays for uptake and insertion. Results presented here demonstrate the effects of several mutations in the amino terminal domain of the mature apoprotein. Deletion of amino acid residues 12–58 greatly affected import into chloroplasts, while deletion or alteration of the hydrophobic region E⁶⁵VIHARWAM⁷³ led to rapid degradation of the mutant LHCP. We suggest that this amino-proximal region is essential for the stability of the LHCP and its ability to integrate into the thylakoid membranes. A structural/functional relationship of this region to a previously examined hydrophobic carboxy-proximal domain [Kohorn and Tobin (1989), The Plant Cell 1, 159–166] is proposed.Abbreviations BSA bovine serum albumin faction V - ELIPs early light-inducible proteins - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - LHCP light-harvesting chlorophyll a/b-protein - LHC IIb light-harvesting complex associated with Photosystem II - pLHCP precursor to LHCP - Rubisco ribulose 1,5-biphosphate carboxylase-oxygenase - SDS-PAGE sodium dodecyl sulfate-poly-acrylamide gel electrophoresis     

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.     

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.     

Role of spermidine in the stabilization of the apoprotein of the light-harvesting chlorophyll a/b-protein complex of photosystem II during leaf senescence process

Barley leaf discs maintained in dark accumulated a massive amount of putrescine (Put), lost chlorophyll and senescenced rapidly. At the same time RNase activity increased significantly. Exogenous spermidine (Spd) inhibited RNase activity, the loss of chlorophyll and degradation of the proteins from thylakoid membranes. Using SDS-PAGE and immunoblot analysis it was shown that spermidine was effective in the retardation of the loss of LHCPII observed in water-treated detached leaves. Analysis of PSII particles isolated from leaf fragments floated in water in the dark revealed the presence of Put, Spd and Spm. In spermidine treated leaves the level of this polyamine in photosystem II was above 5-fold higher than in control. The experimental findings obtained in this study provide evidence that applied spermidine interacts directly with thylakoid membranes so that they become more stable to degradation during senescence.     

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.     

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.     

Fluorescence decay kinetics of mutants of corn deficient in photosystem I and photosystem II

The fast fluorescence decay kinetics of two photosynthetic mutants of corn (Zea mays) have been compared with those of normal corn. The fluorescence of normal corn can be resolved into three exponential decay components of lifetime 900–1500 ps (slow), 300–500 ps (middle) and 50–120 ps (fast), the yields of which are affected by light intensity and Mg²⁺ levels. The Photosystem II-(PS II)-defective mutant hcf-3 has similar decay lifetimes (approx. 1200, 450 and 100 ps) but is not affected by light intensity, reflecting the absence of PS II charge recombination. However, yields do respond to Mg²⁺ in a fashion typical of normal corn, which may be correlated with the presence of normal levels of light-harvesting chlorophyll a + b complex (LHCP). The PS I mutant hcf-50 also shows three-component decay kinetics. In conjunction with the results on the LHCP-deficient mutant of barley presented in a recent paper (Karukstis, K.K. and Sauer, K. (1984) Biochim. Biophys. Acta 766, 148–155), these data suggest that the slow component of normal chloroplasts is kinetically controlled by the decay processes of the LHCP and that the energy comes from one of two sources: (a) charge recombination in the reaction centre or (b) energy transferred within or between LHCP units only. The fast component appears to originate from both PS I and PS II. The complex response of the middle component to cations and light intensity, and its presence in all of the mutants, suggests that it also may have multiple origins.     

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.     

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.     

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