Genome-wide identification and expression analysis of carotenoid cleavage oxygenase genes in Litchi (Litchi chinensis Sonn.)

Carotenoid cleavage oxygenases (CCOs) include the carotenoid cleavage dioxygenase (CCD) and 9-cis-epoxycarotenoid (NCED), which can catalize carotenoid to form various apocarotenoids and their derivatives, has been found that play important role in the plant world. But little information of CCO gene family has been reported in litchi (Litchi chinensis Sonn.) till date. In this study, a total of 15 LcCCO genes in litchi were identified based on genome wide lever. Phylogeny analysis showed that LcCCO genes could be classified into six subfamilies (CCD1, CCD4, CCD7, CCD8, CCD-like, and NCED), which gene structure, domain and motifs exhibited similar distribution patterns in the same subfamilies. MiRNA target site prediction found that there were 32 miRNA target sites in 13 (86.7%) LcCCO genes. Cis-elements analysis showed that the largest groups of elements were light response related, following was plant hormones, stress and plant development related. Expression pattern analysis revealed that LcCCD4, LcNCED1, and LcNCED2 might be involving with peel coloration, LcCCDlike-b might be an important factor deciding fruit flavor, LcNCED2 and LcNCED3 might be related to flower control, LcNCED1 and LcNCED2 might function in fruitlet abscission, LcCCD4a1, LcCCD4a2, LcCCD1, LcCCD4, LcNCED1, and LcNCED2 might participate in postharvest storage of litchi. Herein, Genome-wide analysis of the LcCCO genes was conducted in litchi to investigate their structure features and potential functions. These valuable and expectable information of LcCCO genes supplying in this study will offer further more possibility to promote quality improvement and breeding of litchi and further function investigation of this gene family in plant.

and their derivatives to perform important biological functions in plants. The CCOs can be divided into two forms, one named as CCD (Carotenoid Cleavage Dioxygenase) and the other is NCED (9-cis-epoxycarotenoid dioxygenase) depending on whether the substrates are epoxidated [4].
The CCO gene family has been reported commonly involved in the formation of flavor and scent, coloration, even growth and development, ecological adaptation in plants through regulating the carotenoids pathway. In Arabidopsis thaliana, AtCCO genes family includes 4 CCD and 5 NECD genes [5]. At the beginning of the previous work, the CCO genes were divided into five categories, which included CCD1, CCD4, CCD7, CCD8, and NCED [6,7]. Recently, another category of CCO genes called CCD-like (CCDL) was found in grape (Vitis vinifera), tomato (Solanum lycopersicum), apple (Malus × domestica) and Sugar cane (Saccharum) [8][9][10][11]. In general, different categories of CCO genes exhibit different roles. CCD1 can catalyze carotenoids into several metabolites like α-ionone, β-ionone, and geranylacetone, which play an important role in the formation of the flavor and scent of horticultural plants [12,13]. CmCCD4a gene contributes to white color formation in chrysanthemum petalsonly (Chrysanthemum morifolium Ramat.) by catalyzing the carotenoids to colorless compound [14]. GmCCD4 in soybeans was also found to be a negative regulator of carotenoid content [15]. Natural Variation in CCD4 gene promoter is a major genetic determinant of natural variation in C30 apocarotenoids which is responsible for red coloration of citrus peel [16]. The CCD7 and CCD8 enzymes are involved in the biosynthesis of the strigolactone (a relatively novel apocarotenoid hormone), which could control shoot branching and reproductive development and regulate plant responses to drought and salt stress [7,[17][18][19]. NCED is the key enzyme for the biosynthesis of abscisic acid (ABA), which is closely involved in the fruit development, ripening and senescence. Such as FaNCED1 RNAi in strawberry (Fragaria × ananassa) fruits could decline the ABA content significantly and resulted in uncolored fruits [20]. The application of exogenous ABA could accelerate the accumulation of anthocyanin by increasing the expression of NCED genes to promote the coloration of strawberry [21], grape [22], sweet cherry [23] and litchi fruits [24]. Some reports showed that ABA might be correlate with the fruit abscission of the citrus, apple and litchi [44][45][46]. The ABA content increased by uniconazole spraying was helpful to the flower control and fruit retention of litchi [48,49]. ABA was considered to play key role during the fruit senescence, which was the most important factor that deciding the shelf life of fruit [53,54]. Additionally, ABA is also reported to be related to the bud dormancy, leaf abscission, and responses to diverse environmental stresses [25]. Together, these studies showed that CCO genes play an important role in plant world.
Litchi (Litchi chinensis Sonn.) is a member of the sapindaceae family and an important subtropical and tropical economic fruit which is famous by its attractive skin colour and exotic flavour. But there are still some challenges in the litchi planting industry, such as the peel coloration ('stay green' or pigmenting uneven problem in some varities like 'Feizixiao'), fruit abscission, flowering control and postharvest preservation. The CCO gene family have been reported to be involved in important biological functions in the plants [1][2][3]. However, this gene family has not been identified in litchi. In this study, genome-wide identification of CCO gene family had been conducted, and their gene structure, domain, motif, phylogenetic relationship, miRNA target sites, cis-elements, 3D protein structure, and expression patterns were comprehensively analyzed. The study may provide a solid foundation for future functional studies of CCO genes in litchi and other fruit trees.

Identification of LcCCO genes and their physicochemical properties
After homology search, a total of 15 LcCCO genes were identified in litchi. Physicochemical properties analysis found that the largest protein was LcCCD1, which contained 1434 amino acids, the smallest protein was LcC-CD4c1, which contained 303 amino acids. MW of LcCCO proteins ranged from 16.25 kDa to 34.23 kDa, pI ranged from 5.54 to 8.15. Instability index analysis revealed that LcCCD1, LcCCDlike-a, LcCCDlike-b, LcCCD4c1, LcC-CD4c2, and LcCCD8b were stable proteins(Instability index < 40), and the rest were unstable proteins. Aliphatic index of LcCCO proteins ranged from 75.61 to 84.62. Grand Average of Hydropathicity analysis showed that all LcCCO proteins were hydrophilic protein. Subcellular localization prediction exhibited that most of LcCCO proteins (11,73.3%) were located in chloroplast, three LcCCO proteins (LcCCDlike-a, LcCCDlike-b, and LcC-CD4c1) were located in cytoplasm, one LcCCO proteins (LcCCD8b) was located in mitochondrion (Table 1).

Gene structure, domain, motif and chromosomal arrangement analysis
Gene structure analysis showed that the numbers of exon of LcCCO genes ranged from 1 to 32. CCD1 subfamily contained the largest number of exons, following were the CCD7 and CCD8 subfamily, CCD4 and NCED subfamily just had one exon. LcCCO genes in the same subfamilies displayed similar structure distribution patterns (Fig. 2B). Conserved domain analysis exhibited that all of LcCCO genes contained a RPE65 domain (Fig. 2B). Motif analysis showed that all of LcCCO genes had the motif 5 and motif 7, and like conserved domain, displayed similar patterns in the same subfamilies. Such as in NCED family, the distribution pattern of motifs of each member began with motif9, 10,6,8,10,6,1,4,9,3,5,7, and 2 from N-terminal to C-terminal ( Fig. 2C-D). Chromosomal distribution analysis showed that LcCCO genes located in seven chromosomes (Fig. 2E). Chromosomes1 and 5 (Chr1 and Chr5) contained nine (60%) LcCCO genes (five and four respectively). Chr11 had two (13.3%) LcCCO genes. Chr2, 9, 14 and 5 carried only one (6.7%) LcCCO gene. LcCCDlike-b, LcNCED2, and LcCCD4 were located in the regions with high gene density.

Prediction of miRNA target site of LcCCO genes
MiRNA target site prediction showed that a total of 31 miRNA target sites could be found in 13 (86.7%) LcCCO genes with the exception of LcCCDlike-b and LcNCED3 (Table 2). Among all LcCCO genes, LcCCD1 and LcC-CDlike-a existed the most miRNA target sites, which could be targeted by 10 and 7 miRNAs separately. LcC-CD4c1 and LcCCD7 just had one miRNA target site. In the same subfamily, we found that some members could be targeted by a same miRNA. Such as LcCCD4a1, LcC-CD4a2, LcCCD4b, and LcCCD4c2, which belonged to the CCD4 subfamily, could by targeted by Lc-miRN23 simultaneously. LcCCD1 and LcCCDlike-a, which belonged to CCD1 subfamily, could be targeted by Lc-miRN58 concurrently, but the LcCCDlike-a existed two different Lc-miRN58 target sites. More generally, LcCCO genes in the same or different subfamilies were targeted by different miRNAs. Such as LcNCED1, LcNCED2, and LcNCED3, which belonged to NCED subfamily, there were no common miRNA targets.

Cis-regulatory elements analysis of LcCCO genes
Cis-regulatory elements analysis found that a total of 411 cis-elements could be identified in the promoter region of LcCCO genes with the exception of common elements like TATA-box and CAAT-box and some unknown functional elements ( Fig. 3    development related, possessed 25 (6.08%) elements, such as endosperm expression (GCN4_motif ), meristem expression (CAT-box), MYB binding site involved in flavonoid biosynthesis (MBSI) and seed specific regulatory element (RY-element).

GO enrichment analysis of LcCCO genes
In order to predict the exact functions the litchi genes, GO enrichment analysis of LcCCO genes had been conducted in study. The result showed that the function of LcCCO genes functioned in moleculler function, cellular component and biological process (Fig. 5A and Table S4).
When comes to the biological process, it was clearly that LcCCO genes were involving in the process of fruit ripening, pollination, flower development, catabolic process, response to endogenous stimulus, signal transduction, response to abiotic stimulus, response to light stimulus, reproduction so on.

Expression patterns analysis of LcCCO genes by RNA-seq data
In order to investigate the potential function of LcCCO genes, the expression pattern of LcCCO genes related to peel coloration, fruit abscission, flowering control, and postharvest preservation of litchi were analysed based on the RNA-seq data supplied by our research group (not published) and other groups published online ( Fig. 5B-G). During the peel coloration inhibition experiment of 'Feizixiao' litchi induced by exogenous CPPU treatment ( Fig. 5B and Table S5), compared to the complete green stage of fruit, LcCCD1, LcNCED1, and LcNCED2 exhibited down-regulated expression in the best edible stage (this stage of 'Feizixiao' litchi which existed 'stay green' phenomenon) between control and treatment groups, but much more obviously in the treatment groups (decreased by 1.49, 5.44, and 6.24 times in control groups and 1.33, 2.38, and 5.58 times in the treatment groups    Table  S6). These results suggested that LcCCDlike-b, LcCCD1, LcCCD4, LcNCED1, and LcNCED2 might play an important role in the fruit maturation of 'Feizixiao' litchi. Compared to 'Feizixiao' litchi, 'Nuomici' litchi fruit could complete coloring [28]. LcCCD1 and LcCCD4 exhibited down-regulated expression in the yellow and red stage of fruit, LcNCED1 showed up-regulated expression after green stage, and reached the peak in the red stage. LcNCED2 displayed down-regulated expression at yellow stage and up-regulated expression in the red stage ( Fig. 5D and Table S7). These finding suggest that LcCCD1, LcCCD4, LcNCED1, and LcNCED2 might function during the fruit maturation of 'Nuomici' litchi.
Uniconazole treatment of litchi inflorescences can control flowering and improve fruit-setting in litchi [29]. LcCCD4, LcCCD4c2, LcCCD8a, LcNCED2, and LcNCED3 showed down-regulated expression obviously in the entire inflorescences after uniconazole spraying, decreased by 1.72, 1.53, 15.97, 2.80, and 3.18 times separately. No apparent change found in other genes ( Fig. 5E and Table S8), indicated that the above genes might be involved in the flowering control and fruit-setting improvement of litchi.
In the fruitlet samples during fruit abscission of 'Wuye' litchi induced by girlding plus defoliation treatment [30], LcCCD1, LcCCD4a2, LcCCD8a, and LcNCED1 showed down-regulated expression on the second day after treatment, and LcCCD4a2 decreased most significantly (11.07 times). LcCCD4 and LcNCED2 displayed down-regulated expression on the fourth day after treatment, decreased by 1.15 and 1.13 times separately. LcNCED3 exhibited down-regulated expression on the seventh day after treatment, LcCCD4 showed up-regulated expression on the seventh day after treatment (Fig. 5F and Table S9). In the abscission zone samples during fruitlet abscission of 'Feizixiao' litchi caused by exogenous ethephon treatment [31], LcCCD4a2, LcCCD4b, LcCCD8a, and LcC-CD8b showed down-regulated expression evidently on the first, second and third day. LcCCD1, LcCCD4, and LcNCED1 displayed down-regulated expression on the first and second day and up-regulated expression on the third day after treatment. LcNCED2 exhibited upregulated expression evidently during the whole times ( Fig. 5G and Table S10). These results indicated the above genes might be related in the fruitlet abscission of litchi.
In the peel samples of 'Huaizhi' litchi on 0d and 4d after stored at room temperature and 0 h, 24 h, and 48 h stored at room temperature after precooling for 14 days [32], LcCCD1, LcCCD4, LcNCED1, and LcNCED2 showed relative higher expression on 0d in the sample which stored at room temperature without precooling treatment, but expression inhibition could be found obviously in the samples which do the precooling treatment. All of the above four genes showed down-regulated expression on 4d after stored at room temperature without precooling treatment, up-regulated expression on 14 h and 48 h after stored at room temperature treated by precooling. It was interesting that LcCCD4a1 and LcCCD4a2 showed significantly up-regulated expression in 0 h stored at room temperature after precooling (Fig. 5H and Table  S11). These data suggested that the above genes might be involved in the rapid fruit senescence induced by precold storage.

Identification of expression patterns of LcCCO genes by quantitative qRT-PCR
In order to further explore the potential function of LcCCO genes, the samples which involving in the inhibition of peel coloration of 'Feizixiao' litchi induced by exogenous CPPU, the natural peel coloration of 'Nuomici' litchi, fruitlet abscission of litchi produced by girdling plus defoliation treatment and exogenous ethephon treatment were collected. The expression patterns of these pivotal LcCCO genes obtained by the RNA-seq data were assessed by quantitative qRT-PCR (Fig. 6).
The results showed that the expression pattern of most of LcCCO genes were consistent with their expression patterns in the RNA-seq data described above (Figs. 5B, D, F-G, and 6) excepted for the expression of LcNCED1 during the coloration of 'Feizixiao' litchi and the expression of LcCCD4 during the coloration of 'Nuomici' litchi (Figs. 5B, D, 6A-B). Interestingly, there were also some differences of the expression of LcCCO genes between the experiment of fruitlet abscission of 'Feizixiao' litchi and 'Wuye' litchi produced by girdling plus defoliation treatment (Fig. 6C). This might be caused by the variety differences between 'Wuye' litchi and 'Feizixiao' litchi. But there were still some LcCCO genes shared the similar expression patterns, Such as LcCCD4, LcCCD4a2, LcC-CD8a, and LcNCED1.

Identification of LcCCO genes
Compared to the MYB, bZIP and bHLH gene family, CCO is a relatively small gene family in plant. In our study, a total of 15 LcCCO genes were identified in litchi and could be divided into six (CCD1, CCD4, CCD7, CCD8, CCD-like, and NCED) subfamilies based on the phylogenetic relationships analysis with Arabidopsis thaliana, Solanum lycopersicum, Malus × domestica and Litchi chinensis Sonn (Fig. 1), which was consistent with the previous work [10,35]. Physicochemical properties analysis showed that the length of most of LcCCO proteins ranged from 500 to 600aa (Table 1), which displayed similarity with other plants [6,36,37]. RPE65 domain is a specific conserved domain in CCO protein, which is the key to the enzymatic oxidation activity cleavage of carotenoids [38]. Conserved domain analysis showed that all of LcCCO proteins contained a RPE65 domain and which exhibited similar distribution pattern in the same subfamily. Like distribution of RPE65 domain, gene structure and motif showed high similarity of distribution pattern in the same subfamily too ( Fig. 2A-C). These results indicated that the genes in the same subfamily which held the similar function probably. Motif 5 and motif 7 were located in all LcCCO protein, implied that they were important characteristics and may be responsible for common functions between them. Subcellular localization analysis can help to understand the site where the protein will function. In the study, 11 (73.3%) LcCCO proteins were predicted to be located in the chloroplast, suggested that these genes might participate in chlorophyll photosynthesis. 3 (20.0%) LcCCO proteins located in cytoplasm and 1 (6.7%) LcCCO protein located in mitochondria ( Table 1), indicated that these genes functioned in cytoplasm and mitochondria, and might not be involved in chlorophyll photosynthesis. These results are consistent with the previous study [35].
MiRNA is considered as a kind of post transcriptional regulator, and play a critical role during the development of plant [39,39,40]. In our result, 13 (86.7%) LcCCO genes obtained 31 miRNA target sites predicted combined with the litchi miRNAs described previously [41], suggested that post transcriptional regulation of LcCCO genes by miRNA might be functioning during the development of litchi. Cis-regulatory elements located in the gene promoter region which could regulated the gene expression on transcriptional level [42]. Cis-regulatory elements analysis found that a large number of cis-elements which involving in light responsive, plant hormones and stress (biological and abiotic stress related) and pant growth and development could be detected (Fig. 3A-C and Table S2). It was interesting that ABRE element which related to ABA response were the largest group in the plant hormones related cis-elements. These indicated that the transcription of LcCCO genes could be in response to light, plant hormones, biological and abiotic stress and pant growth and development. In order to investigate their function during the development period of litchi, RNA-seq data and quantitative qRT-PCR analyses related to peel coloration, flowering control, fruit abscission, and postharvest preservation were used to do further analysis.

LcCCO genes might be involved in the coloration and flavor of litchi
The colour of horticultural produce, is a key factor that can decide and enhance their economic value. The carotenoids metabolism pathway of CCD4 had been proved to be related to the color formation in plant species like chrysanthemum petalsonly and citrus by affecting the catalytic degradation pathway of carotenoids [14,16]. ABA was considered as the critical hormone related to the coloration in plant, including strawberry [21], grape [22], sweet cherry [23] and litchi fruit [24]. NCED is a key regulator of ABA biosynthesis. FaNCED1 RNAi resulted in uncolored strawberry fruits by declining the ABA and anthocyanin content significantly (Fragaria × ananassa) [20]. 'Feizixiao'litchi existed in 'stayed green'category at the best edible stage. RNA-seq data and qRT-PCR analysis showed that the expression of LcCCD4, LcNCED1, and LcNCED2 could be inhibited obviously by exogenous CPPU treatment during fruit maturation of 'Feizixiao' litchi. LcCCD4 reached a peak at 50d and 57d after anthesis (the best edible stage) and then declined, LcNCED1 and LcNCED2 reached the peak at 43d and 35d after anthesis separately, but kept relative low expression (Figs. 5B, 6A, Table S5). RNAseq data showed that LcCCD4, LcNCED1, and LcNCED2 exhibited up-regulated expression apparently during light-regulated anthocyanin biosynthesis to promote the coloration of 'Feizixiao' litchi after removing bag, and the TPM value of them was much higher than the experiment samples treated by CPPU (Fig. 5B-C and Table S5-6). Compared to 'Feizixiao' variety, 'Nuomici' litchi fruit could fulfil coloration. RNA-seq data and qRT-PCR analysis showed that LcCCD4 exhibited relative high expression before 67d after anthesis and reached the peak at 60d (yellow stage). LcNCED1 exhibited relative higher expression than 'Feizixiao' litchi samples described above and reached a peak at 60d after anthesis and then declined, but still kept relative higher expression at 67d (Red stage). LcNCED2 exhibited two peaks at 41d (green stage) and 67d (red stage) after anthesis (Figs. 5D, 6B and Table S7). Among the above results, the higher expression of LcCCD4, LcNCED1, and LcNCED2 during the early stage of fruit maturation of 'Nuomici' litchi might contribute to the carotenoid degradation and anthocyanin accumulation to complete coloration. The relative low expression of LcCCD4, LcNCED1, and LcNCED2 in 'Feizixiao' litchi might be the key factor to induce the 'stay green' phenomenon. In addition to participating in coloring, CCO genes liked CCD1 was reported to be involving in the formation of the flavor and scent in plants by catalyzing degradation of carotenoids [12,43]. We could clearly find that LcCCD1 showed down expression during the fruit maturation in the both of 'Feizixiao' and 'Nuomici' varieties simultaneously, and LcCCDlike-b displayed down expression significantly in 'Feizixiao' variety but could not be detected in the 'Nuomici' variety (Fig. 5B, D and Table S5, S7). Although the dynamic of carotenoid content all showed a downtrend, but displayed some differences during fruit maturation of 'Feizixiao'and 'Nuomici' varieties (Supplement Fig. 1). These implied that LcCCDlike-b might be an important factor which involving in the different flavor between these two varieties. But these conjectures need further works to confirmed them.

LcCCO genes might be involved in fruitlet abscission of litchi
ABA was also reported to be related to plant organ abscission [44,45]. Litchi is an important subtropical and tropical economic fruit. It is reported that there are 3-5 fruit drop waves (I, II, III, IV, V) between different cultivars, and high ABA lever is regarded one of most important physiological reasons for the fruit drop wave II, III, and V [46]. Based on the public RNA-seq data and qRT-RCR analysis, we found that the expression of LcNCED1, LcNCED2, and other LcCCO genes were enhanced in fruitlet and abscission zone samples during fruitlet abscission induced by GPD and exogenous ETH treatment in 'Feizixiao' variety separately (Figs. 5F-G, 6C-D and Table S9-10). These implied that LcNCED1 and LcNCED2 might function in accelerating the ABA content to promote the fruitlet abscission of litchi, the function of other LcCCO genes needed further work to investigate. Contrary to the result of 'Feizixiao' variety, the expression of LcNCED1, LcNCED2 other CCO genes in 'Wuye' variety were inhibited in the fruitlet samples during fruitlet abscission induced by GPD treatment (Fig. 5F and Table S9). The differences of gene expression between 'Wuye' variety and 'Feizixiao' variety treated by GPD, which might be caused by the difference between these two varieties, but needed further works to demonstrate this conjecture.

LcCCO genes might be involved in flower control of litchi
Easily flowering and without controlling of the inflorescence development and flowering can lead to low fruitsetting percentage or even zero yield by consuming excessive amounts of accumulated nutrients during the production of litchi [47]. Uniconazole spraying which can regulate the changes of endogenous hormone levels (reducing GA content and increased ABA content) to function in the flower control and fruit retention of litchi, is considered as an effective chemical method [48,49]. Another research proved that uniconazole was a strong competitive inhibitor of ABA 8'-hydroxylase to effectively inhibit ABA catabolism in Arabidopsis plants [50]. In this study, the RNA-seq data published by Wei et al. [29] showed that LcNCED2 and LcNCED3 displayed low expression in litchi inflorescences treated by uniconazole ( Fig. 5E and Table S8). These suggested that the ABA accumulation is the result of the joint action of biosynthesis regulated by LcNCED2 and LcNCED3 and catabolism inhibition by uniconazole. The later might be much more important during the flowering controlling to improve the litchi fruit-setting. but also need more experiment to confirm it.

LcCCO genes might be involved in the postharvest preservation of litchi
Litchi is a non-climacteric tropical and subtropical fruit which is highly perishable, and typical symptoms is pericarp browning and loss of flavor after harvest. The shelf life of litchi fruit could be prolonged up to a month storage in cold environment, but the fruit senescence of fruits which were at ambient temperatures after pre-cold storage treatment could be accelerated, only 1-2 days, much less than the fruit under ambient conditions, which is approximately 4-6 days [32,51,52]. ABA was considered as one of key factors which contributed the fruit senescence [53,54]. In this study, The RNA-seq data published by Yun et al. [32] showed that LcCCD4a1 and LcCCD4a2 displayed significantly up-regulated expression at 0 h, LcCCD1, LcCCD4, LcNCED1, and LcNCED2 displayed up-regulation at 14 h and 48 h stored at room temperature after treated by precooling ( Fig. 5H and Table S11). These data suggested that the above genes might be involved in the rapid fruit senescence induced by pre-cold storage by enhanced the ABA content and accelerate the carotenoid degradation, but need further experiments to confirmed it.

Conclusions
In conclusion, CCO gene family were identified comprehensively in litchi. A total of 15 LcCCO genes were identified and could be classified into five subfamilies based on the phylogenetic relationships with other several species. And then the physicochemical properties, the distribution of gene structure, conserved domain and motif, ciselements, miRNA target sites, 3D protein structure were further analysed. In addition, RNA-seq data and qRT-PCR analysis revealed that LcCCO genes might be related to the peel coloration, fruit flavor, flower control, fruit abscission and postharvest storage of litchi. Our result not only will help lay the foundation for the function identification of CCO gene family in litchi, but also will help us understand the role of this gene family in other plant species.

Identification of LcCCO genes
The genome and gene annotation files of litchi was supplied by College of Horticulture, South China Agricultural University, Guangzhou, China (data unpublished yet). The CCO protein sequences of Arabidopsis thaliana were downloaded from TAIR database (https:// www. arabi dopsis. org/). Homology search was conducted by the TBtools software [26], and then confirmed the RPE65 (retinal pigment epithelial membrane protein) domain used the Pfam database (http:// pfam. xfam. org/) and Hmmer2.3 database (http:// hmmer. janel ia. org/). The sequences did not contain the RPE65 domain would be deleted, and the rest proteins were considered as the litchi CCO (LcCCO) proteins. Physicochemical properties of LcCCO proteins like molecular weight (MW), Pi, instability index, aliphatic index was analyzed by ExPASY website (http:// web. expasy. org/ protp aram/), Subcellular localization prediction was conducted by the BUSCA website (http:// busca. bioco mp. unibo. it/), signal peptide and transmembrane structure were predicted by the MBC website (http:// cello. life. nctu. edu. tw).

Phylogenetic analysis
The CCO protein sequences of Arabidopsis thaliana, Solanum lycopersicum, Malus × domestica and Litchi chinensis Sonn. were used to do phylogenetic analysis. Phylogenetic tree was conducted by Clustal X2 and MEGA 6 software using maximum-likelihood (ML) methods with the following parameter settings: poisson model, partial deletion and 1000 bootstrap replicates. At last, the LcCCO proteins were renamed depended on which subfamily they belonged to.

Gene structure, conserved domain, conserved motif and chromosomal arrangement analysis
The structure information of LcCCO genes can be acquired by the gff file of litchi genome. The conserved domain identification was conducted by NCBI cdsearch website (https:// www. ncbi. nlm. nih. gov/ Struc ture/ bwrpsb/ bwrpsb. cgi) and Pfam website (http: //www. sanger. ac. uk/ softw are/ Pfam/). The motif analysis was executed by MEME tools (http:// meme-suite. org/ tools/ meme). Phylogenetic tree, gene structure, conserved domain and motif of LcCCO genes were displayed by TBtools software [26].

miRNA target site prediction
Litchi miRNAs sequences could be obtained from the previous works  and the LcCCO genes sequences were adopted to do the miRNA target site prediction by the psRNATarget website (https:// www. zhaol ab. org/ psRNA Target/ analy sis? funct ion=3) with default parameter settings.

Cis-regulatory elements analysis
The 2000 bp upstream sequences from the translation initiation codon ' ATG' of LcCCO genes were extracted by the TBtools software [26] and then used to do the cis-regulatory elements analysis by Plant Care database (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/).

Gene Ontology (GO) enrichment analysis
Firstly, all litchi genes was blasted to the uniprot_sprot. fasta file downlorded from Swissprot database (https:// www. unipr ot. org/). Then GO annotation and enrichment analysis was conducted by the TBtools software [26].

Expression analysis of LcCCO genes identified by quantitative qRT-PCR
To further investigate the function of LcCCO genes, four sets of litchi samples with 3 biological replicates in each group were collected for the quantitative qRT-PCR analysis as followed: (1) The peel samples of different development stages (on 35d (complete green stage, corresponed to the T1 and CK1 treatment used in the RNA-seq data),