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.

Analysis of the pigment stoichiometry of pigment-protein complexes from barley (Hordeum vulgare). The xanthophyll cycle intermediates occur mainly in the light-harvesting complexes of photosystem I and photosystem II.

The carotenoid zeaxanthin has been implicated in a nonradiative dissipation of excess excitation energy. To determine its site of action, we have examined the location of zeaxanthin within the thylakoid membrane components. Five pigment-protein complexes were isolated with little loss of pigments: photosystem I (PSI); core complex (CC) I, the core of PSI; CC II, the core of photosystem II (PSII); light-harvesting complex (LHC) IIb, a trimer of the major light-harvesting protein of PSII; and LHC IIa, c, and d, a complex of the monomeric minor light-harvesting proteins of PSII. Zeaxanthin was found predominantly in the LHC complexes. Lesser amounts were present in the CCs possibly because these contained some extraneous LHC polypeptides. The LHC IIb trimer and the monomeric LHC II a, c, and d pigment-proteins from dark-adapted plants each contained, in addition to lutein and neoxanthin, one violaxanthin molecule but little antheraxanthin and no zeaxanthin. Following illumination, each complex had a reduced violaxanthin content, but now more antheraxanthin and zeaxanthin were present. PSI had little or no neoxanthin. The pigment content of LHC I was deduced by subtracting the pigment content of CC I from that of PSI. Our best estimate for the carotenoid content of a LHC IIb trimer from dark-adapted plants is one violaxanthin, two neoxanthins, six luteins, and 0.03 mol of antheraxanthin per mol trimer. The xanthophyll cycle occurs mainly or exclusively within the light-harvesting antennae of both photosystems.

Thirty years of fun with antenna pigment-proteins and photochemical reaction centers: A tribute to the people who have influenced my career.

The author summarizes the research contributions to photosynthesis made by him, his graduate and postdoctoral students, visiting scientists and by his collaboration with other photosynthesis workers during 1964-1994. The development of isolation procedures and biochemical/biophysical characterization of antenna pigment-proteins and photochemical reaction centers are described together with the author's education and experiences as a scientific researcher. Some anecdotes hopefully add insight into what it was like to be in this area of science during the period.

Assembly of the Light-Harvesting Complexes (LHCs) of Photosystem II (Monomeric LHC IIb Complexes Are Intermediates in the Formation of Oligomeric LHC IIb Complexes).

The light-induced assembly of light-harvesting complex (LHC) II has been followed during the biogenesis of the plastid. Seedlings grown in intermittent light (IML) accumulate only small amounts of chlorophyll b. The minor LHC II apoproteins are present; however, the apoprotein levels of the major LHC II complex, LHC IIb, are severely depressed after exposure to IML. The levels of all LHC II apoproteins increase rapidly upon exposure to continuous illumination. The 25-kD, type 3 LHC IIb subunit appears to be more abundant during the early hours of greening in relation to its level in mature thylakoids. The LHC IIb apoproteins are initially associated with pigments to form monomeric pigment-protein complexes. The abundance of monomeric LHC IIb complexes gradually decreases during exposure to continuous light and a concomitant increase occurs in the amount of the trimeric and higher-order oligomeric forms. Pulse-chase experiments verify that labeled LHC IIb monomeric complexes are intermediates in the formation of trimeric and higher-order oligomeric LHC IIb-pigmented complexes. Therefore, the assembly of LHC II occurs via the initial pigmentation of the apoproteins to form monomeric complexes and proceeds in a sequential manner.

Organization of the Light-Harvesting Complex of Photosystem I and Its Assembly during Plastid Development.

Photosystem I (PSI) holocomplexes were fractionated to study the organization of the light-harvesting complex I (LHC I) pigment-proteins in barley (Hordeum vulgare) plastids. LHC Ia and LHC Ib can be isolated as oligomeric, presumably trimeric, pigment-protein complexes. The LHC Ia oligomeric complex contains both the 24- and the 21.5-kD apoproteins encoded by the Lhca3 and Lhca2 genes and is slightly larger than the oligomeric LHC Ib complex containing the Lhca1 and Lhca4 gene products of 21 and 20 kD. The synthesis and assembly of LHC I during light-driven development of intermittent light-grown plants occurs rapidly upon exposure to continuous illumination. Complete PSI complexes are detected by nondenaturing Deriphat (disodium N-dodecyl-[beta]-iminodipropionate-160)-PAGE after 2 h of illumination, and their appearance correlates with that of the 730- to 740-nm emission characteristic of assembled LHC I. However, the majority of the newly synthesized LHC I apoproteins are present as monomeric complexes in the thylakoids during the early hours of greening. We propose that during development of the protochloroplast the LHC I apoproteins are first assembled into monomeric pigmented complexes that then aggregate into trimers before becoming attached to the pre-existing core complex to form a complete PSI holocomplex.

Biochemical and spectroscopic characterization of the reaction center-LH1 complex and the carotenoid-containing B820 subunit of Chromatium purpuratum.

Two complexes, the reaction center light-harvesting complex 1 (RC-LH1) and the B820 subunit of the LH1, have been isolated and characterized from the purple-sulfur photosynthetic bacterium Chromatium purpuratum. The RC-LH1 consists of the B870 antenna and a P-870 RC with an associated tetraheme cytochrome. This complex can be further fractionated to yield the B820 subunit of the LH1. The C. purpuratum B820 subunit is the first isolated from a purple-sulfur bacterium. It is also the first that retains its carotenoid absorption properties. CD spectra in the Qy region of bacteriochlorophyll a in both the RC-LH1 and the B820 subunit are bathochromically shifted as compared to other such complexes. Comparison of the sequence of the LH1 beta polypeptide to other LH1 beta s reveals the presence of additional aromatic amino acids in the vicinity of both of the conserved histidines in the C. purpuratum beta polypeptide. The CD spectra of these C. purpuratum pigment-protein complexes can be interpreted in terms of exciton interaction between bacteriochlorophylls in the B820 subunit of the LH1 and in the B870, with additional spectral characteristics arising from interactions of the pigments with their protein environment.

Purification and characterization of the peripheral antenna of the purple-sulfur bacterium Chromatium purpuratum: evidence of an unusual pigment-protein composition.

The purification and characterization of the peripheral antenna and the preliminary characterization of a carotenoid-protein complex from the purple-sulfur bacterium Chromatium purpuratum are described. The peripheral antenna of C. purpuratum is unusual among purple bacteria in that it can be resolved by SDS-PAGE into six subunits, the largest number observed thus far for a spectrally pure antenna complex. N-terminal sequence analyses of these subunits suggest that they may have an additional bacteriochlorophyll-binding site located outside the transmembrane domain. The results of pigment-protein quantification are also consistent with additional pigment-binding sites in the C. purpuratum LH2. Furthermore, CD measurements and sequence analysis suggest the presence of considerable beta-type in addition to alpha-helical secondary structure. Thus, the secondary and quaternary structures of this complex differ significantly from light-harvesting complexes of other purple photosynthetic bacteria. A carotenoid-protein complex is also described; it is an apparent association of three proteins and carotenoid and is closely associated with the peripheral antenna. The purple-sulfur bacteria are evolutionarily older than the relatively better characterized purple-nonsulfur organisms. The phenotypic features described here of the C. purpuratum photosynthetic apparatus are related to those of other purple bacteria and green-sulfur bacteria and may reflect the evolutionary position of this organism.

Identification of a novel light-harvesting complex II protein (LHC IIc').

The caroteno-chlorophyll-protein, LHC IIc, is a relatively minor component of the PS II antenna. Isolated LHC IIc contains a major protein of 28 kDa along with a 26 kDa subunit in lower abundance. Previously, it was not known if the 26 kDa protein was closely related to the 28 kDa LHC IIc protein or if it was a comigrating LHC IIb contaminating subunit. A sequence of 20 amino acid residues was obtained by direct protein micro-sequencing of an internal cyanogen bromide-derived peptide fragment of the 26 kDa protein isolated from barley. The sequence shows, and antibody reactions confirm, that the 26 kDa protein is similar but distinct from both the 28 kDa LHC IIc and LHC IIb protein sequences, indicating that there remains at least one more cab gene to be identified in higher plants. Furthermore, it is difficult to interpret the data in any way other than that there is a novel LHC II pigment-protein (LHC IIc') that co-migrates with LHC IIc.

Light-induced biogenesis of light-harvesting complex I (LHC I) during chloroplast development in barley (hordeum vulgare). Studies using cDNA clones of the 21- and 20-kilodalton LHC I apoproteins.

The light-harvesting complex (LHC) Ib pigment-proteins form the major component of the LHC I complex in higher plants. They comprise chlorophylls a and b, xanthophylls, and at least two polypeptide subunits of 21 and 20 kD in barley (Hordeum vulgare). We have identified two cDNA clones, LHC Ib-21 and LHC Ib-20, encoding the 21- and 20-kD LHC Ib apoproteins, respectively. N-terminal protein sequences of the purified LHC Ib polypeptides were used for the unequivocal correlation of these clones to their respective apoproteins. The cDNA clones encode two proteins that have strong sequence similarity to other LHC I and LHC II pigment-binding polypeptides of photosystems I and II. The 21-kD polypeptide contains 201 amino acid residues (22.14 kD), and the 20-kD polypeptide contains 200 amino acid residues (22.18 kD). The biogenesis of the LHC Ib apoproteins and the pigmented LHC I during the light-induced development of the chloroplast was studied. Accumulation of the two LHC Ib mRNAs is induced by light, and their amount is regulated by phytochrome. LHC Ib polypeptide accumulation in the thylakoid membrane temporally lags behind transcript accumulation. The rates of accumulation of LHC Ib transcripts and of their apoproteins lag behind those of the major LHC II component, LHC IIb. Complete assembly of the LHC Ib pigment-protein, as observed by low-temperature fluorescence spectroscopy, requires exposure of dark-grown seedlings to 72 h or more of light.

Identification and Analysis of a Barley cDNA Clone Encoding the 31-Kilodalton LHC IIa (CP29) Apoprotein of the Light-Harvesting Antenna Complex of Photosystem II.

The light-harvesting complex (LHC) of photosystem II is composed of several different pigment-binding apoproteins. We have identified a cDNA clone LHCIIa-1 encoding the 31-kilodalton LHC IIa (CP29, Chl a/b-P1) apoprotein of barley (Hordeum vulgare). Direct protein microsequencing of an internal peptide fragment from the LHC IIa apoprotein has been used to identify unequivocally the cDNA clone as that coding for the LHC IIa apoprotein. Microsequencing of the 28-kilodalton LHC IIc protein (CP26) showed only minor sequence similarity to the LHC IIa protein, indicating that they are two different gene products. LHCIIa-1 codes for a protein of 286 amino acid residues (molecular weight, 31,308), which displays strong similarities to other pigment-binding LHC proteins, and yet contains an additional 42 amino acid residue segment. Two regions of strong intramolecular sequence similarity are also observed.

Correlation of apoproteins with the genes of the major chlorophyll a/b binding protein of photosystem II in Arabidopsis thaliana. Confirmation for the presence of a third member of the LHC IIb gene family.

The major light-harvesting complex in higher plants is LHC IIb. The LHC IIb of Arabidopsis thaliana contains 2 pigment-binding apoproteins of 28 and 25 kDa. To determine the relationship between them and the LHC IIb gene family members, each protein was purified to homogeneity, subjected to direct protein sequencing, and the sequences compared with those deduced from LHC IIb genes in this organism. The 28 kDa protein is the product of Type I LHC IIb genes. The 25 kDa LHC IIb component is distinctly different from the 28 kDa LHC IIb protein, and is more closely related to the type III LHC IIb gene product of barley. Type III gene products lack the first 9-11 residues found in proteins of the Type I and II genes, a region that contains a phosphorylatable threonine residue. The lack of the N-terminal residues explains why this LHC IIb apoprotein has never been seen to be phosphorylated, and partly or wholly why it is smaller. The implication of the missing N-terminus on uptake of LHC II precursor proteins into the plastid and of the relative organization of the LHC IIb subunits in the PS II antenna is discussed.

Biochemical composition and organization of higher plant photosystem II light-harvesting pigment-proteins.

The light-harvesting complex (LHC) of barley photosystem II (PS II) was fractionated by Deriphat-polyacrylamide gel electrophoresis into five different pigmented components: one subcomplex (LHC IIb) and four pigment-proteins (LHC IIa, -c, -d, and -e). No loss of chorophyll from the components occurred during fractionation, and violaxanthin is the only photosynthetic pigment that apparently occurs in thylakoids free of association with protein. Each LHC II component has a distinct stoichiometry of neoxanthin, violaxanthin, lutein, chlorophyll a, and chlorophyll b. LHC IIa, -d, and -e were obtained as monomeric pigment-proteins, each of which contained one apoprotein Mr 31,000, 21,000, and 13,000, respectively. LHC IIc was also isolated as a monomer but it contained two poly-peptides of Mr 29,000 and 26,500, whereas the trimeric LHC IIb subcomplex (Mr 72,000) contained three subunits of Mr 28,000, 27,000, and 25,000, but not in equal stoichiometry. How the LHC II subunits are organized in PS II was examined. We isolated PS II subcomplexes which contained four of the LHC II subunits and the core complex (CC II) in unit stoichiometry; the relative strengths of association of the LHC II subunits with CC II are: LHC IIa greater than LHC IIc greater than LHC IIb greater than LHC IId. The LHC II subunits were associated with the native dimeric and not with the derived monomeric form of CC II. In addition, a multimeric LHC II sub-complex composed of the LHC IIa, LHC IIb, and LHC IId pigment-proteins was isolated. We propose that this LHC II subcomplex, which contained the Mr 28,000 and 25,000 subunits but lacked the Mr 27,000 subunit of LHC IIb and CC II. An LHC IIb pigmented fraction of LHC IIb and CC II. An LHC IIb pigmented fraction of Mr 250,000 was isolated which contained only the Mr 28,000 and 27,000 subunits. The LHC IIb subunits in this complex were the most highly phosphorylated on a protein basis. These data together with analyses of the chlorophyll b-less barley chlorina f2 mutant were used to construct a model for the LHC II pigment-protein arrangement in higher plant PS II.

The use of non-denaturing Deriphat-polyacrylamide gel electrophoresis to fractionate pigment-protein complexes of purple bacteria.

The suitability of Deriphat-polyacrylamide gel electrophoresis as a method for separating purple bacterial pigment-protein complexes has been tested. When appropriate non-denaturing detergents are used to solubilize chromatophores, this method provides a rapid, easy and microscale procedure for analyzing the composition of the bacterial photosynthetic apparatus with minimal disruption of individual pigment-proteins. Its usefulness is further illustrated by employing it to test for suitable detergents with which to solubilize purple bacterial chromatophores, and as an assay to study variation in the composition of the photosynthetic unit of bacterial cultures grown under different conditions.

Amino-terminal sequence of the 21 kDa apoprotein of a minor light-harvesting pigment-protein complex of the Photosystem II antenna (LHC IId/CP24).

The 21 kDa apoprotein of LHC IId, a minor light-harvesting antenna component of Photosystem II, has been isolated and subjected to N-terminal protein sequencing. A sequence of 66 residues was obtained which contains regions of considerable homology to both those reported for LHC II and LHC I, but which is obviously distinct from them. The proposed occurrence of an identical 21 kDa LHC subunit in both photosystems I and II is shown to be incorrect.

Correlation of some published amino acid sequences for photosystem I polypeptides to a 17 kDa LHCI pigment-protein and to subunits III and IV of the core complex.

Photosystem I (PSI) in barley consists of at least 11 polypeptides of which three have apparent sizes of 15-19 kDa. Two of these polypeptides (subunits III and IV) are constituents of the core complex (CCI), the third is a component of the light-harvesting complex (LHCI). After fractionation of PSI into its CCI and LHCI components, each of the polypeptides has been isolated and its N-terminal region sequenced. We conclude that the gene sequence published for subunit IV of spinach [(1988) FEBS Lett. 237, 108-112] is not that of subunit IV but rather that of the 17 kDA LHCIc pigment protein. We confirm that the published sequence for subunit III [(1988) Curr. Genet. 14, 511-518] is indeed that of subunit III; seemingly conflicting identifications, based on apparent sizes on SDS-PAGE, of which polypeptides are subunits III and IV are probably explained by subunit III's electrophoretic migration rate being dependent on the solvent.

Post-transcriptional control of plastid mRNA accumulation during adaptation of chloroplasts to different light quality environments.

The adaptation of germinating spinach seedlings to yellow and red light was studied and compared with plants grown in white light. Spinach chloroplasts isolated from cotyledons and leaves of yellow and white light-grown plants showed similar membrane structures and compositions, while chloroplasts from plants grown in red light have significant adaptive changes. Based on an equal amount of chlorophyll, these changes include a reduction in the number of photosystem I complexes, an increase of photosystem II antenna size, and an increased ratio of stacked to unstacked membranes in red light-adapted chloroplasts. The decrease in the number of photosystem I complexes per unit of chlorophyll in these chloroplasts was qualitatively correlated with an approximately 10-fold decrease in the level of the psaA mRNA encoding the photosystem I 65-kilodalton to 70-kilodalton chlorophyll apoprotein, as well as with a differential decrease in mRNA levels of other photosynthetic proteins. Light quality adaptations do not significantly affect the plastid to nuclear DNA ratio or the overall chloroplast transcription activity. The relative transcriptional activities of 10 plastid genes, as determined by run-on transcription assays, are similar in chloroplasts from cotyledons and leaves of plants grown under the three light qualities. Only the psaA gene shows a 30% to 40% decrease in transcription activity in chloroplasts of plants adapted to red light. This decrease in psaA transcription activity, however, cannot fully account for the decrease of its mRNA level. We conclude, therefore, that post-transcriptional mechanisms are primarily responsible for the control of differential chloroplast mRNA accumulation in light quality adaptations.