Natural overexpression of CAROTENOID CLEAVAGE DIOXYGENASE 4 in tomato alters carotenoid flux.

Carotenoids and apocarotenoids function as pigments and flavor volatiles in plants that enhance consumer appeal and offer health benefits. Tomato (Solanum lycopersicum.) fruit, especially those of wild species, exhibit a high degree of natural variation in carotenoid and apocarotenoid contents. Using positional cloning and an introgression line (IL) of Solanum habrochaites "LA1777', IL8A, we identified carotenoid cleavage dioxygenase 4 (CCD4) as the factor responsible for controlling the dark orange fruit color. CCD4b expression in ripe fruit of IL8A plants was ∼8,000 times greater than that in the wild type, presumably due to 5' cis-regulatory changes. The ShCCD4b-GFP fusion protein localized in the plastid. Phytoene, ζ-carotene, and neurosporene levels increased in ShCCD4b-overexpressing ripe fruit, whereas trans-lycopene, ß-carotene, and lutein levels were reduced, suggestive of feedback regulation in the carotenoid pathway by an unknown apocarotenoid. Solid-phase microextraction-gas chromatography-mass spectrometry analysis showed increased levels of geranylacetone and ß-ionone in ShCCD4b-overexpressing ripe fruit coupled with a ß-cyclocitral deficiency. In carotenoid-accumulating Escherichia coli strains, ShCCD4b cleaved both ζ-carotene and ß-carotene at the C9-C10 (C9'-C10') positions to produce geranylacetone and ß-ionone, respectively. Exogenous ß-cyclocitral decreased carotenoid synthesis in the ripening fruit of tomato and pepper (Capsicum annuum), suggesting feedback inhibition in the pathway. Our findings will be helpful for enhancing the aesthetic and nutritional value of tomato and for understanding the complex regulatory mechanisms of carotenoid and apocarotenoid biogenesis.

Subfunctionalization of D27 Isomerase Genes in Saffron.

Chromoplasts and chloroplasts contain carotenoid pigments as all-trans- and cis-isomers, which function as accessory light-harvesting pigments, antioxidant and photoprotective agents, and precursors of signaling molecules and plant hormones. The carotenoid pathway involves the participation of different carotenoid isomerases. Among them, D27 is a ß-carotene isomerase showing high specificity for the C9-C10 double bond catalyzing the interconversion of all-trans- into 9-cis-ß-carotene, the precursor of strigolactones. We have identified one D27 (CsD27-1) and two D27-like (CsD27-2 and CsD27-3) genes in saffron, with CsD27-1 and CsD27-3, clearly differing in their expression patterns; specifically, CsD27-1 was mainly expressed in the undeveloped stigma and roots, where it is induced by Rhizobium colonization. On the contrary, CsD27-2 and CsD27-3 were mainly expressed in leaves, with a preferential expression of CsD27-3 in this tissue. In vivo assays show that CsD27-1 catalyzes the isomerization of all-trans- to 9-cis-ß-carotene, and could be involved in the isomerization of zeaxanthin, while CsD27-3 catalyzes the isomerization of all-trans- to cis-ζ-carotene and all-trans- to cis-neurosporene. Our data show that CsD27-1 and CsD27-3 enzymes are both involved in carotenoid isomerization, with CsD27-1 being specific to chromoplast/amyloplast-containing tissue, and CsD27-3 more specific to chloroplast-containing tissues. Additionally, we show that CsD27-1 is co-expressed with CCD7 and CCD8 mycorrhized roots, whereas CsD27-3 is expressed at higher levels than CRTISO and Z-ISO and showed circadian regulation in leaves. Overall, our data extend the knowledge about carotenoid isomerization and their implications in several physiological and ecological processes.

Enzymatic isomerization of ζ-carotene mediated by the heme-containing isomerase Z-ISO.

Carotenoids are a large and diverse class of isoprenoid compounds synthesized by plants, algae, some bacteria, arthropods, and fungi. These pigments contribute to plant growth and survival by protecting plants from photooxidative stress and serving as precursors of plant hormones and other signaling compounds. In humans, carotenoids are essential components of the diet and contribute anti-oxidant and provitamin A activities. Carotenoids are synthesized in the membranes of plant plastids where phytoene is converted into all trans lycopene by a biosynthetic pathway that was only recently completed by the discovery of the new enzyme, 15-cis-ζ-carotene isomerase (Z-ISO), which controls carotenoid pathway flux to products necessary for plant development and function. Z-ISO catalysis of the cis to trans isomerization of the 15-cis double bond in 15-cis-ζ-carotene is mediated by a unique mechanism dependent on the redox-state of a heme b cofactor. This chapter describe methods for the functional analysis of Z-ISO, including complementation of Z-ISO in engineered E. coli, separation of Z-ISO enzyme substrate and products, ζ-carotene isomers, by high pressure liquid chromatography (HPLC), expression and purification of Z-ISO and in vitro enzymatic reactions.

Genomics-based strategies toward the identification of a Z-ISO carotenoid biosynthetic enzyme suitable for structural studies.

Over the past 20years, structural genomics efforts have proven enormously successful for the determination of integral membrane protein structures, particularly for those of prokaryotic origin. However, traditional genomic expansion screens have included up to hundreds of targets, necessitating the use of robotics and other automation not available to most laboratories. Moreover, such large-scale screens of eukaryotic targets are not easily performed at such a scale. To have broader appeal, traditional structural genomic approaches need to be modified and improved such that they are feasible for most laboratories and especially so for proteins from eukaryotic organisms. One such refinement, termed "microgenomic expansion," has been recently described. This approach improves the process of target selection by making target screening a two-step process, with a minimal number of targets tested at each step. Microgenomic expansion methods are applied here theoretically to a project that has the objective of acquiring a structure for the plant 15-cis-ζ-carotene isomerase, Z-ISO.

ClZISO mutation leads to photosensitive flesh in watermelon.

KEY MESSAGE: The mutation of ClZISO identified in EMS-induced watermelon leads to photosensitive flesh in watermelon. Watermelon (Citrullus lanatus) has a colorful flesh that attracts consumers and benefits human health. We developed an ethyl-methanesulfonate mutation library in red-fleshed line '302' to create new flesh color lines and found a yellow-fleshed mutant which accumulated ζ-carotene. The initial yellow color of this mutant can be photobleached within 10 min under intense sunlight. A long-term light-emitting diode (LED) light treatment turned flesh color from yellow to pink. We identified this unique variation as photosensitive flesh mutant ('psf'). Using bulked segregant analysis, we fine-mapped an EMS-induced G-A transversion in 'psf' which leads to a premature stop codon in 15-cis-ζ-carotene isomerase (ClZISO) gene. We detected that wild-type ClZISO is expressed in chromoplasts to catalyze the conversion of 9,15,9'-tri-cis-ζ-carotene to 9,9'-di-cis-ζ-carotene. The truncated ClZISOmu protein in psf lost this catalytic function. Light treatment can partially compensate ClZISOmu isomerase activity via photoisomerization in vitro and in vivo. Transcriptome analysis showed that most carotenoid biosynthesis genes in psf were downregulated. The dramatic increase of ABA content in flesh with fruit development was blocked in psf. This study explores the molecular mechanism of carotenoid biosynthesis in watermelon and provides a theoretical and technical basis for breeding different flesh color lines in watermelon.

ζ-Carotene Isomerase Suppresses Tillering in Rice through the Coordinated Biosynthesis of Strigolactone and Abscisic Acid.

Rice tillering is an important agronomic trait affecting grain yield. Here, we identified a high-tillering mutant tillering20 (t20), which could be restored to the wild type by treatment with the strigolactone (SL) analog rac-GR24. T20 encodes a chloroplast ζ-carotene isomerase (Z-ISO), which is involved in the biosynthesis of carotenoids and their metabolites, SL and abscisic acid (ABA). The t20 mutant has reduced SL and ABA, raising the question of how SL and ABA biosynthesis is coordinated, and whether they have overlapping functions in tillering. We discovered that rac-GR24 stimulated T20 expression and enhanced all-trans-ß-carotene biosynthesis. Importantly, rac-GR24 also stimulated expression of Oryza sativa 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (OsNCED1) through induction of Oryza sativa HOMEOBOX12 (OsHOX12), promoting ABA biosynthesis in shoot base. On the other hand, ABA treatment significantly repressed SL biosynthesis and the ABA biosynthetic mutants displayed elevated SL biosynthesis. ABA treatment reduced the number of basal tillers in both t20 and wild-type plants. Furthermore, while ABA-deficient mutants aba1 and aba2 had the same number of basal tillers as wild type, they had more unproductive upper tillers at maturity. This work demonstrates complex interactions in the biosynthesis of carotenoid, SLs and ABA, and reveals a role for ABA in the regulation of rice tillering.

Oxygenic Phototrophs Need ζ-Carotene Isomerase (Z-ISO) for Carotene Synthesis: Functional Analysis in Arthrospira and Euglena.

For carotenogenesis, two biosynthetic pathways from phytoene to lycopene are known. Most bacteria and fungi require only phytoene desaturase (PDS, CrtI), whereas land plants require four enzymes: PDS (CrtP), ζ-carotene desaturase (ZDS, CrtQ), ζ-carotene isomerase (Z-ISO) and cis-carotene isomerase (CrtISO, CrtH). The gene encoding Z-ISO has been functionally identified in only two species, Arabidopsis thaliana and Zea mays, and has been little studied in other organisms. In this study, we found that the deduced amino acid sequences of Arthrospira Z-ISO and Euglena Z-ISO have 58% and 62% identity, respectively, with functional Z-ISO from Arabidopsis. We studied the function of Z-ISO genes from the cyanobacterium Arthrospira platensis and eukaryotic microalga Euglena gracilis. The Z-ISO genes of Arthrospira and Euglena were transformed into Escherichia coli strains that produced mainly 9,15,9'-tri-cis-ζ-carotene in darkness. In the resulting E. coli transformants cultured under darkness, 9,9'-di-cis-ζ-carotene was accumulated predominantly as Z-ISO in Arabidopsis. This indicates that the Z-ISO genes were involved in the isomerization of 9,15,9'-tri-cis-ζ-carotene to 9,9'-di-cis-ζ-carotene in darkness. This is the first functional analysis of Z-ISO as a ζ-carotene isomerase in cyanobacteria and eukaryotic microalgae. Green sulfur bacteria and Chloracidobacterium also use CrtP, CrtQ and CrtH for lycopene synthesis as cyanobacteria, but their genomes did not comprise Z-ISO genes. Consequently, Z-ISO is needed in oxygenic phototrophs, whereas it is not found in anoxygenic species.

Dissection of tomato lycopene biosynthesis through virus-induced gene silencing.

Lycopene biosynthesis in tomato (Solanum lycopersicum) fruits has been proposed to proceed through a poly-cis pathway catalyzed by phytoene synthase (PSY), two desaturases (phytoene desaturase [PDS] and ζ-carotene desaturase [ZDS]), and two cis-trans isomerases (ζ-carotene isomerase [ZISO] and prolycopene isomerase [CrtISO]). The mechanism of action of these enzymes has been studied in Escherichia coli, but a systematic study of their in vivo function is lacking. We studied the function of nine candidate genes (PSY1, PSY2, PSY3, PDS, ZDS, ZISO, CrtISO, CrtISO-Like1, and CrtISO-Like2) using virus-induced gene silencing (VIGS) coupled to high-resolution liquid chromatography coupled with diode array detector and mass spectrometry, which allowed the identification and quantitation of 45 different carotenoid isomers, including linear xanthophylls. The data confirm the confinement of the VIGS signal to the silenced fruits and the similarity of the phenotypes of PSY1- and CrtISO-silenced fruits with those of the yellow flesh and tangerine mutants. Light was able to restore lycopene biosynthesis in ZISO-silenced fruits. Isomeric composition of fruits silenced at different metabolic steps suggested the existence of three functional units, comprising PSY1, PDS/ZISO, and ZDS/CrtISO, and responsible for the synthesis of 15-cis-phytoene, 9,9'-di-cis-ζ-carotene, and all-trans-lycopene, respectively. Silencing of a desaturase (PDS or ZDS) resulted in the induction of the isomerase in the same functional unit (ZISO or CrtISO, respectively). All-trans-ζ-carotene was detectable in nonsilenced fruits, greatly increased in ZDS-silenced ones, and disappeared in CrtISO-Like1-/CrtISO-Like2-silenced ones, suggesting the existence of a metabolic side branch, comprising this compound and initiated by the latter enzymes.

Contribution of two ζ-carotene desaturases to the poly-cis desaturation pathway in the cyanobacterium Nostoc PCC 7120.

The presence of two completely unrelated ζ-carotene desaturases CrtQa and CrtQb in some Nostoc strains is unique. CrtQb is the ζ-carotene desaturase, which was acquired by almost all cyanobacteria. The additional CrtQa can be regarded as an evolutionary relict of the CrtI desaturase present in non-photosynthetic bacteria. By reconstruction of the carotene desaturation pathway, we showed that both enzymes from Nostoc PCC 7120 were active. However, they differed in their preferred utilization of ζ-carotene Z isomers. CrtQa converted ζ-carotene isomers that were poorly metabolized by CrtQb. In this respect, CrtQa complemented the reactions of CrtQb, which is an advantage avoiding dead ends in the poly-cis desaturation pathway. In addition to ζ-carotene desaturation, CrtQa still possesses the Z to E isomerase function of the ancestral desaturase CrtI. Biochemical characterization showed that CrtQb is an enzyme with one molecule of tightly bound FAD and acts as a dehydrogenase transferring hydrogen to oxidized plastoquinone.

Structure and function characterization of the phytoene desaturase related to the lutein biosynthesis in Chlorella protothecoides CS-41.

Phytoene desaturase is the key enzyme involved in the biosynthesis pathway of lutein. The unicellular microalga, Chlorella protothecoides CS-41, had been selected for the heterotrophic production of high concentrations of lutein. In this study, a cDNA copy of the pds gene from C. protothecoides was obtained using the rapid amplification of cDNA ends (RACE) technique. Phylogenetic analysis of the deduced amino acid sequence revealed that the phytoene desaturases derived from the algal family. Expression of the pds gene in Escherichia coli produced a single protein of 61 kDa. The PDS activity of the expressed protein was confirmed by the production of ζ-carotene as the result from the action of the enzyme's desaturation activity, which was identified by high-performance liquid chromatography and heterologous complementation analysis. Using random and site-directed mutagenesis, a single amino acid mutation (N144D) was identified and confirmed. This mutant encodes an inactive enzyme, which implies that amino acid 144 is crutial to the activity of the PDS enzyme. Therefore, by gene cloning and expression in prokaryotic cells, the gene for ζ-carotene production or as part of the biosynthetic pathway of lutein had been characterized from Chlorella protothecoides CS-41.

Biosynthesis of fucoxanthin and diadinoxanthin and function of initial pathway genes in Phaeodactylum tricornutum.

The biosynthesis pathway to diadinoxanthin and fucoxanthin was elucidated in Phaeodactylum tricornutum by a combined approach involving metabolite analysis identification of gene function. For the initial steps leading to ß-carotene, putative genes were selected from the genomic database and the function of several of them identified by genetic pathway complementation in Escherichia coli. They included genes encoding a phytoene synthase, a phytoene desaturase, a ζ-carotene desaturase, and a lycopene ß-cyclase. Intermediates of the pathway beyond ß-carotene, present in trace amounts, were separated by TLC and identified as violaxanthin and neoxanthin in the enriched fraction. Neoxanthin is a branching point for the synthesis of both diadinoxanthin and fucoxanthin and the mechanisms for their formation were proposed. A single isomerization of one of the allenic double bounds in neoxanthin yields diadinoxanhin. Two reactions, hydroxylation at C8 in combination with a keto-enol tautomerization and acetylation of the 3'-HO group results in the formation of fucoxanthin.

Maize Y9 encodes a product essential for 15-cis-zeta-carotene isomerization.

Carotenoids are a diverse group of pigments found in plants, fungi, and bacteria. They serve essential functions in plants and provide health benefits for humans and animals. In plants, it was thought that conversion of the C40 carotenoid backbone, 15-cis-phytoene, to all-trans-lycopene, the geometrical isomer required by downstream enzymes, required two desaturases (phytoene desaturase and zeta-carotene desaturase [ZDS]) plus a carotene isomerase (CRTISO), in addition to light-mediated photoisomerization of the 15-cis-double bond; bacteria employ only a single enzyme, CRTI. Characterization of the maize (Zea mays) pale yellow9 (y9) locus has brought to light a new isomerase required in plant carotenoid biosynthesis. We report that maize Y9 encodes a factor required for isomerase activity upstream of CRTISO, which we term Z-ISO, an activity that catalyzes the cis- to trans-conversion of the 15-cis-bond in 9,15,9'-tri-cis-zeta-carotene, the product of phytoene desaturase, to form 9,9'-di-cis-zeta-carotene, the substrate of ZDS. We show that recessive y9 alleles condition accumulation of 9,15,9'-tri-cis-zeta-carotene in dark tissues, such as roots and etiolated leaves, in contrast to accumulation of 9,9'-di-cis-zeta-carotene in a ZDS mutant, viviparous9. We also identify a locus in Euglena gracilis, which is similarly required for Z-ISO activity. These data, taken together with the geometrical isomer substrate requirement of ZDS in evolutionarily distant plants, suggest that Z-ISO activity is not unique to maize, but will be found in all higher plants. Further analysis of this new gene-controlled step is critical to understanding regulation of this essential biosynthetic pathway.

zeta-Carotene cis isomers as products and substrates in the plant poly-cis carotenoid biosynthetic pathway to lycopene.

The plant carotenoid biosynthetic pathway to cyclic carotenes proceeds via carotene precursors in cis configuration. Involvement of individual isomers was elucidated by genetic complementation of desaturations and in vitro reactions of the corresponding enzyme. Determination of substrate and product specificity of phytoene and zeta-carotene desaturase revealed that 15-cis-phytoene is converted to 9,15,9'-tricis-zeta-carotene with 15,9'-dicis-phytofluene as intermediate by the first desaturase. Prior to a subsequent conversion by zeta-carotene desaturase, the 15-cis double bond of 9,15,9'-tricis-zeta-carotene has to be (photo)isomerized to all-trans. Then, the resulting 9,9'-dicis-zeta-carotene is utilized by zeta-carotene desaturase via 7,9,9'-tricis-neurosporene to 7,9,7',9'-tetracis-lycopene. Other zeta-carotene isomers that are assumed to be spontaneous isomerization products were not converted, except for the asymmetric 9-cis-zeta-carotene. This isomer is desaturated only to 7,9-dicis-neurosporene resembling a dead-end of the pathway. Prolycopene, the product of the desaturation reactions, is finally isomerized by a specific isomerase to all-trans-lycopene, which is a prerequisite for cyclization to beta-carotene. The 5-cis-lycopene and the 9-cis-and 13-cis-beta-carotene isomers detected in leaves are thought to originate independently from cis precursors by non-enzymatic isomerization of their all-trans forms.