Summary
To better understand the mechanisms regulating plant carotenoid metabolism in staple crop, we report the map‐based cloning and functional characterization of the Glycine max carotenoid cleavage dioxygenase 4 (GmCCD4) gene, which encodes a carotenoid cleavage dioxygenase enzyme involved in metabolizing carotenoids into volatile β‐ionone. Loss of GmCCD4 protein function in four Glycine max increased carotenoid content (gmicc) mutants resulted in yellow flowers due to excessive accumulation of carotenoids in flower petals. The carotenoid contents also increase three times in gmicc1 seeds. A genome‐wide association study indicated that the GmCCD4 locus was one major locus associated with carotenoid content in natural population. Further analysis indicated that the haplotype‐1 of GmCCD4 gene was positively associated with higher carotenoid levels in soybean cultivars and accumulated more β‐carotene in engineered E. coli with ectopic expression of different GmCCD4 haplotypes. These observations uncovered that GmCCD4 was a negative regulator of carotenoid content in soybean, and its various haplotypes provide useful resources for future soybean breeding practice.
Keywords: GmCCD4 gene, genetic mapping, carotenoids, yellow flower, soybean
Introduction
Carotenoids are widely distributed in plants, algae and bacteria, with more than 750 members in nature. Carotenoids are important for the human diet as they are the only precursors for vitamin A biosynthesis (Giuliano et al., 2008). In humans, carotenoids promote antioxidant activity and reduce age‐related macular degeneration of the eye (Davies, 2007; Fraser and Bramley, 2004; Krinsky and Johnson, 2005). Previous studies have shown that carotenoids dissolved in oil or aqueous dispersions are efficiently absorbed (>50%) (Blomstrand and Werner, 1967; Goodman et al., 1966), while carotenoids in uncooked vegetables, such as β‐carotene in carrots or lycopene in tomato juice, are poorly absorbed (<3%) (Chug‐Ahuja et al., 1993; Khachik et al., 1992; Stahl and Sies, 1992). Given the significance of carotenoids in the prevalence of vitamin A deficiency in developing countries, a better understanding of the mechanisms regulating plant carotenoid composition is essential, especially in edible seeds of staple crops (Chandler et al., 2013; Gonzalezjorge et al., 2013; Shewmaker et al., 1999).
Animals are unable to synthesize carotenoids de novo and must obtain these compounds via ingestion (Zhai et al., 2016). Plant carotenoid biosynthesis pathway has been elucidated from previous studies, Ɛ‐carotene desaturase (ZDS), carotenoid isomerases (ZISO and CRTISO) and cyclase (LCY) are four key regulatory enzymes of twelve enzymes in this pathway (Fantini et al., 2013; Nisar et al., 2015). ZISO, ZDS and CRTISO catalyse Ɛ‐carotene to all‐trans‐lycopene, the latter could be cyclized to α‐carotene and β‐carotene to originate two biosynthesis sub‐pathways of lutein and neoxanthin, respectively. α‐carotene is further hydroxylated by β‐OHases (encoded by LUT5) and Ɛ‐OHases (encoded by LUT1) to produce zeinoxanthin and lutein; β‐carotene is further hydroxylated by β‐Ohases to produce zeaxanthin (Cazzaniga et al., 2012; Kim et al., 2009). Zeaxanthin epoxidase (ZEP) hydroxylates the β‐rings of zeaxanthin to yield antheraxanthin and then violaxanthin; this reaction can be inverted by violaxanthin de‐epoxidase (VDE) (Hieber et al., 2000). Violaxanthin is converted to neoxanthin by neoxanthin synthase (NSY) in the final step of the core carotenoid biosynthetic pathway (Nisar et al., 2015).
Steady‐state carotenoid accumulation depends on the metabolic equilibrium between carotenoid biosynthesis/storage and carotenoid degradation in plants (Hannoufa and Hossain, 2012; Li and Yuan, 2013). Thus, the catalytic activity of carotenoid cleavage oxygenases (CCOs), which results in the enzymatic breakdown of C40 carotenoids into apocarotenoids, is critical for regulating carotenoid accumulation. The CCO exists in two forms, namely CCD (Carotenoid Cleavage Dioxygenase) and NCED (Nine‐Cis Epoxycarotenoid Dioxygenase) (Auldridge et al., 2006; Priya et al., 2019). In Arabidopsis, four CCDs (CCD1, CCD4, CCD7, CCD8) and five NCEDs (NCED2, NCED3, NCED5, NCED6, NCED9) were identified (Bouvier et al., 2005). CCD1 and CCD4 cleavage carotenoids to form volatile small apocarotenoids (Hou et al., 2016; Nacke et al., 2012; Pu et al., 2020; Yahyaa et al., 2015; Zhang et al., 2015; Zheng et al., 2019). CCD7/MAX3 and CCD8/MAX4 are involved in the synthesis of strigolactones (SLs) (Alder et al., 2012; Gomez‐Roldan et al., 2008; Wang et al., 2019; Wang et al., 2020). The five members of the NCED sub‐group are exclusively involved in cleavage of violaxanthin and neoxanthin to form ABA (Fantini et al., 2013).
Recent studies have increased our understanding of carotenoid metabolism, genetic regulation and genetic manipulation for high‐carotenoid cultivars in higher plants (Ashraf et al., 2015; Bai et al., 2016; Pu et al., 2020; Sui et al., 2016; Zeng et al., 2015; Zheng et al., 2019). Soybean (Glycine max) products are increasingly consumed by humans worldwide, as this protein‐ and oil‐dense food meets human nutritional needs and improves human living standards. While there are plenty of carotenoid metabolism pathways that remain to be learned in higher plants, the genetic underpinnings understood to date have been successfully translated to significantly elevate levels of carotenoids in soybean, transgenic seeds accumulated carotenoid 60–741 µg/gr seed by transforming the phytoene synthase (crtB) gene from Pantoea ananatis (Pierce et al., 2015), 845 µg/gr seed by overexpressing a seed‐specific bacterial phytoene synthase gene from Pantoea ananatis (Schmidt et al., 2014), over 800 µg/g seed by expressing the maize phytoene synthase and 500 µg/g seed by expressing the β‐carotene hydroxylase (CrtZ), β‐carotene ketolase (CrtW) genes from Brevundimonas sp., along with the maize phytoene synthase (Park et al., 2017), which the latter, includes the synthesis of the high value carotenoid astaxanthin in the seed. However, the function of soybean gene in carotenoid metabolism is still lacking. The only reported studies are limited to the correlation between the expression level of GmCCD1/GmCCD4 and lutein content (Kanmaru et al., 2009), and the transcriptional response of carotenoid oxygenase genes to abiotic stresses (Wang et al., 2013). In this study, to assess the genetic control of carotenoid levels in soybean, a forward genetic strategy was employed to identify mutants with yellow flower and increased carotenoid content in a γ‐irradiated mutant population. We found that GmCCD4 was a negative regulator of carotenoids accumulation in soybean, and the strong association between polymorphisms in the GmCCD4 gene and carotenoid content among soybean cultivars. This work illustrated that Glycine max carotenoid cleavage dioxygenase 4 (GmCCD4), encoding CCD4 in soybean, catalysed the cleavage of carotenoids to regulate the carotenoids turnover.
Results
Identification of soybean yellow flower mutants
We screened 100,000 γ‐ray‐induced mutants, generated from Williams 82, for yellow flowers, and identified four mutants with flowers of different shades of yellow (Figure 1a). A slightly differences in seed coat or cotyledon colour were observed between the four mutants and WT (Figure S1). The mutants, in addition to yellow flowers, exhibited considerably increased carotenoid contents compared with the WT (Figure 1b,c). The four mutants were termed Glycine max increased carotenoid content 1, 2, 3 and 4 (gmicc1, 2, 3, and 4). The total carotenoid concentrations of the flowers and seeds in the gmicc mutants were over threefold and twofold higher than the WT, respectively (Table S1). Compared with WT, all kinds of carotenoids in flowers of gmicc mutants were significantly increased except zeinoxanthin, with 13‐fold increase in zeaxanthin, threefold in antheraxanthin, 19‐fold in lutein, threefold in violaxanthin, sixfold in neoxanthin and threefold in α‐carotene (Figure 1b). In comparison with WT, gmicc seeds had higher levels of β‐carotene, lutein and zeaxanthin in seeds, with 11‐fold increase in β‐carotene, threefold in lutein and twofold in zeinoxanthin (Figure 1c). The increased carotenoid contents in the gmicc seeds as compared to the WT were primarily due to increases in the β‐carotene and lutein concentrations (Figure 1b,c; Table S1). Genetic allelic tests were conducted by crossing each of these three mutants with the gmicc1 mutant, and the all three types of intercrossed F1 hybrids exhibited the mutated yellow flower phenotype. This demonstrated that gmicc1, 2, 3 and 4 were genetically allelic to each other (Table S2).
Cloning and characterization of the candidate gene of gmicc mutants
In order to identify the mutation responsible for the observed phenotypic changes, Hedou 12, a Chinese soybean cultivar with purple flowers (Song et al., 2015), was crossed with the gmicc1 mutant to obtain an F2 population of 976 individuals. Of these 976 plants, 706 exhibited the WT white flower phenotype and 270 exhibited the mutant yellow flower phenotype. The segregating 2.61:1 ratio between white and yellow flower phenotypes indicated that these phenotypic changes were controlled by a single recessive locus (fitting the 3:1 ratio of one gene mutation segregation, χ2 test, p = 0.18).
The candidate locus was mapped using 270 F2 individual mutants with the mutant yellow flower phenotype. Using 165 previously developed INDEL markers (Song et al., 2015), the candidate locus was mapped to a genomic region between MOL0885 and MOL0857 on Chromosome 1; this region is 6.6 Mb in the soybean reference genome sequence (G. max Wm82.a2.v1) (Schmutz et al., 2010) (Figure 2a). The candidate locus was further located in a 3‐kb region between MOL3492 and MOL3452, with 27 recombinants (Figure 2a; Table S3). This region harbours one gene, Glyma.01G154900.1, based on the WT reference genome (G. max Wm82.a2.v1). This gene and its flanking sequences were sequenced within the defined Glyma.01G154900.1 region of the WT and gmicc1 mutant, and a single nucleotide deletion in the coding region of Glyma.01G154900.1 (the 877th G in the first exon) was identified (Figure 2b). This deletion caused a frameshift leading to a premature stop codon and truncated protein (Figure 2c). The Glyma.01G154900.1 protein contains a RPE65 domain, which was affected by the mutation in the mutant gmicc1 protein and caused the loss of two of the four catalytic sites (Figure 2c). The mutations did not cause significant alterations in the expression levels of the Glyma.01G154900.1 gene in the flowers of the gmicc mutants except gmicc2 (Figure S2a) suggesting that disruption in the protein function caused the gmicc mutant phenotype.
These results suggested that Glyma.01G154900.1 was the candidate gene. In order to verify this supposition, the genomic regions of the Glyma.01G154900.1 gene in the gmicc1, 2,3 and 4 mutants were amplified (Figure S2b). In the gmicc2 mutant, there was a large deletion abolishing the entire Glyma.01G154900.1 gene. In the gmicc3 mutant, there were two single nucleotide mutations (from C to T at position + 445 bp and from C to G at position + 1,918 bp) and three single nucleotide insertions (the A at position + 1,887 bp, A at position + 1,897 bp and T at position + 1,926 bp), which leading to 15 amino acids substitutes (Figure 2b,c). In the gmicc4 mutant, there was a single nucleotide mutation (from G to A at position + 407 bp), which leading to one amino acid substitute in the Retinal Pigment Epithelium 65 kDa protein (RPE65) domain (Figure 2b,c) (Chander et al., 2012; Kiser et al., 2012; Kloer, 2005; Priya et al., 2016). All these mutations caused either complete loss of the entire protein or functional loss of the Glyma.01G154900.1 protein (Figure 2c). In combination, the sequencing results and the allelism tests revealed that independent mutations in Glyma.01G154900.1 always resulted in yellow flowers and increased carotenoid content.
In order to confirm these results, a 7 kb genomic fragment, including 3 kb upstream and 1 kb downstream sequences of the Glyma.01G154900.1 gene, was introduced into the gmicc1 mutant using Agrobacterium tumefaciens‐mediated transformation (Yamada et al., 2010). Three independent complementation transgenic lines in the gmicc1 background exhibited the white flower colour and reduced carotenoid contents in both seeds and flowers same as WT (Figure 2d, Figure S3a, b and Table S1). These transgenic plants were verified by detection of inserted genes (Figure S3c,d), bar protein immunoassay (Figure S3d) and exogenous transcripts (Figure S3e). The transcripts of both exogenous transformed wild‐type and mutated gmicc1 were found in these transgenic plants in the gmicc1 background (Figure S3e), and there was no significant difference in the expression levels of the Glyma.01G154900.1 among WT, gimicc1 and transgenic plants (Figure S3f). This result also demonstrated that Glyma.01G154900.1 was indeed the candidate gene.
The mutated candidate gene encoded GmCCD4
Previous bioinformatic study assigned the Glyma.01G154900.1 gene as GmCCD4 in soybean (Kanmaru et al., 2009), we followed this pre‐existing name as the target gene of our mutants in this study. In order to determine the phylogenetic relationships and functional conservation of this protein across different species, 42 homologs of the AT4G19170 (CCD4) gene from nine species were aligned and used to construct a neighbour‐joining (NJ) phylogenetic tree (Table S4). The phylogenetic analysis of the GmCCD4 and GmCCD4‐like proteins revealed that GmCCD4 was most likely a single copy gene with high identity to the Arabidopsis CCD4 gene (Figure 3a). A synteny analysis of the 87,000 bp region surrounding AtCCD4 (beginning at 10,450,000 bp and ending at 10,537,000 bp on A. thaliana chromosome 4) was performed against the G. max genome using the webtool MCScanX (Wang et al., 2012). The synteny plot showed that the gene arrangement of the AtCCD4 flanking sequence was highly syntenic to the gene arrangements of G. max chromosomes 1 and 11. However, the GmCCD4 homologue was lost from the homologous region of chromosome 11 (Figure S4a). Putative copies of GmCCD4 were searched for in wild and cultivar soybean genomes and proteomes (Kim et al., 2010; Li et al., 2014; Zhou et al., 2015); these searches revealed that GmCCD4 was a single copy gene among these sequenced soybeans.
The gene expression levels of GmCCD4 were quantified in both vegetative and reproductive organs of the WT plants, including the roots, stems, leaves, stem apical meristems (SAMs), inflorescences, unopened flowers, opened flowers, pods and seeds using real‐time quantitative PCR (qPCR). GmCCD4 was most highly expressed in opened flowers, followed by pods and unopened flowers, while GmCCD4 gene was expressed the lowest in roots (Figure S4b). Besides the seven carotenoid cleavage dioxygenases, three 9‐cis epoxycarotenoid dioxygenases were identified in the soybean (Figure S5). The expression levels of the carotenoid cleavage dioxygenases and the 9‐cis epoxycarotenoid dioxygenases were detected in the opened flowers of the WT, gmicc1 mutant and transgenic complementation lines. Of the nine GmCCD‐like genes, GmCCD1‐1, GmCCD7‐1, GmCCD7‐2, GmNCED2, GmNCED3‐1 and GmNCED3‐2 were the most highly expressed in opened flowers of the gmicc1 mutant compared with the WT and complementation lines (Figure 3b). The significant increase of GmCCD7‐1 expression might due to the substrate similarity between GmCCD4 and GmCCD7‐1. This suggested that lack of GmCCD4 function induced the expression of other GmCCD‐like genes in the gmicc1 mutant.
GmCCD4 catalysed the degradation of β‐ and/or α‐carotenes
Most plant CCD4s catalyse the oxidative cleavage of carotenoids into volatile apocarotenoids at the double‐bond position 9, 10 (9’, 10’) (Huang et al., 2009; Rubio et al., 2008; Zhang et al., 2015). In order to determine the cleavage reaction of carotenoids catalysed by GmCCD4, volatile apocarotenoids were analysed in the petals of the WT, gmicc1 mutant and complementation lines. A major peak, with a retention time of 14.42 min, was detected in the volatile compounds released from the white petals of the WT and T2 complementation lines (Figure 4a; Figure S6a‐c); the mass spectrum of this peak was consistent with that of β‐ionone (Figure 4b). In contrast, the yellow petals of the gmicc1 mutant did not release β‐ionone (Figure 4a).
In order to investigate the enzymatic characteristics of GmCCD4 and mutation proteins, GmCCD4, gmicc1, gmicc3 and gmicc4 genes were introduced into Escherichia coli strains previously engineered to accumulate β‐carotene. The yellow colour was retained in E. coli cells transformed with the pET32a‐gmicc1, pET32a‐gmicc3 and pET32a‐gmicc4, while the yellow colour was lost in E. coli cells transformed with pET32a‐GmCCD4 (Figure 4c). β‐carotene accumulated in E. coli cells carrying the mutated GmCCD4 proteins, indicating that gmicc proteins did not cleave this carotenoid. In contrast, lycopene accumulated in E. coli carrying either GmCCD4 or gmicc (Figure 4d), indicating that neither of these proteins cleaved lycopene. These results indicated that GmCCD4 encodes a functional CCD enzyme and the yellow flower phenotype of the gmicc mutants was caused by the inhibition of CCD enzymatic activity.
GC‐MS analysis of the headspace of E. coli cells harbouring GmCCD4 and gmicc1 indicated that these cells generated β‐methylionone. In addition, β‐methylionone was present only in cells harbouring GmCCD4; β‐methylionone was absent in cells harbouring gmicc1 and the pET32a plasmid (Figure S6d–f). Based on the chemical structures of β‐ and/or α‐carotenes, the β‐ionone released in the WT and complementation lines were the product of β‐ and/or α‐carotenes cleaved preferentially at the double‐bond position 9, 10 by GmCCD4 (Figure S6g). We speculated that the generation of β‐methylionone in E. coli cells might have been the result of the microbial transformation of β‐ionone in bacteria.
Genome‐wide association study of carotenoids and variations of GmCCD4
A genome‐wide association study (GWAS) of carotenoid contents with four million markers generated through genome‐resequencing among 182 cultivars was conducted (Table S5). The distribution of β‐carotenoid contents among these cultivars looked approximately normal and seemed appropriate for GWAS (Figure S7). As shown in the Manhattan plots and quantile–quantile for β‐carotenoid content, we found 3 notable positive association (p ≤ 7.66×10‐ 7 in the standard mixed linear model) loci, including the GmCCD4 locus (Figure 5a,b). Further analysis showed that the GmCCD4 locus explained about 16.84% of the variance in β‐carotenoid content across the entire population of 182 cultivars. Of the SNPs identified in the GmCCD4 locus, 11, including five non‐synonymous, were located in the exon region, one was located in the 5’UTR, and 20 were located in the intron region (Table S6 and S7). These results further demonstrated that GmCCD4 was a possible candidate gene of genetic variation in carotenoid content across the examined soybean germplasms.
To further investigate whether GmCCD4 polymorphism was related to the carotenoid content, we identified four haplotypes among the 182 soybean germplasms, based on the five non‐synonymous SNPs in the GmCCD4 locus (Figure 5c). These five SNPs were located in the first exon of GmCCD4 gene, SNP1 and SNP3 caused significant hydrophobicity changes among haplotypes (Table S7), which could influence various noncovalent bonds, such as the interaction between enzyme and substrate, antibody, and antigen (Bloemendal et al., 1989). Even though SNP4 and SNP5 were in the PRR65 domain, but the hydrophobicity changes were smaller than SNP1 and SNP3 (Table S7). The hydrophobicity change of SNP2 was similar as SNP4 and SNP5; however, it was co‐segregated with SNP3. Among these five non‐synonymous SNPs, SNP2 and SNP3 were positively associated with higher carotenoid content in seeds (Figure 5c). According to SNP2 and SNP3 polymorphisms, the four haplotypes were divided into two subgroups, H1 and H2/3/4. Among H2/3/4, the characteristic polymorphism variation of H2 was SNP4 and SNP5, and H3 was SNP1, respectively (Figure 5c). Interestingly, large proportions of the cultivars carried haplotype 1 and had higher levels of β‐carotenoids (Figure 5d; Figure S8).
Analysis of genotype and phenotype relationship of GmCCD4
Pedigree linkage analysis were also performed in an F2 population that was derived from a cross between low‐carotenoid line Heinong 46 (haplotype 2) and high‐carotenoid line Hefeng 39 (haplotype 1). A total 119 F2 individuals were collected for genotyping and phenotyping. The F2 plants were genotyped as three groups according to the GmCCD4 genotype: haplotype 1, haplotype 2, and heterozygous. The β‐carotene contents of plants in the different groups were significantly different. That is, the F2 individuals carrying haplotype 1 had the highest levels of β‐carotene, while the F2 individuals carrying haplotype 2 had the lowest levels of β‐carotene; the heterozygous F2 individuals had intermediate levels of β‐carotene (Figure 6a). These results further indicated that haplotype 1 of GmCCD4 was positively associated with higher carotenoid content.
To investigate the enzymatic characteristics of the GmCCD4 proteins in cultivars with different haplotypes, GmCCD4 genes with different haplotype were cloned from cultivars Heihe 43 (H1), Heinong 67 (H2), Hefeng 7 (H3) and Hudou 9765 (H4), and introduced into E. coli strains previously engineered to accumulate β‐carotene. The yellow colour was retained in E. coli cells transformed with the pET32a vector while the yellow colour was lost in E. coli cells transformed with four haplotypes of GmCCD4 (Figure 6b). Absorbance was measured every 40 min at 453 nm to monitor changes in β‐carotene levels. The E. coli cells transformed with haplotype‐1 of GmCCD4 accumulated more β‐carotene than cells transformed with any of the other three GmCCD4 haplotypes (Figure 6c). This indicated that haplotype‐1 of GmCCD4 exhibited the lowest enzymatic activity in degrading β‐carotene. However, the enzyme activities of four different haplotypes of GmCCD4 showed no significant difference in vitro. These observations indicated that four kinds of GmCCD4 encode functional CCD4 enzyme in cleaving β‐carotene with no significant changes in enzyme activity in vitro.
GmCCD4 affected the dynamic equilibrium between carotenes and xanthophylls
The expression of GmLUT5 and GmLUT1 increased 50% and 130% in the gmicc1 mutant compared with the WT and transgenic complementation lines (Figure 7). The expression of GmZEP‐2 decreased 122% in the gmicc1 mutant compared with the WT and transgenic complementation lines; the expression level of GmZEP‐1 and GmVDE exhibited no significant changes between the gmicc1 mutant and WT (Figure 7). The expression of GmNSY increased 40% in the gmicc1 mutant compared with the WT and transgenic complementation lines (Figure 7). It suggested that accumulation of antheraxanthin and violaxanthin in mutant might due to the expression changes of GmZEP‐2 and GmNSY or the mutation of GmCCD4. Nevertheless, the metabolites of carotenoids were preferentially influenced by the mutation of GmCCD4.
Furthermore, there were no significant differences in yield indices (i.e. seed number, plant height, seed weight and plant height) between the WT and gmicc mutants (Figure 8). These observations indicated that losing the function of GmCCD4 had limited influences on yield and other metabolite pathways, therefore, the GmCCD4 gene could be a candidate bioengineering gene used for the propagation and proliferation of more nutritional soybeans.
Discussion
Carotenoids endow flowers and fruits with distinct colours (e.g. yellow, orange and red colours) (Grotewold, 2006), which then attract animals for pollination or seed dispersal (Kevan and Baker, 1983). Carotenoids are also photo protectants, antioxidants and accessory pigments in photosynthesis (Grotewold, 2006; Walter and Strack, 2011). Compared with other members of the CCD family, disruption of CCD4 often causes colour variations in flowers, fruits and tubers. The functional CCD4 gene is likely to be the major determinant in flower colour related to carotenoid accumulation (Brandi et al., 2011; Campbell et al., 2010; Ohmiya et al., 2009; Ohmiya et al., 2012). In Chrysanthemum, CmCCD4a is specifically transcribed in flowers; loss of function of CmCCD4 leads to the change of flower colour from white to yellow (Ohmiya et al., 2006). In Brassica, four variations, including two INDELs and two insertions of transposable elements in the Bnac3.CCD4, disrupt the function of BnaC3.CCD4 and change the petal colour from white to yellow (Zhang et al., 2015). In Lonicera japonica, LjCCD4 plays important roles in dynamic flower coloration by cleaving carotenoids to apocarotenoids, and the expression pattern of LjCCD4 is negatively correlated with the carotenoid content (Pu et al., 2020). CCD4s also contribute to the divergence of yellow and white colour in potato tubers and peach fruit flesh (Brandi et al., 2011; Campbell et al., 2010; Fantini et al., 2013). Consistent with previous studies, loss of GmCCD4 protein function in four gmicc mutants resulted in yellow flower colour in our study.
Until now, we firstly report yellow flower mutants in soybean, which are not existed in nature. Our four yellow flower mutants were selected from our laboratory 100,000 soybeans mutant population during the past ten years, we speculate that the huge amount of mutant screening is one of important reasons to find this novel phenotype. Interestingly, we also notified that some of the cultivars with much higher β‐carotene than gmicc mutants in seeds, but their flowers remain white colour. This implies that different regulation networks of carotenoid turnover might be employed in flower and seed. The conflict of flower colour and seed carotenoids accumulation was also observed in Arabidopsis, the petals of the knockout mutant ccd4‐1 still kept white colour (personal correspondence from Professor Dean DellaPenna), even though the carotenoid content in seeds of the ccd4‐1 mutant increased relative to the wild type (Gonzalezjorge et al., 2013). Besides GmCCD4 locus, two other loci were also detected associating with seed carotenoid content in GWAS analysis experiment, which indicated that at least three regulators are involved in seed carotenoid content regulation. This suggests that various factors are involved in turnover of carotenoid in different organs, and the yellow flower phenotype might also appear in specific genetic background.
In C. sativus, the transcriptome analysis indicates that various TFs in carotenoid‐accumulating tissues possibly participate in the regulation of CsCCD expression in a TF‐dependent manner (Bouvier et al., 2003; Sui et al., 2016). In citrus, a 5’ cis‐regulatory change at CCD4b is a major genetic determinant of natural variation in C30 apocarotenoids responsible for red coloration of citrus peel (Zheng et al., 2019). Based on five non‐synonymous SNPs, four haplotypes of GmCCD4 were identified among cultivars, which is tightly related to the carotenoid content. However, the enzyme activities of four different haplotypes of GmCCD4 showed no significant difference in vitro. This suggests that these SNP polymorphisms could not directly regulate the enzyme activity of GmCCD4 to control the accumulation of carotenoids, which might be likely through response to some cis‐ and trans‐factors as above. It is worthy to investigate the regulation network of carotenoid turnover mechanism in soybean in the future, which could bring more target genes for soybean improvement in the future.
Experimental procedures
Plant materials, DNA extraction and genetic mapping
Two soybean cultivars Hedou 12 and Williams 82, the WT, were obtained from the Chinese Academy of Agricultural Sciences (Beijing, China). Four gmicc mutants were identified by screening for yellow flowers among 100,000 WT γ‐irradiated mutants (Cheng et al., 2016). The plant heights were measured at R8 stage; the seed number per plant, total‐seed weight and weight of hundred seeds were measured after harvesting. Genomic DNA was extracted from both cultivars and from gmicc mutants using a DNeasy Plant Mini Kit (Qiagen, CA). Anchor markers used for primary mapping were retrieved from Song et al. (2015). Fine mapping markers of the GmCCD4 region were developed using INDELs between Hedou 12 and WT following previously described methods (Song et al., 2015). All primers used are provided in Table S3.
Plasmid construction and transformation
The GmCCD4 gene (including 3 kb upstream and 1 kb downstream sequences) was amplified using the primers OL6826F and OL6826R from WT and inserted into the pCAMBIA3301 (Cambia) vector between restriction endonuclease SacI and BamHI sites to generate the ProGmCCD4: GmCCD4 plasmid. The ProGmCCD4: GmCCD4 plasmid was introduced into Agrobacterium tumefaciens strain EHA105 and then transformed to gmicc1 mutants following the Agrobacterium‐mediated transformation protocol previously described by Yamada et al. (2010). PAT/bar quick test was conducted according the manufacturer (PAT/bar EPSPS LFD Strips, Youlong Biotech, China).
Phylogenetic analysis
CCD protein sequences similar to the GmCCD4 enzyme were identified in various plant species using Phytozome V12 (Schmutz et al., 2010). In total, 42 peptide sequences were selected and aligned using CLUSTALW (Thompson et al., 1994). All positions with <5% alignment gaps, missing data and ambiguous bases were tolerated at any position. The final alignment had 309 positions. Then, an NJ phylogenetic tree was constructed based on this alignment using MEGA7 (Kumar et al., 2016; Saitou and Nei, 1987). The bootstrap consensus tree, inferred from 1,000 replicates, represented the evolutionary history of the taxa analysed (Felsenstein, 1985). Branches corresponding to partitions reproduced in ≤ 50% of all bootstrap replicates were collapsed. Evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965).
RNA isolation and real‐time quantitative PCR analysis
Total RNA was isolated from soybean SAMs, leaves, stems, roots, inflorescences, unopened flowers, opened flowers, pods and seeds using TRNzol (TIANGEN, Beijing, China) following the manufacturer's instructions. cDNA was synthesized using a FastQuant RT Kit (with gDNase) (TIANGEN, Beijing, China), following the manufacturer's instructions. Relative gene expression was quantified with RT‐qPCR using a FastStart Universal SYBR Green Master (ROX) (Roche, Mannheim, Germany) in a Stratagene Mx3005P sequence Detection System (Applied Biosystems, Waldbrann, Germany) following the manufacturer's instructions. Three biological replicates were analysed to quantify the levels of gene expression, and three technical replicates were performed. Relative gene expression was calculated using the ATP‐binding cassette transporter gene (Cons4, Glyma.12G020500) as a control (Liu et al., 2016; Ping et al., 2014).
Co‐expression of GmCCD4 and carotenoid biosynthetic enzymes in E. coli
The full‐length cDNAs of GmCCD4, gmicc1, gmicc3 and gmicc4 were amplified from the WT, gmicc1, gmicc3 and gmicc4 mutants, respectively, using the primers OL6116F and OL6116R (containing the restriction enzyme sites EcoRI and HindIII, respectively). Amplified cDNAs were digested and cloned into the pET32a expression vector. The full‐length cDNAs of CCD4 and CCD1 were amplified from the flowers of Arabidopsis Col‐0. CCD4 was amplified with the primers, OL6117F and OL6117R, which contained the restriction enzyme sites EcoRI and SalI, respectively. CCD1 was amplified with the primers, OL6118F and OL6118R, which contained the restriction enzyme sites EcoRI and BamHI, respectively. Amplified cDNAs were digested and cloned into the pET32a expression vector for expression in E. coli strains.
GmCCD4/gmicc1/gmicc3/gmicc4/AtCCD4/AtCCD1 and the carotenoid biosynthetic enzymes, β‐carotene‐pACCAR16ΔcrtX and lycopene‐pACCRT‐EIB, were co‐expressed following previously described methods (Huang et al., 2009; Tian et al., 2014) with slight modifications. Briefly, the plasmids of the pET32a vector (pET32a‐AtCCD4, pET32a‐AtCCD1, pET32a‐GmCCD4, pET32a‐gmicc1, and pET32a‐gmicc3 and pET32a‐gmicc4) were introduced into E. coli strain, BL21 (DE3), which had been previously transformed with the carotenoid biosynthetic genes encoding β‐carotene (pACCAR16ΔcrtX) or lycopene (pACCRT‐EIB) (Misawa et al., 1995). The E. coli strain was inoculated in 100 mL of Luria‐Bertani (LB) medium and cultured at 37 °C until the optical density at a wavelength of 600 nm (OD600) reached 0.4–0.6. Then, 1 mM isopropyl‐β‐D‐thiogalactopyranoside was added to LB solid medium and recombinant proteins were induced at 28°C for 4 h. GmCCD4 genes with different haplotype were cloned from cultivars Heihe 43 (H1), Heinong 67 (H2), Hefeng 7 (H3) and Hudou 9765 (H4), introduced into Escherichia coli strains and expressed.
Extraction and quantification of carotenoids
In order to measure carotenoid concentration, seeds and flower petals were weighed, freeze‐dried for 17.5 h and ground with a Mixer Mill MM 400 (Retsch, Haan, Germany). Carotenoids were extracted following previously described methods (Yang et al., 2015). Then, the compounds were separated in 10 µL aliquots extract using an Exion UPLC coupled to a QTRAP 6500 PLUS (Sciex) equipped with a Luna silicagel column (3 μm; 150 mm × 2.0 mm) (Phenomenex) at 40°C. The mobile phases were acetonitrile (ACN):methanol:dichloromethane (80:15:5 [v:v:v]; A) and ACN:methanol:dichloromethane (30:20:50 [v:v:v]; B), with a flow rate of 0.8 mL/min at the following gradient: 0–18 min, 5% buffer B increased to 70%; 19–20 min, buffer B reduced to 5%; and 20–22 min, buffer B held at 5%. Compounds in the carotenoids were identified based on characteristic absorption spectra, typical retention times and standards published by CaroNature Co. (Bern, Switzerland). Carotenoids were quantified with standard calibration curves, and cholesterol‐d6 was used as an internal recovery control. The β‐carotenoid content in cultivars was calculated using the absorbance value measured in a Nanophotometer (IMPLEM, Germany) at 453 nm.
Analysis of volatile compounds in petals and bacteria
The identification of volatile compounds released from petals and bacteria carrying GmCCD4 and gmicc1 was conducted following the methods previously described by Tian et al. (2014) and Zhang et al. (2015). The volatile compounds released in the headspace were analysed using headspace solid‐phase microextraction gas chromatography–mass spectrometry (SPME‐GC‐MS).
Population genetics analysis and GWAS/Genetic diversity analysis
The original sequences of 182 cultivars were filtered using the Fastp software (Chen et al., 2018) and aligned with the reference genome of soybean (version: Gmax_275_Wm82.a2.v1) using the Burrows‐Wheeler Aligner (BWA) tool (Li and Durbin, 2009). SNP data (unpublished) were performed using the HapoltypeCaller method provided by GATK software (Mckenna et al., 2010) and used for the GWAS. SNPs with> 10% missing data or with minor allele frequencies (MAFs) of < 5% were removed. Genome‐wide association mapping was implemented in TASSEL following the mixed linear model (Bradbury et al., 2007). We used Storey and Tibshirani method (Storey and Tibshirani, 2003) to calculate Q value based on p value of GWAS analysis result. Then, the threshold value is determined by Q value ≤ 0.1. Finally, the threshold was set as p ≤ 7.66×10‐ 7 (‐log10 p = 6.12).
Conflict interest
The authors declare there is no conflict interest.
Author contributions
X.Z.F. and S.X.Y. conceived and designed the research study, experiments and wrote the manuscript. J.S.G. conducted experiments and contributed to the experimental design under the supervision of X.Z.F. X.G. and B.L. participated in preparation of the E. coli strains engineered to accumulate β‐carotene. Lastly, K.Q.T., S.D.W. and G.L. analysed the genome data.
Supporting information
Acknowledgements
This work was supported by the National Key Research and Development Project from the Ministry of Science and Technology of China (No. 2016YFD0101900), Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA0801010502) and programs from National Natural Science of China (No. 31571692 and 31471743). This work was also supported by the National Postdoctoral Program for Innovative Talents (No. BX201700242) and China Postdoctoral Science Foundation (No. 2017M621226).
Gao, J. , Yang, S. , Tang, K. , Li, G. , Gao, X. , Liu, B. , Wang, S. and Feng, X. (2021) GmCCD4 controls carotenoid content in soybeans. Plant Biotechnol. J., 10.1111/pbi.13506
References
- Alder, A. , Jamil, M. , Marzorati, M. , Bruno, M. , Vermathen, M. , Bigler, P. , Ghisla, S. et al. (2012) The path from β‐carotene to carlactone, a strigolactone‐like plant hormone. Science 335, 1348–1351. [DOI] [PubMed] [Google Scholar]
- Ashraf, N. , Jain, D. and Vishwakarma, R. (2015) Identification, cloning and characterization of an ultrapetala transcription factor CsULT1 from Crocus: a novel regulator of apocarotenoid biosynthesis. BMC Plant Biol. 15, 25. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Auldridge, M.E. , Mccarty, D.R. and Klee, H.J. (2006) Plant carotenoid cleavage oxygenases and their apocarotenoid products. Curr. Opin. Plant Biol. 9, 315–321. [DOI] [PubMed] [Google Scholar]
- Bai, C. , Capell, T. , Berman, J. , Medina, V. , Sandmann, G. , Christou, P. and Zhu, C. (2016) Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor‐product balance. Plant Biotechnol. J. 14, 195–205. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bloemendal, M. , Marcus, Y. , Sijpkes, A.H. and Somsen, G. (1989) Role of hydrophobicity in protein structure is overestimated. Int. J. Pept. Protein Res. 34, 405–408. [DOI] [PubMed] [Google Scholar]
- Blomstrand, R. and Werner, B. (1967) Studies on the intestinal absorption of radioactive beta‐carotene and vitamin A in man. Conversion of beta‐carotene into vitamin A. Scand. J. Clin. Lab. Invest 19, 339–345. [DOI] [PubMed] [Google Scholar]
- Bouvier, F. , Dogbo, O. and Camara, B. (2003) Biosynthesis of the food and cosmetic plant pigment bixin. Science 300, 2089–2091. [DOI] [PubMed] [Google Scholar]
- Bouvier, F. , Isner, J.C. , Dogbo, O. and Camara, B. (2005) Oxidative tailoring of carotenoids: a prospect towards novel functions in plants. Trends Plant Sci. 10, 187–194. [DOI] [PubMed] [Google Scholar]
- Bradbury, P.J. , Zhang, Z. , Kroon, D.E. , Casstevens, T.M. , Ramdoss, Y. and Buckler, E.S. (2007) TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633–2635. [DOI] [PubMed] [Google Scholar]
- Brandi, F. , Bar, E. , Mourgues, F. , Horvath, G. , Turcsi, E. , Giuliano, G. , Liverani, A. et al. (2011) Study of 'Redhaven' peach and its white‐fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism. BMC Plant Biol. 11, 24. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Campbell, R. , Ducreux, L.J.M. , Morris, W.L. , Morris, J.A. , Suttle, J.C. , Ramsay, G. , Bryan, G.J. et al. (2010) The Metabolic and developmental roles of carotenoid cleavage Dioxygenase4 from potato. Plant Physiol. 154, 656–664. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cazzaniga, S. , Li, Z. , Niyogi, K.K. , Bassi, R. and Dallosto, L. (2012) The Arabidopsis szl1 mutant reveals a critical role of β‐carotene in photosystem I photoprotection. Plant Physiol. 159, 1745–1758. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chander, P. , Gentleman, S. , Poliakov, E. and Redmond, T.M. (2012) Aromatic residues in the substrate cleft of RPE65 protein govern retinol isomerization and modulate its progression. J. Biol. Chem. 287, 30552–30559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chandler, K. , Lipka, A.E. , Owens, B.F. , Li, H. , Buckler, E.S. , Rocheford, T. and Gore, M.A. (2013) Genetic analysis of visually scored orange kernel color in maize. Crop Sci. 53, 189–200. [Google Scholar]
- Chen, S. , Zhou, Y. , Chen, Y. and Gu, J. (2018) fastp: an ultra‐fast all‐in‐one FASTQ preprocessor. Bioinformatics 34, 884–890. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng, W. , Gao, J.S. , Feng, X.X. , Shao, Q. , Yang, S.X. and Feng, X.Z. (2016) Characterization of dwarf mutants and molecular mapping of a dwarf locus in soybean. J. Integ. Agricult. 15, 60345–60347. [Google Scholar]
- Chug‐Ahuja, J.K. , Holden, J.M. , Forman, M.R. , Mangels, A.R. , Beecher, G.R. and Lanza, E. (1993) The development and application of a carotenoid database for fruits, vegetables, and selected multicomponent foods. J. Am. Diet. Assoc. 93, 318–323. [DOI] [PubMed] [Google Scholar]
- Davies, K.M. (2007) Genetic modification of plant metabolism for human health benefits. Mut. Res.‐Fund. Mol. Mechan. Mutagenesis 622, 122–137. [DOI] [PubMed] [Google Scholar]
- Fantini, E. , Falcone, G. , Frusciante, S. , Giliberto, L. and Giuliano, G. (2013) Dissection of tomato lycopene biosynthesis through virus‐induced gene silencing. Plant Physiol. 163, 986–998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Felsenstein, J. (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39, 783–791. [DOI] [PubMed] [Google Scholar]
- Fraser, P.D. and Bramley, P.M. (2004) The biosynthesis and nutritional uses of carotenoids. Prog. Lipid Res. 43, 228–265. [DOI] [PubMed] [Google Scholar]
- Giuliano, G. , Tavazza, R. , Diretto, G. , Beyer, P. and Taylor, M.A. (2008) Metabolic engineering of carotenoid biosynthesis in plants. Trends Biotechnol. 26, 139–145. [DOI] [PubMed] [Google Scholar]
- Gomez‐Roldan, V. , Fermas, S. , Brewer, P.B. , Puech‐Pagès, V. , Dun, E.A. , Pillot, J.‐P. , Letisse, F. et al. (2008) Strigolactone inhibition of shoot branching. Nature 455, 189–194. [DOI] [PubMed] [Google Scholar]
- Gonzalezjorge, S. , Ha, S.H. , Magallaneslundback, M. , Gilliland, L.U. , Zhou, A. , Lipka, A.E. , Nguyen, Y.N. et al. (2013) Carotenoid cleavage dioxygenase4 is a negative regulator of β‐carotene content in Arabidopsis seeds. Plant Cell 25, 4812–4826. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goodman, D.S. , Blomstrand, R. , Werner, B. , Huang, H.S. and Shiratori, T. (1966) The intestinal absorption and metabolism of vitamin A and beta‐carotene in man. J. Clin. Investig. 45, 1615–1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grotewold, E. (2006) The genetics and biochemistry of floral pigments. Annu. Rev. Plant Biol. 57, 761–780. [DOI] [PubMed] [Google Scholar]
- Hannoufa, A. and Hossain, Z. (2012) Regulation of carotenoid accumulation in plants. Biocatal. Agric. Biotechnol. 1, 198–202. [Google Scholar]
- Hieber, A.D. , Bugos, R.C. and Yamamoto, H.Y. (2000) Plant lipocalins: violaxanthin de‐epoxidase and zeaxanthin epoxidase. Biochem. Biophys. Acta. 1482, 84–91. [DOI] [PubMed] [Google Scholar]
- Hou, X. , Rivers, J. , León, P. , Mcquinn, R.P. and Pogson, B.J. (2016) Synthesis and function of apocarotenoid signals in plants. Trends Plant Sci. 21, 792–803. [DOI] [PubMed] [Google Scholar]
- Huang, F.C. , Molnár, P. and Schwab, W. (2009) Cloning and functional characterization of carotenoid cleavage dioxygenase 4 genes. J. Exp. Bot. 60, 3011–3022. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kanmaru, K. , Hayase, T. , Yamada, T. and Kitamura, K. (2009) Characterization of candidate genes involved in catabolism of lutein in soybean seeds and metabolic engineering for generation of lutein rich soybean. Soy Protein Res. Japan 12, 46–50. [Google Scholar]
- Kevan, P. and Baker, H. (1983) Insects as flower visitors and pollinators. Annu. Rev. Entomol. 28, 407–453. [Google Scholar]
- Khachik, F. , Goli, M.B. , Beecher, G.R. , Holden, J. , Lusby, W.R. , Tenorio, M.D. and Barrera, M.R. (1992) Effect of food preparation on qualitative and quantitative distribution of major carotenoid constituents of tomatoes and several green vegetables. J. Agricult. Food Chem. 40, 390–398. [Google Scholar]
- Kim, M.y , Lee, S. , Van, K. , Kim, T‐h , Jeong, S‐c , Choi, I‐y , Kim, D‐s et al. (2010) Whole‐genome sequencing and intensive analysis of the undomesticated soybean (Glycine soja Sieb. and Zucc.) genome. Proc. Natl Acad. Sci. USA 107, 22032–22037. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim, J. , Smith, J.J. , Tian, L. and Dellapenna, D. (2009) The evolution and function of carotenoid hydroxylases in Arabidopsis . Plant Cell Physiol. 50, 463–479. [DOI] [PubMed] [Google Scholar]
- Kiser, P.d , Farquhar, E.r , Shi, W. , Sui, X. , Chance, M.r and Palczewski, K. (2012) Structure of RPE65 isomerase in a lipidic matrix reveals roles for phospholipids and iron in catalysis. Proc. Natl Acad. Sci. USA 109, 2747–2756. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kloer, D.P. (2005) The structure of a retinal‐forming carotenoid oxygenase. Science 308, 267–269. [DOI] [PubMed] [Google Scholar]
- Krinsky, N.I. and Johnson, E.J. (2005) Carotenoid actions and their relation to health and disease. Mol. Aspects Med. 26, 459–516. [DOI] [PubMed] [Google Scholar]
- Kumar, S. , Stecher, G. and Tamura, K. (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li, H. and Durbin, R. (2009) Fast and accurate short read alignment with Burrows‐Wheeler transform. Bioinformatics 25, 1754–1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li, L. and Yuan, H. (2013) Chromoplast biogenesis and carotenoid accumulation. Arch. Biochem. Biophys. 539, 102–109. [DOI] [PubMed] [Google Scholar]
- Li, Y.‐h. , Zhou, G. , Ma, J. , Jiang, W. , Jin, L.‐g. , Zhang, Z. , Guo, Y. et al. (2014) De novo assembly of soybean wild relatives for pan‐genome analysis of diversity and agronomic traits. Nat. Biotechnol. 32, 1045–1052. [DOI] [PubMed] [Google Scholar]
- Liu, Y. , Zhang, D. , Ping, J. , Li, S. , Chen, Z. and Ma, J. (2016) Innovation of a regulatory mechanism modulating semi‐determinate stem growth through artificial selection in soybean. PLoS Genet. 12, e1005818. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McKenna, A. , Hanna, M. , Banks, E. , Sivachenko, A. , Cibulskis, K. , Kernytsky, A. , Garimella, K. et al. (2010) The genome analysis toolkit: a mapreduce framework for analyzing next‐generation DNA sequencing data. Genome Res. 20, 1297–1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Misawa, N. , Satomi, Y. , Kondo, K. , Yokoyama, A. , Kajiwara, S. , Saito, T. , Ohtani, T. et al. (1995) Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J. Bacteriol. 177, 6575–6584. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nacke, C. , Hüttmann, S. , Etschmann, M.M.W. and Schrader, J. (2012) Enzymatic production and in situ separation of natural β‐ionone from β‐carotene. J. Indust. Microbiol. Biotechnol. 39, 1771–1778. [DOI] [PubMed] [Google Scholar]
- Nisar, N. , Li, L. , Lu, S. , Khin, N.C. and Pogson, B.J. (2015) Carotenoid metabolism in plants. Mol. Plant 8, 68–82. [DOI] [PubMed] [Google Scholar]
- Ohmiya, A. , Kishimoto, S. , Aida, R. , Yoshioka, S. and Sumitomo, K. (2006) Carotenoid cleavage dioxygenase (CmCCD4a) contributes to white color formation in chrysanthemum petals. Plant Physiol. 142, 1193–1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ohmiya, A. , Sumitomo, K. and Aida, R. (2009) “Yellow Jimba”: suppression of carotenoid cleavage dioxygenase (CmCCD4a) expression turns white chrysanthemum petals yellow. J. Japan. Soc. Horticult. Sci. 78, 450–455. [Google Scholar]
- Ohmiya, A. , Toyoda, T. , Watanabe, H. , Emoto, K. , Hase, Y. and Yoshioka, S. (2012) Mechanism behind petal color mutation induced by heavy‐ion‐beam irradiation of recalcitrant chrysanthemum cultivar. J. Japan. Soc. Horticult. Sci. 81, 269–274. [Google Scholar]
- Park, H. , Weier, S. , Razvi, F. , Peña, P.A. , Sims, N.A. , Lowell, J. , Hungate, C. et al. (2017) Towards the development of a sustainable soya bean‐based feedstock for aquaculture. Plant Biotechnol. J. 15, 227–236. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pierce, E.C. , Lafayette, P.R. , Ortega, M.A. , Joyce, B.L. , Kopsell, D.A. and Parrott, W.A. (2015) Ketocarotenoid production in soybean seeds through metabolic engineering. PLoS One 10, e0138196. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ping, J. , Liu, Y. , Sun, L. , Zhao, M. , Li, Y. , She, M. , Sui, Y. et al. (2014) Dt2 is a gain‐of‐function MADS‐domain factor gene that specifies semideterminacy in soybean. Plant Cell 26, 2831–2842. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Priya, R. , Dass, J.F.P. and Siva, R. (2016) Gene expression prediction and hierarchical clustering analysis of plant CCD genes. Plant Mol. Biol. Rep. 34, 618–627. [Google Scholar]
- Priya, R. , Sneha, P. , Dass, J.F.P. , Doss C, G.P. , Manickavasagam, M. and Siva, R. (2019) Exploring the codon patterns between CCD and NCED genes among different plant species. Comp. Biol. Med. 114, 103449. [DOI] [PubMed] [Google Scholar]
- Pu, X. , Li, Z. , Tian, Y. , Gao, R. , Hao, L. , Hu, Y. , He, C. et al. (2020) The honeysuckle genome provides insight into the molecular mechanism of carotenoid metabolism underlying dynamic flower coloration. New Phytol. 227, 930–943. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rubio, A. , Rambla, J.L. , Santaella, M. , Gómez, M.D. , Orzaez, D. , Granell, A. and Gómez‐Gómez, L. (2008) Cytosolic and plastoglobule‐targeted carotenoid dioxygenases from Crocus sativus are both involved in beta‐ionone release. J. Biol. Chem. 283, 24816–24825. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saitou, N. and Nei, M. (1987) The neighbor‐joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. [DOI] [PubMed] [Google Scholar]
- Schmidt, M.A. , Parrott, W.A. , Hildebrand, D.F. , Berg, R.H. , Cooksey, A. , Pendarvis, K. , He, Y. et al. (2014) Transgenic soya bean seeds accumulating β‐carotene exhibit the collateral enhancements of oleate and protein content traits. Plant Biotechnol. J. 13, 590–600. [DOI] [PubMed] [Google Scholar]
- Schmutz, J. , Cannon, S.B. , Schlueter, J. , Ma, J. , Mitros, T. , Nelson, W. , Hyten, D.L. et al. (2010) Genome sequence of the palaeopolyploid soybean. Nature 463, 178–183. [DOI] [PubMed] [Google Scholar]
- Shewmaker, C.K. , Sheehy, J.A. , Daley, M. , Colburn, S. and Ke, D.Y. (1999) Seed‐specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects. J. Cell Sci. 20, 401–412. [DOI] [PubMed] [Google Scholar]
- Song, X. , Wei, H. , Cheng, W. , Yang, S. , Zhao, Y. , Li, X. , Luo, D. et al. (2015) Development of INDEL markers for genetic mapping based on whole genome resequencing in soybean. G3 5, 2793–2799. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stahl, W. and Sies, H. (1992) Uptake of lycopene and its geometrical isomers is greater from heat‐processed than from unprocessed tomato juice in humans. J. Nutr. 122, 2161–2166. [DOI] [PubMed] [Google Scholar]
- Storey, J.D. and Tibshirani, R. (2003) Statistical significance for genomewide studies. Proc. Natl. Acad. Sci. USA 100, 9440–9445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sui, X. , Zhang, J. , Golczak, M. , Palczewsk, K. and Kiser, P.D. (2016) Key residues for catalytic function and metal coordination in a carotenoid cleavage dioxygenase. J. Biol. Chem. 291, 19401–19412. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thompson, J.D. , Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions‐specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tian, X. , Ji, J. , Wang, G. , Jin, C. , Guan, C. and Wu, G. (2014) Molecular cloning and characterization of a novel carotenoid cleavage dioxygenases 1 from Lycium chinense . Biotechnol. Appl. Biochem. 62, 772–779. [DOI] [PubMed] [Google Scholar]
- Walter, M.H. and Strack, D. (2011) Carotenoids and their cleavage products: biosynthesis and functions. Nat. Product Rep. 28, 663–692. [DOI] [PubMed] [Google Scholar]
- Wang, J.Y. , Haider, I. , Jamil, M. , Fiorilli, V. , Saito, Y. , Mi, J. , Baz, L. et al. (2019) The apocarotenoid metabolite zaxinone regulates growth and strigolactone biosynthesis in rice. Nat. Commun. 10, 810. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang, Y. , Shang, L. , Yu, H. , Zeng, L. , Hu, J. , Ni, S. , Rao, Y. et al. (2020) A strigolactone biosynthesis gene contributed to the green revolution in rice. Mol. Plant 13, 1–10. [DOI] [PubMed] [Google Scholar]
- Wang, Y. , Tang, H. , DeBarry, J.d , Tan, X. , Li, J. , Wang, X. , Lee, T‐h et al. (2012) MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 40, e49. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang, R.K. , Wang, C.E. , Fei, Y.Y. , Gai, J.Y. and Zhao, T.J. (2013) Genome‐wide identification and transcription analysis of soybean carotenoid oxygenase genes during abiotic stress treatments. Mol. Biol. Rep. 40, 4737–4745. [DOI] [PubMed] [Google Scholar]
- Yahyaa, M. , Berim, A. , Isaacson, T. , Marzouk, S. and Ibdah, M. (2015) Isolation and functional characterization of carotenoid cleavage dioxygenase‐1 from Laurus nobilis L. (Bay Laurel) Fruits. J. Agricult. Food Chem. 63, 8275–8282. [DOI] [PubMed] [Google Scholar]
- Yamada, T. , Watanabe, S. , Arai, M. , Harada, K. and Kitamura, K. (2010) Cotyledonary node pre‐wounding with a micro‐brush increased frequency of Agrobacterium‐mediated transformation in soybean. Plant Tissue Cult. Lett. 27, 217–220. [Google Scholar]
- Yang, S. , Zhou, Q. , Yang, L. , Xue, Y. , Xu, J. and Xue, C. (2015) Effect of thermal processing on astaxanthin and astaxanthin esters in pacific white shrimp Litopenaeus vannamei. J. Oleo Sci. 64, 243–253. [DOI] [PubMed] [Google Scholar]
- Zeng, J. , Wang, X. , Miao, Y. , Wang, C. , Zang, M. , Chen, X. , Li, M. et al. (2015) Metabolic engineering of wheat provitamin A by simultaneously overexpressing CrtB and silencing carotenoid hydroxylase (TaHYD). J. Agricult. Food Chem. 63, 9083–9092. [DOI] [PubMed] [Google Scholar]
- Zhai, S. , Xia, X. and He, Z. (2016) Carotenoids in staple cereals: metabolism, regulation, and genetic manipulation. Front. Plant Sci. 7, 1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang, B. , Liu, C. , Wang, Y. , Yao, X. , Wang, F. , Wu, J. , King, G.J. et al. (2015) Disruption of a Carotenoid Cleavage Dioxygense 4 gene converts flower colour from white to yellow in Brassica species. New Phytol. 206, 1513–1526. [DOI] [PubMed] [Google Scholar]
- Zheng, X. , Zhu, K. , Sun, Q. , Zhang, W. , Wang, X. , Cao, H. , Tan, M. et al. (2019) Natural variation in CCD4 promoter underpins species‐specific evolution of red coloration in citrus peel. Mol. plant 12, 1294–1307. [DOI] [PubMed] [Google Scholar]
- Zhou, Z. , Jiang, Y. , Wang, Z. , Gou, Z. , Lyu, J. , Li, W. , Yu, Y. et al. (2015) Resequencing 302 wild and cultivated accessions identifies genes related to domestication and improvement in soybean. Nat. Biotechnol. 33, 408–414. [DOI] [PubMed] [Google Scholar]
- Zuckerkandl, E. and Pauling, L. (1965) Evolutionary divergence and convergence in proteins. New York: Academic Press. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.