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 plastid membranes of barley (Hordeum vulgare). Light-induced appearance of mRNA coding for the apoprotein of the light-harvesting chlorophyll a/b protein

Illumination of dark-grown barley plants induces a massive insertion of the light-harvesting chlorophyll a/b protein into the developing thylakoid membrane. In addition to the onset of chlorophyll synthesis, light induces specifically the appearance of a prominent mRNA species which codes for a polypeptide of Mr 29500. This component was identified as a precursor of the apoprotein of the light-harvesting chlorophyll a/b protein. The precursor has an Mr larger than the authentic protein by approximately 4000. Studies of the chlorophyll-b-less mutant chlorina f2 of barley offer the first clue to the mechanism which controls the light-dependent mRNA formation. The induction of the mRNA coding for the aproprotein of the light-harvesting chlorophyll a/b protein does not seem to be linked directly to the assembly process of the light-harvesting structure and does not require chlorophyll b. It is proposed that light exerts its influence on the mRNA formation by a reaction which is different from the phototransformation of protochlorophyll(ide) to chlorophyll(ide).     

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.     

Excitation energy transfer in the light-harvesting chlorophyll a/b.protein

The "light-harvesting chlorophyll a/b.protein" described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600-700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitiation dependence of the fluorescence polarization shows a minimum polarization of 1.9% at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8% at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a double structure in the chlorophyll b absorption band which suggest an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the So leads to S1 transition moments of the chlorophyll molecules within the protein.     

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     

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     

Exchange of pigment-binding amino acids in light-harvesting chlorophyll a/b protein

Four amino acids in the major light-harvesting chlorophyll (Chl) a/b complex (LHCII) that are thought to coordinate Chl molecules have been exchanged with amino acids that presumably cannot bind Chl. Amino acids H68, Q131, Q197, and H212 are positioned in helixes B, C, A, and D, respectively, and, according to the LHCII crystal structure [Kühlbrandt, W., et al. (1994) Nature 367, 614-621], coordinate the Chl molecules named a(5), b(6), a(3), and b(3). Moreover, a double mutant was analyzed carrying exchanges at positions E65 and H68, presumably affecting Chls a(4) and a(5). All mutant proteins could be reconstituted in vitro with pigments, although the thermal stability of the resulting mutant versions of recombinant LHCII varied significantly. All complexes reconstituted with the mutant proteins contained fewer chlorophyll molecules per two lutein molecules than complexes reconstituted with the wild-type protein. However, the chlorophyll-binding amino acids could not be unambiguously assigned to binding either chlorophyll a or b, as in most cases more than one chlorophyll molecule was lost due to the mutation. The changes in Chl stoichiometries suggest that in LHCII some chlorophyll positions can be filled with either Chl a or b. Only some of the point mutations in LHCII affected the ability of the apoprotein to assemble into trimeric LHCII upon insertion into isolated thylakoid membranes. Among these were exchanges of H68 with either F or L, suggesting that the stability of the LHCII trimer significantly depends on this amino acid or the Chl molecule named a(5) that is attached to it and is located close to the center of the trimeric complex. The ion pair bridge between E65 and R185 in LHCII does not appear to be essential for the proper folding of the protein.     

Assembly of the barley light-harvesting chlorophyll a/b proteins in barley etiochloroplasts involves processing of the precursor on thylakoids

A barley gene encoding the major light-harvesting chlorophyll a/b-binding protein (LHCP) has been sequenced and then expressed in vitro to produce a labelled LHCP precursor (pLHCP). When barley etiochloroplasts are incubated with this pLHCP, both labelled pLHCP and LHCP are found as integral thylakoid membrane proteins, incorporated into the major pigment-protein complex of the thylakoids. The presence of pLHCP in thylakoids and its proportion with respect to labelled LHCP depends on the developmental stage of the plastids used to study the import of pLHCP. The reduced amounts of chlorophyll in a chlorophyll b-less mutant of barley does not affect the proportion of pLHCP to LHCP found in the thylakoids when import of pLHCP into plastids isolated from the mutant plants is examined. Therefore, insufficient chlorophyll during early stages of plastid development does not seem to be responsible for their relative inefficiency in assembling pLHCP. A chase of labelled pLHCP that has been incorporated into the thylakoids of intact plastids, by further incubation of the plastids with unlabelled pLHCP, reveals that the pLHCP incorporated into the thylakoids can be processed to its mature size. Our observations strongly support the hypothesis that after import into plastids, pLHCP is inserted into thylakoids and then processed to its mature size under in vivo conditions.     

Carotenoid-deficient maize seedlings fail to accumulate light-harvesting chlorophyll a/b binding protein (LHCP) mRNA

Yellow leaves of chlorophyll-deficient seedlings and white leaves of carotenoid-deficient seedlings contain no detectable light-harvesting chlorophyll a/b binding proteins (LHCP). Chlorophyll-deficient leaves contain plastids which are arrested in development prior to chloroplast formation [Mascia, P.N. and Robertson, D.S. (1978) Planta (Berl.) 143, 207-211] while carotenoid-deficient leaves contain plastids which are arrested in development at a rudimentary stage [Bachmann, M. D., Robertson, D.S., Bowen, C.C., and Anderson, I.C. (1967) J. Ultrastruc. Res. 21, 41-60]. Chlorophyll-deficient leaves have normal levels of nuclear-encoded LHCP mRNA while carotenoid-deficient leaves contain only trace amounts of LHCP mRNA. Similar results were obtained with carotenoid deficiencies caused by nuclear gene mutations and by treatment with the herbicide norflurazon which blocks carotenoid biosynthesis. We conclude that events at early stages of plastid development influence the accumulation of a nuclear-encoded mRNA.     

Evidence for a light-harvesting chlorophyll a-protein complex in a chlorophyll b-less barley mutant

Chloroplasts of a chlorophyll (Chl) b-less barley mutant were solubilized with digitonin and fractionated by polyacrylamide gel electrophoresis with sodium deoxycholate in the running buffer. By this procedure, in contrast to using sodium dodecylsulfate (SDS) for solubilization, a Chl a-protein analogous to the major light-harvesting Chl a-b protein complex from wildtype chloroplasts was recovered. This mutant Chl a-protein comprises about fifty percent of the total Chl a, and is very similar in carotenoid, amino acid, protein and polypeptide composition to the major wildtype antenna Chl a-b protein. The only major differences we have found is its instability in the presence of SDS and sensitivity to protease action. Even with deoxycholate, the mutant Chl a complex often dissociates during electrophoresis into two green bands. The lack of Chl b appears to affect the normal organization of Chl a and protein in such a way as to render the complex more unstable.CIW-DPB No. 917.     

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.     

Insertion of light-harvesting chlorophyll a/b protein into the thylakoid topographical studies.

The major light-harvesting chlorophyll a/b-binding protein (Lhcb1,2) of photosystem II is inserted into the thylakoid via the signal recognition particle dependent pathway. However, the mechanism by which the protein enters the membrane is at this time unknown. In order to define some topographical restrictions for this process, we constructed several recombinant derivatives of Lhcb1 carrying hexahistidine tags at either protein terminus or in the stromal loop domain. Additionally, green fluorescent protein (GFP) was fused to either terminus. None of the modifications significantly impair the pigment-binding properties of the protein in the in vitro reconstitution of LHCII. With the exception of the C-terminal GFP fusion, all mutants stably insert into isolated thylakoids in the absence of Ni2+ ions. The addition of low concentrations of Ni2+ ions abolishes the thylakoid insertion of C-terminally His-tagged mutants whereas the other His-tagged proteins fail to insert only at higher Ni2+ concentrations. The C-terminus of Lhcb1 must cross the membrane during protein insertion whereas the other sites of Lhcb1 modification are positioned on the stromal side of LHCII. We conclude that a Ni2+-complexed His tag and fusion to GFP inhibit translocation of the protein C-terminus across the thylakoid. Our observations indicate that the N-terminal and stromal domain of Lhcb1 need not traverse the thylakoid during protein insertion and are consistent with a loop mechanism in which only the C-terminus and the lumenal loop of Lhcb1 are translocated across the thylakoid.     

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.     

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.     

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     

Immunological evidence for apoproteins of the light-harvesting chlorophyll-protein complex in a mutant of barley lacking chlorophyll b

A barley mutant lacking chlorophyll b and the pigmented light-harvesting chlorophyll-protein of photo-system 2 is shown by several criteria to contain functional apoproteins of the light-harvesting complex. 1. Electrophoretic comparison of thylakoid polypeptide patterns, and the effects of trypsin treatment on these, suggests that the mutant contains several polypeptides equivalent in mobility to those of the wild-type complex. 2. An antibody monospecific for the light-harvesting complex agglutinated both wild-type and mutant thylakoids. 3. 'Western blot' immunoelectrophoretic analysis indicated that of four distinct subunits of the light-harvesting complex in the wild-type thylakoids, three are detectable in the mutant. 4. As in wild-type lamellae at least one of the light-harvesting complex polypeptides is phosphorylated by the endogenous protein kinase. The results are considered in terms of a general role for the light-harvesting complex polypeptides in membrane appression and the regulation of excitation energy distribution within thylakoids.     

Localization of the N-terminal domain in light-harvesting chlorophyll a/b protein by EPR measurements

The conformational distribution of the N-terminal domain of the major light-harvesting chlorophyll a/b protein (LHCIIb) has been characterized by electron-electron double resonance yielding distances between spin labels placed in various domains of the protein. Distance distributions involving residue 3 near the N terminus turned out to be bimodal, revealing that this domain, which is involved in regulatory functions such as balancing the energy flow through photosystems (PS) I and II, exists in at least two conformational states. Models of the conformational sub-ensembles were generated on the basis of experimental distance restraints from measurements on LHCIIb monomers and then checked for consistency with the experimental distance distribution between residues 3 in trimers. Only models where residue 3 is located above the core of the protein and extends into the aqueous phase on the stromal side fit the trimer data. In the other state, which consequently is populated only in monomers, the N-terminal domain extends sideways from the protein core. The two conformational states may correspond to two functional states of LHCIIb, namely trimeric LHCIIb associated with PSII in stacked thylakoid membranes and presumably monomeric LHCIIb associated with PSI in nonstacked thylakoids. The switch between these two is known to be triggered by phosphorylation of Thr-6. A similar phosphorylation-induced conformational change of the N-terminal domain has been observed by others in bovine annexin IV which, due to the conformational switch, also loses its membrane-aggregating property.     

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