Carotenoid Isomerase Is Key Determinant of Petal Color of Calendula officinalis*

Background: Reddish 5-cis-carotenoids accumulate in the orange but not yellow petals of calendula. Results: A CRTISO in orange petals of calendula lacks an isomerase activity. Conclusion: CRTISO activity is a key factor in determining calendula petal color. Significance: Cys-His-His at position 462 and Gly at position 450 of CoCRTISO are important for the isomerase activity. Orange petals of calendula (Calendula officinalis) accumulate red carotenoids with the cis-configuration at the C-5 or C-5′ position (5-cis-carotenoids). We speculated that the orange-flowered calendula is a carotenoid isomerase (crtiso) loss-of-function mutant that impairs the cis-to-trans conversion of 5-cis-carotenoids. We compared the sequences and enzyme activities of CRTISO from orange- and yellow-flowered calendulas. Four types of CRTISO were expressed in calendula petals. The deduced amino acid sequence of one of these genes (CoCRTISO1) was different between orange- and yellow-flowered calendulas, whereas the sequences of the other three CRTISOs were identical between these plants. Analysis of the enzymatic activities of the CoCRTISO homologs showed that CoCRTISO1-Y, which was expressed in yellow petals, converted carotenoids from the cis-to-trans-configuration, whereas both CoCRTISO1-ORa and 1-ORb, which were expressed in orange petals, showed no activity with any of the cis-carotenoids we tested. Moreover, the CoCRTISO1 genotypes of the F2 progeny obtained by crossing orange and yellow lines linked closely to petal color. These data indicate that CoCRTISO1 is a key regulator of the accumulation of 5-cis-carotenoids in calendula petals. Site-directed mutagenesis showed that the deletion of Cys-His-His at positions 462–464 in CoCRTISO1-ORa and a Gly-to-Glu amino acid substitution at position 450 in CoCRTISO1-ORb abolished enzyme activity completely, indicating that these amino acid residues are important for the enzymatic activity of CRTISO.

and tomato (Solanum lycopersicum) mutant tangerine (18). These mutants accumulate prolycopene and other cis-isomers of its upstream precursors. Isaacson et al. (19) demonstrated that tomato CRTISO isomerizes adjacent cis-double bonds at C-7 and C-9 pairwise into the trans-configuration and functions in the carotenoid biosynthesis pathway by converting cis-lycopenes to all-trans-lycopene, a prerequisite for lycopene cyclization.
Accumulation of cis-carotenoids in the orange petals of calendula led us to speculate that the orange-flowered calendula is a crtiso loss-of-function mutant. Here, we cloned genes encoding CRTISO and compared their sequences in orangeand yellow-flowered calendulas. We found four types of CRTISO genes, one of which was expressed in a petal colorspecific manner. We examined the enzymatic activity of the proteins encoded by the CRTISO homologs and showed that CRTISO is a key factor in the accumulation of 5-cis-carotenoids in calendula petals. We also provide useful information regarding a region of CRTISO that is important for its enzymatic activity.

EXPERIMENTAL PROCEDURES
Plant Materials-Four orange-flowered cultivars (Alice Orange, Pompom Orange, Orange Star, and Orange Gem) and four yellow-flowered cultivars (Alice Yellow, Pompom Yellow, Gold Star, and Golden Gem) of calendula (C. officinalis L.) were grown in greenhouses at the National Institute of Floricultural Science (Tsukuba, Ibaraki, Japan) (supplemental Fig. S1).
Cloning of CRTISO cDNAs from Calendula Petals-Total RNA was isolated from the petals of fully opened flowers and mature leaves by using the cetyltrimethylammonium bromide method (21). cDNAs were synthesized by using the SuperScript first strand synthesis system (Invitrogen).
Partial length cDNAs encoding CRTISO and actin were amplified by means of RT-PCR with primers that corresponded to the highly conserved amino acid sequences among eudicot plants. Primer sequences for RT-PCR are shown in supplemental Table S1. cDNAs obtained from the petals of Alice Orange (orange flower) and Alice Yellow (yellow flower) served as PCR templates. Amplified PCR products of appropriate length were cloned into a pCR2.1 vector (TA cloning kit; Invitrogen) and sequenced with a Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA).
We amplified the 5Ј cDNA ends of the gene from the petals of Alice Orange and Alice Yellow with the SMART RACE cDNA amplification kit (Clontech) according to the supplier's protocols. Primers were designed from partial cDNA sequences using the Oligo computer software (Molecular Biology Insights, Cascade, CO). Then we designed 5Ј end primers based on the 5Ј RACE sequence and used them to amplify the full-length cDNAs encoding CRTISO. For full-length cDNA cloning, 3Ј RACE cDNAs were used as templates. Primer sequences for 5Ј RACE and the 5Ј end of CRTISO are shown in supplemental Table S2. Four types of CRTISO homologs were identified using this cloning procedure (designated CoCRTISO1, CoCRTISO2, CoCRTISO3, and CoCRTISO4).
Phylogenetic Analysis and Prediction of the Presence of a Chloroplast/Chromoplast Transit Peptide-Multiple alignments of the deduced amino acid sequences of CRTISO were produced with a Web-based version of ClustalW. The phylogenetic tree was calculated by using the neighbor-joining method and bootstrap analysis (1000 replicates) using PHYLIP via the same Web site and was visualized with Treeview version 1.6.6. Prediction of the presence of chloroplast/chromoplast transit peptides (cTP) in the protein sequences and the location of potential cTP cleavage sites were carried out using the ChloroP 1.1 server (22).
Quantitative Real Time PCR Analysis-We performed real time quantitative PCR to quantify the mRNAs of the CoCRTISO homologs in petals and leaves. Total RNAs were isolated from the petals of fully opened flowers and mature leaves by means of the cetyltrimethylammonium bromide method (21). cDNAs were synthesized by using the SuperScript first strand synthesis system (Invitrogen) from total RNA treated with DNase I. Transcript levels were analyzed with the SYBR Premix Ex Taq kit (TaKaRa, Shiga, Japan) and a Thermal Cycler Dice real time system (TaKaRa) according to the manufacturer's instructions. Primers specific to each CRTISO homolog for PCR were designed using the Oligo software based on the full-length cDNA sequences, avoiding homologous regions among the CoCRTISO homologs, and were checked for specific product formation by using PCR. Actin primers were also designed from the partial cDNA sequence. Primer sequences are shown in supplemental Table  S3. The concentration of actin mRNA in each sample was determined to normalize the values and account for differences in the amount of total RNA.
Site-directed Mutagenesis of CRTISO and Construction of Expression Vectors-The coding regions of CoCRTISO homologs were amplified by using PCR with 5Ј end primers with the BstZ17I restriction site, 3Ј end primers with the SalI restriction site, and Prime STAR HS DNA polymerase (TaKaRa). Primer sequences are shown in supplemental Table  S4. The open reading frame of each gene was shortened by deleting the predicted transit peptide sequence. The fragments were cloned into a pBlunt-TOPOII vector (Invitrogen) and digested with BstZ17I and SalI. Then the fragments were subcloned into a pMAL-c2x vector (New England Biolabs, Ipswich, MA) digested with XmnI and SalI. The recombinant proteins contained a maltose-binding protein at the C terminus.
Additionally, we made a CoCRTISO1 site-directed mutant series, named m1 to m11 (see Table 1 and Fig. 1). The mutations were introduced by use of overlap extension PCR (23): fragments containing 20 -50-bp overlap regions were amplified by using PCR (first PCR) with plasmid DNAs as templates and with mutagenic primers. The fragments were mixed and subjected to PCR amplification by using a 5Ј end primer with a BstZ17I restriction site and a 3Ј end primer with a SalI restriction site for CoCRTISO1 (second PCR). Primer sequences and the template and primer combinations for the first PCR are shown in supplemental Tables S5 and S6. The amplified products were subcloned into a pBlunt-TOPOII vector and digested with BstZ17I and SalI. The fragments were then inserted into a pMAL-c2x vector digested with XmnI and SalI.
Expression and Extraction of CoCRTISO in Escherichia coli-E. coli cells of the strain BL21 carrying the pMAL-CoCRTISO plasmids were grown in LB medium containing ampicillin (100 mg liter Ϫ1 ) at 37°C until absorbance at 550 nm reached 0.5. Isopropyl thio-␤-D-galactoside was then added (0.5 mM) for 12-15 h at 25°C to induce the expression of the recombinant genes. Bacterial cells were harvested, resuspended in one-tenth volume of cold incubation buffer (1% Triton-X, 100 mM Tris, 10 mM MgCl 2 , pH 7.4), and ruptured by using an ultrasonic cell disruptor at 10 -15 W m Ϫ2 (Microson XL; Misonix, Farmingdale, NY). The lysate was centrifuged at 13,000 ϫ g for 15 min at 4°C. The clear supernatant (soluble fraction) was collected and kept on ice, either for the in vitro assay or for protein purification. Affinity chromatography of recombinant proteins was carried out by use of an amylose resin column (New England Biolabs) according to the manufacturer's instructions.
In Vitro Enzyme Assay-An in vitro enzyme assay was carried out basically as described by Isaacson et al. (19). Carotenoids (ϳ1 mg ml Ϫ1 ) were dissolved in incubation buffer by sonication. To test the activity of CoCRTISO1 and its mutant enzymes, we incubated 500 l of crude enzyme extracts containing ϳ20 g of substrate at 30°C in the dark for 3 h. To test the enzymatic activity and substrate specificity of CoCRTISO1, -2, -3, and -4, we incubated 200 g of purified enzymes, 25 mg of catalase, 150 mg of glucose oxidase, 750 mg of glucose, and ϳ5 g of substrate in a total volume of 500 l of incubation buffer at 30°C in the dark for 3 h. After the reaction, 200 l of acetone and 200 l of diethyl ether were added to the reaction mixture, which was then centrifuged at 10,000 ϫ g for 5 min. The organic phase was collected, dried in vacuo, dissolved in 100 l of methanol, and subjected to HPLC analysis.
Reciprocal Crossing between Orange-and Yellow-flowered Calendula-To investigate the inheritance of flower color, we crossed orange-and yellow-flowered calendulas for two generations. Calendula is allogamous, so no seeds are set by selfpollination. Therefore, we crossed the cultivars as follows: first, we created eight lines by crossing orange-flowered with orangeflowered cultivars (Alice Orange ϫ Orange Star, Alice Orange ϫ Orange Gem, and Orange Star ϫ Alice Orange), yellow-flowered with orange-flowered cultivars (Alice Yellow ϫ Alice Orange, Gold Star ϫ Orange Star, and Alice Orange ϫ Alice Yellow), and yellow-flowered with yellow-flowered cultivars (Golden Gem ϫ Alice Yellow and Alice Yellow ϫ Golden Gem) and analyzed the flower color of the progeny (F 1 generation). Next, we created an F 2 generation to analyze the segregation of flower color (supplemental Fig. S2). It was difficult to obtain sufficient quantities of F 2 seeds from the crosses between the above-mentioned F 1 progenies because of inbreeding depression; therefore, we used two lines, the orange-flowered progeny of Alice Orange ϫ Orange Star and the yellowflowered progeny of Alice Yellow ϫ Golden Gem, as parents for the crossing test. The progeny of these lines were crossed with each other, and the seeds obtained were analyzed as the F 2 generation.
Isolation of Genomic DNA and Genomic PCR to Identify the Genotype of CoCRTISO1-The genomic DNA of F 2 plants was isolated from immature leaves by using Nucleon PhytoPure (GE Healthcare) according to the manufacturer's instructions. To identify the genotypes of the F 2 plants and the calendula cultivars, we performed genomic PCR. Primer sequences specific to each CoCRTISO1 homolog are shown in supplemental Table S7. PCR amplification was carried out in a TP-3000 thermal cycler (TaKaRa) with Z-Taq DNA polymerase (TaKaRa) according to the manufacturer's instructions.

RESULTS
Cloning of CRTISO Genes from Calendula Petals-We performed RT-PCR-mediated cloning of CRTISO genes by using primers that corresponded to the highly conserved region among eudicot plants and isolated four CRTISO homologs from an orange-flowered Alice Orange cultivar and from a yellow-flowered Alice Yellow cultivar (supplemental Fig. S3). The full-length sequence of one of these homologs (designated CoCRTISO1) differed between Alice Orange and Alice Yellow. The sequences of the other homologs (designated CoCRTISO2, -3, and -4) were identical between the two cultivars. In addition, we isolated full-length CoCRTISO1 cDNA clones from orangeand yellow-flowered cultivars. All of the CoCRTISO1 sequences from the yellow-flowered cultivars were identical (designated CoCRTISO1-Y). In contrast, the CoCRTISO sequences from the orange-flowered cultivars were of two types (designated CoCRTISO1-ORa and CoCRTISO1-ORb). CoCRTISO1-ORa was isolated from Alice Orange and Orange Star; CoCRTISO1-ORb was isolated from Pompom Orange and Orange Gem (Fig. 1). The deduced amino acid sequences of CoCRTISO1-Y, -1-ORa, and -1-ORb consisted of 648, 645, and 648 amino acids, respectively. The polypeptide of CoCRTISO1-ORa includes four amino acid substitutions and the deletion of a three-amino acid sequence (Cys-His-His), whereas the polypeptide of CoCRTISO1-ORb includes four amino acid substitutions compared with CoCRTSO1-Y. The deduced amino acid sequence of CoCRTISO1-Y showed 96, 88, and 87% similarity with those of CoCRTISO2, CoCRTISO3, and CoCRTISO4, respectively. CoCRTISO1-Y also showed 78% similarity with CmCRTISO, found in chrysanthemum (Chrysanthemum morifolium) (supplemental Fig. S3), 74% with AtCRTISO from arabidopsis, and 73% similarity with SlCRTISO from tomato. CoCRTISO homologs contained cTPs at the N terminus, indicating that they would likely be transported to the chromoplast. The predicted lengths of the cTPs were 49 amino acids in CoCRTISO1, -2, and -3 and 69 amino acids in CoCRTISO4.
Expression of CoCRTISO in Petals and Leaves-We designed primer sets specific to each CoCRTISO homolog for real time quantitative PCR analysis and compared the expression levels of these homologs between orange-and yellow-flowered cultivars. We found that CoCRTISO1-Y was expressed only in the petals and leaves of yellow-flowered cultivars and that CoCRTISO1-OR was expressed only in the petals and leaves of orange-flowered cultivars (Fig. 2). On the other hand, CoCRTISO2, -3, and -4 were expressed in the petals of both orange-and yellow-flowered cultivars. The expression levels of CoCRTISO2, -3, and -4 were very low compared with those of CoCRTISO1; they were ϳ1/150 to 1/2000 of those of CoCRTISO1-Y. All homologs, except CoCRTISO2, were expressed at higher levels in petals than in leaves.
In Vitro Enzymatic Activity of CoCRTISO1 and Its Mutant Polypeptides-We examined the enzymatic activity of CoCRTISO1-Y, -1-ORa, and -1-ORb expressed in E. coli. E. coli cells carrying pMAL-CoCRTISO1-Y, -1-ORa, or -1-ORb accumulated a polypeptide of an apparent molecular mass of ϳ100 kDa, matching the predicted size of the mature CoCRTISO1 polypeptide (58 kDa) plus the maltose-binding protein (42 kDa) (supplemental Fig. S4). Crude enzyme extracts (soluble fraction of an E. coli lysate) were used to assay the enzymatic activity of CoCRTISO1 in vitro. Equivalent extracts of E. coli cells transformed with the empty vector pMAL-c2x served as controls. Carotenoids extracted from tangerine tomato fruits (which contain mainly prolycopene) and from the orange petals of calendula (which contain 5-cis-carotenoids) served as substrates. Compared with the control, CoCRTISO1-Y caused an increase in the percentage of (all-E)-lycopene in the extracts from both tangerine tomato and calendula, indicating that CoCRTISO1-Y functioned as an isomerase for the cis-to-trans conversion (Fig. 3, Table 1, and supplemental Table S8). On the other hand, reaction mixtures of CoCRTISO1-ORa and -1-ORb produced the same HPLC chromatograms as that of the control, indicating that these proteins had no isomerase activity.

In Vitro Enzymatic Activity and Substrate Specificity of
CoCRTISO-We analyzed differences in the enzyme activity and substrate specificity of the CoCRTISO homologs. Enzymes were purified with an amylose resin from crude cell extracts of E. coli carrying pMAL-CoCRTISO1, -2, -3, and -4. Western blot analysis showed that the purified fractions contained the desirable polypeptides of an apparent molecular mass of ϳ100 kDa (supplemental Fig. S4).
Inheritance of Petal Color and the Relationship between CoCRTISO1 Genotype and Petal Color-To determine the inheritance of petal color of calendula, we performed crossing experiments. All of the F 1 progenies obtained from crosses between yellow-and yellow-flowered cultivars and between yellow-and orange-flowered cultivars had yellow petals, and all of the F 1 progenies obtained from crosses between orange-and orange-flowered cultivars had orange petals. The yellow-flowered progenies in the crosses between yellow-and orange-flowered lines were crossed with each other, and the petal color of the F 2 progenies was analyzed (supplemental Fig. S2). The 146 F 2 progenies segregated for 102 yellow-flowered and 44 orangeflowered individuals. These results indicate that the yellow petal color was dominant over orange and was a monogenic character ( Table 2).
Genomic PCR with primers specific to either CoCRTISO1-Y or -1-OR was performed to examine the genotypes of CoCRTISO in the F 2 progenies. All of the orange-flowered F 2 progenies showed DNA amplification only with primers specific to CoCRTISO1-OR (Table 2 and Fig. 5). On the other hand,  Table 1). Percentages of the total peak area are shown in supplemental Table S8. Peak 1, prolycopene ((7Z,9Z,7ЈZ,9ЈZ)-lycopene); peak 2, (all-E)-lycopene.

Orange-flowered Calendula Is a crtiso Mutant
of the 102 yellow-flowered F 2 progenies, 69 showed DNA amplification with primers specific to both CoCRTISO1-Y and -1-OR, and 33 progenies showed DNA amplification only with primers specific to CoCRTISO1-Y. Additionally, in the yellowflowered cultivars tested, amplification was detected only with primers specific to CoCRTISO1-Y; in the orange-flowered cultivars tested, amplification was detected only with primers specific to CoCRTISO1-OR. These results indicate that the orange-flowered progenies had a homozygous genotype of CoCRTISO1-OR and that the yellow-flowered progenies had a homozygous genotype of CoCRTISO1-Y or a heterozygous genotype of CoCRTISO1-Y and -1-OR. All of the orange-and yellow-flowered cultivars tested had homozygous genotypes of CoCRTISO1-OR and CoCRTISO1-Y, respectively.

DISCUSSION
Our previous work demonstrated that the orange petal color of calendula is due to the accumulation of reddish cis-carotenoids, mainly 5-cis-lycopenes (12). Based on the assumption that a difference in CRTISO enzyme activity between yellowand orange-flowered calendula was responsible for the difference in petal color, we analyzed nucleotide sequences and enzymatic activities of CoCRTISO homologs between yellow-and orange-flowered calendula. Recently, CRTISO sequences from various plant species have been deposited in DNA databases. However, no plant in the databases appears to have multiple types of CRTISO genes, except maize (24). Here, we showed that calendula CRTISO forms a small multigene family of at least four CRTISO homologs (CoCRTISO1, -2, -3, and -4) that were expressed in petals (supplemental Fig. S3). Deduced amino acid sequences of CoCRTISO2, -3, and -4 were completely identical between orange-and yellow-flowered calendulas, whereas there were several differences between CoCRTISO1-Y from yellow-flowered calendula and    CoCRTISO1-ORa and -1-ORb from orange-flowered calendula. Real time quantitative PCR analysis showed that CoCRTISO1-Y was expressed only in yellow-flowered cultivars and that CoCRTISO1-OR (total of -1-ORa and -1-ORb) was expressed only in orange-flowered cultivars. These results suggest that CoCRTISO1 is a key determinant of the petal color of calendula.
CoCRTISO2, -3, and -4 were expressed in all cultivars regardless of petal color; however, the expression levels of these homologs in petals were extremely low compared with that of CoCRTISO1: 1/150 to 1/2000 of CoCRTISO1-Y. These results indicate that, in yellow petals, most of the CRTISO activity is derived from CoCRTISO1-Y, and most 5-cis-carotenoids are converted to trans-carotenoids. By contrast, in orange petals, the total CRTISO activity is low because of the lack of the isomerase activity of both CoCRTISO1-ORa and -1-ORb, which may result in the accumulation of 5-cis-carotenoids.
To test the hypothesis, we crossed orange-and yellow-flowered cultivars (supplemental Fig. S2). All of the F 1 progenies obtained by reciprocal crosses between orange-and yellowflowered cultivars had yellow petals, indicating that yellow petal color is dominant over orange. Moreover, the F 2 progenies showed an ϳ3:1 (yellow:orange) segregation ratio ( Table 2). In addition, genomic PCR analysis of F 2 progenies with primers specific to either CoCRTISO1-Y or -1-OR (Fig. 5) showed that all of the CoCRTISO1 genotypes were closely linked to the petal color of the F 2 progenies: orange-flowered progenies had a homozygous genotype of CoCRTISO1-OR, and yellow-flowered progenies had either a homozygous genotype of CoCRTISO1-Y or a heterozygous genotype of CoCRTISO1-Y and -1-OR. These results support the hypothesis that CoCRTISO1-Y and -1-OR determine the petal color of calendula.
To clarify the cause of the loss-of-function of CoCRTISO1-ORa and -1-ORb, we performed site-directed mutagenesis of the CoCRTISO1 homologs. The polypeptide of CoCRTISO1-ORa includes four amino acid substitutions and the deletion of a three-amino acid sequence, whereas that of CoCRTISO1-ORb includes four amino acid substitutions compared with CoCRTISO1-Y (Fig. 1). We then constructed a series of 11 sitedirected mutant genes and tested the enzymatic activities of their translation products to determine which divergent amino acids cause the inactivation of CoCRTISO1-ORa and -1-ORb (Table 1). We found that deletion of Cys-His-His at positions 462-464 in CoCRTISO1-ORa and a Gly-to-Glu substitution at position 450 in CoCRTISO1-ORb completely abolished the enzyme activity. These amino acid residues are highly conserved from cyanobacteria to higher plants, suggesting that they are essential for CRTISO enzyme activity. This is the first report demonstrating the importance of those amino acid residues for CRTISO activity.
In conclusion, we propose that the mechanism regulating the amount of 5-cis-carotenoids in calendula petals is as follows: in yellow petals, 5-cis-carotenoids, which are produced during the course of carotenoid biosynthesis, are changed into trans-carotenoids mainly by CoCRTISO1-Y. The trans form ends of the polyene chain are immediately cyclized and hydroxylated, and finally these carotenoids accumulate as yellow xanthophylls. On the other hand, the orange petals have low CRTISO activity because CoCRTISO1-OR, a major CoCRTISO homolog expressed in these petals, is inactive. Total CRTISO activity, which is derived from CoCRTISO2, -3, and -4, is insufficient for the cis-to-trans conversion of the substantial amount of 5-ciscarotenoids in orange petals; therefore, only a fraction of the 5-cis-carotenoids is converted to the trans form, leaving the rest of the 5-cis-carotenoids to accumulate. The results obtained in this study demonstrate that the conversion of lycopene from the cis-to the trans-configuration is an important process for lycopene cyclization. The question of how 5-cis-carotenoids are synthesized remains. Further research is needed on the function of PDS and ZDS to fully understand carotenoid biosynthesis in the petals of calendula.