Genome-wide identification and expression profiling of the carotenoid cleavage dioxygenase (CCD) gene family in Brassica napus L

Abstract

Carotenoid cleavage dioxygenase (CCD), a key enzyme in carotenoid metabolism, cleaves carotenoids to form apo-carotenoids, which play a major role in plant growth and stress responses. CCD genes had not previously been systematically characterized in Brassica napus (rapeseed), an important oil crop worldwide. In this study, we identified 30 BnCCD genes and classified them into nine subgroups based on a phylogenetic analysis. We identified the chromosomal locations, gene structures, and cis-promoter elements of each of these genes and performed a selection pressure analysis to identify residues under selection. Furthermore, we determined the subcellular localization, physicochemical properties, and conserved protein motifs of the encoded proteins. All the CCD proteins contained a retinal pigment epithelial membrane protein (RPE65) domain. qRT-PCR analysis of expression of 20 representative BnCCD genes in 16 tissues of the B. napus cultivar Zhong Shuang 11 (‘ZS11’) revealed that members of the BnCCD gene family possess a broad range of expression patterns. This work lays the foundation for functional studies of the BnCCD gene family.

Introduction

“Carotenoids” is the general term for the class of natural pigments widely present in animals, plants, and microorganisms. Carotenoids are lipid-soluble isoprene-like compounds that contain 40 carbon molecules and comprise more than 750 pigments with different structures [1]. These pigments have numerous important biological functions; for example, they are photo-protective and are indispensable components of photosynthesis [2, 3]. They also can scavenge free radicals and have antioxidant properties [4]. Furthermore, carotenoids are components of cell membranes, potential anti-cancer agents, and can interact with proteins [5]. Carotenoids are exploited as coloring agents in flowers and fruits to attract pollinators and agents of seed dispersal [6–8]. In addition, carotenoids are precursors of important phytohormones, such as abscisic acid and strigolactones, which regulate plant development and plant–environment interactions [9–11].

The carotenoid biosynthesis pathway in plants has been fully elucidated; the catalytic oxidative cracking of carotenoids is a key process in this pathway. Multiple conjugated double bonds exist within the carotenoid center chain, which can be specifically cleaved by carotenoid cleavage dioxygenases (CCDs) to form a variety of apo-carotenoids, and some apo-carotenoids can be further degraded into small biologically active molecules [12]. In plants, the addition of two oxygen atoms to the cleavage product means that CCDs possess the characteristics of a dioxygenase [13]. Moreover, CCDs are also a class of non-heme oxygenases, whose catalytic activity requires Fe²⁺ as a cofactor [14, 15]. CCD proteins contain four highly conserved histidine residues bound to Fe²⁺ and all contain a retinal pigment epithelial membrane protein (RPE65) domain that is characteristic of enzymes involved in carotenoid cleavage [16, 17].

In plants, CCD proteins are encoded by an ancient gene family. CCD gene family consists of two subfamilies: carotenoid cleavage dioxygenases (CCDs) and 9-cis epoxycarotenoid dioxygenases (NCEDs) [12]. Through analysis of a novel ABA-deficient mutant of maize, the first protein found to specifically cleave carotenoids, viviparous14 (VP14), was identified by Schwartz et al [14]. Vallabhaneni et al. described the characteristics of the CCD gene family in the grass species maize (Zea mays), rice (Oryza sativa), and sorghum (Sorghum bicolor) [18]. During water stress and seed dormancy, TaNCED of Triticum aestivum might play a primary role in regulation of ABA content [19]. Besides, drought stress can induce expression of NCED3 and accumulation of endogenous ABA in Nicotiana tabacum [20]. Wei et al. found at least seven CCD genes in Solanum lycopersicum genome sequence and analyzed their expression patterns [21]. CCD genes have also been identified or functionally expressed in a variety of other plant species, such as Glycine max [22], Gossypium hirsutum, Solanum tuberosum [23], Cucurbita pepo [24], Saccharum officinarum [25], Crocus sativus, Osmanthus fragrans [26], Vitis vinifera [27], Mangifera indica [28] and Amygdalus persica [29].

In Arabidopsis thaliana, the CCD gene family consists of nine members: four CCD genes (AtCCD1, 4, 7, and 8) and five NCED genes (AtNCED2, 3, 5, 6 and 9) [30]. Carotenoid cleavage dioxygenase homologs in other plant species are named according to the system used for Arabidopsis CCD family members.

The CCD1 and CCD4 enzymes catabolize a variety of carotenoids and produce volatile apo-carotenoids, which are important for the biosynthesis of aromas and flavors of flowers and fruits, respectively [31, 32]. The carotenoid content of mature seeds of AtCCD1 mutants was higher than that in the wild type, indicating a role for CCD1 in carotenoid catabolism [33]. CCD4 regulates carotenoid homeostasis and CmCCD4a is specifically expressed in white petals of Chrysanthemum morifolium [34]. RNAi-mediated inhibition of CmCCD4a expression in white-petaled plants results in the production of yellow flowers [35]. In line with this observation, loss or downregulation of CmCCD4a function led to an increase in carotenoid content in petals [12]. CCD7 and CCD8 function in strigolactone biosynthesis, regulate axillary bud growth, and inhibit branching [36, 37]. Using RNAi to reduce Actinidia chinensis CCD8 expression increases in branch development and delays leaf senescence [38].

The reaction catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) is a rate-limiting step in ABA biosynthesis, and thus influences plant tolerance to diverse abiotic stresses [39]. AtNCED5, AtNCED6 and AtNCED9 are the dominant contributors to developmentally regulated ABA synthesis in seeds and thereby regulate seed embryo maturation and dormancy [30]. Overexpression of OsNCED3 increased drought resistance in rice and caused an increased ABA level [40]. AtNCED2 and AtNCED3 transcripts are abundant in Arabidopsis roots and their encoded proteins function in abscisic acid biosynthesis and thereby regulate lateral root growth [30]. Furthermore, heterologous expression of Brassica napus NCED3 led to ABA accumulation and NO and ROS generation in transgenic Arabidopsis plants, thereby enhancing abiotic stress tolerance [41].

B. napus, an important oilseed crop globally, is an allopolyploid derived from a natural interspecific cross between Brassica rapa (turnip; 2n = 2x = 20) and Brassica oleracea (kohlrabi; 2n = 2x = 18). The important physiological functions of carotenoid cleavage products in plants, such as abscisic acid and strigolactones, have prompted studies of the lyases involved in carotenoid metabolism. Little is known about the BnCCD gene family. The availability of the B. napus genome sequence would enable the identification and analysis of members of this family [42].

In this study, we identified 30 BnCCD genes. In addition to analyzing their gene evolution and structure, chromosomal localization, conserved motifs, and cis-acting promoter elements, we determined their tissue- and organ-specific expression and examined the physicochemical properties of their encoded proteins. The results form a solid basis for further studies on the biological functions of the BnCCD gene family.

Materials and methods

Plant materials

The B. napus cultivar Zhong Shuang 11 (‘ZS11’) was planted in Chongqing, China (29°45’ N, 106°22’ E). To analyze the expression patterns of BnCCD genes, four different tissues of ‘ZS11’ were harvested when plants were in full flower: mature leaves (Le), sepals (Se), flowers (F), and stems (St). The seeds (S) and silique pericarps (Sp) were harvested at different timepoints following the termination of flowering (25, 30, 35, 40, 45, and 50 days after flowering). Samples were immediately frozen in liquid nitrogen and stored at –80°C for further use.

Identification of CCD genes in Brassica. napus, Brassica. rapa, and Brassica. oleracea

The coding sequences of AtCCD genes were downloaded from TAIR (https://www.arabidopsis.org/) and used as reference sequences. The BnCCD, BrCCD, and BoCCD genes were identified through the BLASTN analysis [43] of AtCCD genes against the Brassica Database (BRAD, http://brassicadb.org/brad/index.php) [44]. The local database was established using Geneious 4.8.5 software (http://www.geneious.com/; Biomatters, Auckland, New Zealand). The genome-wide alignment was verified by the MEGABLAST program [45], and the following screening standards were used: the consistency of aligned sequences with the reference sequence was ≥80% and gene sequences <700 bp were discarded. Because enzymes involved in carotenoid cleavage contain a retinal pigment epithelial membrane protein (RPE65) domain [16, 17], gene sequences from the Pfam database [46] that did not contain this domain were excluded.

Multiple sequence alignment and phylogenetic analysis

CCD amino acid sequences were subjected to multiple sequence alignment using MUSCLE [47] with default parameters. The evolutionary relationships of B. napus CCD proteins with those of A. thaliana, B. oleracea, and B. rapa were analyzed using MEGA7 [45]. To identify the conserved blocks of all predicted sequences, the Gblocks program was used [48]. The substitution saturation was detected using DAMBE [49]. A phylogenetic tree was constructed using the neighbor-joining method implemented in MEGA7. The number of bootstrap replications was 1,000, and paired deletion was performed. The phylogenetic tree was visualized using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).

Protein properties, sequence analysis, and duplication time inference

The chromosomal location of BnCCD genes, which was queried from the Brassica napus genome browser, and the genes were mapped onto chromosomal linkage groups by Mapchart software [50]. The Gene Structure Display Server (GSD 2.0) (http://gsds.cbi.pku.edu.cn/index.php) was used to portray the exon–intron structures of the BnCCD genes. The ExPASy proteomics server database (http://expasy.org/) [51]was used to predict the relative molecular weight, theoretical isoelectric point, protein stability, and aliphatic amino acid content of BnCCD proteins. The conserved motifs were identified using the MEME Version 5.0.5 online tool (http://meme-suite.org/tools/meme) [52] and the maximum motif retrieval value was set to 20; other parameters used the default settings. Annotations of the identified motifs were obtained from InterProScan (www.ebi.ac.uk/Tools/InterProScan/) [53].

To determine whether the CCD protein-coding sequences are under selective pressure, the ratios of synonymous substitution rate (ks) and non-synonymous substitution rate (ka) of homologous gene pairs were calculated using TBtools software [54]. The approximate date of duplication events was inferred by substituting Ks values into the formula $\mathrm{T}=Ks/\left(2\times 1.5\times {10}^{-8}\right)\times {10}^{-6}$million years ago (MYA) [55].

Predicted subcellular localization of BnCCD proteins and promoter analysis of BnCCD genes

The subcellular localization of BnCCD proteins were predicted using WoLF PSORT (http://www.genscript.com/tools/wolf-psort) [56]. The SOPMA secondary structure prediction method (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) was used to predict the secondary structure of BnCCD proteins and the presence of transmembrane helices within BnCCD proteins were predicted using TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The promoter sequences (2.0-kb region immediately upstream of the translation start sites) of the BnCCD genes were obtained from the B. napus genome database (http://www.genoscope.cns.fr/brassicanapus/) [57]. PlantCARE (Error! Hyperlink reference not valid.webtools/plantcare/html/) [58] was used to predict the presence of cis-acting sequences within each promoter.

RNA-seq analysis

To analyze the tissue-specific expression of the BnCCD genes, publically available B.napus RNA-sequencing (RNA-seq) data PRJNA358784 (BioProject) were downloaded from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) [59]. All BnCCD genes expression levels were quantified in terms of FPKM (fragments per kilobase of exon per million mapped fragments) using Cufflinks with default parameters [60], and then extracted from RNA-seq data according to their B. napus code, including expression data from thirteen different organs (roots, stems, leaves, buds, anthocauli, calyxes, petals, pistils, stamens, anthers, capillaments, seeds and silique pericarps) at different developmental stages in B.napus cultivar ZS11. The heatmap for BnCCD genes was constructed using TBtools software.

RNA extraction and quantitative real-time PCR

Total RNA was extracted using the RNeasy Extraction Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Contaminating genomic DNA was removed with DNase I. Total RNA (1 μg) was used to synthesize cDNA by reverse transcriptase (TaKaRa). The gene-specific primer pairs used to analyze BnCCD genes expression by qRT-PCR were designed using Primer Premier 5 [61] and the ACTIN 7 gene was used as an endogenous reference gene. All the primers were listed in S1 Table.

Quantitative RT-PCR was carried out using TB Green Premix Ex Taq on a Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The reaction mixture included 10 μL TB Green Premix Ex Taq, 0.5 μL forward primer, 0.5 μL reverse primer, 2 μL cDNA template, and 7 μL nuclease-free H₂O in a total volume of 20 μL. The PCR cycling conditions were as follows: 95°C for 30s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Following PCR amplification, the dissolution curve was analyzed to ensure the specificity of the amplified products. Three biological replicates and three technical replicates were performed for each reaction. The relative gene expression levels were calculated using the 2^−ΔΔCt method [61]. All the results were plotted as the mean ± standard error of mean (SEM) from three independent biological replicates using Graph Pad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA).

Results

Identification and characterization of BnCCD genes

Using the nine AtCCD coding sequences as a BLASTN query, we identified 30 B. napus CCD genes (BnCCDs), 16 originating from B. rapa (BrCCDs) and 14 from B. oleracea (BoCCDs)(S2 Table). All the genes contained sequences encoding the RPE65 domain. RPE65 belongs to a family of carotenoid oxygenases in plant, bacterial, and animal systems that typically oxidatively cleave conjugated double bonds in the polyene backbone of carotenoids [16, 17]. In addition to light related functions, the function and mechanism of RPE65 in plants have not been elucidated. To determine the evolutionary relationships among 60 CCD proteins, we constructed a phylogenetic tree by the neighbor-joining method. Based on the topology of the phylogenetic tree, CCD proteins could be grouped into nine distinct subgroups (I, II, III, IV, V, VI, VII, VIII and IX), each of which contained one of the AtCCD proteins and the corresponding subgroup members of B. rapa, B. oleracea, and B. napus (Fig 1).

Fig 1. Phylogenetic analysis of 60 CCD protein sequences from A. thaliana, B. rapa, B. oleracea, and B. napus.

Sixty CCD protein sequences were used to construct the neighbor-joining tree using Mega 7 with 1,000 bootstrap replicates. The CCD proteins were devided into nine phylogenetic subgroups (I, II, III, IV, V, VI, VII, VIII and IX) and are marked with different background colors.

The physical and chemical properties of the BnCCD proteins are summarized in Table 1. The length of BnCCD proteins ranged from 255 amino acids (aa) (BnNCED3f) to 668 aa (BnCCD7a), with a mean length of 551 aa. The predicted molecular weights varied from 28.65 kDa (BnNCED3f) to 74.79 kDa (BnCCD7a) and the theoretical isoelectric point (PI) ranged from 4.75 (BnNCED3f) to 8.83 (BnNCED3e). Out of the 30 BnCCD proteins, 27 had PI values of less than 7, and the values of the remaining three BnCCD proteins were greater than 7. Based on the hydrophilicity index of amphoteric proteins between –0.5 ~ +0.5 (a negative GRAVY value indicates hydrophilicity and a positive value indicates hydrophobicity), all BnCCD proteins are amphiphilic proteins.

Table 1. The genes and encoded protein features of the 30 BnCCDs identified in this study.

Gene name	Gene ID	Subgroup	Chromosome	Gene length (bp)	Gene position		Number of exons	Protein length (aa)	Molecular wt. (kDa)	pI	GRAVY	Subcellular location
					Start	End
BnCCD1a	BnaA09g41150D	I	A09	4466	28783045	28787510	13	483	55.24182	6.36	−0.175	chlo: 5, cyto: 2, vacu: 2, E.R.: 2, nucl: 1, pero: 1
BnCCD1b	BnaAnng12520D	I	Ann_random	3193	13541287	13544479	13	525	59.31523	5.70	−0.256	cyto: 7, chlo: 3, nucl: 1, mito: 1, plas: 1
BnCCD1c	BnaC04g20610D	I	C04	3267	21697508	21700774	14	525	59.35633	5.94	−0.257	cyto: 10, chlo: 2, plas: 1
BnCCD1d	BnaC08g33680D	I	C08	8770	32058916	32067685	14	612	69.62388	6.95	−0.367	chlo: 9, mito: 3, nucl: 1
BnCCD1e	BnaC08g33690D	I	C08	7652	32077226	32084877	15	611	69.52093	6.41	−0.289	chlo: 14
BnNCED2a	BnaA01g09090D	II	A01	1746	4447853	4449598	1	581	64.52248	5.33	−0.224	chlo: 9.5, chlo_mito: 5.5, cyto: 1, plas: 1, vacu: 1
BnNCED2b	BnaA03g58230D	II	A03_random	1752	1600131	1601882	1	583	64.95409	5.49	−0.237	chlo: 6, cyto: 5, plas: 1.5, cysk_plas: 1.5
BnNCED2c	BnaC01g10770D	II	C01	1746	6588592	6590337	1	581	64.51950	5.31	−0.214	chlo: 9, cyto: 4
BnNCED2d	BnaC07g35240D	II	C07	1752	37711814	37713565	1	583	64.82201	5.55	−0.243	chlo: 7, cyto: 4, plas: 1.5, cysk_plas: 1.5
BnNCED3a	BnaA01g29390D	III	A01	1662	20329204	20331002	2	553	60.99077	5.28	−0.287	mito: 10, chlo: 4
BnNCED3b	BnaA03g33390D	III	A03	1191	16162114	16163388	2	396	44.24510	4.99	−0.242	cyto: 6, cysk: 6, mito: 1
BnNCED3c	BnaA05g25030D	III	A05	1794	18611263	18613467	1	597	65.80847	5.94	−0.297	mito: 8, chlo: 6
BnNCED3d	BnaC01g36910D	III	C01	1797	36059468	36061576	1	598	65.72044	5.89	−0.276	mito: 8, chlo: 6
BnNCED3e	BnaC05g39180D	III	C05	948	37757359	37758306	1	315	34.06851	8.83	−0.291	mito: 11, chlo: 3
BnNCED3f	BnaC05g39190D	III	C05	768	37758388	37759155	1	255	28.64751	4.75	−0.217	cyto: 8, cysk: 4, plas: 1
BnNCED3g	BnaC05g39200D	III	C05	1794	37769098	37770891	1	597	65.84553	6.01	−0.318	mito: 9, chlo: 5
BnNCED3h	BnaCnng32080D	III	Cnn_random	1194	30423020	30424656	2	396	44.29825	5.09	−0.219	cysk: 8, cyto: 4, mito: 1
BnCCD4a	BnaA01g09900D	IV	A01	2020	4881104	4883125	1	596	65.70485	6.33	−0.236	chlo: 12, mito: 1.5, cyto_mito: 1.5
BnCCD4b	BnaA08g09110D	IV	A08	1782	8767981	8769762	3	532	58.57082	6.16	−0.129	chlo: 12, cyto: 1
BnCCD4c	BnaC01g11550D	IV	C01	2067	7220281	7222347	1	593	65.43358	6.45	−0.234	chlo: 12, mito: 1.5, cyto_mito: 1.5
BnNCED5a	BnaA09g26450D	V	A09	1170	19605914	19607683	1	589	65.33387	5.37	−0.324	chlo: 11, nucl: 2
BnNCED5b	BnaCnng38510D	V	Cnn_random	1170	37143600	37145369	1	589	65.45993	5.42	−0.320	chlo: 11, nucl: 1, mito: 1
BnNCED6a	BnaA07g06050D	VI	A07	1758	6379220	6380977	1	585	65.00222	6.07	−0.321	chlo: 7.5, chlo_mito: 6.5, mito: 4.5
BnNCED6b	BnaC07g07580D	VI	C07	1758	11978406	11980163	1	585	65.06919	6.00	−0.329	pero: 8, chlo: 3, cyto: 2
BnCCD7a	BnaA04g26000D	VII	A04	5803	18550548	18556350	10	668	74.78965	5.77	−0.381	chlo: 9, plas: 3, mito: 1
BnCCD7b	BnaC04g50070D	VII	C04	3912	47874505	47878416	9	648	72.69485	6.19	−0.303	chlo: 8, plas: 3, mito: 1, vacu: 1
BnCCD8a	BnaA01g04140D	VIII	A01	3287	1908477	1911763	6	569	63.98902	7.13	−0.341	chlo: 10, mito: 3
BnCCD8b	BnaC01g05600D	VIII	C01	12036	2963042	2975077	7	574	64.98737	8.50	−0.452	chlo: 7, cyto: 4, nucl: 2
BnNCED9a	BnaA07g39250D	IX	A07_random	1809	2059180	2060988	1	602	66.75458	5.90	−0.299	chlo: 14
BnNCED9b	BnaC06g38870D	IX	C06	1809	36264799	36266607	1	602	66.79169	6.11	−0.281	chlo: 14

chlo: chloroplast; cyto: cytoplasmic; vacu: vacuolar; E.R: Endoplasmic Reticulum; pero: peroxisomal; mito: mitochondria; nucl: nucleus; plas: plasma membrane.

Chromosomal localization and gene structure

By Mapchart, an analysis of the chromosomal distribution of the BnCCD loci showed that these genes were not evenly distributed across the chromosomes (Fig 2). The 30 BnCCD loci are distributed on 13 chromosomes in B. napus; the 14 BnCCDs are located on the A subgenome and the 16 BnCCDs are located on the C subgenome. Chromosomes A01 and C01 possess the most BnCCD loci, each containing four BnCCDs. Chromosomes A03, A04, A05, A07, A08, C06, A03-random, A07-random, and Ann-random each contain only one BnCCD locus, whereas chromosomes A09, C04, C07, C08, and Cnn-random each contain two BnCCD loci. BnNCED3e, BnNCED3f, and BnNCED3g (subgroup III) are located on chromosome C05.

Fig 2. Genomic distribution of BnCCD genes.

The chromosomal location of each BnCCD locus was mapped to the B. napus genome: the chromosome number is indicated above each chromosome. The scale is in megabases (Mb). Ann and Cnn are pseudo-molecule chromosomes. Random means that the specific location of the gene is unknown.

The positions of exons and introns in members of a gene family might have played crucial roles during evolution [62]. Therefore, we next analyzed the gene structure (exon–intron organization) of the 30 BnCCDs (Fig 3). Genes with similar structures were closely related. BnNCED2, BnNCED5, BnNCED6, and BnNCED9 subgroups were intron-less. The BnCCD1 subgroup contained 13–15 exons; two genes contained 13 exons, two genes contained 14 exons, and only BnCCD1e possessed 15 exons. All BnCCD4 subgroup members contained one exon, except for BnCCD4b, which contained three. BnCCD8b contained 6 introns, one more than BnCCD8a.

Fig 3. Exon–intron structures of the BnCCD genes, arranged based on their phylogenetic relationships.

An unrooted phylogenetic tree was constructed based on the full-length sequences of BnCCD proteins using the N-J method in MEGA7. Bootstrap values based on 1,000 replications were calculated. The exon–intron structure of the BnCCD genes was analyzed using the GSDS online tool. The lengths of the exons and introns for each BnCCD gene are drawn to scale.

Conserved motifs and protein profiles of BnCCD proteins

Twenty putative BnCCD protein motifs were predicted by the MEME program (Fig 4). The BnNCED2, NCED5, NCED6, and NCED9 proteins all contain motifs 1–17. Except for BnNCED3e, all other BnNCED3 proteins contain motifs 2, 6, 7, 10, 12, 16, and 17. Motifs 1, 2, 7, and 15 are present in all BnCCD1, BnCCD4, and BnCCD8 proteins and all BnCCD7 members contain motifs 1, 4, 10, 12, 15, 18, and 19. Using the InterProScan program, we searched for annotations for the conserved motifs. Motifs 1–9 and 19 were associated with carotenoid oxygenase (IPR004294) and the remaining motifs had not been annotated.

Fig 4. The conserved motifs of the BnCCD proteins, arranged based on their phylogenetic relationships.

The conserved motifs of the BnCCD proteins were identified by MEME. Gray lines represent non-conserved sequences and each motif is indicated by a colored box (numbered at the bottom). The lengths of the motifs in each protein are drawn to scale.

Prediction of secondary structure using SOPMA showed that the main structure of the BnCCD proteins was a random coil. In addition to BnNCED3e, the alpha helix was more than the extension chain. The alpha helix was equal to the extension chain in BnCCD7a. The secondary structure characteristics of other BnCCD proteins ranged from more to less: random curl, extended strand, alpha helix, beta turn. Analysis using the TMHMM Server v. 2.0 showed that no BnCCD proteins possessed a transmembrane domain.

Nineteen of the 30 BnCCD proteins were predicted to be localized to the chloroplast, five to the cytoplasm, and five to the mitochondrion. BnNCED6b was predicted to be localized to the peroxisome.

Ka and Ks calculation for orthologous CCD genes between Brassica napus and Arabidopsis thaliana

To determine whether the CCD protein-coding genes in B. napus and A. thaliana are under selective pressure, the Ka/Ks ratios for 30 pairs of orthologous genes were calculated using TBtools software (Table 2). The Ka/Ks ratios of all the gene pairs were considerably lower than 1, indicating that the CCD gene family has undergone purifying selection. The Ks values for B. napus relative to A. thaliana ranged from 0.28852 to 0.65433, suggesting that gene duplications occurred approximately 9.62–21.81 million years ago (MYA).

Table 2. The non-synonymous (Ka) and synonymous substitution rate (Ks) for orthologous CCD gene pairs between B. napus and A. thaliana.

Orthologous gene pairs		Ka	Ks	Ka/Ks	Duplication date (MYA)
AtCCD1	BnCCD1a	0.08151	0.36209	0.22510	12.07
AtCCD1	BnCCD1b	0.04122	0.32012	0.12875	10.67
AtCCD1	BnCCD1c	0.03384	0.28852	0.11730	9.62
AtCCD1	BnCCD1d	0.05913	0.38774	0.15250	12.92
AtCCD1	BnCCD1e	0.07611	0.35530	0.21422	11.84
AtNCED2	BnNCED2a	0.06843	0.46502	0.14716	15.50
AtNCED2	BnNCED2b	0.04285	0.41304	0.10374	13.77
AtNCED2	BnNCED2c	0.06108	0.47780	0.12784	15.93
AtNCED2	BnNCED2d	0.04606	0.43371	0.10620	14.46
AtNCED3	BnNCED3a	0.04945	0.56001	0.08829	18.67
AtNCED3	BnNCED3b	0.05042	0.55742	0.09045	18.58
AtNCED3	BnNCED3c	0.05052	0.51718	0.09768	17.24
AtNCED3	BnNCED3d	0.04962	0.52187	0.09509	17.40
AtNCED3	BnNCED3e	0.05472	0.45159	0.12116	15.05
AtNCED3	BnNCED3f	0.05271	0.58288	0.09043	19.43
AtNCED3	BnNCED3g	0.04581	0.51353	0.08921	17.12
AtNCED3	BnNCED3h	0.05693	0.55439	0.10268	18.48
AtCCD4	BnCCD4a	0.07819	0.59005	0.13252	19.67
AtCCD4	BnCCD4b	0.06912	0.50381	0.13720	16.79
AtCCD4	BnCCD4c	0.07461	0.60924	0.12246	20.31
AtNCED5	BnNCED5a	0.04001	0.38163	0.10484	12.72
AtNCED5	BnNCED5b	0.03847	0.40056	0.09605	13.35
AtNCED6	BnNCED6a	0.08570	0.57248	0.14970	19.08
AtNCED6	BnNCED6b	0.09078	0.57371	0.15824	19.12
AtCCD7	BnCCD7a	0.07106	0.45309	0.15683	15.10
AtCCD7	BnCCD7b	0.07166	0.42570	0.16834	14.19
AtCCD8	BnCCD8a	0.05711	0.42701	0.13374	14.23
AtCCD8	BnCCD8b	0.09405	0.49007	0.19191	16.34
AtNCED9	BnNCED9a	0.07262	0.61567	0.11796	20.52
AtNCED9	BnNCED9b	0.06799	0.65433	0.10391	21.81

The cis-acting elements predicted to be present in BnCCD promoters

The cis-acting elements present in the promoters are essential for transcriptional gene regulation. In total, 87 types of cis-acting elements were predicted to be present in 30 BnCCD promoters (S3 Table), using the online software PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). In addition to CAAT and TATA boxes, each promoter contained more than 10 cis-acting elements. Among these, 26 different elements are associated with responses to light, with 206 occurrences across all promoters. The remaining elements are associated with hormonal and stress responses and tissue-specific expression. Hormone-responsive cis-elements account for a large proportion of the total and include ABRE (abscisic acid response element), AuxRR-core and TGA-elements (auxin response), ERE (ethylene-response element), TCA-element (salicylic acid response), P-box and GARE-motif (gibberellin-response), and the CGTCA and TGACG motifs (MeJA-response). Notably, all BnCCDs contain ABRE, CGTCA, and TGACG motifs, suggesting that this gene family functions in hormone response pathways. Additional cis-elements related to stress response were identified; for example, LTR (low-temperature responsiveness) is present in all BnCCD promoters except for BnNCED3f and BnCCD8a. Additional elements include the drought-inducible MBS element, TC-rich repeats, a cis-acting element involved in defense and stress responsiveness, and the WUN-motif, a wound-responsive element.

Some cis-acting elements in BnCCD promoters might determine the tissue-specific expression pattern, such as the CAT-box, which is associated with meristematic expression; GCN4_motif, involved in endosperm expression; HD-Zip 1, involved in differentiation of the palisade mesophyll cells; and RY-element, which confers seed-specific expression.

RNA-seq analysis

We analyzed the expression of 30 BnCCDs at different developmental stages and in various tissues from the B. napus cultivar ZS11 and constructed an expression heat map (Fig 5). Whereas no BnNCED3f and BnNCED3g transcripts were detected in any tissue, the remaining 28 BnCCDs showed tissue- and development-specific expression levels. BnCCD1b and BnCCD1c were highly expressed in leaves, stems, buds, flowers, seeds, and silique pericarp, but expression was relatively low in roots. BnNCED9a and BnNCED9b had similar expression patterns and were highly expressed in seeds. Genes belonging to the same subgroup had a wide range of expression patterns: BnCCD4a and BnCCD4c were highly expressed in leaves, stems, and pericarps; however, BnCCD4b, which belongs to the BnCCD4 subgroup, had a low expression level in leaves, stems, and pericarps but was highly expressed in anthers, suggesting that it might function in stamen growth and development.

Fig 5. Heatmap of the expression patterns of selected BnCCD genes in different tissues and at different developmental stages.

The expression data were obtained from publically available RNA-sequening (RNA-seq) data. Ro: Roots; St: Stems; Le: Leaves; LeY: Young leaves; LeO: Old leaves; Bu: Buds; Ao: Anthocauli; Cal: Calyxes; Pe: Petals; Pi: Pistils; Sta: Stamens; At: Anthers; Cap: Capillaments; Se: Seeds; Sp: Silique pericarps; _s: At seedling stage; _b: In the bud stage; _i: At the initial flowering stage; _f: At the flourishing flowering stage; _3d: 3 days after flowering; _7,10,13,21,24,27,30,35,43d: 7,10,13, 21,24,27,30,35,43days after flowering; Ro_s: Roots at seedling stage.

Expression profiling of BnCCDs in different organs

To gain insight into the biological functions of BnCCDs, we analyzed their expression patterns in flowers, leaves, sepals, stems, seeds, and silique pericarps, and for seeds and silique pericarps also at different developmental stages (25, 30, 35, 40, 45, and 50 days after flowering) by qRT-PCR. Several BnCCDs, including BnCCD1b, BnCCD1c, BnCCD1d, BnNCED2a, BnNCED2c, BnNCED3a, BnNCED3c, BnNCED3d, BnCCD4a, BnCCD4c, BnNCED5a, BnNCED5b, BnNCED6a, BnNCED6b, BnCCD7a, BnCCD7b, BnCCD8a, BnCCD8b, BnNCED9a, and BnNCED9b, had diverse expression patterns in different tissues (Fig 6). Except for BnCCD8b, the expression level of CCD genes was low in the stem. BnNCED5b and BnCCD8a transcripts were abundant in flowers, BnNCED5a and BnNCED5b transcripts were abundant in sepals, and BnNCED9a and BnNCED9b transcripts were abundant in seeds. However, the expression patterns of some genes within the same family differed: the transcript levels of BnCCD1b and BnCCD1c in leaves were higher than those in other tissues, whereas BnCCD1d was most highly expressed in sepals. In addition, BnNCED2a and BnNCED2c transcript levels were higher in seeds at 40–50 days after flowering (40S, 45S, and 50S) than at 25–35 days after flowering (25S, 30S and 35S). BnCCD4a and BnCCD4c transcript levels were abundant in silique pericarps collected at the 25 days after flowering (25Sp), 35Sp, and 45Sp stages, but were present at low levels at the 30Sp, 40Sp, 45Sp and 50Sp stages.

Fig 6. The expression patterns of selected BnCCD genes in different tissues and at different developmental stages.

The expression patterns of 20 BnCCD genes in B.napus cultivar Zhong Shuang 11 (ZS11) were analyzed by qRT-PCR. F: flowers; Le: leaves; Se: sepals; St: stems; 25S, 30S, 35S, 40S, 45S, 50S: seeds collected at 25, 30, 35, 40, 45and 50 days after flowering, respectively. 25Sp, 30Sp, 35Sp, 40Sp, 45Sp, 50Sp: silique pericarps collected at 25, 30, 35, 40, 45 and 50 days after flowering, respectively. Values represented the mean ± standard error of mean (SEM) of three biological replicates with three technical replicates at each developmental stage. Error bars indicated the SEM among three experiments.

Discussion

Arabidopsis contains nine CCD genes. Brassicaceae, including Arabidopsis, underwent an ancient α, β and γ whole-genome duplication polyploidization events, and also experienced an additional recent genome-wide tripling (WGT) event [63]. Because it is an allotetraploid formed by interspecific hybridization between B. rapa and B. oleracea, B. napus would be expected to contain six copies of each Arabidopsis thaliana gene (i.e., 54) [64]. Because of genome shrinkage and gene loss, the number of BnCCDs identified in this study was a little lower (i.e., 30), with only 2–6 copies of each Arabidopsis gene present. The B. rapa and B. oleracea genomes contain 16 BrCCD and 14 BoCCD genes, respectively, and the number of BnCCDs is the sum of these. However, only two members of the BnNCED9 subgroup and three of the BnCCD4 subgroup were identified in this study, whereas two members were found in the corresponding subgroups of both B. rapa and B. oleracea, indicating that gene loss might have occurred among subgroups BnCCD4 and BnNCED9.

The molecular characteristics of all BnCCD protein members were found to differ, but proteins in the same subgroup had similar molecular weights and isoelectric points. The position of exon–intron boundaries reflects the evolution of these genes [62]. Subcellular localization predictions suggested that BnCCD proteins are localized to the chloroplast, mitochondrion, cytoplasm, and peroxisome. BnCCD1b, BnCCD1c, BnNCED3b, BnNCED3f, and BnNCED3h were predicted to be cytoplasmic, suggesting that BnCCD1 and BnNCED3 might interact in the cytoplasm. It also showed that these genes may not participate in chlorophyll photosynthesis, which was consistent with the research by Zhang et al. [28]. BnNCED2 subgroup is located in the chloroplast. Studies have shown that histidine residues in NCED amino acids can bind Fe ²⁺ to make NCED proteins function [65], and there is a chloroplast transit peptide structure in them [66]. Wang et al. [67] found that NCED2 protein in Camellia sinensis is localized in chroloplast and has N-terminal chloroplast targeting signal peptide sequences, which further proves that CsNCED2 has the biological activity of cleaving epoxy carotenoids to generate ABA precursors in plastids. Besides, in both sugarcane and rice, CCD8 are proteins located in chloroplast [25, 68]. Through online analysis we found that BnCCD8 is also localized in the chloroplast.

Seven cis-acting elements associated with various stress responses were predicted among the promoters of the 30 BnCCDs; LTR, MBS, TC-rich repeats, and nine cis-acting elements were associated with responses to abscisic acid, MeJA, salicylic acid, and auxin. CCD1, 4, and 8 and NCED2 and 9 are transcriptionally upregulated following treatment of B. rapa with the phytohormones ABA and SL [16]. Furthermore, Nicotiana tabacum plants that heterologously expressed NCED1 of Stylosanthes guianensis had increased tolerance to light, oxidative, drought, salt, and cold stress, indicating that CCD might regulate plant tolerance against these abiotic stresses [69]. The G-Box, GT1, and ACE motifs, which are transcriptionally responsive to light, are present in the BnCCD gene promoters. In addition, BnCCD promoters contain circadian response elements, RY-elements, and MSA-like and MBSI elements, indicating that most CCD genes function in plant growth and development and stress responses.

Based on RNA-seq data (S4 Table), we constructed an expression heatmap, which demonstrated that the 30 BnCCDs were differentially expressed in different B. napus tissues. Members of the same gene subgroup occasionally exhibited different expression patterns, such as BnCCD1 and BnCCD4, potentially reflecting new functionalization among the gene family during evolution. Genes of the CCD1 and CCD4 subgroups function in the formation of a variety of apo-carotenoids, which confer unique colors, tastes, and aromas [70–72]. A CCD1 loss-of-function mutant showed a decreased level of β-ionone in tomato fruit (Solanum lycopersicum) [31] and petunia flowers (Petunia hybrida) [73]. Carotenoid homeostasis is regulated by CCD4 in different tissues, such as Arabidopsis seeds [74] and potato tubers [23]. BnCCD4b was mainly expressed in stamens, suggesting that it might function in stamen growth and development. Species such as Solanumly copersicum, Prunus persica, and Crocus, were also reported to preferentially express CCD4 in floral organs, indicating that the evolution of CCD4 genes might have been adaptive and have enhanced specific physiological traits unique to flowering plants [21, 75, 76]. CCD4 with normal function or lack of function will change the color of fruit and flower organs. Previous studies have suggested CCD4 gene can fade the yellow petals of Rhododendron japonicum and Eustoma grandiflorum [77, 78], because its ability of cleaving carotenoids. Inactivation of CCD4 will change the color of B.napus and Chrysanthemum morifolium petals from white to yellow [79, 80]. Controlling the expression of CCD gene family provides a way to change the color of plant petals.

Conclusion

In conclusion, we performed a comprehensive study of CCD gene family in B.napus. We identified 30 putative BnCCDs that were classed into nine subgroups (I-IX) on the basis of their phylogenetic relationships. The length of the BnCCD proteins ranged from 255 to 668 aa, and the Ks values for B. napus relative to A. thaliana ranged from 0.28852 to 0.65433. In addition, RNA-seq data and qRT-PCR analysis revealed that BnCCDs were differentially expressed in different tissues and organs, and had tissue/organ specificity and expression preference, suggesting that BnCCDs had clear function differentiation. Our results will help lay the foundation for the functional characterization of the CCD gene family and better understand the structural and functional relationships among these family members.

Supporting information

S1 Table. qRT-PCR primers used to analyze BnCCDs expression.

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S2 Table. List of identified CCD genes in B.napus, B.rapa, B.oleracea and A. thaliana.

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S3 Table. Cis-acting elements in the promoter regions of BnCCDs.

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S4 Table. RNA-seq data of 30 BnCCDs.

(XLSX)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

JNL was funded to undertake research by Ministry of Science and Technology of the People´s Republic of China (http://www.most.gov.cn/) (The National Key Research and Development Plan, 2018YFD0100504-05, 2016YFD0101305) (The National Natural Science Foundation of China, 31830067, U1302266). JNL was funded to undertake research by Ministry of Education of the People's Republic of China (http://www.moe.gov.cn/) (The 111 Project, B12006). XTZ was funded to do research by Ministry of Education of the People's Republic of China (National Undergraduate Training Program for Innovation and Entrepreneurship, 201810635080). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.