Intragenic Enhancers and Suppressors of Phytoene Desaturase Mutations in Chlamydomonas reinhardtii

Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. A light-sensitive, phytoene-accumulating mutant, pds1-1, was isolated in Chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase (PDS) gene. PDS catalyzes the second step in carotenoid biosynthesis-the conversion of phytoene to ζ-carotene. Decreased accumulation of downstream colored carotenoids suggested that the pds1-1 mutant is leaky for PDS activity. A screen for enhancers of the pds1-1 mutation yielded the pds1-2 allele, which completely lacks PDS activity. A second independent null mutant (pds1-3) was identified using DNA insertional mutagenesis. Both null mutants accumulate only phytoene and no other carotenoids. All three phytoene-accumulating mutants exhibited slower growth rates and reduced plating efficiency compared to wild-type cells and white phytoene synthase mutants. Insight into amino acid residues important for PDS activity was obtained through the characterization of intragenic suppressors of pds1-2. The suppressor mutants fell into three classes: revertants of the pds1-1 point mutation, mutations that changed PDS amino acid residue Pro64 to Phe, and mutations that converted PDS residue Lys90 to Met. Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 90 and 143 are in close contact in the active PDS enzyme and have important roles in its structural stability and/or activity.     

Identification of the carotenoid isomerase provides insight into carotenoid biosynthesis, prolamellar body formation, and photomorphogenesis

Carotenoids are essential photoprotective and antioxidant pigments synthesized by all photosynthetic organisms. Most carotenoid biosynthetic enzymes were thought to have evolved independently in bacteria and plants. For example, in bacteria, a single enzyme (CrtI) catalyzes the four desaturations leading from the colorless compound phytoene to the red compound lycopene, whereas plants require two desaturases (phytoene and zeta-carotene desaturases) that are unrelated to the bacterial enzyme. We have demonstrated that carotenoid desaturation in plants requires a third distinct enzyme activity, the carotenoid isomerase (CRTISO), which, unlike phytoene and zeta-carotene desaturases, apparently arose from a progenitor bacterial desaturase. The Arabidopsis CRTISO locus was identified by the partial inhibition of lutein synthesis in light-grown tissue and the accumulation of poly-cis-carotene precursors in dark-grown tissue of crtISO mutants. After positional cloning, enzymatic analysis of CRTISO expressed in Escherichia coli confirmed that the enzyme catalyzes the isomerization of poly-cis-carotenoids to all-trans-carotenoids. Etioplasts of dark-grown crtISO mutants accumulate acyclic poly-cis-carotenoids in place of cyclic all-trans-xanthophylls and also lack prolamellar bodies (PLBs), the lattice of tubular membranes that defines an etioplast. This demonstrates a requirement for carotenoid biosynthesis to form the PLB. The absence of PLBs in crtISO mutants demonstrates a function for this unique structure and carotenoids in facilitating chloroplast development during the first critical days of seedling germination and photomorphogenesis.     

Comparison of carotenoid content,gene expression and enzyme levels in tomato (Lycopersicon esculentum) leaves

Physiological conditions which lead to changes in total carotenoid content in tomato plantlets were identified. Carotenoid levels were found to increase after the onset of a dark period during a normal 24 h cycle. This rapid initial increase is followed by a steady decrease in carotenoid content throughout the night. A decrease in the expression of several carotenogenic genes, namely pds, zds (carotenoid desaturases) and ptox (plastid terminal oxidase), was observed following the removal of the light (when carotenoid content is at its highest). An increase in gene expression was observed before the return to light for pds and zds (when carotenoid levels were at their lowest), or following the return to light for ptox. The phytoene desaturation inhibitor norflurazon leads to a decrease coloured carotenoid content and, in the light, this correlated with pds and zds gene induction. In the dark, norflurazon treatment led to only a weak decrease in carotenoid content and only a small increase in pds and zds gene expression. The striking absence of phytoene accumulation under norflurazon treatment in the dark suggests a down-regulation of carotenoid formation in darkness However, prolonged dark conditions, or treatment with photosynthetic inhibitors, surprisingly led to higher carotenoid levels, which correlated with decreased expression of most examined genes. In addition to light, which acts in a complex way on carotenoid accumulation and gene expression, our results are best explained by a regulatory effect of carotenoid levels on the expression of several biosynthetic genes. In addition, monitoring of protein amounts for phytoene desaturase and plastid terminal oxidase (which sometimes do not correlate with gene expression) indicate an even more complex regulatory pattern.     

Bansformation of tobacco with a mutated cyanobacterial phytoene desaturase gene confers resistance to bleaching herbicides

Carotenoids are constituents of the photosynthetic apparatus and essential for plant survival because of their involvement in protection of chlorophylls against photooxidation. Certain classes of herbicides are interfering with carotenoid biosynthesis leading to pigment destruction and a bleached plant phenotype. One important target site for bleaching herbicides is the enzyme phytoene desaturase catalysing the desaturation of phytoene in zeta-carotene. This enzymatic reaction can be inhibited by norflurazon or fluridone. We have transformed tobacco with a mutated cyanobacterial phytoene desaturase gene (pds) derived from the Synechococcus PCC 7942 mutant NFZ4. Characterization of the resulting transformants revealed an up to 58 fold higher norflurazon resistance in comparison to wild type controls. The tolerance for fluridone was also increased 3 fold in the transgenics. Furthermore, the transformed tobacco maintained a higher level of D1 protein of photosystem II indicating a lower susceptibility to photooxidative damage in the presence of norflurazon. In contrast, the genetic manipulation did not confer herbicide resistance against zeta-carotene desaturase inhibitors.     

Dual role of the plastid terminal oxidase in tomato

The plastid terminal oxidase (PTOX) is a plastoquinol oxidase whose absence in tomato (Solanum lycopersicum) results in the ghost (gh) phenotype characterized by variegated leaves (with green and bleached sectors) and by carotenoid-deficient ripe fruit. We show that PTOX deficiency leads to photobleaching in cotyledons exposed to high light primarily as a consequence of reduced ability to synthesize carotenoids in the gh mutant, which is consistent with the known role of PTOX as a phytoene desaturase cofactor. In contrast, when entirely green adult leaves from gh were produced and submitted to photobleaching high light conditions, no evidence for a deficiency in carotenoid biosynthesis was obtained. Rather, consistent evidence indicates that the absence of PTOX renders the tomato leaf photosynthetic apparatus more sensitive to light via a disturbance of the plastoquinone redox status. Although gh fruit are normally bleached (most likely as a consequence of a deficiency in carotenoid biosynthesis at an early developmental stage), green adult fruit could be obtained and submitted to photobleaching high light conditions. Again, our data suggest a role of PTOX in the regulation of photosynthetic electron transport in adult green fruit, rather than a role principally devoted to carotenoid biosynthesis. In contrast, ripening fruit are primarily dependent on PTOX and on plastid integrity for carotenoid desaturation. In summary, our data show a dual role for PTOX. Its activity is necessary for efficient carotenoid desaturation in some organs at some developmental stages, but not all, suggesting the existence of a PTOX-independent pathway for plastoquinol reoxidation in association with phytoene desaturase. As a second role, PTOX is implicated in a chlororespiratory mechanism in green tissues.     

The cyanobacterium Gloeobacter violaceus PCC 7421 uses bacterial-type phytoene desaturase in carotenoid biosynthesis

Carotenoid composition and its biosynthetic pathway in the cyanobacterium Gloeobacter violaceus PCC 7421 were investigated. beta-Carotene and (2S,2'S)-oscillol 2,2'-di(alpha-L-fucoside), and echinenone were major and minor carotenoids, respectively. We identified two unique genes for carotenoid biosynthesis using in vivo functional complementation experiments. In Gloeobacter, a bacterial-type phytoene desaturase (CrtI), rather than plant-type desaturases (CrtP and CrtQ), produced lycopene. This is the first demonstration of an oxygenic photosynthetic organism utilizing bacterial-type phytoene desaturase. We also revealed that echinenone synthesis is catalyzed by CrtW rather than CrtO. These findings indicated that Gloeobacter retains ancestral properties of carotenoid biosynthesis.     

Functional analysis of genes from Streptomyces griseus involved in the synthesis of isorenieratene,a carotenoid with aromatic end groups,revealed a novel type of carotenoid desaturase

The biosynthesis of the aromatic carotene isorenieratene is restricted to green photosynthetic bacteria and a few actinomycetes. Among them Streptomyces griseus has been used to study the genes involved in this pathway. Five genes out of seven of two adjacent operons in one cluster could be identified to be sufficient for the synthesis of isorenieratene. Stepwise deletions of these genes demonstrated their participation in phytoene synthesis, phytoene desaturation and lycopene cyclization. The novel gene crtU was assigned to encode a unique desaturase responsible for the conversion of β-carotene via β-isorenieratene to isorenieratene by a desaturation/methyltransferation mechanism. Sequence analysis of crtU revealed two conserved regions, one at the N-terminus and the other at the C-terminus of the protein which is universal to different types of carotene desaturases. In addition, the sequence comprises a motif typically found in methyltransferases. The deletion of the two remaining genes of the cluster left the carotenoid biosynthetic pathway unaffected.     

The <Emphasis Type="Italic">pds2</Emphasis> mutation is a lesion in the <Emphasis Type="Italic">Arabidopsis</Emphasis> homogentisate solanesyltransferase gene involved in plastoquinone biosynthesis

Plastoquinone plays critical roles in photosynthesis, chlororespiration and carotenoid biosynthesis. The previously isolated pds2 mutant from Arabidopsis was deficient in tocopherol and plastoquinone accumulation, and the biochemical phenotype of this mutant could not be reversed by externally applied homogentisate, suggesting a later step in tocopherol and/or plastoquinone biosynthesis had been disrupted. Recently, the protein encoded by At3g11950 (AtHST) was shown to condense homogentisate with solanesyl diphosphate (SDP), the substrate for plastoquinone synthesis, but not phytyl diphosphate (PDP), the substrate for tocopherol biosynthesis. We have sequenced the AtHST allele in the pds2 mutant background and identified an in-frame 6 bp (2 aa) deletion in the gene. The pds2 mutation could be functionally complemented by constitutive expression of AtHST, demonstrating that the molecular basis for the pds2 mutation is this 6 bp-lesion in the AtHST gene. Confocal microscopy of EGFP tagged AtHST suggested that AtHST is localized to the chloroplast envelope, supporting the hypothesis that plastoquinone synthesis occurs in the plastid.     

Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and zeta-carotene in Capsicum chromoplasts.

In plants, zeta-carotene is the first visible carotenoid formed in the biosynthetic pathway through the following two-step desaturation reaction: phytoene-->phytofluene--> zeta-carotene. Using Capsicum annuum chromoplast membranes and the reconstitution system previously described [Camara, B., Bardat, F. & Monéger, R. (1982) Eur. J. Biochem. 127, 255-258], we have attempted to purify the desaturase(s) catalyzing these reactions. The two activities were coincidental during all the purification procedures. Only a single polypeptide with 56 +/- 2 kDa was detected by SDS/PAGE of all active fractions. The enzyme contained protein-bound FAD. Antibodies raised against the purified polypeptide selectively precipitated the phytoene and the phytofluene desaturase activities, thus demonstrating that the enzyme is a bifunctional flavoprotein. The antibodies were used to isolate a full-length cDNA clone from which was deduced the primary structure of the desaturase which contains a characteristic dinucleotide-binding site. Overexpression of the cDNA in Escherichia coli allowed the production of a recombinant desaturase which had all the properties of the chromoplast desaturase. The phytoene/phytofluene desaturase mRNA levels were extremely low in green fruits and increased slightly before detectable carotenoid synthesis and remained constant throughout ripening. However, the desaturase activity and protein levels were found to increase significantly during the chloroplast to chromoplast transition in C. annuum fruits.     

新型白化型除草剂靶标酶对羟苯基丙酮酸双加氧酶及其耐性转基因植物研究进展*

对羟苯基丙酮酸双加氧酶(ρ-hydroxyphenylpyruvate dioxygenase,HPPD;EC 1.13.11.27)催化生物体内对羟苯基丙酮酸与O2作用形成尿黑酸的反应,是植物体中质体醌和生育酚生物合成途径的关键酶。当其活性受到抑制时,植物体中作为类胡萝卜素生物合成途径中最终电子受体和光合链电子传递体的质体醌的生物合成受阻,进而导致类胡萝卜素合成减少,光合链电子传递受阻,致使植物体出现白化症状。目前已经开发了多种以HPPD为靶标的除草剂,该类除草剂及抗除草剂转基因植物研究具有广阔的前景。对这一新型白化型除草剂靶标酶以及耐该类除草剂转基因植物的研究进展作了简要综述。     

Light-Stimulated Carotenoid Biosynthesis during Transformation of Maize Etioplasts Is Regulated by Increased Activity of Isopentenyl Pyrophosphate Isomerase

Light-stimulated carotenoid biosynthesis associated with the transformation of etioplasts to chloroplasts was investigated after dark-grown maize (Zea mays) seedlings were transferred into light. These studies focused on the enzymes of the pathway to detect those enzyme activities that were stimulated in the light and thus that were responsible for increased biosynthesis of carotenoids. In preliminary experiments, norflurazon, an inhibitor of phytoene desaturase, was used to prevent phytoene being further metabolized to carotenoids. Light-dependent stimulation of phytoene accumulation indicated that the light-regulated steps are located in the pathway leading to phytoene synthesis. The use of the 14C- labeled precursors mevalonic acid, isopentenyl pyrophosphate, and farnesyl pyrophosphate pointed to increased activity of an enzyme involved in the biosynthetic steps between isopentenyl pyrophosphate and farnesyl pyrophosphate. Determination of the activities of all five enzymes of the pathway involved in the sequence from mevalonic acid to phytoene revealed that the only enzyme activity stimulated by light was isopentenyl pyrophosphate isomerase. Over a 3-h period of illumination, this enzyme activity, like carotenoid biosynthesis, was stimulated 2.8-fold.     

Maize w3 disrupts homogentisate solanesyl transferase (ZmHst) and reveals a plastoquinone‐9 independent path for phytoene desaturation and tocopherol accumulation in kernels

Maize white seedling 3 (w3) has been used to study carotenoid deficiency for almost 100 years, although the molecular basis of the mutation has remained unknown. Here we show that the w3 phenotype is caused by disruption of the maize gene for homogentisate solanesyl transferase (HST), which catalyzes the first and committed step in plastoquinone‐9 (PQ‐9) biosynthesis in the plastid. The resulting PQ‐9 deficiency prohibits photosynthetic electron transfer and eliminates PQ‐9 as an oxidant in the enzymatic desaturation of phytoene during carotenoid synthesis. As a result, light‐grown w3 seedlings are albino, deficient in colored carotenoids and accumulate high levels of phytoene. However, despite the absence of PQ‐9 for phytoene desaturation, dark‐grown w3 seedlings can produce abscisic acid (ABA) and homozygous w3 kernels accumulate sufficient carotenoids to generate ABA needed for seed maturation. The presence of ABA and low levels of carotenoids in w3 nulls indicates that phytoene desaturase is able to use an alternate oxidant cofactor, albeit less efficiently than PQ‐9. The observation that tocopherols and tocotrienols are modestly affected in w3 embryos and unaffected in w3 endosperm indicates that, unlike leaves, grain tissues deficient in PQ‐9 are not subject to severe photo‐oxidative stress. In addition to identifying the molecular basis for the maize w3 mutant, we: (1) show that low levels of phytoene desaturation can occur in w3 seedlings in the absence of PQ‐9; and (2) demonstrate that PQ‐9 and carotenoids are not required for vitamin E accumulation.     

Cloning and knockout of phytoene desaturase gene in <Emphasis Type="Italic">Sphingomonas elodea</Emphasis> ATCC 31461 for economic recovery of gellan gum

A gene encoding phytoene desaturase (crtI) in the carotenoid biosynthetic pathway of Sphingomonas elodea ATCC 31461, an industrial gellan gum-producing strain, was cloned and identified. This gene is predicted to encode a 492-amino acid protein with significant homology to the phytoene desaturase of other carotenogenic organisms. Knockout of crtI gene blocked yellow carotenoid pigment synthesis and resulted in the accumulation of colorless phytoene, confirming that it encodes phytoene desaturase. Further research indicates that the yield of gellan gum production by crtI gene knockout mutants is almost the same as that by the wild-type strain. In addition, a recovery method based on the colorless fermentation broth of the crtI gene knockout mutant was investigated. Compared to the volume of alcohol for the parent strain, much less alcohol (30%) is required in this recovery process; thus, the costs of downstream purification of gellan gum can be substantially reduced.     

Complementation of the Arabidopsis pds1 Mutation with the Gene Encoding p-Hydroxyphenylpyruvate Dioxygenase

Plastoquinone and tocopherols are the two major quinone compounds in higher plant chloroplasts and are synthesized by a common pathway. In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. Mutation of the PDS1 locus disrupts the activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the first committed step in the synthesis of both plastoquinone and tocopherols in plants. Although plants homozygous for the pds1 mutation could be rescued by growth in the presence of homogentisic acid, the product of HPPDase, we were unable to determine if the mutation directly or indirectly disrupted HPPDase activity. This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli. pHPPD encodes a 50-kD polypeptide with homology to previously identified HPPDases, including 37 highly conserved amino acid residues clustered in the carboxyl region of the protein. Expression of pHPPD in E. coli catalyzes the accumulation of homogentisic acid, indicating that it encodes a functional HPPDase enzyme. Mapping of pHPPD and co-segregation analysis of the pds1 mutation and the HPPD gene indicate tight linkage. Constitutive expression of pHPPD in a pds1 mutant background complements this mutation. Finally, comparison of the HPPD genomic sequences from wild type and pds1 identified a 17-bp deletion in the pds1 allele that results in deletion of the carboxyterminal 26 amino acids of the HPPDase protein. Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene.     

Phytoene desaturase is localized exclusively in the chloroplast and up-regulated at the mRNA level during accumulation of secondary carotenoids in Haematococcus pluvialis (Volvocales, chlorophyceae)

The unicellular green alga Haematococcus pluvialis Flotow is known for its massive accumulation of ketocarotenoids under various stress conditions. Therefore, this microalga is one of the favored organisms for biotechnological production of these antioxidative compounds. Astaxanthin makes up the main part of the secondary carotenoids and is accumulated mostly in an esterified form in extraplastidic lipid vesicles. We have studied phytoene desaturase, an early enzyme of the carotenoid biosynthetic pathway. The increase in the phytoene desaturase protein levels that occurs following induction is accompanied by a corresponding increase of its mRNA during the accumulation period, indicating that phytoene desaturase is regulated at the mRNA level. We also investigated the localization of the enzyme by western-blot analysis of cell fractions and by immunogold labeling of ultrathin sections for electron microscopy. In spite of the fact that secondary carotenoids accumulate outside the chloroplast, no extra pathway specific for secondary carotenoid biosynthesis in H. pluvialis was found, at least at this early stage in the biosynthesis. A transport process of carotenoids from the site of biosynthesis (chloroplast) to the site of accumulation (cytoplasmatic located lipid vesicles) is implicated.     

Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis

Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene （PDS3） encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarfphenotypes ofthepds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in thepds3 mutant. Further analysis showed that high level ofphytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype ofthepds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.     

Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum

The green sulfur bacterium Chlorobium tepidum is a strict anaerobe and an obligate photoautotroph. On the basis of sequence similarity with known enzymes or sequence motifs, nine open reading frames encoding putative enzymes of carotenoid biosynthesis were identified in the genome sequence of C. tepidum, and all nine genes were inactivated. Analysis of the carotenoid composition in the resulting mutants allowed the genes encoding the following six enzymes to be identified: phytoene synthase (crtB/CT1386), phytoene desaturase (crtP/CT0807), zeta-carotene desaturase (crtQ/CT1414), gamma-carotene desaturase (crtU/CT0323), carotenoid 1',2'-hydratase (crtC/CT0301), and carotenoid cis-trans isomerase (crtH/CT0649). Three mutants (CT0180, CT1357, and CT1416 mutants) did not exhibit a discernible phenotype. The carotenoid biosynthetic pathway in C. tepidum is similar to that in cyanobacteria and plants by converting phytoene into lycopene using two plant-like desaturases (CrtP and CrtQ) and a plant-like cis-trans isomerase (CrtH) and thus differs from the pathway known in all other bacteria. In contrast to the situation in cyanobacteria and plants, the construction of a crtB mutant completely lacking carotenoids demonstrates that carotenoids are not essential for photosynthetic growth of green sulfur bacteria. However, the bacteriochlorophyll a contents of mutants lacking colored carotenoids (crtB, crtP, and crtQ mutants) were decreased from that of the wild type, and these mutants exhibited a significant growth rate defect under all light intensities tested. Therefore, colored carotenoids may have both structural and photoprotection roles in green sulfur bacteria. The ability to manipulate the carotenoid composition so dramatically in C. tepidum offers excellent possibilities for studying the roles of carotenoids in the light-harvesting chlorosome antenna and iron-sulfur-type (photosystem I-like) reaction center. The phylogeny of carotenogenic enzymes in green sulfur bacteria and green filamentous bacteria is also discussed.     

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.