Search

Several dinoflagellate species have plastids that more closely resemble those of an unrelated algal group, the haptophytes, suggesting these plastids have been obtained by tertiary endosymbiosis. Because both groups are photosynthetic, all of the genes for nuclear-encoded plastid proteins might be supplied by the dinoflagellate host or some of them might have been replaced by haptophyte genes. Sequences of the conserved nuclear psbO gene were obtained from the haptophyte Isochrysis galbana, the peridinin-containing dinoflagellate Heterocapsa triquetra, and the 19'hexanoyloxy-fucoxanthin-containing dinoflagellate Karenia brevis. Phylogenetic analysis of the oxygen-evolving-enhancer (PsbO) proteins confirmed that in K. brevis the original peridinin-type plastid was replaced by that of a haptophyte, an alga which had previously acquired a red algal chloroplast by secondary endosymbiosis. It showed clearly that during this tertiary symbiogenesis the original psbO gene in the dinoflagellate nucleus was replaced by a psbO gene from the haptophyte nucleus. The phylogenetic analysis also confirmed that the origin of the peridinin-type dinoflagellate plastid was indeed a red alga.

The light-harvesting complexes (LHCs) are a superfamily of chlorophyll-binding proteins present in all photosynthetic eukaryotes. The Lhc genes are nuclear-encoded, yet the pigment-protein complexes are localized to the thylakoid membrane and provide a marker to follow the evolutionary paths of plastids with different pigmentation. The LHCs are divided into the chlorophyll a/b-binding proteins of the green algae, euglenoids, and higher plants and the chlorophyll a/c-binding proteins of various algal taxa. This work examines the phylogenetic position of the LHCs from three additional taxa: the rhodophytes, the cryptophytes, and the chlorarachniophytes. Phylogenetic analysis of the LHC sequences provides strong statistical support for the clustering of the rhodophyte and cryptomonad LHC sequences within the chlorophyll a/c-binding protein lineage, which includes the fucoxanthin-chlorophyll proteins (FCP) of the heterokonts and the intrinsic peridinin-chlorophyll proteins (iPCP) of the dinoflagellates. These associations suggest that plastids from the heterokonts, haptophytes, cryptomonads, and the dinoflagellate, Amphidinium, evolved from a red algal-like ancestor. The Chlorarachnion LHC is part of the chlorophyll a/b-binding protein assemblage, consistent with pigmentation, providing further evidence that its plastid evolved from a green algal secondary endosymbiosis. The Chlorarachnion LHC sequences cluster with the green algal LHCs that are predominantly associated with photosystem II (LHCII). This suggests that the green algal endosymbiont that evolved into the Chlorarachnion plastid was acquired following the emergence of distinct LHCI and LHCII complexes.

The prochlorophytes are oxygenic prokaryotes differing from other cyanobacteria by the presence of a light-harvesting system containing both chlorophylls (Chls) a and b and by the absence of phycobilins. We demonstrate here that the Chl a/b binding proteins from all three known prochlorophyte genera are closely related to IsiA, a cyanobacterial Chl a-binding protein induced by iron starvation, and to CP43, a constitutively expressed Chl a antenna protein of photosystem II. The prochlorophyte Chl a/b protein (pcb) genes do not belong to the extended gene family encoding eukaryotic Chl a/b and Chl a/c light-harvesting proteins. Although higher plants and prochlorophytes share common pigment complements, their light-harvesting systems have evolved independently.

A fucoxanthin-chlorophyll protein (FCP) cDNA from the raphidophyte Heterosigma carterae encodes a 210-amino acid polypeptide that has similarity to other FCPs and to the chlorophyll a/b-binding proteins (CABs) of terrestrial plants and green algae. The putative transit sequence has characteristics that resemble a signal sequence. The Heterosigma fcp genes are part of a large multigene family which includes members encoding at least two significantly different polypeptides (Fcp1, Fcp2). Comparison of the FCP sequences to the recently determined three-dimensional structure of the pea LHC II complex indicates that many of the key amino acids thought to participate in the binding of chlorophyll and the formation of complex-stabilizing ionic interactions are well conserved. Phylogenetic analyses of sequences of light-harvesting proteins shows that the FCPs of several chromophyte phyla form a natural group separate from the intrinisic peridinin-chlorophyll proteins (iPCPs) of the dinoflagellates: Although the FCP and CAB genes shared a common ancestor, these lineages diverged from each other prior to the separation of the CAB LHC I and LHC II sequences in the green algae and terrestrial plants.

The 22-kDa chlorophyll a/b-binding protein (CAB) (psbS gene product) is associated with photosystem II and related to the CAB gene family. Here we report that the PSII-S protein unlike other chlorophyll-binding proteins is stable in the absence of pigments. It is present in etiolated spinach plants and accumulates in the dark progressively with the cellular development of the seedlings. Furthermore, it is present in several pigment-deficient mutants. Analysis of the pigment composition of the PSII-S protein isolated from etiolated plants suggests that neither carotenoids nor chlorophyll precursors are involved in its stabilization in the dark. Exposure of etiolated spinach to light leads to further accumulation of the PSII-S protein, which appears more early than other chlorophyll-binding proteins. Accumulation of the PSII-S protein in green plants is developmentally regulated and restricted to photosynthetic tissues. It is suggested that the function of the PSII-S protein may not be light-harvesting but it could act as a ligand chaperone required for transient binding of pigments during biogenesis or turnover of chlorophyll-binding proteins. Such function would be essential for coordination between pigment biosynthesis and ligation as well as avoiding toxic effects of non-bound chlorophyll molecules.

The intrinsic 22 kDa polypeptide associated with photosystem II (psbS protein) was found to be able to bind chlorophyll. Extraction of isolated photosystem II membranes with octyl-thioglucopyranoside, followed by repetitive electrophoresis under partially denaturing conditions gave only one green band. It contained both chlorophyll a and chlorophyll b, exhibited an absorption maximum at 674 nm and a 77 K fluorescence peak at 675 nm. The chlorophyll-protein band contained a single polypeptide of 22 kDa. Based on these results and on previous protein sequence comparisons, it is suggested that the psbS protein is a chlorophyll a/b binding polypeptide and should thus be denoted CP22.

The three closely related intrinsic membrane polypeptides of the photosystem II light-harvesting complex (LHC II) were successfully resolved on a PRP-1 poly(styrene-divinylbenzene) column using a three-stage linear water-acetonitrile gradient containing 0.1% trifluoroacetic acid. The hydrophobic proteins of photosystem I (PS I-200) and photosystem II core particles were also separated by this method, showing that membrane proteins of different sizes and hydrophobicities can be resolved in this system.

An intrinsic 22 kDa polypeptide is found associated with the oxygen-evolving photosystem II (PSII) core complex in all green plants and cyanobacteria so far examined, although it does not appear to be required for oxygen evolution. Amino acid sequence information obtained from the purified 22 kDa protein was used to construct a probe that was employed to isolate a full-length cDNA clone encoding the 274-residue precursor of the 22 kDa protein. Hydropathy plot analysis predicts the existence of four membrane-spanning helices in the mature protein. The two halves of the approximately 200-residue mature protein show high sequence similarity to each other, suggesting that the psbS gene arose from an internal gene duplication. The 22 kDa protein has some sequence similarity to chlorophyll a/b-binding proteins.

Using an improved SDS-PAGE system, the polypeptides of the major chlorophyll a/b light-harvesting complex of PSII (LHCII) from tomato leaves were resolved into five polypeptide bands. All the polypeptides were matched with the genes encoding them by comparing amino acid sequences of tryptic peptides with gene sequences. The two major LHCII bands (usually comigrating as a '27 kDa' polypeptide) were encoded by cab1 and cab3 (Type I LHCII) genes. A third strong band of about 25 kDa was encoded by cab4 (Type II) genes. Polypeptides from two minor bands of 23-24 kDa were not N-terminally blocked; their N-terminal sequences showed they were Type III LHCII proteins. One complete cDNA clone and several incomplete clones for Type III polypeptides were sequenced. Combined with the peptide sequences, the results indicate that there are at least four different Type III genes in tomato, encoding four almost identical polypeptides. Thus, all the LHCII CAB polypeptides have been identified, and each type of LHCII polypeptide is encoded by distinct gene or genes in tomato.

CP29, the core chlorophyll a/b (CAB) antenna complex of Photosystem II (PSII), has two nuclear-encoded polypeptides of approximately 26 and 28 kDa in tomato (Lycopersicon esculentum). Cab9, the gene for the Type I (26 kDa) CP29 polypeptide was cloned by immunoscreening a tomato leaf cDNA library. Its identity was confirmed by sequencing tryptic peptides from the mature protein. Cab9 is a single-copy gene with five introns, the highest number found in a CAB protein. In vitro transcription-translation gave a 31 kDa precursor which was cleaved to about 26 kDa after import into isolated tomato chloroplasts. The Cab9 polypeptide has the two highly conserved regions common to all CAB polypeptides, which define the members of this extended gene family. Outside of the conserved regions, it is only slightly more closely related to other PSII CABs than to PSI CABs. Sequence analysis of tryptic peptides from the Type II (28 kDa) CP29 polypeptide showed that it is also a member of the CAB family and is very similar or identical to the CP29 polypeptide previously isolated from spinach. All members of the CAB family have absolutely conserved His, Gln and Asn residues which could ligate the Mg atoms of the chlorophylls, and a number of conserved Asp. Glu, Lys and Arg residues which could form H-bonds to the polar groups on the porphyrin rings. The two conserved regions comprise the first and third predicted trans-membrane helices and the stroma-exposed segments preceding them.

A large proportion of the chlorophyll in a plant is engaged in harvesting light energy and transferring it to the photochemical reaction centres. These 'antenna' chlorophylls are non-covalently bound to specific proteins to form chlorophyll-protein complexes. The chlorophyll a/b-binding (CAB) polypeptides are encoded by an extended family of nuclear genes. It has recently been discovered that other proteins not known to bind chlorophyll, the early light-inducible proteins (ELIPs), are also related and could be considered part of this family. We suggest that the latter proteins may be involved in pigment biosynthesis or in assembly of the thylakoid membrane.

Tryptic peptide sequences from the 22 kDa polypeptide of tomato LHCI were used to construct a probe for gene cloning. The two genes cloned, cab11 and cab12, encode proteins of 251 and 250 residues that are 88% identical in overall amino acid sequence and 93% identical in the deduced mature protein. Each gene is present in a single copy per haploid genome; cab11 on chromosome 3 and cab12 on chromosome 6, and each has 2 introns located in similar positions to introns in other members of the Chl a/b-binding (CAB) protein gene family. Comparison of the amino acid sequences of LHCI, LHCII, CP29 and CP24 polypeptides confirms that all CABs share two regions of very high similarity which include the first and third transmembrane helices and the stroma-exposed sequences preceding them. However, near the N-terminus and between the conserved regions, the LHCI polypeptides have sequence motifs which appear to be PSI-specific.