Light-harvesting Complex Apoproteins Research Articles

The role of chlorophyll b in photosynthesis: Hypothesis

BackgroundThe physico-chemical properties of chlorophylls b and c have been known for decades. Yet the mechanisms by which these secondary chlorophylls support assembly and accumulation of light-harvesting complexes in vivo have not been resolved.PresentationBiosynthetic modifications that introduce electronegative groups on the periphery of the chlorophyll molecule withdraw electrons from the pyrrole nitrogens and thus reduce their basicity. Consequently, the tendency of the central Mg to form coordination bonds with electron pairs in exogenous ligands, a reflection of its Lewis acid properties, is increased. Our hypothesis states that the stronger coordination bonds between the Mg atom in chlorophyll b and chlorophyll c and amino acid sidechain ligands in chlorophyll a/b- and a/c-binding apoproteins, respectively, enhance their import into the chloroplast and assembly of light-harvesting complexes.TestingSeveral apoproteins of light-harvesting complexes, in particular, the major protein Lhcb1, are not detectable in leaves of chlorophyll b-less plants. A direct test of the hypothesis – with positive selection – is expression, in mutant plants that synthesize only chlorophyll a, of forms of Lhcb1 in which weak ligands are replaced with stronger Lewis bases.ImplicationsThe mechanistic explanation for the effects of deficiencies in chlorophyll b or c points to the need for further research on manipulation of coordination bonds between these chlorophylls and chlorophyll-binding proteins. Understanding these interactions will possibly lead to engineering plants to expand their light-harvesting antenna and ultimately their productivity.

Light-induced biogenesis of chlorophyll-protein complexes in developing wheat thylakoids.

It is shown that the apoproteins of core complexes (CC) I and II, the alpha- and beta-subunits of CF1 ATP-synthase complexes, are present in seedlings grown under intermittent light (IML). The levels of light-harvesting complex (LHC) apoproteins in the 30- to 18-kD region increase rapidly upon exposure to continuous light (CL). The newly synthesized LHC apoproteins appear to be present predominantly in monomeric forms that later assemble into higher-order oligomeric forms. During the early stages of greening of wheat seedlings, polypeptides in the 20.5-19 and 17.5-15.5 kD regions (so-called "early light-induced proteins" (ELIP)) are observed, but they disappear fully after 6 h. As greening proceeds, the 727-nm band in low-temperature fluorescence spectra (77 K) gradually shifts to longer wavelength (740-742 nm), which clearly demonstrates the light-driven biogenesis of LHC I and its assembly with CC I.

Antisense inhibition of the beta-carotene hydroxylase enzyme in Arabidopsis and the implications for carotenoid accumulation, photoprotection and antenna assembly.

The xanthophylls are oxygenated carotenoids and are important structural components of the photosynthetic apparatus. Xanthophylls contribute to the assembly and stability of light-harvesting complex apoproteins (LHC) and contribute to photoprotection via non-photochemical quenching of chlorophyll fluorescence (NPQ) in oxygenic photosynthetic organisms. Previously, mutations have been described that disrupt many steps in the xanthophyll biosynthetic pathway. However, there are no definitive reports of a lesion that effects the beta-hydroxylase enzyme, which catalyzes hydroxylation of the beta-rings of beta-carotene and alpha-carotene, and is thus necessary for synthesis of essentially all xanthophylls of higher plant chloroplasts. We have utilized an antisense approach to effectively reduce levels of beta-hydroxylase in Arabidopsis thaliana in order to examine how a reduction in this enzyme impacts carotenoid biosynthesis and plant viability. Expression of the antisense beta-hydroxylase transgene resulted in a maximal reduction in violaxanthin of 64% and a maximal reduction in neoxanthin of 41%. This reduction was reflected in a 22% increase in beta-carotene and a reduction in the total carotenoid pool, whereas lutein levels were relatively unaltered. Despite the reduction in violaxanthin and neoxanthin, the antisense beta-hydroxylase plants had a wild-type complement of chlorophylls and LHCs on a fresh weight basis. Under high light stress, the unconverted pool of violaxanthin was the same size as in wild type and thus there was an even greater proportional reduction in zeaxanthin of 75%. Despite this marked decrease in zeaxanthin, NPQ only declined by 16%.

Chlorophyll synthesis modulates retention of apoproteins of light-harvesting complex II by the chloroplast in Chlamydomonas reinhardtii

Chlorophyll synthesis modulates retention of apoproteins of light-harvesting complex II by the chloroplast in Chlamydomonas reinhardtii

Secretory granules in the cytoplasm of a wall-less mutant of Chlamydomonas reinhardtii contain processed light-harvesting complex apoproteins and HSP70

Large, electron dense granules are present within vacuoles in the cytoplasm of Chlamydomonas reinhardtii cwl15 arg7A cells. Immunocytochemical electron microscopy revealed labeling of these granules when sections of cells were incubated with antibodies specific for apoproteins (Lhcb) of the light-harvesting chlorophyll a b complex (LHCII). The granules were also labeled with antibodies against the cytosolic molecular chaperone Hsp70. Purified granules contained Hsp70 and processed, mature forms of Lhcb as minor constituents. Appearance of processed Lhcb in the vacuole suggests that these proteins were products of retrograde movement resulting from aborted import into the chloroplast. Immunoreactive granules were released from the cell following fusion of the vacuolar and plasma membranes.

A transcription factor with a leucine-zipper motif involved in light-dependent inhibition of expression of the puf operon in the photosynthetic bacterium Rhodobacter sphaeroides.

In the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides the synthesis of components of the photosystem is regulated in response to oxygen tension and light intensity. We have purified and cloned a trans-acting protein (SPB) that binds to the promoter region of the puf operon, which encodes the apoproteins of light-harvesting complex I and the reaction center. The SPB was composed of a single polypeptide with an apparent molecular mass of 15.0 kDa. The nucleotide sequence of the spb gene was determined. The gene encoded 104 amino acid residues, which correspond to a molecular mass of 11.5 kDa. SPB exhibited 53% homology to HvrA in Rhodobacter capsulatus. The deduced amino acid sequence indicated that SPB contained a region with homology to the leucine-zipper motif of c-JUN, a transcription factor in eukaryotes, and SPB also had a DNA-binding domain on the amino-terminal side of the leucine-zipper motif. The leucine-zipper motif of SPB might contribute to the formation of a dimer. Northern analysis indicated that spb was constitutively and monocistronically transcribed in R. sphaeroides, irrespective of growth conditions. Structural and functional differences between SPB and HvrA are discussed.

Localization of light-harvesting complex apoproteins in the chloroplast and cytoplasm during greening ofChlamydomonas reinhardtii at 38°C

Assembly of the major light-harvesting complex (LHC II) and development of photosynthetic function were examined during the initial phase of thylakoid biogenesis inChlamydomonas reinhardtii cells at 38°C. Continuous monitoring of LHC II fluorescence showed that these processes were initiated immediately upon exposure of cells to light. However, mature-size apoproteins of LHC II (Lhcb) increased in amount in an alkali-soluble (non-membrane) fraction in parallel with the increase in the membrane fraction. Alkali-soluble Lhcb were not integrated into membranes when protein synthesis was inhibited, suggesting that they were not active intermediates in LHC II assembly, nor were they recovered in a purified chloroplast preparation. Immunocytochemical analysis of greening cells revealed Lhcb inside the chloroplast near the envelope and in clusters deeper in the organelle. Antibody binding also detected Lhcb in granules within vacuoles in the cytosol, and Lhcb were recovered in granules purified from greening cells. Our results suggest that the cytosolic granules serve as receptacles of Lhcb synthesized in excess of the amount that can be accommodated by thylakoid membrane formation within the plastid envelope.

Effects of water stress on light-harvesting complex II (LHCII) and expression of a gene encoding LHCII in Zea mays

Summary Ten-day-old Zea mays L. Xinyu No. 4 seedlings were naturally stressed by stopping irrigation. Water content and water potential in attached leaves were reduced after treatments of 48 and 72 h, which represent moderate and severe water stress, respectively. At moderate water stress, the apoproteins of light-harvesting complex II (LHCII) decreased more rapidly than the content of chlorophyll. However, the contents of both chlorophyll and apoproteins decreased markedly in severe water-stressed maize leaves. Northern blotting showed that the loss of apoproteins of LHCII in water-stressed maize leaves is partially due to the decline of the steady state level of mRNA for LHCII, which indicated that the expression of LHCII under water stress is regulated at the transcript level. Circular dichroism measurements of LHCII in thylakoids isolated from water-stressed maize leaves revealed that the LHCII was dissociated and the conformation of LHCII in thylakoids was also altered by water stress.

Stability of the Apoproteins of Light-Harvesting Complex I and II during Biogenesis of Thylakoids in the Chlorophyll b-less Barley Mutant Chlorina f2

Transcription and translation of Lhc (cab) genes have been compared in the chlorina f2 mutant of barley (Hordeum vulgare) and its wild type to study the effect of chlorophyll b's absence on the regulation of assembly of the light-harvesting complexes (LHC). All tested genes were transcribed and the amount of their respective mRNAs increased rhythmically upon illumination of etiolated mutant plants. The synthesis of individual LHC apoproteins also had a rhythmic pattern when total leaf protein extracts were examined, whereas they increased gradually in the thylakoid. Only some LHC pigment-proteins present in wild-type thylakoids were found in mature mutant membranes. Thus, only the 25-kD (type 3) apoprotein of the three apoproteins of the major LHC IIb complex survived. The amount of the minor LHC II pigment-proteins was considerably reduced but not to zero. Photosystem I had some of the two LHC la apoproteins but had little of those of LHC lb. This was reflected in a shift of the 77-K emission maximum of whole leaves from 741 to 732 nm. It is concluded that the two largest LHC IIb and the LHC Ib apoproteins need chlorophyll b for stable integration into the membrane and that posttranslational regulation plays a major role in LHC assembly.

Formation of light-harvesting complexes of photosystem II in Scenedesmus

The patterns of accumulation of photosynthetic pigments, mRNAs transcribed from the genes encoding chlorophyll a/b-binding proteins (Lhc mRNAs), and light-harvesting complex (LHC) apoproteins have been compared in dark- and light-grown cells of wild-type (WT) Scenedesmus obliquus and the mutants WT-LHC1, C-6D, C-6E, C-2A′, C-2A′-LHC1 and C-2A′-LHC2. In contrast to the WT, which developed the complete photosynthetic apparatus in darkness, mutant strains exhibited either a lack of chlorophyll (Chl) b, carotenoids or LHC formation, or a light requirement for the synthesis of Chls or carotenoids. Accumulation of LHC apoproteins strictly depended on the presence of Chl a and carotenoids. The formation of a functional light-harvesting complex additionally required Chl b. Amounts of Lhc mRNAs of dark-grown mutant cells exceeded those of the WT. Thus, accumulation of Lhc mRNAs was independent of Chl and carotenoid synthesis and of chloroplast development. Transcripts of 1.4 and 1.6 kb accumulated constitutionally during growth in darkness. Illumination increased the amounts of these mRNAs and, additionally, a 1.8-kb Lhc mRNA appeared. Hence, light altered the expression of Lhc genes both quantitatively and qualitatively. Ratios of Chl a/Lhc mRNA, Chl a/LHC apoprotein and Lhc mRNA/LHC apoprotein were identical in the greening mutants C-6D, C-2A′, C-2A′-LHC1, and C-2A′-LHC2 after 8 h illumination. Light-independent accumulation of Lhc mRNAs, by contrast, was not correlated with the amounts of either Chl a or LHC apoproteins.

Formation of light-harvesting complexes of photosystem II in Scenedesmus

The patterns of accumulation of photosynthetic pigments, mRNAs transcribed from the genes encoding chlorophyll a/b-binding proteins (Lhc mRNAs), and light-harvesting complex (LHC) apoproteins have been compared in dark- and light-grown cells of wild-type (WT) Scenedesmus obliquus and the mutants WT-LHC 1 , C-6D, C-6E, C-2A', C-2A'-LHC 1 and C-2A'-LHC 2 . In contrast to the WT, which developed the complete photosynthetic apparatus in darkness, mutant strains exhibited either a lack of chlorophyll (Ch1) b, carotenoids or LHC formation, or a light requirement for the synthesis of Chls or carotenoids. Accumulation of LHC apoproteins strictly depended on the presence of Ch1 a and carotenoids. The formation of a functional light-harvesting complex additionally required Ch1 b

Two Sweetclover (Melilotus alba Desr.) Mutants Temperature Sensitive for Chlorophyll Expression.

The nonallelic sweetclover (Melilotus alba Desr.) mutants U371 (ch10/ch10 genotype) and U372 (ch11/ch11 genotype) are derived from the U389 (+/+ genotype) parental strain. Growth of the U389 strain at a temperature of 17 or 26[deg]C results in plants normally green in appearance. The U371 and U372 mutant plants grown at 26[deg]C are slightly to moderately chlorophyll (Chl) deficient and have decreased Chl b/a ratios. Growth of the mutants at 17[deg]C results in plants severely deficient in Chl a, with markedly reduced levels of carotenoids except for violaxanthin, and with negligible amounts of Chl b or apoproteins for the light-harvesting complex of photosystem II. If mutant plants grown at 17[deg]C are transferred to 26[deg]C, during the next 20 d the amount of Chl per fresh weight will increase 5-fold and both the Chl b/a ratio and the expression of the light-harvesting complex apoproteins will progressively increase. Studies of the U371 mutant during the temperature-induced greening demonstrate progressive changes in chloroplast ultra-structure and leaf carbon isotope fractionation that parallel the increases in Chl. Changes observed in the leaf carbon isotope fractionation in the mutant suggest that, in addition to the already known effects of various abiotic factors, structural and metabolic internal factors can also influence whether the limitation in CO2 fixation is at the level of diffusion or carboxylation. Such temperature-initiated progressive greening in these and similar mutants may make them useful tools to elucidate not only the biosynthesis and assembly of the photosynthetic apparatus, but also physiological phenomena such as the influence of light-driven energy production on the overall carbon isotope fractionation during photosynthesis.

The 20 kDa apoprotein of the CP24 complex of photosystem II: An alternative model to study import and intra‐organellar routing of nuclear‐encoded thylakoid proteins

SummaryThe 20 kDa polypeptide, the apoprotein of the chlorophyll a/b antenna complex CP24 associated with photosystem II, is a remote relative of light‐harvesting complex (LHC) apoproteins and thus a member of the extended cab gene family. LHC apoproteins are poly‐topic integral components of the thylakoid membrane with probably three transmembrane segments which originate in nuclear genes and are made in the cytosol as precursors. They possess exclusively stroma‐targeting transit peptides for import into the organelle and integrate into the thylakoid membrane via uncleaved hydrophobic domains of the mature protein. The CP24 apoprotein displays intriguing structural differences to LHC apoproteins with a potential impact on the routing and targeting processes during biogenesis. In particular, it lacks a pronounced second hydrophobic segment in the mature polypeptide chain found in LHCPs, and carries a transit peptide that is reminiscent of thylakoid‐targeting transit peptides. We have used in organello assays with isolated intact chloroplasts and the authentic precursor of the 20 kDa apoprotein from spinach, or appropriate chimaeric polypeptides consisting of a transit peptide and the mature part of various nuclear‐encoded thylakoid proteins of known location and targeting epitopes, in order to resolve the characteristics of its targeting properties, as well as to determine the contribution of the individual parts of the precursor molecule to its import and subsequent intra‐organellar routing. Our experiments demonstrate that the transit peptide of the CP24 apoprotein is required only for the import of the protein into the organelle. All subsequent steps, such as the integration of the protein into the thylakoid membrane, binding of chlorophyll, assembly into the CP24 complex and migration to the grana lamellae, still take place if the authentic transit peptide is replaced by a targeting signal of a nuclear‐encoded stromal protein.

Correlation between pigment composition and apoproteins of the light‐harvesting complex II (LHC II) in wheat (Triticum aestivum)

Seedlings of wheat (Triticum aestivum L. cv. Walde, Weibull) grown in continuous weak red light (16 mW m−2) with or without SAN‐9789, contained significantly lower amounts of chlorophylls and carotenoids compared to untreated plants grown in a greenhouse. The Chl alb ratios were 3.6 in the greenhouse‐grown plants, 5.1 in untreated and ca 16 in SAN‐treated plants grown in weak red light, respectively. The main difference in polypeptide composition of thylakoids isolated from red light‐grown plants, compared to those grown in the greenhouse, was a lower amount of proteins of the light‐harvesting complex (LHC) II. The amount of apo‐LHC and LHC were correlated to the xanthophyll to β‐carotene ratios in these plants. The absence of grana and the absence of proteins of the light‐harvesting complex 11 in SAN‐treated plants, support the general dogma that these proteins are involved in the formation of grana. Since the amount of apo‐LHC and LHC could be correlated to the presence of carotenoids as well as the chlorophylls, it is concluded that the carotenoids are necessary for the correct assembly and stabilization of the apoproteins of LHC II in the thylakoid membranes.

Changes in phosphorylation of thylakoid membrane proteins in light-harvesting complex mutants from Chlamydomonas reinhardtii

Phosphorylation of thylakoid membrane proteins was assessed in vivo in the BF3 and BF4 mutants from Chlamydomonas reinhardtii which are deficient in light-harvesting complex (LHC). Thylakoid membranes from the BF3 mutant, which was lacking in chlorophyll b, nevertheless contained significant amounts of LHC apoproteins. The phosphorylation pattern of thylakoid membrane proteins in the mutant was similar to that in the wild type. Incubation of BF3 cells in reducing conditions induced an increase in LHC phosphorylation and a fluorescence quenching at room temperature, thereby indicating that kinase-controlled state transitions still occurred in this mutant. In contrast to the BF3 mutant, the BF4 mutant still contained chlorophyll b but lacked most of the LHC apoproteins. It contained no LHC phosphoproteins and did not undergo state transitions as shown by the absence of fluorescence quenching in reducing conditions in vivo. Surprisingly, phosphorylation of the PS II subunits D2 and 6 (CP IV apoprotein) was totally abolished in this mutant. However, the two PS II subunits of low molecular weight, L5 and L6, were still slightly phosphorylated.

Light-dependent accumulation and localization of photosystem II proteins in maize.

We have raised antibodies against several major components of photosystem II. These antisera, which are directed against the apoproteins of two chlorophyll-binding proteins (CPa-1 and CPa-2), the apoprotein of light-harvesting complex II and the 33-kDa extrinsic protein of the oxygen-evolving complex, were used to examine the light regulation of photosystem II assembly in maize. The principal findings of this study are as follows. The 33-kDa protein is present in dark-grown maize and the content increases 5-10-fold upon illumination. The level of the protein is mediated at least in part by phytochrome and is independent of the accumulation of chlorophyll. In contrast, none of the three chlorophyll-binding proteins examined was detectable in leaves of maize grown in darkness or under other light regimes where chlorophyll does not accumulate. Even in the absence of photosystem II assembly, the 33-kDa protein is properly transported across the thylakoid into the lumen. However, the protein does not attach in the normal way to the inner surface of the membrane under these conditions.

Mutants of Sweetclover (Melilotus alba) Lacking Chlorophyll b

Mutants of sweetclover (Melilotus alba) with defects in the nuclear ch5 locus were examined. Using thin-layer chromatography and absorption spectroscopy, three of these mutants were found to lack chlorophyll (Chl) b. One of these three mutants, U374, possessed thylakoid membranes lacking the three Chl b-containing pigment-protein complexes (AB-1, AB-2, and AB-3) while still containing A-1 and A-2, Chl a complexes derived from photosystems I and II, respectively. Complete solubilization and denaturation of the thylakoid proteins from this mutant revealed very little apoprotein from the Chl b-containing light-harvesting complexes, the major thylakoid proteins in normal plants. The normal and mutant sweetclover plants had active thylakoid protein kinase activities and numerous polypeptides were labeled following incubation with [gamma-(32)P]ATP. With the U374 mutant, however, there was very little detectable label co-migrating with the light-harvesting complex apoproteins on polyacrylamide gels. The Chl b-deficient chlorina-f2 mutant of barley (Hordeum vulgare) also had an active protein kinase activity capable of phosphorylating numerous polypeptides, including ones migrating with the same mobility as the light-harvesting complex apoproteins. These results indicate that the sweetclover mutants may be useful systems for studies on the function and organization of Chl b in thylakoid membranes of higher plants.