<Emphasis Type="Italic">ZmCCD7/ZpCCD7</Emphasis> encodes a carotenoid cleavage dioxygenase mediating shoot branching

Main conclusion

ZmCCD7/ZpCCD7 encodes a carotenoid cleavage dioxygenase that may mediate strigolactone biosynthesis highly responsive to phosphorus deficiency and undergoes negative selection over domestication from Zea ssp. parviglumis to Zea mays.Carotenoid cleavage dioxygenase 7 (CCD7) functions to suppress shoot branching by controlling strigolactone biosynthesis. However, little is known about CCD7 and its functions in maize and its ancestor (Zea ssp. parviglumis) with numerous shoot branches. We found that ZmCCD7 and ZpCCD7 had the same coding sequence, indicating negative selection of the CCD7 gene over domestication from Zea ssp. parviglumis to Zea mays. CCD7 expression was highly responsive to phosphorus deficiency in both species, especially in the meristematic zone and the pericycle of the elongation zone of maize roots. Notably, the crown root had the strongest ZmCCD7 expression in the meristematic zone under phosphorus limitation. Transient expression of GFP tagged ZmCCD7/ZpCCD7 in maize protoplasts indicated their localization in the plastid. Further, ZmCCD7/ZpCCD7 efficiently catalyzed metabolism of six different linear and cyclic carotenoids in E. coli, and generated β-ionone by cleaving β-carotene at the 9,10 (9′,10′) position. Together with suppression of shoot branching in the max3 mutant by transformation of ZmCCD7/ZpCCD7, our work suggested that ZmCCD7/ZpCCD7 encodes a carotenoid cleavage dioxygenase mediating strigolactone biosynthesis in maize and its ancestor.

     

New target carotenoids for CCD4 enzymes are revealed with the characterization of a novel stress-induced carotenoid cleavage dioxygenase gene from Crocus sativus

Apocarotenoid compounds play diverse communication functions in plants, some of them being as hormones, pigments and volatiles. Apocarotenoids are the result of enzymatic cleavage of carotenoids catalyzed by carotenoid cleavage dioxygenase (CCD). The CCD4 family is the largest family of plant CCDs, only present in flowering plants, suggesting a functional diversification associated to the adaptation for specific physiological capacities unique to them. In saffron, two CCD4 genes have been previously isolated from the stigma tissue and related with the generation of specific volatiles involved in the attraction of pollinators. The aim of this study was to identify additional CCD4 members associated with the generation of other carotenoid-derived volatiles during the development of the stigma. The expression of CsCCD4c appears to be restricted to the stigma tissue in saffron and other Crocus species and was correlated with the generation of megastigma-4,6,8-triene. Further, CsCCD4c was up-regulated by wounding, heat, and osmotic stress, suggesting an involvement of its apocarotenoid products in the adaptation of saffron to environmental stresses. The enzymatic activity of CsCCD4c was determined in vivo in Escherichia coli and subsequently in Nicotiana benthamiana by analyzing carotenoids by HPLC–DAD and the volatile products by GC/MS. β-Carotene was shown to be the preferred substrate, being cleaved at the 9,10 (9′,10′) bonds and generating β-ionone, although β-cyclocitral resulting from a 7,8 (7′,8′) cleavage activity was also detected at lower levels. Lutein, neoxanthin and violaxanthin levels in Nicotiana leaves were markedly reduced when CsCCD4c is over expressed, suggesting that CsCCD4c recognizes these carotenoids as substrates.     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

Effect of codon optimization on the enhancement of the β-carotene contents in rice endosperm

A β-carotene is the most well-known dietary source as provitamin A carotenoids. Among β-carotene-producing Golden Rice varieties, PAC (Psy:2A:CrtI) rice has been previously developed using a bicistronic recombinant gene that linked the Capsicum Psy and Pantoea CrtI genes by a viral 2A sequence. To enhance β-carotene content by improving this PAC gene, its codon was optimized for rice plants (Oryza sativa L.) by minimizing the codon bias between the transgene donor and the host rice and was then artificially synthesized as stPAC (stPsy:2A:stCrtI) gene. The GC content (58.7 from 50.9%) and codon adaptation index (0.85 from 0.77) of the stPAC gene were increased relative to the original PAC gene with 76% DNA identity. Among 67 T₁ seeds of stPAC transformants showing positive correlations between transgene copy numbers (up to three) and carotenoid contents, three stPAC lines with a single intact copy were chosen to minimize unintended insertional effects and compared to the representative line of the PAC transgene with respect to their codon optimization effects. Translation levels were stably increased in all three stPAC lines (3.0-, 2.5-, 2.9-fold). Moreover, a greater intensity of the yellow color of stPAC seeds was correlated with enhanced levels of β-carotene (4-fold, 2.37 μg/g) as well as total carotenoid (2.9-fold, 3.50 μg/g) relative to PAC seeds, suggesting a β-branch preference for the stPAC gene. As a result, the codon optimization of the transgene might be an effective tool in genetic engineering for crop improvement as proven at the enhanced levels of translation and carotenoid production.     

Alteration of flower colour in <Emphasis Type="Italic">Ipomoea nil</Emphasis> through CRISPR/Cas9-mediated mutagenesis of <Emphasis Type="Italic">carotenoid cleavage dioxygenase 4</Emphasis>

Japanese morning glory, Ipomoea nil, exhibits a variety of flower colours, except yellow, reflecting the accumulation of only trace amounts of carotenoids in the petals. In a previous study, we attributed this effect to the low expression levels of carotenogenic genes in the petals, but there may be other contributing factors. In the present study, we investigated the possible involvement of carotenoid cleavage dioxygenase (CCD), which cleaves specific double bonds of the polyene chains of carotenoids, in the regulation of carotenoid accumulation in the petals of I. nil. Using bioinformatics analysis, seven InCCD genes were identified in the I. nil genome. Sequencing and expression analyses indicated potential involvement of InCCD4 in carotenoid degradation in the petals. Successful knockout of InCCD4 using the CRISPR/Cas9 system in the white-flowered cultivar I. nil cv. AK77 caused the white petals to turn pale yellow. The total amount of carotenoids in the petals of ccd4 plants was increased 20-fold relative to non-transgenic plants. This result indicates that in the petals of I. nil, not only low carotenogenic gene expression but also carotenoid degradation leads to extremely low levels of carotenoids.     

Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.     

Candidate gene sequencing and validation of SNP markers linked to carotenoid content in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava is a widely grown staple in Sub-Saharan Africa and consumed as a cheap source of calories, but the crop is deficient in micronutrients including pro-vitamin A carotenoids. This challenge is currently being addressed through biofortification breeding that relies on phenotypic selection. Gene-based markers linked to pro-vitamin A content variation are expected to increase the rate of genetic gain for this critical trait. We sequenced four candidate carotenoid genes from 167 cassava accessions representing the diversity of elite breeder lines from IITA. Total carotenoid content was determined using spectrophotometer and total β-carotene was quantified by high-performance liquid chromatography. Storage root yellowness due to carotenoid pigmentation was assessed. We carried out candidate gene association analysis that accounts for population structure and kinship using genome-wide single nucleotide polymorphisms (SNPs) generated through genotyping-by-sequencing. Significant SNPs were used to design competitive allele-specific PCR assays and validated on the larger population for potential use in marker-assisted selection breeding. Candidate gene sequencing of the genes β-carotene hydroxylase (crtRB), phytoene synthase (PSY2), lycopene epsilon cyclase (lcyE), and lycopene beta cyclase (lcyB) yielded a total of 37 SNPs. Total carotenoid content, total β-carotene, and color parameters were significantly associated with markers in the PSY2 gene. The SNPs from lcyE were significantly associated with color while those of lcyB and crtRB were not significantly associated with carotenoids or color parameters. These validated and breeder-friendly markers have potential to enhance the efficiency of selection for high β-carotene cassava, thus accelerating genetic gain.     

Change of carotenoid composition in crabs during embryogenesis

Changes of the qualitative and quantitative compositions of carotenoids are studied at various development stages of the external hard roe, determined based on color differences, for the species C. opilio, P. camtschaticus, and P. platypus. It has been revealed that the major carotenoids of the new egg are astaxanthin and β-carotene. Intermediate products of transformation of β-carotene into astaxanthin are identified: echinenone, canthaxanthine, and phenicoxanthine. The carotenoid content per embryo for the new hard roe of C. opilio (the orange egg) amounted to 22.7 ng, of P. camtschaticus and P. platypus (the violet egg)—to 49.2 and 23.3 ng, respectively. In the hard roe at the later development stage (the brown egg) the carotenoid content was decreased to 13.1 ng in C. opilio and to 20.1 ng in P. camtschaticus. Development of embryos is accompanied by accumulation of esterified carotenoids and a decrease of β-carotene and astaxanthine concentrations in all studied species.     

Genetic analysis of provitamin A carotenoid β-cryptoxanthin concentration and relationship with other carotenoids in maize grain (<Emphasis Type="Italic">Zea mays</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Provitamin A (proVA) carotenoids are converted into retinol (vitamin A) in the human body, are the subject of human nutrition studies, and are targets for biofortification of staple crops. β-Carotene has been the principal target for enhancing levels of proVA. There is recent interest in enhancing the proVA carotenoid β-cryptoxanthin since it has excellent bioavailability, and in maize may be nearly as effective as β-carotene in providing retinol to humans. This study was designed to enhance our understanding of the genetic control of: levels of β-cryptoxanthin, conversion of β-carotene into β-cryptoxanthin and zeaxanthin, conversion of β-cryptoxanthin into zeaxanthin, and flux into and within the β-branch of carotenoid pathway. A biparental population derived from two inbreds with relatively high levels of β-cryptoxanthin and different ratios of β-carotene to β-cryptoxanthin and β-cryptoxanthin to zeaxanthin was studied. Three field replications of this F_2:3 population were grown, grain analyzed by liquid chromatography (LC), and composite interval mapping (CIM) performed to identify 90 quantitative trait loci (QTL) for carotenoids. We detected QTL for β-carotene/(β-cryptoxanthin + zeaxanthin) and (β-carotene + β-cryptoxanthin)/zeaxanthin ratios that contain candidate gene hydroxylase 4 (hyd4), which has not been previously associated with QTL for carotenoids in maize grain. Two color assessment methods, visual score and chromameter reading, were used to phenotype one replicate of the population for initial assessment as simple alternative measuring procedures. A common finding for LC and chromameter analysis included QTL on chromosome 5 that contain candidate gene lycopene β cyclase (lcyβ).     

Engineering of the carotenoid pathway in <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis> leading to the synthesis of zeaxanthin

Zeaxanthin is an essential nutrient for prevention of macular degeneration. However, it is limited in our diet. For the production of zeaxanthin, we have engineered zeaxanthin synthesis into a carotenoid mutant of Xanthophyllomyces dendrorhous which is blocked in astaxanthin synthesis and accumulates β-carotene instead. Two strategies were followed to reach high-yield zeaxanthin synthesis. Total carotenoid synthesis was increased by over-expression of genes HMGR, crtE, and crtYB encoding for limiting enzymes in the pathway leading to and into carotenoid biosynthesis. Then bacterial genes crtZ were used to extend the pathway from β-carotene to zeaxanthin in this mutant. The increase of total carotenoids and the formation of zeaxanthin is dependent on the number of gene copies of crtYB and crtZ integrated into the X. dendrorhous upon transformation. The highest zeaxanthin content around 500 μg/g dw was reached by shaking flask cultures after codon optimization of crtZ for Xanthophyllomyces. Stabilization of carotenoid and zeaxanthin formation in the final transformant in the absence of selection agents was achieved after passing through a sexual cycle and germination of basidiospores. The values for the transformant before and after stabilization were very similar resembling about 70 % of total carotenoids and corresponding to a conversion rate of 80 % for hydroxylation of β-carotene to zeaxanthin. The stabilized transformant allowed experimental small-scale fermentation yielding X. dendrorhous cells with a zeaxanthin content similar to the shaking flask cultures. Our result demonstrates the potential of X. dendrorhous for its development as a zeaxanthin producer and its suitability for large-scale fermentation.     

Effects of light and nitrogen starvation on the content and composition of carotenoids of the green microalga <Emphasis Type="Italic">Parietochloris incisa</Emphasis>

The changes in pigment content and composition of the unicellular alga Parietochloris incisa comb. nov (Trebouxiophyceae, Chlorophyta) were studied. This alga is unique in its ability to accumulate high amounts of arachidonic acid in the cell during cultivation under different irradiances and nitrogen availability in the medium. Under low irradiance of 35 μE/(m₂ s) photosynthetically active radiation the P. incisa cultures possessed slow growth and a relatively low carotenoid-to-chlorophyll ratio. At higher irradiances (200 and 400 μE/(m₂ s)) on complete medium, the alga displayed higher growth rate and an increase in the carotenoid content, especially that of β-carotene and lutein. Both on nitrogen-free (regardless of illumination intensity) and nitrogen-replete medium (under high light), a considerable increase in the ratio of carotenoid and chlorophyll contents was recorded. Predominant accumulation of xanthophylls took place in thylakoid membranes, whereas β-carotene deposition occurred mainly in the cytoplasmic lipid globules (oil bodies); lower amounts of carotenoids were accumulated in the absence of nitrogen. Under high light and nitrogen-deficiency conditions, an increase in violaxanthin de-epoxidation and nonphotochemical quenching was recorded together with a decline in variable chlorophyll fluorescence (F _v/F _m) level. A possible photoprotective role of carotenoids in adaptation of P. incisa to high light under nitrogen starvation conditions is discussed.     

Construction of an alternative glycerol-utilization pathway for improved β-carotene production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glycerol, which is an inevitable by-product of biodiesel production, is an ideal carbon source for the production of carotenoids due to its low price, good availability and chemically reduced status, which results in a low requirement for additional reducing equivalents. In this study, an alternative carbon-utilization pathway was constructed in Escherichia coli to enable more efficient β-carotene production from glycerol. An aldehyde reductase gene (alrd) and an aldehyde dehydrogenase gene (aldH) from Ralstonia eutropha H16 were integrated into the E. coli chromosome to form a novel glycerol-utilization pathway. The β-carotene specific production value was increased by 50% after the introduction of alrd and aldH. It was found that the glycerol kinase gene (garK), alrd and aldH were the bottleneck of the alternative glycerol metabolic pathway, and modulation of garK gene with an mRS library further increased the β-carotene specific production value by 13%. Finally, co-modulation of genes in the introduced aldH–alrd operon led to 86% more of β-carotene specific production value than that of the strain without the alternative glycerol-utilization pathway and the glycerol-utilization rate was also increased. In this work, β-carotene production of E. coli was significantly improved by constructing and optimizing an alternative glycerol-utilization pathway. This strategy can potentially be used to improve the production of other isoprenoids using glycerol as a cheap and abundant substrate, and therefore has industrial relevance.     

Bioinformatic and expression analyses on carotenoid dioxygenase genes in fruit development and abiotic stress responses in <Emphasis Type="Italic">Fragaria vesca</Emphasis>

Carotenoid dioxygenases, including 9-cis-epoxycarotenoid dioxygenases (NCEDs) and carotenoid cleavage dioxygenases (CCDs), can selectively cleave carotenoids into various apocarotenoid products that play important roles in fleshy fruit development and abiotic stress response. In this study, we identified 12 carotenoid dioxygenase genes in diploid strawberry Fragaria vesca, and explored their evolution with orthologous genes from nine other species. Phylogenetic analyses suggested that the NCED and CCDL groups moderately expanded during their evolution, whereas gene numbers of the CCD1, CCD4, CCD7, and CCD8 groups maintained conserved. We characterized the expression profiles of FveNCED and FveCCD genes during flower and fruit development, and in response to several abiotic stresses. FveNCED1 expression positively responded to osmotic, cold, and heat stresses, whereas FveNCED2 was only induced under cold stress. In contrast, FveNCED2 was the unique gene highly and continuously increasing in receptacle during fruit ripening, which co-occurred with the increase in endogenous abscisic acid (ABA) content previously reported in octoploid strawberry. The differential expression patterns suggested that FveNCED1 and FveNCED2 were key genes for ABA biosynthesis in abiotic stress responses and fruit ripening, respectively. FveCCD1 exhibited the highest expression in most stages of flower and fruit development, while the other FveCCDs were expressed in a subset of stages and tissues. Our study suggests distinct functions of FveNCED and FveCCD genes in fruit development and stress responses and lays a foundation for future study to understand the roles of these genes and their metabolites, including ABA and other apocarotenoid products, in the growth and development of strawberry.     

Glutathione antioxidant complex and carotenoid composition in tissues of the bivalve mollusk <Emphasis Type="Italic">Anadara kagoshimensis</Emphasis> (Tokunaga, 1906)

The correlation of the state of glutathione complex composed of reduced glutathione (GSH), glutathione reductase (GR) activity and glutathione peroxidase (GP) and the qualitative composition of carotenoids was investigated in the bivalve mollusk Anadara kagoshimensis (Tokunaga, 1906). Using high-performance liquid chromatography, UV-Vis and mass spectra, 7 types of carotenoids (trans- and cis-pectenolon, alloxanthine, pectenol A, β-carotene, zeaxanthin and diatoxanthin) were identified in tissues of this species and their quantitative ratio was determined. A positive correlation (R ² > 0.9) was established between GSH and most carotenoid levels. A negative correlation was found for the GR–carotenoids (R ² > 0.75) and GP–pectenol A (R ² > 0.988) systems. The cause-and-effect relations of these regularities are discussed.     

Pigmented basidiomycetous yeasts are a promising source of carotenoids and ubiquinone Q<Subscript>10</Subscript>

Strains of basidiomycetous yeasts isolated from different sources were studied in order to determine the content of carotenoid pigments and ubiquinone Q₁₀ for subsequent selection work to obtain producers of these substances. The high specific productivity of carotenoids (600–700 mg/g) was revealed in the representatives of the following species: Cystofilobasidium capitatum, Rhodosporidium diobovatum, R. sphaerocarpum, Rhodotorula glutinis, Rh. minuta, and Sporobolomyces roseus. The ratio of the major pigments (torulene, torularhodine, and β-carotene) in the representatives of different species was studied. Certain specific features of pigment formation in relation to the taxonomic position of the yeasts were determined. Eurybiont species with substantial ecological lability are the most active producers of carotenoids and ubiquinone Q₁₀ among the epiphytes. It is the first time a comparative analysis of the coenzyme Q₁₀ content in different taxa has been performed using several strains of the same species. The maximal coenzyme Q₁₀ production (1.84 mg/g of dry biomass) was found in the yeast species R. sphaerocarpum.     

The qualitative composition of carotenoids and their seasonal dynamics in tissues of the bivalve <Emphasis Type="Italic">Anadara kagoshimensis</Emphasis> (Tokunaga, 1906)

A total of six carotenoids, viz., β-carotene, pectenol A, pectenolone (trans- and cis-isomers), zeaxanthin, diatoxanthin, and alloxanthin, as well as esters of alloand diatoxanthin, have been detected in total carotenoid extracts from the tissues of the bivalve Anadara kagoshimensis (Tokunaga, 1906) using the methods of thin-layer chromatography, high-performance liquid chromatography, mass spectrometry, UV-VIS spectroscopy, and characteristic reactions for the identification of chemical groups. The major group (over 90% of the total carotenoids) is comprised of alloxanthin, pectenolone, and allo- and diatoxanthin esters. Tissues of A. kagoshimensis are typically characterized by cyclic variations in the level of carotenoids over the period from winter to summer, with the maxima in February and June and the minimum in April. The largest contribution to the seasonal carotenoid dynamics is made by the major group of pigments (R ² = 0.75–0.99), which depends on the pattern of succession of diatomic microalgae during the annual cycle. The pathways of metabolic transformation of the carotenoids in tissues of this bivalve are discussed.     

Map-based cloning and functional analysis of the chromogen gene <Emphasis Type="Italic">C</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The chromogen gene C is critical for anthocyanin regulation in rice, and apiculus color is an important agronomic trait in selective breeding and variety purification. Mapbased cloning and in-depth functional analysis of the C gene will be useful for understanding the molecular mechanism of anthocyanin biosynthesis and for rice breeding. Japonica landrace Lijiangxintuanheigu (LTH) has red apiculi and purple stigmas. Genetic analysis showed that red apiculus and purple stigma in LTH co-segregated indicating control by a single dominant gene, or by two completely linked genes. Using 1,851 recessive individuals from two F₂ populations, the target gene OsC was delimited to a 70.8 kb interval on chromosome 6 that contains the rice homologue of the maize anthocyanin regulatory gene C1. When the entire OsC gene and its full-length cDNA cloned from LTH were transformed into japonica cultivar Kitaake with colorless apiculi and stigmas all positive transformants had red apiculi but non-colored stigmas, validating that OsC alone was responsible for the apiculus color and represented the functional C gene. OsC was constitutively expressed in all tissues examined, with strongest expression in leaf blades. These results set a foundation to clarify the regulatory mechanisms of OsC in the anthocyanin biosynthetic pathway.