One ring or two? Determination of ring number in carotenoids by lycopene epsilon-cyclases.

Carotenoids in the photosynthetic membranes of plants typically contain two beta-rings (e.g., beta-carotene and zeaxanthin) or one epsilon- and one beta-ring (e.g., lutein). Carotenoids with two epsilon-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene epsilon-cyclase (LCYe) adds one epsilon-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene beta-cyclase (LCYb) adds two beta-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two epsilon-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic epsilon-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis epsilon-cyclases involved in the determination of ring number were mapped by analysis of chimeric epsilon-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one epsilon-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two epsilon-rings. An R residue in this position also yields a bi-epsilon-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.

Chlorophyll and carotenoid binding in a simple red algal light-harvesting complex crosses phylogenetic lines.

The membrane proteins of peripheral light-harvesting complexes (LHCs) bind chlorophylls and carotenoids and transfer energy to the reaction centers for photosynthesis. LHCs of chlorophytes, chromophytes, dinophytes, and rhodophytes are similar in that they have three transmembrane regions and several highly conserved Chl-binding residues. All LHCs bind Chl a, but in specific taxa certain characteristic pigments accompany Chl a: Chl b and lutein in chlorophytes, Chl c and fucoxanthin in chromophytes, Chl c and peridinin in dinophytes, and zeaxanthin in rhodophytes. The specificity of pigment binding was examined by in vitro reconstitution of various pigments with a simple light-harvesting protein (LHCaR1), from a red alga (Porphyridium cruentum), that normally has eight Chl a and four zeaxanthin molecules. The pigments typical of a chlorophyte (Spinacea oleracea), a chromophyte (Thallasiosira fluviatilis), and a dinophyte (Prorocentrum micans) were found to functionally bind to this protein as evidenced by their participation in energy transfer to Chl a, the terminal pigment. This is a demonstration of a functional relatedness of rhodophyte and higher plant LHCs. The results suggest that eight Chl-binding sites per polypeptide are an ancestral trait, and that the flexibility to bind various Chl and carotenoid pigments may have been retained throughout the evolution of LHCs.

Identification, expression, and substrate specificity of a mammalian beta-carotene 15,15'-dioxygenase.

We have identified from mouse the first mammalian beta-carotene 15,15'-dioxygenase (beta-CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived precursors. beta-CD is related to the retinal pigment epithelium-expressed protein RPE65 and belongs to a diverse family that includes the plant 9-cis-epoxycarotenoid dioxygenase and bacterial lignostilbene dioxygenases. beta-CD expression in Escherichia coli cells engineered to produce beta-carotene led to the accumulation of all-trans-retinal at the expense of beta-carotene, confirming that beta-CD catalyzed the central cleavage of this vitamin A precursor. Purified recombinant beta-CD protein cleaves beta-carotene in vitro with a V(max) of 36 pmol of retinal/mg of enzyme/min and a K(m) of 6 microm. Non-provitamin A carotenoids were also cleaved, although with much lower activity. By Northern analysis, a 2.4-kilobase (kb) message was observed in liver, kidney, small intestine, and testis, tissues important in retinoid/carotenoid metabolism. This message encoded a 63-kDa cytosolic protein expressed in these tissues. A shorter transcript of 1.8 kb was found in testis and skin. Developmentally, the 2.4-kb mRNA was abundant at embryonic day 7, with lower expression at embryonic days 11, 13, and 15, suggesting a critical role for this enzyme in gastrulation. Identification of beta-CD in an accessible model organism will create new opportunities to study vitamin A metabolism.

Evidence of a role for LytB in the nonmevalonate pathway of isoprenoid biosynthesis.

It is proposed that the lytB gene encodes an enzyme of the deoxyxylulose-5-phosphate (DOXP) pathway that catalyzes a step at or subsequent to the point at which the pathway branches to form isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A mutant of the cyanobacterium Synechocystis strain PCC 6803 with an insertion in the promoter region of lytB grew slowly and produced greenish-yellow, easily bleached colonies. Insertions in the coding region of lytB were lethal. Supplementation of the culture medium with the alcohol analogues of IPP and DMAPP (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol) completely alleviated the growth impairment of the mutant. The Synechocystis lytB gene and a lytB cDNA from the flowering plant Adonis aestivalis were each found to significantly enhance accumulation of carotenoids in Escherichia coli engineered to produce these colored isoprenoid compounds. When combined with a cDNA encoding deoxyxylulose-5-phosphate synthase (dxs), the initial enzyme of the DOXP pathway, the individual salutary effects of lytB and dxs were multiplied. In contrast, the combination of lytB and a cDNA encoding IPP isomerase (ipi) was no more effective in enhancing carotenoid accumulation than ipi alone, indicating that the ratio of IPP and DMAPP produced via the DOXP pathway is influenced by LytB.

Isopentenyl diphosphate isomerase deficiency in Synechocystis sp. strain PCC6803.

Isopentenyl diphosphate isomerase (IPP isomerase) in many organisms and in plastids is central to isoprenoid synthesis and involves the conversion between IPP and dimethylallyl diphosphate (DMAPP). It is shown that Synechocystis PCC6803 is deficient in IPP isomerase activity, consistent with the absence in its genome of an obvious homologue for the enzyme. Incorporation of [1-(14)C]IPP in cell extracts, primarily into C(20), occurs only upon priming with DMAPP in Synechocystis PCC6803 and in Synechococcus PCC7942. Isoprenoid synthesis in these cyanobacteria does not appear to involve interconversion of IPP and DMAPP, raising the possibility that they are not within the plastid evolutionary lineage.

Identification of multi-gene families encoding isopentenyl diphosphate isomerase in plants by heterologous complementation in Escherichia coli.

Two cDNAs encoding isopentenyl diphosphate isomerase (IPI) in Adonis aestivalis, Arabidopsis thaliana, and Lactuca sativa, and single examples from Oryza sativa and Tagetes erecta were identified. An analysis of these and other ipi leads us to suggest a separate origin for green algal and plant genes and propose that a single gene encodes plastid and cytosolic IPI in plants.

Characterization of the Porphyridium cruentum Chl a-binding LHC by in vitro reconstitution: LHCaR1 binds 8 Chl a molecules and proportionately more carotenoids than CAB proteins.

The Porphyridium cruentum light harvesting complex (LHC) binds Chl a, zeaxanthin and beta-carotene and comprises at least 6 polypeptides of a multigene family. We describe the first in vitro reconstitution of a red algal light-harvesting protein (LHCaR1) with Chl a/carotenoid extracts from P. cruentum. The reconstituted pigment complex (rLHCaR1) is spectrally similar to the native LHC I, with an absorption maximum at 670 nm, a 77 K fluorescence emission peak at 677 nm (ex. 440 nm), and similar circular dichroism spectra. Molar ratios of 4.0 zeaxanthin, 0.3 beta-carotene and 8.2 Chl a per polypeptide for rLHCaR1 are similar to those of the native LHC I complex (3.1 zeaxanthin, 0.5 beta-carotene, 8.5 Chl a). The binding of 8 Chl a molecules per apoprotein is consistent with 8 putative Chl-binding sites in the predicted transmembrane helices of LHCaR1. Two of the putative Chl a binding sites (helix 2) in LHCaR1 were assigned to Chl b in Chl a/b-binding (CAB) LHC II [Kühlbrandt et al. (1994) Nature 367: 614-21]. This suggests either that discrimination for binding of Chl a or Chl b is not very specific at these sites or that specificity of binding sites evolved separately in CAB proteins. LHCaR1 can be reconstituted with varying ratios of carotenoids, consistent with our previous observation that the carotenoid to Chl ratio is substantially higher in P. cruentum grown under high irradiance. Also notable is that zeaxanthin does not act as an accessory light-harvesting pigment, even though it is highly likely that it occupies the position assigned to lutein in the CAB LHCs.

A phylogenetic assessment of the eukaryotic light-harvesting antenna proteins, with implications for plastid evolution.

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.

Methodological pluralism: a framework for psychotherapy research.

A new level of sophistication is advocated for psychotherapy research--methodological pluralism. This pluralism recognizes the number of unexamined and untested assumptions that are embedded within any method, including traditional scientific methods. These assumptions restrict the questions that can be asked and the variables that can be tested before any investigation of psychotherapy has occurred. Such premature closure is ultimately unscientific and hinders truly creative investigations of psychotherapy. Consequently, a framework is described that allows multiple methods--with multiple underlying philosophies--to complement one another in psychotherapy research. Five essential benefits of this pluralistic framework are shown: advancement of science, increased objectivity, a level playing field, greater scientific freedom, and an end to the burgeoning "method wars" of psychotherapy research.

Differential expression of two isopentenyl pyrophosphate isomerases and enhanced carotenoid accumulation in a unicellular chlorophyte.

The enzyme isopentenyl pyrophosphate (IPP) isomerase catalyzes the reversible isomerization of IPP to produce dimethylallyl pyrophosphate, the initial substrate leading to the biosynthesis of carotenoids and many other long-chain isoprenoids. Expression of IPP isomerase, and of two enzymes specific to the carotenoid pathway (lycopene beta-cyclase and beta-carotene-C-4-oxygenase), was followed in the green unicellular alga Haematococcus pluvialis after exposure to high illumination. This alga uniquely accumulates carotenoids in the cytoplasm and in late developmental stages turns deep-red in color because of accumulation of ketocarotenoids in the cytosol. The carotenoid/chlorophyll ratio increased 3-fold in wild type and 6-fold in a precocious carotenoid-accumulating mutant (Car-3) within 24 h after increasing the illumination from 20 to 150 micromol photon m-2.s-1. Two cDNAs encoding IPP isomerase in Haematococcus, ipiHp1 and ipiHp2, were identified. Although otherwise highly similar (95% identity overall), the predicted sequence of ipiHp1 contained a 12-aa region not found in that of ipiHp2. This was reflected by a size difference between two polypeptides of 34 and 32.5 kDa, both of which reacted with an antibody to the product of ipiHp1. We suggest that the 32.5-kDa form is involved with the carotenoid accumulation in the cytoplasm, since the 32.5-kDa polypeptide was preferentially up-regulated by high light preceding the carotenoid increase and only this form was detected in red cysts.

GENES AND ENZYMES OF CAROTENOID BIOSYNTHESIS IN PLANTS.

Carotenoids are integral and essential components of the photosynthetic membranes in all plants. Within the past few years, genes encoding nearly all of the enzymes required for the biosynthesis of these indispensable pigments have been identified. This review focuses on recent findings as to the structure and function of these genes and the enzymes they encode. Three topics of current interest are also discussed: the source of isopentenyl pyrophosphate for carotenoid biosynthesis, the progress and possibilities of metabolic engineering of plants to alter carotenoid content and composition, and the compartmentation and association of the carotenogenic enzymes. A speculative schematic model of carotenogenic enzyme complexes is presented to help frame and provoke insightful questions leading to future experimentation.

LhcaR1 of the red alga Porphyridium cruentum encodes a polypeptide of the LHCI complex with seven potential chlorophyll a-binding residues that are conserved in most LHCs.

The accessory light-harvesting polypeptides associated with photosystem I (LHCI) in Porphyridium cruentum bind chlorophyll a, zeaxanthin and beta-carotene. A cDNA library of P. cruentum was screened with an antiserum specific to the LHCI polypeptides, and an 0.9 kb fragment was identified as coding for an LHCI polypeptide. This cDNA, which we named LhcaR1, has an open reading frame encoding 222 amino acid residues including a putative transit peptide of 28 amino acids. Hydropathy analysis suggests that there are three transmembrane helices in the mature polypeptide. Each of the amino acid residues that bind chlorophyll (six residues) and serve in stabilizing the helices in higher-plant LHCs are conserved in helices 1 and 3 of P. cruentum LhcaR1. The N-terminal flanking regions of these two helices also show high sequence conservation with other LHCs. Helix 2 contains a seventh putative chlorophyll-binding site, but resembles helix 2 of higher-plant LHCs to a lesser degree. A sequence motif of 11 residues found near the N-terminus and in each of the three helices suggests the possibility that the red algal LhcaR1 derives from a gene duplication. Polypeptides of the expected molecular weight in six other red algae (Achrochaetium, Bangia, Callithamnion, Cyanidium, Polysiphonia, Spermothamnion) were recognized by the antiserum to P. cruentum LHCI, indicating a wide distribution of LHCI in rhodophytes.

Cloning and functional analysis of the beta-carotene hydroxylase of Arabidopsis thaliana.

An Arabidopsis thaliana cDNA encoding the enzyme beta-carotene hydroxylase was identified by functional complementation in Escherichia coli. The product of this cDNA adds hydroxyl groups to both beta rings of the symmetrical beta-carotene (beta,beta-carotene) to form zeaxanthin (beta,beta-carotene-3,3'-diol) and converts the monocyclic beta-zeacarotene (7',8'-dihydro-beta,psi-carotene) to hydroxy-beta-zeacarotene (7',8'-dihydro-beta,psi-carotene-3-ol). The epsilon rings of delta-carotene (epsilon,psi-carotene) and alpha-zeacarotene (7',8'-dihydro-epsilon,psi-carotene) are poor substrates for the enzyme. The predicted amino acid sequence of the A. thaliana enzyme resembles the four known bacterial beta-carotene hydroxylase enzymes (31-37% identity) but is much longer, with an N-terminal extension of more than 130 amino acids. Truncation of the cDNA to produce a polypeptide lacking the first 69 amino acids does not impair enzyme activity in E. coli. Truncation to yield a polypeptide of a length comparable with the bacterial enzymes (lacking 129 N-terminal amino acids) resulted in the accumulation of the monohydroxy intermediate beta-cryptoxanthin (beta,beta-carotene-3-ol), predominantly, when beta-carotene was provided as the substrate. It is suggested that amino acid residues 70-129 of the A. thaliana enzyme may play a role in formation of a functional homodimer.

Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation.

Carotenoids with cyclic end groups are essential components of the photosynthetic membranes in all plants, algae, and cyanobacteria. These lipid-soluble compounds protect against photooxidation, harvest light for photosynthesis, and dissipate excess light energy absorbed by the antenna pigments. The cyclization of lycopene (psi, psi-carotene) is a key branch point in the pathway of carotenoid biosynthesis. Two types of cyclic end groups are found in higher plant carotenoids: the beta and epsilon rings. Carotenoids with two beta rings are ubiquitous, and those with one beta and one epsilon ring are common; however, carotenoids with two epsilon rings are rare. We have identified and sequenced cDNAs that encode the enzymes catalyzing the formation of these two rings in Arabidopsis. These beta and epsilon cyclases are encoded by related, single-copy genes, and both enzymes use the linear, symmetrical lycopene as a substrate. However, the epsilon cyclase adds only one ring, forming the monocyclic delta-carotene (epsilon, psi-carotene), whereas the beta cyclase introduces a ring at both ends of lycopene to form the bicyclic beta-carotene (beta, beta-carotene). When combined, the beta and epsilon cyclases convert lycopene to alpha-carotene (beta, epsilon-carotene), a carotenoid with one beta and one epsilon ring. The inability of the epsilon cyclase to catalyze the introduction of a second epsilon ring reveals the mechanism by which production and proportions of beta,beta- and beta, epsilon-carotenoids may be controlled and adjusted in plants and algae, while avoiding the formation of the inappropriate epsilon,epsilon-carotenoids.

Pigment protein complexes and the concept of the photosynthetic unit: Chlorophyll complexes and phycobilisomes.

The photosynthetic unit includes the reaction centers (RC 1 and RC 2) and the light-harvesting complexes which contribute to evolution of one O2 molecule. The light-harvesting complexes, that greatly expand the absorptance capacity of the reactions, have evolved along three principal lines. First, in green plants distinct chlorophyll (Chl) a/b-binding intrinsic membrane complexes are associated with RC 1 and RC 2. The Chl a/b-binding complexes may add about 200 additional chromophores to RC 2. Second, cyanobacteria and red algae have a significant type of antenna (with RC 2) in the form of phycobilisomes. A phycobilisome, depending on the size and phycobiliprotein composition adds from 700 to 2300 light-absorbing chromophores. Red algae also have a sizable Chl a-binding complex associated with RC 1, contributing an additional 70 chromophores. Third, in chromophytes a variety of carotenoid-Chl-complexes are found. Some are found associated with RC 1 where they may greatly enhance the absorptance capacity. Association of complexes with RC 2 has been more difficult to ascertain, but is also expected in chromophytes. The apoprotein framework of the complexes provides specific chromophore attachment sites, which assures a directional energy transfer whithin complexes and between complexes and reaction centers. The major Chl-binding antenna proteins generally have a size of 16-28 kDa, whether of chlorophytes, chromophytes, or rhodophytes. High sequence homology observed in two of three transmembrane regions, and in putative chlorophyll-binding residues, suggests that the complexes are related and probably did not evolve from widely divergent polyphyletic lines.

Decrease of polypeptides in the PS I antenna complex with increasing growth irradiance in the red alga Porphyridium cruentum.

Thylakoids isolated from cells of the red alga Porphyridium cruentum exhibit an increased PS I activity on a chlorophyll basis with increasing growth irradiance, even though the stoichiometry of Photosystems I and II in such cells shows little change (Cunningham et al. (1989) Plant Physiol 91: 1179-1187). PS I activity was 26% greater in thylakoids of cells acclimated at 280 µmol photons · m(-2) · s(-1) (VHL) than in cells acclimated at 10 µmol photons · m(-2) · s(-1) (LL), indicating a change in the light absorbance capacity of PS I. Upon isolating PS I holocomplexes from VHL cells it was found that they contained 132±9 Chl/P700 while those obtained from LL cells had 165±4 Chl/P700. Examination of the polypeptide composition of PS I holocomplexes on SDS-PAGE showed a notable decrease of three polypeptides (19.5, 21.0 and 22 kDa) in VHL-complexes relative to LL-complexes. These polypeptides belong to a novel LHC I complex, recently discovered in red algae (Wolfe et al. (1994a) Nature 367: 566-568), that lacks Chl b and includes at least six different polypeptides. We suggest that the decrease in PS I Chl antenna size observed with increasing irradiance is attributable to changes occurring in the LHC I-antenna complex. Evidence for a Chl-binding antenna complex associated with PS II core complexes is lacking at this point. LHC II-type polypeptides were not observed in functionally active PS II preparations (Wolfe et al. (1994b) Biochimica Biophysica Acta 1188: 357-366), nor did we detect polypeptides that showed immunocross-reactivity with LHC II specific antisera (made to Chlamydomonas and Euglena LHC II).

Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.

Pigment orientation changes accompanying the light state transition in Synechococcus sp. PCC 6301.

Low temperature (77 K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. PCC 6301 and those accompanying chromatic acclimation in Porphyridium cruentum in samples stabilized by glutaraldehyde fixation. In light state 2 compared to light state 1 intact cells of Synechococcus showed an increased alignment of allophycocyanin parallel to the cells' long axis whereas the phycobilisomethylakoid membrane fragments exhibited an increased allophycocyanin alignment parallel to the membrane plane. The phycobilisome-thylakoid membrane fragments showed less alignment of a short wave-length chlorophyll a (Chl a) Qy transition dipole parallel to the membrane plane in state 2 relative to state 1.To aid identification of the observed Chl a orientation changes in Synechococcus, linear dichroism spectra were obtained from phycobilisome-thylakoid membrane fragments isolated from red light-grown (increased number of PS II centres) and green light-grown (increased number of PS I centres) cells of the red alga Porphyridium cruentum. An increased contribution of short wavelength Chl a Qy transition dipoles parallel to the long axis of the membrane plane was directly correlated with increased levels of PS II centres in red light-grown P. cruentum.Our results indicate that the transition to state 2 in cyanobacteria is accompanied by an increase in the orientation of allophycocyanin and a decrease in the orientation of Chl a associated with PS II with respect to the thylakoid membrane plane.

Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of beta-carotene.

Carotenoids with cyclic end groups are essential components of the photosynthetic membrane in all known oxygenic photosynthetic organisms. These yellow pigments serve the vital role of protecting against potentially lethal photo-oxidative damage. Many of the enzymes and genes of the carotenoid biosynthetic pathway in cyanobacteria, algae and plants remain to be isolated or identified. We have cloned a cyanobacterial gene encoding lycopene cyclase, an enzyme that converts the acyclic carotenoid lycopene to the bicyclic molecule beta-carotene. The gene was identified through the use of an experimental herbicide, 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA), that prevents the cyclization of lycopene in plants and cyanobacteria. Chemically-induced mutants of the cyanobacterium Synechococcus sp. PCC7942 were selected for resistance to MPTA, and a mutation responsible for this resistance was mapped to a genomic DNA region of 200 bp by genetic complementation of the resistance in wild-type cells. A 1.5 kb genomic DNA fragment containing this MPTA-resistance mutation was expressed in a lycopene-accumulating strain of Escherichia coli. The conversion of lycopene to beta-carotene in these cells demonstrated that this fragment encodes the enzyme lycopene cyclase. The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are required to produce beta-carotene from lycopene, and prove that this enzyme is a target site of the herbicide MPTA. The cloned cyanobacterial lcy gene hybridized well with genomic DNA from eukaryotic algae, thus it will enable the identification and cloning of homologous genes for lycopene cyclase in algae and plants.

Photosynthetic membrane topography: quantitative in situ localization of photosystems I and II.

An immunolabeling approach was developed for quantitative in situ labeling of photosystems I and II (PSI and PSII). Photosynthetic membranes from the phycobilisome-containing red alga Porphyridium cruentum were isolated from cells in which different photosystem compositions were predetermined by growing cells in green light (GL) or red light (RL). Based on phycobilisome densities per membrane area of 390 per m2 (GL) and 450 per m2 (RL) and the PSI reaction center (P700) and PSII reaction center (QA) content, the photosystem densities per m2 of membrane were calculated to be 2520 PSI in GL and 1580 in RL and 630 PSII in GL and 1890 in RL. PSI was detected in the membranes with 10-nm Au particles conjugated to affinity-purified anti-PSI, and PSII was detected with 15-nm Au particles conjugated to anti-PSII. Distribution of Au particles appeared relatively uniform, and the degree of labeling was consistent with the calculated photosystem densities. However, the absolute numbers of Au-labeled sites were lower than would be obtained if all reaction center monomers were labeled. Specific labeling of PSI was 25% in GL and RL membranes, and PSII labeling was 33% in GL but only 17% in RL membranes. An IgG-Au particle is larger than a monomer of either photosystem and could shield several closely packed photosystems. We suggest that clustering of photosystems exists and that the cluster size of PSI is the same in GL and RL cells, but the PSII cluster size is 2 times greater in RL than in GL cells. Such variations may reflect changes in functional domains whereby increased clustering can maximize the cooperativity between the photosystems, resulting in enhancement of the quantum yield.