Β-carotene Hydroxylase Research Articles

Preferential accumulation of orange-colored carotenoids in Ponkan ( Citrus reticulata) fruit peel following postharvest application of ethylene or ethephon

To avoid winter frost, Ponkan ( Citrus reticulata Blanco cv. Ponkan) fruit in Zhejiang Province, China, is frequently harvested early, while chlorophylls are still present in the peel. Postharvest degreening of Ponkan fruit, through either ethylene fumigation or ethephon dipping, was carried out to study the effects of both treatments on color development and carotenoid accumulation. An earlier and deeper orange color development after treatments was indicated by a decline in hue angle ( H°) and an increase in the citrus color index (CCI). Total carotenoid content increased, while chlorophyll content decreased during storage at 10 °C, and these changes were accelerated by both treatments. The treatments stimulated preferential accumulation of carotenoids with orange color (β-carotene and β-cryptoxanthin) while reducing the abundance of carotenoids with yellow color (lutein, violaxanthin and 9- cis-violaxanthin). Gene expression of β-carotene hydroxylase (BCH) was inhibited by both treatments, which could be related to the changes in composition of these differently colored carotenoids.

Cloning and functional characterization of the maize carotenoid isomerase and β-carotene hydroxylase genes and their regulation during endosperm maturation

In order to gain further insight into the partly-characterized carotenoid biosynthetic pathway in corn (Zea mays L.), we cloned cDNAs encoding the enzymes carotenoid isomerase (CRTISO) and β-carotene hydroxylase (BCH) using endosperm mRNA isolated from inbred line B73. For both enzymes, two distinct cDNAs were identified mapping to different chromosomes. The two crtiso cDNAs (Zmcrtiso1 and Zmcrtiso2) mapped to unlinked genes each containing 12 introns, a feature conserved among all crtiso genes studied thus far. ZmCRTISO1 was able to convert tetra-cis prolycopene to all-trans lycopene but could not isomerize the 15-cis double bond of 9,15,9'-tri-cis-ζ-carotene. ZmCRTISO2 is inactivated by a premature termination codon in B73 corn, but importantly the mutation is absent in other corn cultivars and the active enzyme showed the same activity as ZmCRTISO1. The two bch cDNAs (Zmbch1 and Zmbch2) mapped to unlinked genes each coding sequences containing five introns. ZmBCH1 was able to convert β-carotene into β-cryptoxanthin and zeaxanthin, but ZmBCH2 was able to form β-cryptoxanthin alone and had a lower overall activity than ZmBCH1. All four genes were expressed during endosperm development, with mRNA levels rising in line with carotenoid accumulation (especially zeaxanthin and lutein) until 25 DAP. Thereafter, expression declined for three of the genes, with only Zmcrtiso2 mRNA levels maintained by 30 DAP. We discuss the impact of paralogs with different expression profiles and functions on the regulation of carotenoid synthesis in corn.

Transcript accumulation of carotenoid biosynthesis genes in the cyanobacterium Synechocystis sp. PCC 6803 during the dark-to-light transition is mediated by photosynthetic electron transport

Expression of the genes for carotenoid biosynthesis (crt) is dependent on light, but little is known about the underlying mechanism of light sensing and signalling in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter, Synechocystis). In the present study, we investigated the light-induced increase in the transcript levels of Synechocystiscrt genes, including phytoene synthase (crtB), phytoene desaturase (crtP), ζ-carotene desaturase (crtQ), and β-carotene hydroxylase (crtR), during a dark-to-light transition period. During the dark-to-light shift, the increase in the crt transcript levels was not affected by mutations in cyanobacterial photoreceptors, such as phytochromes (cph1, cph2 and cph3) and a cryptochrome-type photoreceptor (ccry), or respiratory electron transport components NDH and Cyd/CtaI. However, treatment with photosynthetic electron transport inhibitors significantly diminished the accumulation of crt gene transcripts. Therefore, the light induction of the Synechocystis crt gene expression is most likely mediated by photosynthetic electron transport rather than by cyanobacterial photoreceptors during the dark-to-light transition.

Extraction of High-Quality RNA and Construction of a cDNA Library from Fruits of Lycium Barbarum Linnaeus (Fructus Lycii)

ABSTRACTHigh-quality RNA isolation is the prerequisite for molecular analysis. In the present study, we used the RNAiso™ for Polysaccharide-rich Plant Tissue solution to extract high-quality RNA from fruits of Lycium barbarum Linnaeus (Fructus Lycii), a popular traditional Chinese herbal medicine used for centuries. The RNA was illustrated to be intact, without protein contamination. Based on the RNA isolated, the gene encoding β-carotene hydroxylase (BCH) was partially cloned by performing reverse transcription-polymerase chain reaction (RT-PCR). In addition, a high quality cDNA library with 6.0×106 clones in total was successfully constructed. The results indicated that this method could be reliably used in isolating high-purity RNA from Fructus Lycii. The outcome will be helpful to the studies of carotenoids or biosynthesis other pharmaceutical elements in Fructus Lycii.

From QTL to candidate gene: Genetical genomics of simple and complex traits in potato using a pooling strategy

BackgroundUtilization of the natural genetic variation in traditional breeding programs remains a major challenge in crop plants. The identification of candidate genes underlying, or associated with, phenotypic trait QTLs is desired for effective marker assisted breeding. With the advent of high throughput -omics technologies, screening of entire populations for association of gene expression with targeted traits is becoming feasible but remains costly. Here we present the identification of novel candidate genes for different potato tuber quality traits by employing a pooling approach reducing the number of hybridizations needed. Extreme genotypes for a quantitative trait are collected and the RNA from contrasting bulks is then profiled with the aim of finding differentially expressed genes.ResultsWe have successfully implemented the pooling strategy for potato quality traits and identified candidate genes associated with potato tuber flesh color and tuber cooking type. Elevated expression level of a dominant allele of the β-carotene hydroxylase (bch) gene was associated with yellow flesh color through mapping of the gene under a major QTL for flesh color on chromosome 3. For a second trait, a candidate gene with homology to a tyrosine-lysine rich protein (TLRP) was identified based on allele specificity of the probe on the microarray. TLRP was mapped on chromosome 9 in close proximity to a QTL for potato cooking type strengthening its significance as a candidate gene. Furthermore, we have performed a profiling experiment targeting a polygenic trait, by pooling individual genotypes based both on phenotypic and marker data, allowing the identification of candidate genes associated with the two different linkage groups.ConclusionsA pooling approach for RNA-profiling with the aim of identifying novel candidate genes associated with tuber quality traits was successfully implemented. The identified candidate genes for tuber flesh color (bch) and cooking type (tlrp) can provide useful markers for breeding schemes in the future. Strengths and limitations of the approach are discussed.

Metabolic Engineering of Saccharomyces cerevisiae for Astaxanthin Production and Oxidative Stress Tolerance

The red carotenoid astaxanthin possesses higher antioxidant activity than other carotenoids and has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. In this study, we produced astaxanthin in the budding yeast Saccharomyces cerevisiae by introducing the genes involved in astaxanthin biosynthesis of carotenogenic microorganisms. In particular, expression of genes of the red yeast Xanthophyllomyces dendrorhous encoding phytoene desaturase (crtI product) and bifunctional phytoene synthase/lycopene cyclase (crtYB product) resulted in the accumulation of a small amount of beta-carotene in S. cerevisiae. Overexpression of geranylgeranyl pyrophosphate (GGPP) synthase from S. cerevisiae (the BTS1 gene product) increased the intracellular beta-carotene levels due to the accelerated conversion of farnesyl pyrophosphate to GGPP. Introduction of the X. dendrorhous crtS gene, encoding astaxanthin synthase, assumed to be the cytochrome P450 enzyme, did not lead to astaxanthin production. However, coexpression of CrtS with X. dendrorhous CrtR, a cytochrome P450 reductase, resulted in the accumulation of a small amount of astaxanthin. In addition, the beta-carotene-producing yeast cells transformed by the bacterial genes crtW and crtZ, encoding beta-carotene ketolase and hydroxylase, respectively, also accumulated astaxanthin and its intermediates, echinenone, canthaxanthin, and zeaxanthin. Interestingly, we found that these ketocarotenoids conferred oxidative stress tolerance on S. cerevisiae cells. This metabolic engineering has potential for overproduction of astaxanthin and breeding of novel oxidative stress-tolerant yeast strains.

Expression of carotenogenic genes and carotenoid production in Haematococcus pluvialis under the influence of carotenoid and fatty acid synthesis inhibitors

Regulation of carotenogenesis in Haematococcus pluvialis was studied using various carotenoid and fatty acid synthesis inhibitors under high light in nutrient sufficient (NS/HL) and nutrient deficient (ND/HL) condition. The results showed inhibition of astaxanthin by all inhibitors studied except cerulenin in ND/HL. Total carotenoids were reduced by inhibitors except DPA in ND/HL. Lycopene accumulation was observed with nicotine treatment while β-carotene accumulation was observed with DPA. Under NS/HL condition, expression of carotenogenic gene PSY (phytoene synthase) was enhanced by all inhibitors studied except DPA while PDS (phytoene desaturase) expression was reduced by all inhibitors tested except nicotine. Expression of LCY (lycopene cyclase) was enhanced by inhibitors. Reduced expression of BKT (β-carotene ketolase) and CHY (β-carotene hydroxylase) were observed under DPA and NF treatment while enhanced expression for BKT was observed under nicotine treatment and CHY expression under nicotine and cerulenin. ND/HL exposure enhanced carotenogenic genes expression when compared to respective inhibitors of NS/HL in most cases. Reduced expression of LCY by nicotine resulted in increased lycopene accumulation while PSY and LCY by cerulenin resulted in decreased β-carotene and lutein. The results indicate the involvement of other regulatory factors besides transcriptional regulation of carotenogenesis in H. pluvialis.

Simple functional analysis of key genes involved in astaxanthin biosynthesis using Arabidopsis cultured cells

Although several biotechnological researchers have reported the production of useful carotenoids in transgenic plants, the acquirement of genetically stable transgenic plants and subsequent examinations for effects of transgenes on the plants require a lot of time and efforts. Here, we show a simple and efficient approach to evaluate key genes involved in carotenoid biosynthesis using Arabidopsis thaliana suspension-cultured cell line T87. A plasmid for the expression of six key-gene candidates for astaxanthin production was constructed and introduced into A. thaliana T87 cells via Agrobacterium-mediated transformation. Five among twenty transgenic cell lines were isolated as lines resistant to a breaching herbicide norflurazon, and the expression of all the six transgenes in the lines was confirmed by quantitative transcriptional analysis. These five transgenic cell lines were shown to accumulate 4.3 to 21.9 μg g−1 fresh weight of astaxanthin, in addition to other ketocarotenoids containing adonirubin, canthaxanthin, echinenone and 3′-hydroxyechinenone. The amount of the ketocarotenoids was estimated to be 36% to 53% of the total carotenoids. The total carotenoid amount was also increased to 4.7 to 13.9 times that of control cells. We further employed microarray analysis to evaluate effects of the transgenes on endogenous gene expression in the transgenic cells. Transcriptional levels of many endogenous carotenogenic genes, e.g., genes for β-carotene hydroxylase, neoxanthin cleavage enzymes, abscisic aldehyde oxidase, 1-deoxy-D-xylulose 5-phosphate synthase, endoplasmic reticulum-targeted and mitochondria-targeted geranyl-geranyl diphosphate synthase were significantly elevated by the expression of the six genes for astaxanthin biosynthesis. These data suggest that transgenic cells adjust their whole cellular isoprenoid-metabolic system to direct the high-level production of the carotenoids including the ketocarotenoids in the plastids.

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize

Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants, which we have used to dissect and modify a complex metabolic pathway. To demonstrate the principle, we transferred 5 carotenogenic genes controlled by different endosperm-specific promoters into a white maize variety deficient for endosperm carotenoid synthesis. We recovered a diverse population of transgenic plants expressing different enzyme combinations and showing distinct metabolic phenotypes that allowed us to identify and complement rate-limiting steps in the pathway and to demonstrate competition between beta-carotene hydroxylase and bacterial beta-carotene ketolase for substrates in 4 sequential steps of the extended pathway. Importantly, this process allowed us to generate plants with extraordinary levels of beta-carotene and other carotenoids, including complex mixtures of hydroxycarotenoids and ketocarotenoids. Combinatorial transformation is a versatile approach that could be used to modify any metabolic pathway and pathways controlling other biochemical, physiological, or developmental processes.

Substrate Specificities and Availability of Fucosyltransferase and β-Carotene Hydroxylase for Myxol 2′-Fucoside Synthesis in Anabaena sp. Strain PCC 7120 Compared with Synechocystis sp. Strain PCC 6803

To elucidate the biosynthetic pathways of carotenoids, especially myxol 2'-glycosides, in cyanobacteria, Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120) and Synechocystis sp. strain PCC 6803 deletion mutants lacking selected proposed carotenoid biosynthesis enzymes and GDP-fucose synthase (WcaG), which is required for myxol 2'-fucoside production, were analyzed. The carotenoids in these mutants were identified using high-performance liquid chromatography, field desorption mass spectrometry, and (1)H nuclear magnetic resonance. The wcaG (all4826) deletion mutant of Anabaena sp. strain PCC 7120 produced myxol 2'-rhamnoside and 4-ketomyxol 2'-rhamnoside as polar carotenoids instead of the myxol 2'-fucoside and 4-ketomyxol 2'-fucoside produced by the wild type. Deletion of the corresponding gene in Synechocystis sp. strain PCC 6803 (sll1213; 79% amino acid sequence identity with the Anabaena sp. strain PCC 7120 gene product) produced free myxol instead of the myxol 2'-dimethyl-fucoside produced by the wild type. Free myxol might correspond to the unknown component observed previously in the same mutant (H. E. Mohamed, A. M. L. van de Meene, R. W. Roberson, and W. F. J. Vermaas, J. Bacteriol. 187:6883-6892, 2005). These results indicate that in Anabaena sp. strain PCC 7120, but not in Synechocystis sp. strain PCC 6803, rhamnose can be substituted for fucose in myxol glycoside. The beta-carotene hydroxylase orthologue (CrtR, Alr4009) of Anabaena sp. strain PCC 7120 catalyzed the transformation of deoxymyxol and deoxymyxol 2'-fucoside to myxol and myxol 2'-fucoside, respectively, but not the beta-carotene-to-zeaxanthin reaction, whereas CrtR from Synechocystis sp. strain PCC 6803 catalyzed both reactions. Thus, the substrate specificities or substrate availabilities of both fucosyltransferase and CrtR were different in these species. The biosynthetic pathways of carotenoids in Anabaena sp. strain PCC 7120 are discussed.

Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can grow and produce the ketocarotenoid astaxanthin in the dark with glucose as sole carbon and energy source. In the present study, we reported that glucose, mannose, fructose, sucrose, galactose and lactose could differentially support the cell growth and astaxanthin biosynthesis. Of the sugars surveyed, glucose and mannose were the best carbon sources for the algal growth in the dark, as indicated by the relatively high specific growth rates (ca. 0.03 h −1) and high cell dry densities (ca. 10 g l −1). Furthermore, the algal cells cultured with glucose and mannose accumulated the highest amounts of astaxanthin (ca. 1 mg g −1), indicating a correlation between cell growth and astaxanthin formation. In addition, various sugars differentially regulated the transcription of three carotenogenic genes encoding phytoene desaturase (PDS), β-carotene ketolase (BKT), and β-carotene hydroxylase (CHYb) respectively. By using glucose fed-batch fermentation, high cell dry weight concentration (ca. 53 g l −1) and high astaxanthin production (ca. 32 mg l −1) were obtained, which are much higher than those ever reported in the alga. The present study suggests that C. zofingiensis is suitable for the production of natural astaxanthin on a massive scale.

Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Haematococcus pluvialis mutants

Growing culture of green alga Haematococcus was exposed to mutagens such as UV, ethyl methane sulphonate (EMS) and 1-methyl 3-nitro 1-nitrosoguanidine (NTG), and further screened over herbicide - glufosinate. The survival rate of cells decreased with increasing concentration of mutagens and herbicides. The mutants exhibited 23–59% increase in total carotenoid and astaxanthin contents. The NTG treated glufosinate resistant mutant showed increased (2.2% to 3.8% w/w) astaxanthin content. The transcript levels of phytoene synthase, phytoene desaturase, lycopene cyclase, β-carotene ketolase and β-carotene hydroxylase enzymes in the mutant cultures were found to be 13-18, 14-17, 3, 3-22 and 6-20 fold higher respectively compared to wild type. The mutant obtained by UV irradiation showed highest lycopene cyclase activity (458 nmole β-carotene formed/mg protein/h) followed by NTG mutant (315 nmole β-carotene formed/mg protein/h) when compared to that of parent strain (105 nmole β-carotene formed/mg protein/h). Expression analysis of carotenoid biosynthetic genes in the mutants exhibited increase in transcript levels compared to wild type.

4-Ketoantheraxanthin, a novel carotenoid produced by the combination of the bacterial enzyme β-carotene ketolase CrtW and endogenous carotenoid biosynthetic enzymes in higher plants

Higher plants do not ordinarily possess ketocarotenoids due to the absence of a carotenoid ketolase enzyme. We expressed genes coding for marine-bacterial enzymes β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ) in tobacco plants ( Nicotiana tabacum) by transplastomic engineering. A novel carotenoid, 4-ketoantheraxanthin, was isolated from the leaves of the tobacco transformants. The structure of 4-ketoantheraxanthin was determined to be (3 S,3′ S,5′ R,6′ S)-5′,6′-epoxy-3,3′-dihydroxy-β,β-caroten-4-one by analysis of the MS, NMR, and CD data. This carotenoid was considered to be synthesized by a 4-ketolation reaction by CrtW of antheraxanthin that had been synthesized by the endogenous carotenoid biosynthetic enzymes present in higher plants and CrtZ. 4-Ketoantheraxanthin was also shown to have potent antioxidative activity against a 1O 2 suppression model.

Conversion of β-carotene into astaxanthin: Two separate enzymes or a bifunctional hydroxylase-ketolase protein?

Astaxanthin is a xanthophyll of great interest in animal nutrition and human health. The market prospect in the nutraceutics industries for this health-protective molecule is very promising. Astaxanthin is synthesized by several bacteria, algae and plants from β-carotene by the sequential action of two enzymes: a β-carotene, 3,3'-hydroxylase that introduces an hydroxyl group at the 3 (and 3') positions of each of the two β-ionone rings of β-carotene, and a β-carotene ketolase that introduces keto groups at carbons 4 and 4' of the β-ionone rings. Astaxanthin is also produced by the yeast-like basidiomycete Xanthophyllomyces dendrorhous. A gene crtS involved in the conversion of β-carotene to astaxanthin has been cloned simultaneously by two research groups. Complementation studies of X. dendrorhous mutants and expression analysis in Mucor circinelloides reveals that the CrtS enzyme is a β-carotene hydroxylase of the P-450 monooxygenase family that converts β-carotene to the hydroxylated derivatives β-cryptoxanthin and zeaxanthin, but it does not form astaxanthin or the ketolated intermediates in this fungus. A bifunctional β-carotene hydroxylase-ketolase activity has been proposed for the CrtS protein. The evidence for and against this hypothesis is analyzed in detail in this review.

The xanthophyll cycle pool size controls the kinetics of non-photochemical quenching in Arabidopsis thaliana

Arabidopsis plants overexpressing β-carotene hydroxylase 1 accumulate over double the amount of zeaxanthin present in wild-type plants. The final amplitude of non-photochemical quenching (NPQ) was found to be the same in these plants, but the kinetics were different. The formation and relaxation of NPQ consistently correlated with the de-epoxidation state of the xanthophyll cycle pool and not the amount of zeaxanthin. These data indicate that zeaxanthin and violaxanthin antagonistically regulate the switch between the light harvesting and photoprotective modes of the light harvesting system and show that control of the xanthophyll cycle pool size is necessary to optimize the kinetics of NPQ.

An investigation of carotenoid biosynthesis in Coffea canephora and Coffea arabica

Carotenoids are essential components of the photosynthetic apparatus in a wide range of organisms. They participate in the adaptation of plastids to changing environmental light conditions and prevent photo-oxidative damage of the photosynthetic apparatus by detoxifying reactive oxygen species. We identified eight cDNAs from the carotenoid biosynthetic pathway (PSY, PDS, ZDS, PTOX, LCY-E, CRTR-B, ZEP and VDE) and two cDNA encoding carotenoid cleavage dioxygenase family members (NCED3 and CCD1) in Coffea canephora. We also obtained cDNA encoding several different fibrillin proteins involved in carotenoid sequestration (FIB). Expression of the coffee carotenoid genes was determined in leaf, branch and flower tissues using quantitative RT-PCR. Expression analysis of these genes in leaf tissue from osmotically stressed plants was also carried out. These experiments showed that the transcript levels of PTOX, CRTR-B, NCED3, CCD1 and FIB1 increased under these stress conditions, while LCY-E decreased, indicating that the metabolic flux towards the xanthophyll cycle branch of the carotenoid biosynthetic pathway may be favoured in leaves under drought conditions. Functional analysis of CcCRTR-B using an in vivo method employing Escherichia coli strains engineered to make carotenoids confirmed that the beta-carotene hydroxylase activity of CcCRTR-B generates beta-cryptoxanthin and zeaxanthin from beta-carotene. A similar approach was also used to show that CcCCD1 encoded a functional 9,10(9'10') carotenoid cleavage dioxygenase, and thus that this enzyme is capable of forming one or more apocarotenoids in vivo. Finally, high-performance liquid chromatography analysis of coffee leaves revealed the presence of alpha-carotene and suggests that Coffea arabica may have higher levels of alpha-carotene than C. canephora.

Elevated Zeaxanthin Bound to Oligomeric LHCII Enhances the Resistance of Arabidopsis to Photooxidative Stress by a Lipid-protective, Antioxidant Mechanism

The xanthophyll cycle has a major role in protecting plants from photooxidative stress, although the mechanism of its action is unclear. Here, we have investigated Arabidopsis plants overexpressing a gene encoding beta-carotene hydroxylase, containing nearly three times the amount of xanthophyll cycle carotenoids present in the wild-type. In high light at low temperature wild-type plants exhibited symptoms of severe oxidative stress: lipid peroxidation, chlorophyll bleaching, and photoinhibition. In transformed plants, which accumulate over twice as much zeaxanthin as the wild-type, these symptoms were significantly ameliorated. The capacity of non-photochemical quenching is not significantly different in transformed plants compared with wild-type and therefore an enhancement of this process cannot be the cause of the stress tolerant phenotype. Rather, it is concluded that it results from the antioxidant effect of zeaxanthin. 80-90% of violaxanthin and zeaxanthin in wild-type and transformed plants was localized to an oligomeric LHCII fraction prepared from thylakoid membranes. The binding of these pigments in intact membranes was confirmed by resonance Raman spectroscopy. Based on the structural model of LHCII, we suggest that the protein/lipid interface is the active site for the antioxidant activity of zeaxanthin, which mediates stress tolerance by the protection of bound lipids.

Expression of a Ketolase Gene Mediates the Synthesis of Canthaxanthin in Synechococcus Leading to Tolerance Against Photoinhibition, Pigment Degradation and UV-B Sensitivity of Photosynthesis¶

The potential of ketocarotenoids to protect the photosynthetic apparatus from damage caused by excess light and UV-B radiation was assessed. Therefore, the cyanobacterium Synechococcus was transformed with a foreign β-carotene ketolase gene under a strong promoter leading to the accumulation of canthaxanthin. This diketo carotenoid is absent in the original strain. Most of the newly formed canthaxanthin was located in the thylakoid membranes. The endogenous β-carotene hydroxylase was unable to interact with the ketolase. Therefore, only traces of astaxanthin were found. The transformant was treated with strong light (500 or 1200 μmol m−2 s−1) and with UV-B radiation. In contrast to a nontransformed strain the overall photosynthesis, measured as oxygen evolution, was protected from inhibition by light of 500 μmol m−2 s−1 and UV-B radiation of 6.8 W m−2. Furthermore, degradation in the light of chlorophyll and carotenoids at an irradiance of 1200 μmol m−2 s−1, which was substantial in the nontransformed control, was prevented. These results indicate that in situ canthaxanthin, which is formed at the expense of zeaxanthin and replaces this hydroxy carotenoid within the photosynthetic apparatus, is a better protectant against solar radiation. These findings are discussed on the basis of the in vitro properties such as inactivating peroxyl radicals, quenching of singlet oxygen and oxidation stability of these different carotenoid structures.

Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii

A carotenoid gene (crtR-B) from the green alga Haematococcus pluvialis, encoding beta-carotene hydroxylase that was able to catalyze the conversion of beta-carotene to zeaxanthin and canthaxanthin to astaxanthin, was cloned into Chlamydomonas reinhardtii chloroplast expression vector p64D to yield plasmid p64DcrtR-B. The vector p64DcrtR-B was transferred to the chloroplast genome of C. reinhardtii using micro-particle bombardment. PCR and Southern blot analyses indicated that crtR-B was integrated into the chloroplast genome of the transformants. RTPCR assays showed that the H. pluvialis crt R-B gene was expressed in C. reinhardtii transformants. The transformants rapidly synthesized carotenoids in larger quantities than the wild-type upon being transferred from moderate to high-intensity white light. This research provides a foundation for further study to elucidate the possible mechanism of photo-protection by xanthophylls and other carotenoids in high light conditions or through exposure to UV radiation.

Characterization of a β-carotene hydroxylase of Adonis aestivalis and its expression in Arabidopsis thaliana

Carotenoids are plant secondary metabolites that comprise two main groups: carotenes and xanthophylls. The latter group includes zeaxanthin which is synthesized by beta-carotene hydroxylase catalyzing the hydroxylation of the beta-rings of beta-carotene molecules. To develop tools to alter carotenoid biosynthesis in plants, we isolated a cDNA clone encoding a candidate beta-carotene hydroxylase, CrtH1, from the flower petals of Adonis aestivalis. CrtH1 protein has homology to beta-carotene hydroxylases from other organisms, and possesses the four histidine motifs conserved in this family of enzymes. Sequence analysis predicted the presence of a putative plastid transit peptide at the amino terminus and four transmembrane helical regions. Southern-blot analysis showed CrtH1 to be encoded by a multicopy gene family with at least three members in A. aestivalis. Analysis of CrtH1 transcript abundance by Northern blotting indicates it is highly expressed in flower petals, roots and stems, with relatively low expression in leaves and developing seeds. CrtH1 was able to catalyze the formation of zeaxanthin and its intermediate precursor beta-cryptoxanthin from beta-carotene in functional assays conducted in E. coli. Expression of CrtH1 in Arabidopsis thaliana wild type and a mutant deficient for endogenous beta-carotene hydroxylases enhanced the biosynthesis of violaxanthin in the seeds.