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.     

Regulation of chlorophyll apoprotein expression and accumulation. Requirements for carotenoids and chlorophyll.

Chlorophyll apoprotein accumulation and expression were examined in mutants of Chlamydomonas reinhardtii blocked at specific steps of carotenoid or chlorophyll synthesis. In the absence of carotenoids: 1) apoproteins of the core and light-harvesting complexes of photosystem I (CCI and LHCI, respectively) and photosystem II (CCII and LHCII, respectively) do not accumulate; 2) mRNAs for the CCI, CCII, and LHCII apoproteins accumulate to normal levels; and 3) synthesis of the chlorophyll apoproteins is differentially affected, or in some cases, not affected. In the absence of chlorophylls: 1) the apoproteins fail to accumulate; 2) mRNA levels for CCI and CCII apoproteins are relatively unchanged; 3) levels of LHCII apoprotein mRNA, but not rates of LHCII mRNA synthesis, are reduced in a light-dependent chlorophyll-synthesis mutant (ya12); and 4) synthesis of chlorophyll apoproteins is differentially affected or not affected in the case of several chloroplast-encoded apoproteins. These results demonstrate a direct role for carotenoids as well as chlorophylls in the stabilization of certain chlorophyll apoproteins and, for others, possibly in their translation. The data also indicate a role for chlorophyll synthesis in the stability of LHCII mRNA.     

Rigid core and flexible terminus: structure of solubilized light-harvesting chlorophyll a/b complex (LHCII) measured by EPR

The structure of the major light-harvesting chlorophyll a/b complex (LHCII) was analyzed by pulsed EPR measurements and compared with the crystal structure. Site-specific spin labeling of the recombinant protein allowed the measurement of distance distributions over several intra- and intermolecular distances in monomeric and trimeric LHCII, yielding information on the protein structure and its local flexibility. A spin label rotamer library based on a molecular dynamics simulation was used to take the local mobility of spin labels into account. The core of LHCII in solution adopts a structure very similar or identical to the one seen in crystallized LHCII trimers with little motional freedom as indicated by narrow distance distributions along and between α helices. However, distances comprising the lumenal loop domain show broader distance distributions, indicating some mobility of this loop structure. Positions in the hydrophilic N-terminal domain, upstream of the first trans-membrane α helix, exhibit more and more mobility the closer they are to the N terminus. The nine amino acids at the very N terminus that have not been resolved in any of the crystal structure analyses give rise to very broad and possibly bimodal distance distributions, which may represent two families of preferred conformations.     

Decreasing the chlorophyll a/b ratio in reconstituted LHCII: structural and functional consequences.

Trimeric (bT) and monomeric (bM) light-harvesting complex II (LHCII) with a chlorophyll a/b ratio of 0.03 were reconstituted from the apoprotein overexpressed in Escherichia coli. Chlorophyll/xanthophyll and chlorophyll/protein ratios of bT complexes and 'native' LHCII are rather similar, namely, 0.28 vs 0. 27 and 10.5 +/- 1.5 vs 12, respectively, indicating the replacement of most chlorophyll a molecules with chlorophyll b, leaving one chlorophyll a per trimeric complex. The LD spectrum of the bT complexes strongly suggests that the chlorophyll b molecules adopt orientations similar to those of the chlorophylls a that they replace. The circular dichroism (CD) spectra of bM and bT complexes indicate structural arrangements resembling those of 'native' LHCII. Thermolysin digestion patterns demonstrate that bT complexes are folded and organized like 'native' trimeric LHCII. Surprisingly, in the bT complexes at 77 K, half of the excitations that are created on either chlorophyll b or xanthophyll are transferred to chlorophyll a. No or very limited triplet transfer from chlorophyll b to xanthophyll appears to take place. However, the efficiency of triplet transfer from chlorophyll a to xanthophyll is close to 100%, even higher than in 'native' LHCII at 77 K. It is concluded from the triplet-minus-singlet and CD results that the single chlorophyll a molecule that on the average is present in each bT complex binds preferably next to a xanthophyll molecule at the interface between the monomers.     

Immunological cross-reactivity between the light-harvesting chlorophyll a/b-proteins of a marine green alga and spinach

Immunoblotting was used to probe the reactivity of rabbit polyclonal antibodies against PS1I and PSI light-harvesting chlorophyll a/b-proteins of spinach ( Spinacea oleracea L.) with the light-harvesting complexes of a siphonaceous marine alga, Codium , that have more chlorophyll b, siphonaxanthin and siphonein instead of the lutein. The spinach LHCII antibodies cross-reacted only with the apoproteins of Cod-ium LHCII. Antisera against the spinach LHCI apoproteins showed strong affinity for the apoproteins of Codium LHCI, and also reacted with the polypeptides of spinach LHCII and Codium LHCII. Our results indicate some similarities in the amino acid sequences between the Codium siphonaxanthin-Chl a/fe-proteins of LHCII and LHCI and the corresponding spinach lutein-chlorophyll a/b-proteins.     

Polypeptides belonging to each of the three major chlorophyll a + b protein complexes are present in a chlorophyll-b-less barley mutant

Polypeptides of the three major chlorophyll a + b protein complexes were detected in a chlorophyll-b-less barley mutant (chlorina f2) using immunological techniques. Antibodies to CP Ia, a photosystem I complex containing both the reaction center (CP I) and the chlorophyll a + b antenna (LHCI), detected substantial amounts of LHCI polypeptides in mutant thylakoids. Some polypeptides of the two photosystem-II-associated chlorophyll a + b complexes, CP 29 and LHCII, were also detected using antibodies raised against these complexes. The CP 29 apoprotein and the minor 25-kDa polypeptide of LHCII were present in amounts that could be seen by Coomassie blue staining. In contrast, the two major polypeptides of LHCII were greatly diminished in amount, and one of them may be completely absent. These data suggest that the absence of chlorophyll b may have differing effects on the synthesis, processing or turnover of the various chlorophyll a + b binding polypeptides. They also show that these polypeptides can be inserted into thylakoids in the absence of Chl b, and that significant amounts of some of them are accumulated in the mutant thylakoids.     

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.     

The N-terminal domain of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) is essential for its acclimative proteolysis

Variations in the amount of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) is essential for regulation of the uptake of light into photosystem II. An endogenous proteolytic system was found to be involved in the degradation of LHCII in response to elevated light intensities and the proteolysis was shown to be under tight regulation [Yang, D.-H. et al. (1998) Plant Physiol. 118, 827-834]. In this study, the substrate specificity and recognition site towards the protease were examined using reconstituted wild-type and mutant recombinant LHCII. The results show that the LHCII apoprotein and the monomeric form of the holoprotein are targeted for proteolysis while the trimeric form is not. The N-terminal domain of LHCII was found to be essential for recognition by the regulatory protease and the involvement of the N-end rule pathway is discussed.     

STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis

During leaf senescence, plants degrade chlorophyll to colorless linear tetrapyrroles that are stored in the vacuole of senescing cells. The early steps of chlorophyll breakdown occur in plastids. To date, five chlorophyll catabolic enzymes (CCEs), NONYELLOW COLORING1 (NYC1), NYC1-LIKE, pheophytinase, pheophorbide a oxygenase (PAO), and red chlorophyll catabolite reductase, have been identified; these enzymes catalyze the stepwise degradation of chlorophyll to a fluorescent intermediate, pFCC, which is then exported from the plastid. In addition, STAY-GREEN (SGR), Mendel's green cotyledon gene encoding a chloroplast protein, is required for the initiation of chlorophyll breakdown in plastids. Senescence-induced SGR binds to light-harvesting complex II (LHCII), but its exact role remains elusive. Here, we show that all five CCEs also specifically interact with LHCII. In addition, SGR and CCEs interact directly or indirectly with each other at LHCII, and SGR is essential for recruiting CCEs in senescing chloroplasts. PAO, which had been attributed to the inner envelope, is found to localize in the thylakoid membrane. These data indicate a predominant role for the SGR-CCE-LHCII protein interaction in the breakdown of LHCII-located chlorophyll, likely to allow metabolic channeling of phototoxic chlorophyll breakdown intermediates upstream of nontoxic pFCC.     

Conformational rearrangements in light-harvesting complex II accompanying light-induced chlorophyll a fluorescence quenching

Light-induced chlorophyll a (Chl a) fluorescence quenching was studied in light-harvesting complex of photosystem II (LHCII). Fluorescence intensity decreased by ca. 20% in the course of 20 min illumination (412 nm, 36 micromol m(-2) s(-1)) and was totally reversible within 30 min dark adaptation. The pronounced quenching was observed only in LHCII in an aggregated form and exclusively in the presence of molecular oxygen. Structural rearrangement of LHCII correlated to the quenching was monitored by measuring changes in UV-Visible light absorption spectra, and by measuring Fourier-transform infrared spectroscopy (FTIR) in the Amide I region of the protein (1600-1700 cm(-1)). The light-induced structural rearrangement of LHCII was interpreted as a partial disaggregation of the complex based on the decrease in the light scattering signal and the characteristic features observed in the FTIR spectra: the relative increase in the intensity of the band at 1653 cm(-1), corresponding to a protein in the alpha-helical structure at the expense of the band centered at 1621 cm(-1), characteristic of aggregated forms. The fact that the light-driven isomerization of the all-trans violaxanthin to the 13-cis form was not observed under the non-oxygenic conditions coincided with the lack of large-scale conformational reorganization of LHCII. The kinetics of this large-scale structural effect does not correspond to the light-induced fluorescence quenching, in contrast to the kinetics of structural changes in LHCII observable at low oxygen concentrations. Photo-conversion of 5% of the pool of all-trans violaxanthin to 9-cis isomer was observed under such conditions. Possible involvement of the violaxanthin isomerization in the process of structural rearrangements and excitation quenching in LHCII is discussed.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl(2), an inhibitor of cytochrome b(6)f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b(6)f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

Participation of Chlorophyll b Reductase in the Initial Step of the Degradation of Light-harvesting Chlorophyll a/b-Protein Complexes in Arabidopsis

The light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) is the most abundant membrane protein in green plants, and its degradation is a crucial process for the acclimation to high light conditions and for the recovery of nitrogen (N) and carbon (C) during senescence. However, the molecular mechanism of LHCII degradation is largely unknown. Here, we report that chlorophyll b reductase, which catalyzes the first step of chlorophyll b degradation, plays a central role in LHCII degradation. When the genes for chlorophyll b reductases NOL and NYC1 were disrupted in Arabidopsis thaliana, chlorophyll b and LHCII were not degraded during senescence, whereas other pigment complexes completely disappeared. When purified trimeric LHCII was incubated with recombinant chlorophyll b reductase (NOL), expressed in Escherichia coli, the chlorophyll b in LHCII was converted to 7-hydroxymethyl chlorophyll a. Accompanying this conversion, chlorophylls were released from LHCII apoproteins until all the chlorophyll molecules in LHCII dissociated from the complexes. Chlorophyll-depleted LHCII apoproteins did not dissociate into monomeric forms but remained in the trimeric form. Based on these results, we propose the novel hypothesis that chlorophyll b reductase catalyzes the initial step of LHCII degradation, and that trimeric LHCII is a substrate of LHCII degradation.     

7-Hydroxymethyl chlorophyll a reductase functions in metabolic channeling of chlorophyll breakdown intermediates during leaf senescence

During natural or dark-induced senescence, chlorophyll degradation causes leaf yellowing. Recent evidence indicates that chlorophyll catabolic enzymes (CCEs) interact with the photosynthetic apparatus; for example, five CCEs (NYC1, NOL, PPH, PAO and RCCR) interact with LHCII. STAY-GREEN (SGR) and CCEs interact with one another in senescing chloroplasts; this interaction may allow metabolic channeling of potentially phototoxic chlorophyll breakdown intermediates. 7-Hydroxymethyl chlorophyll a reductase (HCAR) also acts as a CCE, but HCAR functions during leaf senescence remain unclear. Here we show that in Arabidopsis, HCAR-overexpressing plants exhibited accelerated leaf yellowing and, conversely, hcar mutants stayed green during dark-induced senescence. Moreover, HCAR interacted with LHCII in in vivo pull-down assays, and with SGR, NYC1, NOL and RCCR in yeast two-hybrid assays, indicating that HCAR is a component of the proposed SGR-CCE-LHCII complex, which acts in chlorophyll breakdown. Notably, HCAR and NOL are expressed throughout leaf development and are drastically down-regulated during dark-induced senescence, in contrast with SGR, NYC1, PPH and PAO, which are up-regulated during dark-induced senescence. Moreover, HCAR and NOL are highly up-regulated during greening of etiolated seedlings, strongly suggesting a major role for NOL and HCAR in the chlorophyll cycle during vegetative stages, possibly in chlorophyll turnover.     

Random mutations directed to transmembrane and loop domains of the light-harvesting chlorophyll a/b protein: impact on pigment binding.

The major light-harvesting complex of photosystem II (LHCII) can be reconstituted in vitro by folding its bacterially expressed apoprotein, Lhcb, in detergent solution in the presence of chlorophylls and carotenoids. To compare the impact of alpha-helical transmembrane domains and hydrophilic loop domains of the apoprotein on complex formation and stability, we introduced random mutations into a segment of the protein comprising the stromal loop, the third (C-proximal) transmembrane helix, and part of the amphipathic helix in the C-terminal domain. The mutant versions of Lhcb were screened for the loss of their ability to form stable LHCII upon reconstitution in vitro. Most steps during the screening, including expression of the recombinant protein, its reconstitution with pigments, and the assay for complex formation by measuring energy transfer from chlorophyll b to chlorophyll a, were performed as one-vessel reactions on 96-well microtiter plates. This enabled us to screen several hundred mutant Lhcb versions. Mutants that had lost their ability to form stable LHCII carried between one and four amino acid exchanges. Among the single-point mutations, several were at positions in the C-proximal transmembrane helix, including an amino acid that is thought to be directly involved in chlorophyll binding. However, we also found four point mutations in the stromal loop domain that, in our assay, completely abolished the formation of stable LHCII. These data show that the stromal loop domain has a significant impact on LHCII formation and/or stability in vitro.     

Transbilayer organization of the main chlorophyll a/b-protein of photosystem II of thylakoid membranes

To probe the location of the carboxyl-terminus of the 28 kDa apoprotein of the light-harvesting chlorophyll a/b-protein complex of PS II (LHCII), an antibody was generated against a synthetic octapeptide corresponding to the C-terminal region of LHCII. The high specificity of the octapeptide antiserum was deonstrated by immunoblots and immunogold labelling. The octapeptide antiserum agglutinated destacked thylakoid membranes, but no significant agglutination occurred with inside-out vesicles suggesting that the COOH-terminus is located at the outer, stroma-exposed surface where the NH₂-terminus is also located [(1983) J. Biol. Chem. 258, 9941-9948]. Our results support a model for LHCII with four transmembrane-spanning domains.     

Immunological characterization of chlorophyll a/b-binding proteins of barley thylakoids

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCI) using a photosystem (PS) I preparation (PSI-200) wild-type from barley (Hordeum vulgare L. cv. Svaløf's Bonus) as the antigen. These antibodies cross-reacted with a minor light-harvesting chlorophyll a/b-protein of PSII (Chla/b-P1=CP29), but not with the major one, LHCII (=Chla/b-P2^**). Similarly, a monoclonal antibody to Chla/b-P1, elicited by a PSII preparation as the antigen, cross-reacted with LHCI, but not LHCII. This explains why an antigen consisting of LHCII, free of LHCI, but contaminated with Chla/b-P1, can elicit antibodies which cross-react with LHCI. Immunoblot assays showed that LHCI and Chla/b-P1 have at least two epitopes in common. Immunogold labelling of thin-sectioned wild-type thylakoids confirmed a preferential localisation of Chla/b-P1 in grana partition membranes and LHCI in stroma lamellae. The presence of LHCI was demonstrated in barley mutants lacking the PSI reaction centre (viridis-zb ⁶³) and chlorophyll b (chlorina-f2), and was correlated with the presence of long-wavelength (730 nm) fluorescence emission at 77 K. The mutant viridis-k ²³, which has a 77 K long-wavelength fluorescence peak at 720 nm, was shown by immune-blot assay to lack LHCI, although Chla/b-P1 was present.Abbreviations Chl-P chlorophyll-protein - CM Carlsberg Monoclonal - Da dalton - LHC light-harvesting complex - PAGE polyacrylamide gel electrophoresis - PSI, II photosystem I, II - PSI-200 PSI containing LHCI polypeptides - SDS sodium dodecyl sulphate     

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.     

NaCl-induced phosphorylation of light harvesting chlorophyll a/b proteins in thylakoid membranes from the halotolerant green alga, Dunaliella salina

Light could induce phosphorylation of light harvesting chlorophyll a/b binding proteins (LHCII) in Dunaliella salina and spinach thylakoid membranes. We found that neither phosphorylation was affected by glycerol, whereas treatment with NaCl significantly enhanced light-induced LHCII phosphorylation in D. salina thylakoid membranes and inhibited that in spinach. Furthermore, even in the absence of light, NaCl and several other salts induced LHCII phosphorylation in D. salina thylakoid membranes, but not in spinach thylakoid membranes. In addition, hypertonic shock induced LHCII phosphorylation in intact D. salina under dark conditions and cells adapted to different NaCl concentrations exhibited similar LHCII phosphorylation levels. Taken together, these results show for the first time that while LHCII phosphorylation of D. salina thylakoid membranes resembles that of spinach thylakoid membranes in terms of light-mediated control, the two differ with respect to NaCl sensitivity under light and dark conditions.     

Novel aspects of chlorophyll a/b-binding proteins

The light-harvesting proteins (LHC) constitute a multigene family including, in higher plants, at least 12 members whose location, within the photosynthetic membrane, relative abundance and putative function appear to be very different. The major light-harvesting complex of photosystem II (LHCII) is the most abundant membrane protein in the biosphere and fulfil a constitutive light-harvesting function for photosystem II while the early light-induced proteins (ELIPs) are expressed in low amounts under stress conditions. Primary sequence analysis suggests that all these proteins share a common structure which was resolved at 3.7 Å resolution by electron crystallography in the case of the major LHCII complex: Three transmembrane helices connected by hydrophilic loops coordinate seven chlorophyll a and five chlorophyll b molecules by histidine, glutamine, asparagine lateral chains as well as by charge compensated ionic pairs of glutamic acid and arginine residues; moreover, at least two xantophyll molecules are located at the centre of the structure in close contact with seven porphyrins, tentatively identified as chlorophyll a. The antenna system is also involved in the regulation of excitation energy transfer to reaction centre II. This function has been attributed to three members of the protein family, namely CP29, CP26 and CP24 (also called minor chlorophyll proteins) which have been recently characterised and shown to bind most of the xantophyll cycle carotenoids, thus suggesting that the non-photochemical quenching mechanism is acting in these proteins. Further support to this assignment comes from the recent identification of protonation sites in CP29 and CP26 by covalent dicyclohexhylcarbodiimide binding suggesting that these respond to low lumenal pH. In addition, CP29 is reversibly phosphorylated under light and cold stress conditions, undergoing conformational change, supporting the hypothesis that these subunits, present in low amounts in photosystem II, have a major regulatory role in the light-harvesting function and are thus important in environmental stress resistance.