Spectral and functional comparisons between the carotenoids of the two antenna complexes of Rhodopseudomonas capsulata.

The spectral and functional properties of carotenoids associated with each of the two light-harvesting complexes of the Rhodopseudomonas capsulata photosynthetic antenna system have been distinguished by studying mutants lacking one or the other complex. In mutants containing only the light-harvesting I complex (LH-I), the absorption spectrum of the carotenoids is blue-shifted compared to wild type. Carotenoid absorption in mutants possessing only the light-harvesting II complex (LH-II) complex is red-shifted. The circular dichroism spectrum of carotenoids in each complex is also distinctive. Although carotenoids in each complex function with approximately the same efficiency in harvesting and transmitting light energy for photosynthesis, only the carotenoids associated with LH-II undergo an electrochromic bandshift upon generation of a transmembrane potential. These observations are interpreted to indicate that both the orientation of carotenoid molecules with respect to the plane of the membrane, and the immediate electrochemical environment of these molecules differ in the two light-harvesting complexes.

Transcription of the Rhodobacter capsulatus nifHDK operon is modulated by the nitrogen source. Construction of plasmid expression vectors based on the nifHDK promoter.

We characterized the Rhodobacter capsulatus nifHDK promoter by nucleotide sequencing and nuclease S1 analysis of mRNA-protected DNA probes. Comparison of this promoter to nifP and ntrP promoters from other species reveals extensive homology to the canonical nifP consensus sequence. Using lac fusions we have demonstrated that transcription of the nifHDK operon is totally repressed when the growth medium is supplemented with ammonia, becomes fully derepressed in ammonia-free medium, and proceeds at intermediate levels when other nitrogen sources are used. Based on this information, we constructed plasmid expression vectors in which the rates of transcription from cloned DNA fragments are determined by the nitrogen source used in the growth medium.

A DNA fragment hybridizing to a nif probe in Rhodobacter capsulatus is homologous to a 16S rRNA gene.

We have sequenced the Rhodobacter capsulatus nifH and nifD genes. The nifH gene, which codes for the dinitrogenase reductase protein, is 894 bp long and codes for a polypeptide of predicted Mr 32,412. The nifD gene, which codes for the alpha subunit of dinitrogenase, is 1,500 bp long and codes for a protein of predicted Mr 56,113. A 776-bp BglII-XhoI fragment containing only nif sequences was used as a hybridization probe against R. capsulatus genomic DNA. Two HindIII fragments, 11.8 kb and 4.7 kb in length, hybridize to this probe. Both fragments have been cloned from a cosmid library. The 11.8-kb fragment contains the nifH, D and K genes, as previously demonstrated (Scolnik and Haselkorn, 1984). In this paper we present evidence that suggests that the 4.7-kb HindIII fragment contains a gene coding for 16S rRNA, and that although homology between nif and this fragment can be observed in filter hybridization experiments, a second copy of the nif structural genes seems not to be present in this region.

Characterization of two members (ACS1 and ACS3) of the 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thaliana.

The nucleotide sequences of two highly homologous 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14)-encoding genes, ACS1 and ACS3, of Arabidopsis thaliana (At) have been determined. The sequence analysis shows that ACS3 is a pseudogene representing a truncated version of ACS1. The missing region of ACS3 corresponding to the fourth exon of ACS1 has been shown by Southern analysis to be absent in the At genome. The chromosomal locations of the five members of the At ACS multigene family have been determined. The results show that each family member resides on a different chromosome. This observation suggests that the ACS3 pseudogene originated by a partial inter-chromosomal gene duplication. The ACS1 polypeptide contains all the conserved and characteristic domains found in the ACC synthase isoenzymes from various plant species, but is unable to express ACS activity in Escherichia coli and yeast. The predicted amino-acid sequence of ACS1 is missing the highly conserved tripeptide, Thr-Asn-Pro (TNP), between Ile204 and Ser205. Introduction of TNP into ACS1 restores the ACS activity, whereas its removal from the enzymatically active ACS2 results in a loss of activity. The results suggest that TNP is crucial for expression of ACS activity in E. coli.

Transcription of Two Photosynthesis-Associated Nuclear Gene Families Correlates with the Presence of Chloroplasts in Leaves of the Variegated Tomato ghost Mutant.

Leaves of the tomato ghost mutant show a variegated green/white phenotype due to a somatically unstable genetic block in carotenoid biosynthesis. Colored carotenoids are not synthesized in white leaves; consequently, chlorophyll is destroyed by photooxidation and the plastids formed show little development of internal membrane structures. Carotenoid biosynthesis proceeds to wild type levels in green tissue, thus chlorophyll accumulates and chloroplasts develop normally. The presence of green sectors allows for the production through tissue culture of variegated green/white plants, in which growth is supported by the photosynthetic green tissue. Thus, ghost is the first plant carotenoid mutant that can be grown to maturity. We determined the steady state mRNA levels for two nuclear gene families that code for chloroplast proteins: rbcS, which codes for the small subunit of ribulose-1-5-bisphosphate carboxylase; and cab, which codes for chlorophyll a/b binding protein. In ghost plants grown in light, the steady state mRNA levels for both gene families were low in white leaves but were similar to wild type in green leaves. Light regulation of the transcripts studied was observed in both ghost green and white leaves. Transcription experiments conducted on nuclei isolated from green and white leaves indicate that the low levels of cytoplasmic mRNAs observed in the absence of colored carotenoids and/or light are due to reduced rates of transcription. We conclude that maximum transcription of rbcS and cab genes in leaves of mature tomato plants requires both light and mature chloroplasts.

cDNA cloning, expression during development, and genome mapping of PSY2, a second tomato gene encoding phytoene synthase.

The enzyme phytoene synthase (Psy) catalyzes the formation of phytoene, an intermediate in the carotenoid biosynthesis pathway. Expression of a previously described gene (PSY1) is induced by fruit ripening in tomato (Lycopersicon esculentum). We describe here the cloning of a partial cDNA for PSY2, a gene related to PSY1. A plasmid containing the PSY2 coding region under control of a bacterial promoter complements a Rhodobacter capsulatus phytoene synthase mutant, indicating that this gene has the capacity to encode an active enzyme. We used reverse transcriptase-polymerase chain reaction followed by digestion with a restriction enzyme to determine that both PSY1 and PSY2 are expressed during tomato development. PSY1 transcripts predominate in seedlings and in late stages of fruit ripening, whereas PSY2 transcripts are relatively more abundant in mature leaves. Both genes are expressed under photooxidative conditions induced by treatment with the carotenoid biosynthesis inhibitor Norflurazon. We used polymerase chain reaction-based restriction fragment length polymorphisms and alien addition lines to map PSY2 to chromosome 2. We conclude that PSY2 is a second tomato gene encoding phytoene synthase.

Carotenoid biosynthesis in photosynthetic bacteria. Genetic characterization of the Rhodobacter capsulatus CrtI protein.

Carotenoids are photoprotective pigments present in many photosynthetic and nonphotosynthetic organisms. The desaturation of phytoene into phytofluene is an early step in the biosynthetic pathway that in the photosynthetic bacterium Rhodobacter capsulatus is mediated by the product of the crtI gene. Here we report the sequence of this gene and the identification of CrtI as a membrane protein of approximate Mr 60,000. Mutant strains with 5-fold lower or 10-fold higher levels of CrtI with respect to wild type have only small differences in their carotenoid content, indicating that the cellular concentration of CrtI is not a limiting factor in carotenoid biosynthesis. However, a correlation was found between the levels of CrtI and the formation of a photosynthetic antenna system.

Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata.

Biological nitrogen fixation requires the nitrogenase enzyme complex, ATP, and a strong reductant. Klebsiella pneumoniae contains 15 linked nitrogen fixation (nif) genes, three of which, nifH, nifD and nifK have been sufficiently conserved in evolution that cloned K. pneumoniae nifHDK DNA will hybridize to DNA sequences from every nitrogen-fixing bacterium examined to date, including the purple, non-sulphur bacterium Rhodopseudomonas capsulata, in which one complete nifHDK operon has been mapped. Using cloned K. pneumoniae nifHDK DNA we report here that R. capsulata contains multiple copies of the genes for nitrogenase components. Two regions containing sequences homologous to all three nif structural genes have been identified, and mutations in one region produced a Nif- phenotype. Nif+ pseudorevertants were derived from these mutants, some of which retained the original mutation suggesting that some of the extra nif gene sequences can be functionally activated.

Regulation of carotenoid biosynthesis during tomato development.

Phytoene synthase (Psy) and phytoene desaturase (Pds) are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. We report here the organ-specific and temporal expression of PDS and PSY in tomato plants. Light increases the carotenoid content of seedlings but has little effect on PDS and PSY expression. Expression of both genes is induced in seedlings of the phytoene-accumulating mutant ghost and in wild-type seedlings treated with the Pds inhibitor norflurazon. Roots, which contain the lowest levels of carotenoids in the plant, have also the lowest levels of PDS and PSY expression. In flowers, expression of both genes and carotenoid content are higher in petals and anthers than in sepals and carpels. During flower development, expression of both PDS and PSY increases more than 10-fold immediately before anthesis. During fruit development, PSY expression increases more than 20-fold, but PDS expression increases less than threefold. We concluded that PSY and PDS are differentially regulated by stress and developmental mechanisms that control carotenoid biosynthesis in leaves, flowers, and fruits. We also report that PDS maps to chromosome 3, and thus it does not correspond to the GHOST locus, which maps to chromosome 11.

Abscisic Acid Control of rbcS and cab Transcription in Tomato Leaves.

Leaves of tomato (Lycopersicon esculentum) plants grown in soil in which moisture was lowered from field capacity to levels approaching permanent wilting point show a 10-fold increase in abscisic acid (ABA) and a 60 to 70 percent decrease in rbcS and cab steady-state mRNA levels. As indicated by transcription run-on experiments, the effect occurs primarily at the transcriptional level. Similar water deficit had only a minor effect on ABA level and on rbcS and cab expression in leaves of sitiens, an ABA mutant of tomato. Expression of rbcL, the chloroplast gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, is not affected by water stress. Application of exogenous ABA results in decreased rbcS and cab expression in both wild-type and sitiens leaves. Analysis of the expression of individual members of the rbcS gene family indicates that under water-deficit conditions, expression derives primarily from only three of the five rbcS genes. Effects of dark adaptation and water deficit are additive for cab but not for rbcS expression. These results support the hypothesis that, at least under water-deficit conditions, ABA or a derivative thereof mediates a negative regulation of rbcS and cab transcription in tomato plants.

Genetic and physical map of the structural genes (nifH,D,K) coding for the nitrogenase complex of Rhodopseudomonas capsulata.

Functional genes coding for the structural components of the nitrogenase complex (nifH,D,K) have been cloned on an 11.8-kilobase-pair HindIII fragment of DNA from the photosynthetic bacterium Rhodopseudomonas capsulata. The genes were physically mapped by hybridization of individual cloned nif genes from Klebsiella pneumoniae and Anabaena sp. strain 7120 to Southern blots of HindIII digests of the cloned R. capsulata fragment, after introduction of HindIII sites into the latter at specified locations by insertion of Tn5. Plasmids with the 11.8-kilobase-pair HindIII fragment containing the Tn5 insertions were also used for complementation tests with chromosomal Nif- mutations and for the generation of subfragments to locate those mutations by marker rescue. The R. capsulata nifH,D,K genes comprise a single unit of expression, with the same organization and polarity as found in K. pneumoniae. However, the R. capsulata nifH,D,K fragment did not complement Nif- point mutations in the corresponding Klebsiella genes, and the Klebsiella nif genes did not function in R. capsulata.

The wild-type gene for glutamine synthetase restores ammonia control of nitrogen fixation to Gln- (glnA) mutants of Rhodopseudomonas capsulata.

The wild-type glnA gene, coding for glutamine synthetase, was cloned from the photosynthetic bacterium Rhodopseudomonas capsulata by using a cosmid library to complement the Gln- phenotype of an Escherichia coli glnA deletion strain. The original cosmid plasmid contained 37 kilobase pairs (kbp) of R. capsulata DNA, of which only 2 kbp was necessary for Gln complementation in E. coli. A plasmid containing this 2-kbp insert was mobilized into G29, a Gln- mutant of R. capsulata which is also unable to repress nitrogenase in ammonia-containing media (Nifc phenotype). The 2-kbp fragment restored glutamine-independent growth and ammonia repression of nitrogenase, indicating that in R. capsulata, production of the signal for nitrogen repression of nif depends on the activity of the glnA gene. Repression of nitrogenase was shown, by hybridization of RNA to cloned nif DNA, to occur at the level of transcription in the wild-type and the complemented G29 strains.

Biosynthesis of carotenoids derived from neurosporene in Rhodopseudomonas capsulata.

We have characterized the carotenoids accumulated by a series of mutants of Rhodopseudomonas capsulata as part of a study of the synthesis, structure, and function of the photosynthetic membranes of this bacterium. The carotenoids in this study were identified by visible and mass spectroscopy, chromatography, derivatization, and chemical analyses. We have located a new genetic region, crtF, necessary for the O-methylation of the carotenoids. Mutants with a lesion in crtF accumulate demethylspheroidene as their major carotenoid during anaerobic growth and demethylspheroidenone when grown in the presence of oxygen, a heretofore undescribed phenotype. The genetic region necessary for O-methylation maps adjacent to the known cluster of genes affecting carotenoid biosynthesis. In addition, we have identified methoxyneurosporene as the carotenoid that preferentially binds to the reaction centers of strain Ga, a green mutant of R. sphaeroides which accumulates three neurosporene-like carotenoids. A metabolic grid for carotenoid biosynthesis is proposed, based upon the intermediates accumulated in these mutants.

Two Arabidopsis thaliana carotene desaturases, phytoene desaturase and zeta-carotene desaturase, expressed in Escherichia coli, catalyze a poly-cis pathway to yield pro-lycopene.

We have expressed in Escerichia coli the enzymes geranylgeranyl diphosphate synthase and phytoene synthase, from the soil bacterium Erwinia stewartii, and the two carotene desaturases phytoene desaturase and carotene zeta-carotene desaturase from Arabidopsis thaliana. We show that pro-lycopene (7,9,7',9'-tetra-cis)-lycopene is the main end product of the plant desaturation pathway in these cells. In addition, light is required in this system. Whereas in the dark mainly zeta-carotene, the phytoene desaturase product, accumulates, illumination leads to activation of this intermediate caused by its photoisomerization. zeta-Carotene then meets the stereospecific requirements of zeta-carotene desaturase and pro-lycopene is formed. In contrast, a strain of E. coli carrying geranylgeranyl diphosphate synthase, phytoene desaturase and the bacterial carotene desaturase CrtI, which mediates lycopene formation from phytoene, does not require light, nor is a poly-cis-lycopene species formed. The stereoselectivity of the plant-type desaturation pathway expressed in E. coli is the same as previously shown with chromoplast membranes. As the phytoene desaturase and zeta-carotene desaturase used originate from a system not capable of developing chromoplasts, this indicates that the poly-cis pathway of carotene desaturation may have a wider occurrence than initially believed.

The gene crtI mediates the conversion of phytoene into colored carotenoids in Rhodopseudomonas capsulata.

Carotenoids are membrane pigments present in all photosynthetic organisms, providing essential photoprotective functions. The first carotenoid formed in the pathway is phytoene, a colorless compound which is then converted into colored carotenoids by a series of dehydrogenation reactions. In the photosynthetic bacterium Rhodopseudomonas capsulata mutations that affect carotenoid biosynthesis before colored carotenoids are formed have a "blue-green" phenotype as opposed to the "red" of wild type cells. We have extracted carotenoids from several blue-green mutants and found that two strains (BPY69 and BPY102) accumulate phytoene and no colored carotenoids. These mutants failed to dehydrogenate phytoene in an in vitro assay. However, dehydrogenation of this compound can be achieved in vitro by adding a cell-free extract from another blue-green mutant blocked earlier in the pathway. Genetic complementation and deletion mapping indicate that the gene crtI is responsible for the conversion of phytoene into colored carotenoids in these mutants.

A light-entrained circadian clock controls transcription of several plant genes.

Diurnal oscillations in steady-state mRNA levels and transcription rates were measured for seven transcripts (five of which encode chloroplast-localized proteins) in tomato seedlings: photosystem I and photosystem II chlorophyll a/b binding proteins (CAB/I and CAB/II), small subunit of RuBisCO (RBCS), actin, subunit II of the photosystem I reaction center (PSAD), subunit I of the photosystem II oxygen-evolving enzyme (OEE1), and a biotin-binding protein of unknown function. CAB/II mRNA levels were found to oscillate greater than 20-fold, showing a peak at noon, while only marginal diurnal oscillations are seen in RBCS transcripts. The oscillations are at least partially controlled at the transcriptional level. Transcription rates of both CAB/II and RBCS, measured by nuclear run-on experiments, were found to oscillate, with a peak around 8 a.m. Transcription rates of the 'biotin' clone also oscillated, with a peak around noon. Transfer of plants to constant darkness or constant light conditions alters the amplitude of the transcriptional oscillation, but does not abolish it, suggesting that it is at least partially controlled by a circadian clock. The oscillations are still visible after three days in complete darkness, and have a period very close to 24 h. The oscillator phase can be reset by out-of-phase light treatment.

Genetic mapping of mutations using phenotypic pools and mapped RAPD markers.

Genetic markers facilitate the study of inheritance and the cloning of genes by genetic approaches. Molecular markers detect differences in DNA sequence, and are thus less ambiguous than phenotypic markers, which require gene expression. We have demonstrated a molecular approach to the mapping of mutant genes using RAPD markers and pooling of individuals based on phenotype. To map genes by phenotypic pooling a strain carrying a mutation is crossed to a strain that is homozygous for the wild-type allele of the corresponding gene. A set of primers corresponding to mapped RAPDs distributed throughout the genome and in coupling phase with respect to the wild type parent is then used to amplify DNA from wild type and mutant pools of F2 individuals. Linkage between the mutant gene and the RAPD markers is visualized by the absence of the corresponding RAPD DNA bands in the mutant pool. We developed a mathematical model for calculating the probability of linkage between RAPDs and target genes and we successfully tested this approach with the model plant Arabidopsis thaliana.

A tomato gene expressed during fruit ripening encodes an enzyme of the carotenoid biosynthesis pathway.

In the initial stages of carotenoid biosynthesis in plants the enzyme phytoene synthase converts two molecules of geranylgeranyl diphosphate into phytoene, the first carotenoid of the pathway. We show here that a tomato (Lycopersicon esculentum) cDNA for a gene (Psy1) expressed during fruit ripening directs the in vitro synthesis of a 47-kDa protein which, upon import into isolated chloroplasts, is processed to a mature 42-kDa form. The imported protein is largely associated with membranes, but it can be easily solubilized by dilution or by treatment at high pH. A plasmid construct containing prokaryotic promoter and ribosome-binding sequences fused to the Psy1 cDNA complements the carotenoidless phenotype of a Rhodobacter capsulatus crtB mutant. We conclude that Psy1 encodes phytoene synthase and that this enzyme is a peripheral plastid membrane protein.

Carotenoid desaturases from Rhodobacter capsulatus and Neurospora crassa are structurally and functionally conserved and contain domains homologous to flavoprotein disulfide oxidoreductases.

The characteristic red color of some photosynthetic bacteria and the orange color of Neurospora conidia is due to the presence of carotenoids, photoprotective pigments synthesized by plants, algae, bacteria, and fungi. Generally, carotenoids are tetraterpenes in which absorption of visible light and photoprotection are mediated by a chain of conjugated double bonds, the chromophore, which is formed by successive desaturations of phytoene, a colorless precursor. The genes al-1 and crtI mediate the desaturation of phytoene in Neurospora crassa and Rhodobacter capsulatus, respectively. Here, we report that alignment of the primary sequence of Al-1, CrtI, and CrtD, another carotenoid desaturase, reveals conservation with amino acid residues that mediate FAD-binding and dimerization functions in Azotobacter vinelandii dihydrolipoamide dehydrogenase and human glutathione reductase, two disulfide oxidoreductases. Plasmids containing the coding region of an al-1 cDNA fused to appropriate bacterial transcriptional and translational signals complement crtI mutants. Our results indicate that both structure and function of carotenoid desaturases have been conserved during evolution and suggest that these enzymes are evolutionarily related to disulfide oxidoreductases.