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Article

Identification and Expression Pattern of the Carotenoid Cleavage Oxygenase Gene Family in Lycium Suggest CCOs Respond to Abiotic Stress and Promote Carotenoids Degradation

by
Weinan Li
1,2,
Jiahang Che
1,2,
Qile Lian
1,2,
Cuiping Wang
3,
Guoli Dai
4 and
Jinhuan Chen
1,2,*
1
College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China
2
Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, Beijing Forestry University, Beijing 100083, China
3
School of Biological Science and Engineering, North Minzu University, Yinchuan 750021, China
4
National Wolfberry Engineering Research Center, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 750002, China
*
Author to whom correspondence should be addressed.
Forests 2023, 14(5), 983; https://doi.org/10.3390/f14050983
Submission received: 28 March 2023 / Revised: 5 May 2023 / Accepted: 6 May 2023 / Published: 10 May 2023
(This article belongs to the Special Issue Abiotic Stress in Tree Species)
Browse Figures
Versions Notes

Abstract

:
Carotenoids are key metabolites in goji (Lycium), a traditional Chinese medicine plant; however, the carotenoid content varies in fruits of different goji species, and the mechanism of this variation is not clear. Carotenoids participate in signal transduction and photosynthesis, and function as colorants and photoprotectors. Members of the carotenoid cleavage oxygenase (CCO) gene family are involved in the regulation of phytohormones, pigments, and aromatic substances, such as abscisic acid (ABA), β-carotenoid, and α-ionone, by degrading carotenoids. Some CCO genes are also related to an abiotic stress response. Here, a total of 12 LbCCO genes were identified and analyzed from the L. barbarum genome. CCO genes were divided into six subfamilies based on the constructed phylogenetic tree, including LbNCEDs, LbCCD1, LbCCD3, LbCCD4, LbCCD7, and LbCCD8. Among them, CCD3 was reported for the first time. The gene structure and motif analysis revealed the conservation of CCO subfamilies. Pseudogene generation and the importance of each subfamily in CCOs were revealed by collinearity analysis. The spatiotemporal transcriptomes of L. barbarum and L. ruthenicum were compared, suggesting that CCD4-1 may dominate carotenoid degradation in goji fruits. Cis-acting elements prediction and environment responsive gene expression analyses indicated that salt-alkali stress and photothermal conditions might influence the expression of CCOs in goji. The results of this study enhance our understanding of the carotenoid degradation pathway, and the functions and responses of CCOs in goji species.

1. Introduction

There are more than 80 species of Lycium in the world [1], with seven species found in China [2]. Of these species, L. barbarum and L. ruthenicum contain numerous phytochemicals including polysaccharides, betaine, phenolics, carotenoids, cerebroside, ascorbic acid, beta-sitosterol, flavonoids, anthocyanins, and vitamins, and have been used for medicinal purposes for thousands of years [3]. The fruits of these two goji species contain dramatically varied amounts of carotenoids [4], indicating potential differences in their carotenoid metabolism processes.
Carotenoids are isoprenoid-based pigments with various functions among bacteria, fungi, plants, and animals [5,6], and are considered as important sources of nutrition for humans and other animals. Carotenoids are precursors of vitamin A, photo-protectors, and antioxidants, enhancers of immunity, and contributors to reproduction [7]. In plants, carotenoids participate in signal transduction and photosynthesis and function as colorants and photoprotectors. Carotenoids can be oxidized by carotenoid cleavage oxygenases (CCOs) into bioactive substances with specific functions, such as volatile flavor compounds and hormones including abscisic acid (ABA) and strigolactone (SL) [8,9,10]. ABA is a major phytohormone that regulates plant growth, development, responses to different kinds of stresses, and a variety of adaptation processes, such as seed dormancy, growth restriction, and leaf senescence [6,11,12]. Strigolactone helps plants sense nutrient availability, especially phosphate and minimize shoot growth and increase the root area to accumulate nutrition, and promotes arbuscular mycorrhizal fungi symbiosis to acquire inorganic nutrients [6,13,14].
In previous studies, CCOs were grouped into six subfamilies, including five types of carotenoid cleavage dioxygenases (CCDs, CCD1, 2, 4, 7, 8) and 9-cis-epoxycarotenoid dioxygenases (NCEDs) with various functions [8,15]. CCDs generate apocarotenoids through a carotenoid oxidative cleavage process [9]. CCD1 functions in the cleavage of carotene, zeaxanthin, and apocarotenoids for the synthesis of apocarotenoid flavor and aroma volatiles, such as α-ionone [16,17,18,19]. CCD4 acts to produce β-cyclocitral or β-ionone through the cleavage of β-carotene in different ways, making CCD4 a potential determinant of fruit or flower color [20,21,22,23]. Lycopene and β-xanthophylls (β-cryptoxanthin, zeaxanthin) were identified as substrates of CCD4 [20,21]. CCD7 and CCD8 are involved in the synthesis of strigolactone and then regulate shoot and root branching or interact with soil microbes [22]. NCEDs encode rate-limiting enzymes in the biosynthesis of ABA and cleaved 9-cis-epoxy-carotenoids produce direct precursors of ABA in higher plants [23]. NCEDs regulate plant development, senescence, and response to abiotic stress by regulating ABA accumulation. The overexpression of LeNCED1 can promote salt accumulation in leaves and xylem sap to help minimize plant growth loss under salt stress [24]. Increasing the ABA level was found to be related to the expression of NCED1 under drought stress in plants such as Aristotelia chilensis [25]. The expression of NCED1 was related to carotenoid degradation in kiwifruit [26]. VviNCED2 and VviNCED3 increased the ABA level in response to cold stress [27]. GaNCED3a can enhance the drought resistance of cotton [28]. Through being constitutively expressed in various tissues in Oryza sativa, OsNCED3 can regulate seed germination and seedling growth by controlling ABA content [29].
Although abiotic stress, including salt-alkali and high temperature, can adversely affect plant growth and development, it also promotes the accumulation of functional phytochemicals in plants [30]. Under salt stress, tomato plants can uptake and transport significant amounts of Na+ to the shoot, resulting in enhanced organoleptic properties of the tomato fruits, as evidenced by increases in carotenoids, flavonoids, vitamin C, total phenolic compounds, total soluble solids, and total titratable acids [31]. High light intensity and high temperature stress induced chlorophyll loss and the synthesis of carotenoids in tomato plants [32]. Carrots under salt and simulated drought conditions exhibit an overall or partial increase in carotenoid content [33,34].
L. barbarum is rich in carotenoids, with amounts reported to be as high as 233 μg/g DW in fruit [35]. However, little is known about carotenoid synthesis and the related metabolic pathways in this species. L. barbarum and L. ruthenicum fruits differ significantly in carotenoid content, with L. barbarum being rich in carotenoids in mature fruits and L. ruthenicum fruits containing almost no carotenoids [35]. To date, the genes responsible for this variation in carotenoid synthesis or degradation between L. barbarum and L. ruthenicum have not been determined. A previous study suggested the influences of both carotenoid synthesis and carotenoid degradation processes on the carotenoid content [36]. In this study, we investigated the CCO gene family of L. barbarum and performed the evolutionary analysis to predict the classification of LbCCOs. We then performed a bioinformatics analysis of the structure, conserved domains, motifs, and promoters of the identified genes. Finally, we analyzed and compared the expression patterns of the CCOs in L. barbarum and L. ruthenicum in different spaces and times and analyzed the expression patterns under different environment conditions.

2. Materials and Methods

2.1. Plant Materials

Three-year-old L. barbarum and L. ruthenicum seedling clones were cultivated in 38°38′51″ N, 106°9′29″ E. Stamens, pistils, green fruits, mature fruits, leaves on current year branches, leaves on last year branches, and stem tips were collected. Each kind of material was collected in June 2021 with three biological replicates from three different trees under normal cultivation and management conditions of goji. Three-year-old L. barbarum clones were cultivated in 38°38′51″ N, 106°9′29″ E (Yinchuan, Ningxia) at 1070 m above sea level and 36°26′3″ N, 96°25′35″ E (Nomhon, Qinghai) at 2750 m above sea level. Weather data were recorded in Nomhon and Yinchuan by a weather station with multiple sensors, and included average high and low temperature, hours of daylight and twilight, and average daily incident shortwave solar energy. Green and red (mature) fruits were collected in July 2022 with three biological replicates from three different trees under normal cultivation and management conditions of goji. All samples were immediately frozen in liquid nitrogen after collection and all samples were stored at −80 °C before testing.

2.2. Identification of LbCCO Family Genes

The whole genome sequence of L. barbarum (GenBank assembly accession: GCA_019175385.1) was downloaded from the NCBI database. RPE65 (retinal pigment epithelial membrane protein) is a conserved structural domain of the CCO gene family [37]. To identify all the CCOs, the HMM (hidden Markov model) profile of the RPE65 domain (PF03055 in Pfam protein database), a statistical model of the primary structure consensus of a sequence family, was used to search against the L. barbarum genome in TBtools [38]. RPE65 domain verification for the protein sequences was performed using SMART [39,40] and NCBI-CCD [41] databases, and genes that encoded proteins with less than 150 amino acids in the RPE65 domain were identified as genes with an incomplete conservative domain and then were filtered out.

2.3. Classification and Protein Characterization of LbCCOs

CCO family genes from Arabidopsis thaliana, Nicotiana tabacum, Solanum lycopersicum, L. ruthenicum, and Crocus sativus were collected as reference genes, and their amino acid sequences were downloaded from the NCBI protein (https://www.ncbi.nlm.nih.gov/protein, accessed on 20 April 2022), Swiss-Prot [42], or TAIR [43] databases. A maximum likelihood phylogeny tree (ML tree) was constructed by Tbtools-One Step Build a ML tree with the CCO protein sequences from the identified genes and reference genes using default parameters [38]. The interactive tree of life (iTOL, https://itol.embl.de/, accessed on 20 April 2022) was used to visualize the ML tree. Gene symbols of L. barbarum CCOs were named, referring to the symbols of genes that clustered into the same class in the ML tree.
Physicochemical properties, including the MW (molecular weight), pI (isoelectric point), instability index, aliphatic index, and GRAVY (grand average of hydropathicity) of LbCCOs were calculated and predicted by ExPASy-ProtParam (https://web.expasy.org/protparam/, accessed on 20 April 2022) [44]. Subcellular localizations of CCO proteins were predicted by TargetP-2.0 (https://github.com/JJAlmagro/TargetP-2.0/, accessed on 20 April 2022).

2.4. Analysis of Gene Structure and Cis-Acting Elements of Identified LbCCOs

Conserved motifs and RPE65 domains were analyzed with default parameters in Tbtools-Simple MEME Wrapper and the NCBI-CCD database [38], respectively. Cis-acting elements in the promoter prediction was performed in PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 April 2022) and then visualized by Tbtools software [38].

2.5. Chromosome Localization, Gene Density, and Collinearity Analysis of LbCCOs

Chromosome localization, gene density, and collinearity analysis were constructed and visualized using Tbtools with default parameters [38]. Collinearity analysis of LbCCOs was calculated by One Step MCScanX of Tbtools [38], retaining results with e-values less than 1 × 10−10 and keeping five blast hits.

2.6. Expression Patterns of LbCCOs

L. barbarum and L. ruthenicum fruit developmental RNA-Seq data (green fruit, color changing, and ripening stages) were filtered from raw data obtained from NCBI-SRA (accession number: PRJNA483521) [45]. High-quality reads (clean reads) were obtained by removing adapter sequences and eliminating reads containing more than 10% unknown nucleotides and low-quality reads (reads containing more than 50% bases with a Q-value ≤ 20). L. ruthenicum stress transcriptome RNA-Seq data (raw data) were obtained from NCBI-SRA (accession number: PRJNA285517). Gene expression levels of fruits in Ningxia and Qinghai were determined from transcriptome data.
Gene expression levels of stamens, pistil, green fruit, mature fruit, leaves on current year branches, leaves on last year branches, and stem tips of the two goji species were determined from transcriptome data. Transcriptome analysis was performed as follows: total RNA was extracted from plant samples using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the instructions. The RNA concentration and purity were measured using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). The RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). A total amount of 1 μg RNA per sample was used as input material. Sequencing libraries were generated using NEBNext UltraTm RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA). For this, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations at an elevated temperature. Cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit. To select cDNA fragments that were 240 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Brea, CA, USA). Library quality was assessed on the Agilent Bioanalyzer 2100 system and sequenced on an Illumina platform, and paired-end reads were generated.
Fastp (https://github.com/OpenGene/fastp, accessed on 15 March 2023) was used to obtain clean reads from raw data with default parameters. Kallisto (https://github.com/pachterlab/kallisto, accessed on 15 March 2023) was used to determine the gene expression level with parameters.

2.7. Gene and Promoter Isolation

Plant tissues from L. barbarum and L. ruthenicum seedlings were transferred to mortar, milled to a powder, and mixed with liquid nitrogen immediately. The Plant Genomic DNA Kit (Tiangen, China) and RNAprep Pure Plant Kit (Tiangen, China) were used to extract DNA and RNA, respectively. After checking the RNA quality, the FastKing RT Kit (Tiangen, China) was used to reversely transcribe RNA to cDNA.
The sequences of CCD4-1s and their promoters were obtained from the goji genome or public database and primers (Supplemental Table S1) were designed by Primer3Plus (https://www.primer3plus.com/, accessed on 19 November 2021) to use PCR to amplify the CCD4-1s coding region and CCD4-1 promoters. After purification by TIANgel Purification Kit (Tiangen, China), the PCR products were cloned into the pMD18-T vector and sequenced. The gene and promoter sequences of L. barbarum and L. ruthenicum were compared. Binding site prediction was performed on the Plant Transcriptional Regulatory Map (http://plantregmap.gao-lab.org/binding_site_prediction.php, accessed on 19 August 2022) website using A. thaliana transcription factors as the model.

2.8. Subcellular Localization

LbCCD4-1 and LrCCD4-1 coding regions without stop codons were inserted into the pBI121 vector and then fused with the EGFP gene to construct plasmids encoding CCD4-1-EGFPs. The plasmids were separately introduced into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method and then transformed into Nicotiana benthamiana leaf cells using the agrobacterium infection method. The tobacco plants were grown in the dark for 1 day, with normal light cultivation for 2 day, and then imaged by TCS SP8 X confocal microscopy (Leica, Germany) using a 488-nm laser line to image EGFP and chloroplast signal.

3. Results

3.1. Identification and Classification of LbCCOs

The L. barbarum genome sequence was obtained from the NCBI database (GenBank assembly accession: GCA_019175385.1). The retinal pigment epithelial membrane protein domain (RPE65 domain) is a conserved structure of carotenoid cleavage oxygenase [37]. To identify LbCCO members, an HMM search was conducted with the RPE65 HMM profile downloaded from the Pfam protein database. A total of 19 putative LbCCOs were detected. NCBI-CCD and SMART were then used to filter genes lacking RPE65-conserved domains. Some genes were also filtered out based on variation in gene length and conserved domain length. Finally, a total of 12 LbCCO genes were identified in the L. barbarum genome (Supplemental Table S2).
To classify and identify the evolutionary relationships of the CCO genes in L. Barbarum, a maximum likelihood (ML) phylogenetic tree was constructed. The tree was constructed with 5000 ultrafast bootstrap replications using 50 CCO amino acid sequences of Lycium species (L. Barbarum and L. Ruthenicum), model plants (Solanum lycopersicum, Arabidopsis thaliana, and Nicotiana tabacum), and Crocus sativus (Figure 1). Other than the LbCCOs, these CCO genes were previously identified and documented in the NCBI database. All CCO genes were classified into seven subfamilies including NCED, CCD1, CCD2, CCD3, CCD4, CCD7, and CCD8 based on the similarity and homology of amino acid sequences. The CCD3 subfamily was the first discovered, and this subfamily clustered near CCD1 and CCD2. LbCCOs were designated according to the relationships in the phylogeny tree with three NCEDs, one CCD1, two CCD3s, three CCD4s, one CCD7, and one CCD8 identified in the L. barbarum genome.

3.2. Physicochemical Properties, Subcellular Localization Prediction, and Function Prediction of LbCCOs

Physicochemical properties of the identified L. barbarum CCOs were predicted using amino acid sequences. Protein length, MW (molecular weight), pI (isoelectric point), instability index, aliphatic index, and GRAVY (grand average of hydropathicity) were calculated by ExPASy-ProtParam. The amino acid lengths of LbCCOs ranged from 506 aa (LbNCED6-2) to 1105 aa (LbCCD4-2). The predicted molecular weights ranged from 60.0 kDa (LbNCED6-2, LbNCED1) to 123.4 kDa (LbCCD4-3, LbNCED6-1). The predicted pI values of LbCCOs ranged from 5.54 (LbNCED6-2) to 8.55 (LbCCD4-2). The predicted pI values of LbCCD1 and LbCCD4 were greater than 7, indicating that these may be basic proteins. The predicted instability index values of LbCCOs ranged from 29.6 (LbCCD8) to 43.9 (LbNCED6-1), and the predicted instability index values of LbNCED6-1, LbCCD7, and LbNCED2 were greater than 40, indicating that these are likely unstable proteins. The predicted aliphatic index values ranged from 75.8 (LbNCED1) to 89.67 (LbCCD3-2). The predicted GRAVY values of LbCCOs ranged from −0.403 (LbNCED1) to −0.179 (LbCCD4-2), and all were less than 0, indicating all LbCCOs are likely hydrophilic proteins. The subcellular localization prediction of LbCCOs was conducted by TargetP-2.0, and the result showed that LbCCD4s and LbNCED6-1 can localize to chloroplasts, and the other LbCCOs map to the cytoplasm. Detailed information is displayed in Table 1.

3.3. Analysis of Gene Structure and Cis-Acting Elements of the LbCCOs

A motif is a similar 3D structure conserved among different proteins that serves a similar function [46]. Domains are parts of a protein with specific functions and usually move or function independently of the rest of the protein [41]. Gene structure and the conserved domains and motifs were further analyzed, and a ML phylogenetic tree was constructed with the identified LbCCOs and the CCOs from different species. Proteins from the same subfamily were clustered in one category, indicating the conservation of CCOs. The analysis of phylogeny, motifs (Figure 2A,B), domains (Figure 2C), and gene structure (Figure 2D) by Tbtools indicated similarities and differences among LbCCOs [38]. There were 3~19 and 3~10 conserved motifs and non-duplicate motifs, respectively, in the LbCCOs. One or two RPE65 domains were found in all LbCCOs. The N-terminal sequences were relatively conserved, and the most common N-terminus multi-motif was motif 4, motif 9, and motif 10. LbCCD1, LbCCD3s, LbCCD4s, and LbNCEDs were more similar than LbCCD7 or LbCCD8 subfamilies. LbCCD3s differed from LbCCD1, LbCCD4s, and LbNCEDs in the number and the type of conserved motifs. There were at least nine non-duplicate motifs in LbCCD1, LbCCD4s, and LbNCEDs, but fewer in LbCCD3s, especially in LbCCD8 and LbCCD7. LbCCD7, LbCCD8, and LbCCD3s were also distinguished from other LbCCOs by the absence of the multi-motif of motif 1, motif 8, motif 6, motif 5, motif 10, and motif 3. LbNCEDs, but not LbCCDs, possess highly conserved gene structures, motif types and locations, and numbers of conserved domains. There was more variation in the conserved motifs of LbCCDs than in LbNCEDs. Only one RPE65 domain was identified in LbNCEDs, with one or two in LbCCDs. There was no intron found in LbNCEDs, but there were 1~4 in LbCCD4s, 5~6 in LbCCD7 and LbCCD8, and 12~24 in LbCCD1 and LbCCD3s.
To investigate the potential regulation of LbCCOs, 2000 bp sequences upstream of the ATG translation start site of LbCCOs were extracted as promoter regions from the L. barbarum genome, and then the PlantCARE database was used to predict cis-acting elements. A total of 35 cis-acting elements were discovered with potential environmental- and hormonal-response capabilities (Figure 3A). Environmental response elements, light, anaerobic, low-temperature, and drought response cis-acting elements and hormonal response elements, methyl jasmonate (MeJA), abscisic acid, salicylic acid, auxin, and gibberellin response cis-acting elements were found. There was considerable variation in the categories and distributions of cis-acting elements among LbCCO promoters. A statistical clustering heat map of promoter cis-acting elements indicated that environment changes or stresses and endogenous hormone levels are able to alter the expression of different LbCCOs (Figure 3B). Among all cis-acting elements, light-related elements, including G-box, Box 4, GT1-motif, and TCT-motif, were most abundant in the promoters of LbCCOs, except LbCCD1. This group was followed in abundance by anaerobic related elements (ARE). These elements were found in all LbCCO promoters except those of LbNCED1, LbCCD3s, and LbCCD4-2. MYB-binding sites (MBS) are related to drought response, and were found in the promoters of LbCCD7, LbCCD3-2, and LbCCD4-3. There were fewer MeJA response elements (CGTCA-motif and TGACG-motif) than light-related elements in LbCCO promoters. MeJA response elements were found in all promoters except in those of LbNCED2 and LbNCED4-2. Salicylic acid related elements (TCA-elements) were only found in the promoters of LbCCD1, LbCCD4-3, and LbCCD8.

3.4. Chromosome Localization and Collinearity of LbCCOs

Collinearity analyses were conducted with the genomes of L. barbarum, S. lycopersicum, and A. thaliana (Figure 4). Twelve LbCCOs were mapped to five chromosomes of L. barbarum, with CCO clusters detected on chromosome 4 (LbCCD3-1, LbCCD4-1, LbCCD8), 5 (LbNCED6-1, LbNCED6-2), 6 (LbCCD1, LbCCD3-2, LbCCD7), and 9 (LbCCD4-2, LbCCD4-3). To identify whether the nearby gene is tandem, gene duplication analysis was performed of the L. barbarum genome using the function of one Step MCScanx in Tbtools [38]. The result showed no tandem genes for the identified LbCCOs. Seven LbCCOs, LbNCED1, LbNCED2, LbCCD1, LbCCD3-1, LbCCD4-1, LbCCD7, and LbCCD8, exhibited collinearity with SlCCOs and one LbCCO, LbCCD4-2, showed collinearity with SlCCOs and AtCCOs, indicating early formation. The observed collinearity was consistent with phylogeny. Compared with L. barbarum and A. thaliana, there were more collinear CCO genes between L. barbarum and S. lycopersicum, which both belong to Solanaceae.

3.5. Expression Patterns of CCOs

CCOs are involved in carotenoid metabolism [47]. The fruit of L. ruthenicum has extremely low carotenoid content while that of L. barbarum is rich in carotenoids [35]. An analysis of gene expression was performed using RNAseq transcriptome data to identify potential differences of CCO mRNA accumulation between L. ruthenicum and L. barbarum. Three fruit development stages of green stage, turning color stage, and mature stage were analyzed, and the expression data were plotted into a clustered heat map (Figure 5B). Two expression patterns were identified by clustering clades for the three stages of fruit development. Members in clade Ⅰ include CCD4-1, CCD1, and NCED1 and showed relatively stable expression. CCD4-1 was the most highly expressed gene among all the CCO members. Interestingly, its expression increased during the fruit maturation process of L. ruthenicum but decreased rapidly in that of L. barbarum. CCD1 and NCED1 were expressed at relatively stable levels in both goji species throughout the fruit maturation process. The remaining CCOs were clustered into an unstable expression clade (clade II). CCD4-2 seems to exhibit a similar expression pattern as CCD4-1, with lower expression in L. barbarum and greater expression in L. ruthenicum during fruit development. Other CCOs showed relatively low expression levels (max TPM < 3) during fruit development in the two species, except the expression of NCED2 during the green fruit stage in L. barbarum. A comparison of the expression patterns of all CCOs between the two species indicates that CCD4-1 is likely the key gene that acts to decrease carotenoids in L. ruthenicum fruit.
To further investigate the expression pattern in different tissues and organisms, further transcriptome analysis was conducted (Figure 5C). Two clades indicated clustering of different patterns. Clade Ⅰ, including NCED1, CCD1, CCD4-1, and CCD4-2, was expressed in a more stable manner than other CCOs with expression in every organ or tissue in both goji species. Only CCD4-2 showed lower accumulation in the mature fruit of L. barbarum. CCD1 exhibited higher expression in tissues of pistils, leaves, and stem tips. The highest expressed gene of CCO was CCD4-1, which was found in all organs and tissues, especially in leaves and fruits, in both two Lycium species. The lower expression levels of CCD4-1 were only observed in mature fruit and young L. ruthenicum leaves of L. barbarum. The expression levels of CCD4-2 and NCED1 were lower than those of CCD1 or CCD4-1. Clade II included NCED2, NCED6s, CCD3s, CCD4-3, CCD7, and CCD8. NCED6s showed preferred expression in stamens, pistils, and green fruit, with trace amounts in the L. ruthenicum stem tip. NCED2 showed preferred expression in L. barbarum stamens and L. ruthenicum pistils. The remaining CCOs in Clade II exhibited low expression levels.
The CCO gene family is a family that negatively regulates carotenoids, and its expression abundance would be higher in L. ruthenicum, which is a species with hardly any carotenoid content compared with L. barbarum and other goji species. To further investigate how CCOs respond to salt and saline-alkali in a clearer way, the expression patterns of CCOs in L. ruthenicum leaves were analyzed following salt (300 mM Na+) and saline-alkali (300 mM Na+, pH = 9) treatments (Figure 5D). Based on the expression patterns, the genes were classified into two clades. Genes in clade I, including LrCCD4-1, LrCCD4-2 LrCCD1, and LrNCED1, showed significantly higher expression abundance than those in clade II, where some genes were completely unexpressed, or expressed at trace levels. Only LrNCED1 was upregulated by the two kinds of treatments, and the expression under saline-alkaline conditions was obviously higher compared to that under salt conditions alone.
Expression patterns of the LbCCOs in the green and mature fruits of the L. barbarum in Yinchuan (in Ningxia province) and Nomhon (in Qinghai province) were investigated next (Figure 5E). The results showed that LbCCD1 and LbNCED1, belonging to clade I, exhibited high expression levels. LbCCD1 was stably expressed during the ripening process of L. barbarum, while the expression of LbNCED1 decreased as the fruit ripened in both locations. The expression levels of these two genes in the fruit from Nomhon were significantly higher than those in fruit from Yinchuan. The average high and low temperature, hours of daylight and twilight, average daily incident shortwave solar energy, and other weather information were recorded in Nomhon and Yinchuan. In Nomhon, the average daily high temperature was above 19 °C; the hottest month of the year was July, with an average high of 23 °C and low of 12 °C; the average daily incident shortwave energy per square meter was above 7.1 kWh; the brightest month of the year was June, with an average of 8.0 kWh. In Yinchuan, the average daily high temperature was above 23 °C; the hottest month of the year was July, with an average high of 29 °C and low of 19 °C; the average daily incident shortwave energy per square meter was above 6.3 kWh; the brightest month of the year was June, with an average of 7.2 kWh. Given that the primary differences between the two locations are light and temperature, this suggests that conditions of intense light irradiation and high temperature may influence the gene expression levels of LbCCD1 and LbNCED1.

3.6. CCD4-1s Localization and Promoter Analysis of Two Goji Species

Given the strikingly different expression pattern for CCD4-1s in the two goji species, the CCD4-1s genes and their promoters were cloned and sequenced. The results showed a high similarity in both the coding areas and promoter regions. Only one amino acid varied between the two CCD4-1 proteins (Supplemental Table S4). Subcellular localization showed that driven by the 35S promoter, both two CCD4-1 proteins that fused to the EGFP protein were targeted to chloroplasts (Figure 6), suggesting that this single difference in the coding sequences does not affect the function of CCD4-1.
To predict whether transcription factors can influence the regulation of CCD4-1s, binding site predictions were compared for the two CCD4-1 promoters. The result indicated that 265 and 272 TFBS (transcription factor binding sites) exist in the promoters of LbCCD4-1 and LrCCD4-1, respectively. The numbers of AP2, B3, BBR-BPC, Dof, MIKC_MADS, and NAC binding sites differed between the promoters of two CCD4-1s, and some of these sites may affect CCD4-1s expression (Table 2 and Supplemental Table S5).

4. Discussion

4.1. Different Functions Classification Prediction Based on the Identification of LbCCOs

In this study, twelve LbCCO genes with RPE65 conserved domains were identified based on the L. barbarum genome. The predicted characteristics of the encoded proteins were analyzed, including pI, molecular weight, and subcellular localization. The subcellular localization prediction and experiment showed that LbCCOs target the chloroplasts or the cytoplasm. LbCCOs may function differently due to different substrate distributions. In plants, carotenoid synthesis usually occurs in the plastids, and each type of carotenoid is synthesized and then bound to the membrane of chloroplasts [10,47,48]. According to a previous report, most CCO members were localized to the plastid membrane, except for CCD1. CCD1 generates C13 or C14 apocarotenoids by degrading products transported outside the chloroplast that have undergone degradation by other CCOs, rather than by the direct degradation of C40 carotenoid substrates and producing apocarotenoids [49]. The prediction of CCOs showed that some CCOs located to the cytoplasm, perhaps by the mutation of the CCO chloroplast transit peptide with CCO evolution.
In most plant species, the CCO gene family includes CCD1, CCD4, CCD7, CCD8, and NCED subfamilies [28,37,50]. In some Crocus species, the CCD2 subfamily is also included [51]. In this study, LbCCD1, LbCCD3, LbCCD4, LbCCD7, LbCCD8, and LbNCED were identified in the L. barbarum genome, but LbCCD2 was not found, indicating that this kind of CCO may not be present in this species. We identified and named a new subfamily member, CCD3, in LbCCOs. CCO gene family members have been identified in many species, such as pepper [52], litchi [53], and tobacco [54], but CCD3 was not reported in these species. However, CCD3 may exist in other species. For example, collinear analysis indicated that LbCCD3-1 is collinear with a gene on S. lycopersicum chromosome 8, and these two genes may be orthologs. The CCD3 subfamily may not have been identified previously because the CCD2 subfamily in non-model plant species, such as C. sativus, has rarely been used to build phylogenetic trees. Although the use of a phylogenetic tree can help distinguish subfamilies among species, the result can be easily affected by the selected sequences. For example, C. sativus CCO proteins including CCD2 were used to build a phylogenetic tree in this study, and the result showed that CCD2 and CCD1 clustered to a same clade, and then clustered with CCD3, which was distinguished as a new subfamily. If CCD2 was not included in the phylogenetic tree, CCD2 or CCD3 could have been identified as CCD1. Further analysis showed that LbCCOs can be divided into four groups according to the motif number, type, relative position, and gene structure. Group I included LbNCEDs, group II included LbCCD1 and LbCCD4s, group III included LbCCD7 and LbCCD8, and group IV included LbCCD3s. Different groups may have different functions. NCED participated in ABA synthesis [27]. CCD1 and CCD4 were reported to cut a variety of carotenoid substances to produce aromas or pigments. CCD7 and CCD8 were related to the production, SL [47]. The functions of CCD3 may differ from those of CCOs and CCD2, and this should be a focus of future work.
Although subfamilies of CCOs differed in gene structure, motif number, type, and relative position, their properties are conserved among species within the same subfamilies [8,37,50]. The mutation of key genes may cause death or abnormal development, so the high conservation of CCOs suggests that they play important roles in regulating plant growth, development, and reproduction. The conservation level within the LbCCO subfamily differed. The function of NCEDs is to synthesize ABA, a key hormone determining plant survival and reproduction [27], indicating that NCEDs are a more conserved subfamily than CCDs. This is consistent with the gene structure and motif analysis.

4.2. Gene Conservation and Pseudogene LbCCOs

Tandem duplication allows for the expansion and evolution of gene families. Additionally, the tandem duplication of CCOs has been found in many species [28,37]. The Ka/Ks ratio measures the direction and magnitude of selection [50]. Although clusters were identified, no evidence for the duplication of LbCCOs was found, suggesting that the use of the Ka/Ks ratio to analyze the contraction and expansion of this family is not informative. Some CCOs were identified with a vestigial or no RPE65 domain, suggesting that during the evolution of the Solanaceae, the CCO gene family expanded and pseudogenes were generated, leading to the loss of tandem duplicated CCOs The evolution of a pseudogene can lead to its disappearance or the production of new copies [55,56,57]. These genes generally acquire mutations, insertions, and deletions without any apparent evolutionary pressures [58]. Collinearity analysis showed that S. lycopersicum shares more collinear genes with L. barbarum than A. thaliana. This result is consistent with the phylogenetic relationships, as S. lycopersicum and L. barbarum are Solanaceae plants. In S. lycopersicum and L. barbarum, at least one gene in each gene subfamily is collinear, suggesting that there may be unique functions in each LbCCO subfamily.

4.3. CCD4-1 May Be a Key Gene Dominating the Degradation of Goji Carotenoids

CCOs are considered important genes for carotenoid degradation to short chain apocarotenoids [59]. Phytoene synthase (PSY) is rate limiting for phytoene formation in the biosynthesis of carotenoids [59]. The overexpression of PSY resulted in a dramatic accumulation of mainly β-carotene in roots and non-green calli in leaves of Arabidopsis, with a high level of C13 apocarotenoid glycoside, the product of CCD4 cleavage carotenoid, detected in the leaves [59]. A mutant of CCD4 indicated that the cleavage effect of CCD4 explains the lack of excessive accumulation of carotenoids in the leaves of PSY overexpressing plants [59]. Overall, the results show that CCD4 is a key gene in carotenoid metabolism in Arabidopsis. A previous study of L. barbarum genomics indicated that CCD (Lba03g01270) may be involved in carotenoid degradation [36]. L. barbarum and L. ruthenicum fruits differed significantly in carotenoid content, with L. barbarum rich in carotenoids in mature fruits and L. ruthenicum fruits containing almost no carotenoids [35]. In this study, CCD4-1s (Lba04g00945) was the most highly expressed gene in both Lycium species, with a similar expression pattern among organs and tissues, except fruit. Transcriptome analysis showed that the expression level of CCD4-1 dramatically increased in L. ruthenicum and decreased in L. barbarum during fruit development. This completely opposite expression pattern of CCOs in the fruits of the two goji species indicated that the low expression of CCD4-1 contributed to the degradation of carotenoids, especially in L. ruthenicum. The previously found CCD (Lba03g01270) was not identified as a CCO member in this study because it lacks the RPE65 domain. CCD4-1 rather than CCD (Lba03g01270) may dominate the degradation of carotenoids. By comparing the coding sequences and promoter sequences of the two CCD4-1 genes, we found that the differences in the transcription factor binding sites rather than differences in the coding sequences of the genes may explain the different carotenoid degradation efficiencies between the two goji species.

4.4. Abiotic Stress May Regulate the Expressions of CCD1, NCED1, or Other CCOs Directly or through Hormones in at Least One Species of Lycium

Gene expression analysis indicated LrNCED1 was the only CCO that responded to salt or saline-alkali stresses, with the upregulation of LrNCED1 expression under treatments. The upregulation of LrNCED1 is more significant under saline-alkaline stress compared to solely salt stress. NCEDs were reported to synthesize ABA [23]. The upregulated LrNCED1 may promote plants to accumulate ABA to resist abiotic stress.
In this study, cis-acting elements were predicted by PlantCare with certain predictive accuracy to determine the factors that regulate LbCCOs expression. Among all cis-acting elements, light related elements were the most frequent elements found in CCO members other than LbCCD1, indicating that light may regulate the expression of CCOs to change the content of carotenoids and hormones. Light provides energy for plants to survive but can also cause harm to plants. Photooxidative stress conditions can induce the formation of a variety of volatile short-chain aldehydic or ketonic derivatives of β-carotene, such as β-ionone or β-cyclocitral, which can increase tolerance to photooxidative stress [60]. These products can also be produced enzymatically by CCOs [47]. Although evidence suggests that these products are not produced by CCOs under intense-light stress, the expression of CCOs may be activated by light and then influence the synthesis of these products [61]. The expression of CCD homologs, which may be related to retinal synthesis, increased in the microorganism Chlorogloeopsis fritschii under UV [62]. Light not only provides energy, but also transmits environmental signals. A study of Vaccinium myrtillus fruit development under different light conditions showed that the degradation of carotenoids can be influenced by light, especially red/far-red light wavelengths. Light activated the expression of VmCCD1 and VmNCED1, leading to carotenoid degradation and ABA synthesis [63]. CCD7 and CCD8 are regulated by phyB and hy5-related pathways. As the key red (R)-light photoreceptor, phytochrome B (phyB) activates the key transcription factor, ELONGATED HYPOCOTYL5 (HY5), to regulate photomorphogenesis, root development, and response to stresses in plants. Activated HY5 acts as a crucial hub to integrate light and other signals to regulate the expression of downstream genes. Shoot phyB triggers the accumulation of shoot-derived HY5 in root, promoting the expression of CCD7 and CCD8 and leading to the synthesis and accumulation of strigolactone (SL) in roots [64].
In addition to light response elements, hormones and other environmental response elements were enriched in the promoters of LbCCOs. Hormones and environment stress treatments can alter the expression of CCOs in some plant species [52]. There are many MeJA response cis-acting elements in the promoters of LbCCOs, except LbNCED2 and LbCCD4-2. MeJA treatment can activate the expression of tobacco CCOs [54]. The expression patterns of CCOs differed for different genes and in different gene subfamilies. CCOs with promoters lacking MeJA response cis-acting elements also respond differently to MeJA [54], suggesting the indirect regulation of MeJA. Previous studies have shown that the rapid and massive accumulation of MeJA goji may explain the development of heat tolerance in goji [65]. The CCOs have been reported to be induced by high temperature in other species, and MeJA and high temperature treatment cause some CaCCOs to show similar expression patterns [52], suggesting heat tolerance goji may respond to high temperature stress by regulating MeJA signal.
The expression levels of LbCCOs in L. barbarum planted in different places were investigated. The geographical difference between the Yinchuan and Nomhon goji production areas lies primarily in altitude, which affects both light and temperature. Weather data also indicated differences in temperature and solar radiation between the two places. The high temperature in Yinchuan during July and August limits goji harvesting, while the cooler temperature in Nomhon allows for continuous harvesting. The differential expression of LbCCD1 and LbNCED1 in these two regions may be caused by differences in light and temperature conditions. Overall, these results suggest that the regulation of the expression of CCD1 and NCED1 in goji fruit may be regulated by a complex signaling network triggered by differences in light and temperature.

5. Conclusions

In this study, we investigated 12 LbCCO genes after screening the genome of L. barbarum. We analyzed the physicochemical properties of the 12 LbCCOs and found that all proteins were predicted to be localized to the cytoplasm or chloroplasts. The phylogenetic tree constructed with related species and Crocus sativus classified CCOs into seven subfamilies of NCED, CCD1, CCD2, CCD3, CCD4, CCD7, and CCD8. CCD3 was first identified as a subfamily to distinguish it from CCD1 and CCD2. The clustering results, gene structure, and motif analysis indicated different degrees of conservation for the different LbCCO subfamilies, with the LbNCED subfamily as the most highly conserved. The analysis of the promoter elements of CCO genes showed that the expression of these genes could be affected by environmental and hormonal factors such as light and MeJA. This is consistent with environment responsive gene expression analysis. The transcriptomes analysis indicated that the regulation of the CCO gene by salt-alkali stress and photothermal conditions could occur directly or indirectly (through hormonal pathways) in at least one species of Lycium. Collinearity analysis did not reveal tandem duplication in the LbCCO gene family. However, L. barbarum shares at least one CCO gene in one subfamily with S. lycopersicum, indicating pseudogene generation and the importance of the different CCO subfamilies. The spatiotemporal transcriptome analysis revealed different expression patterns of LbCCOs in different tissues or organs and the fruit developmental process. CCD4-1 showed opposite expression patterns in the fruit development process of the two goji species, suggesting that LbCCD4-1 may dominate the degradation of carotenoids in goji fruit.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14050983/s1, Supplemental Table S1: Cloning primers; Supplemental Table S2: CDS sequence of LbCCOs from L. barbarum; Supplemental Table S3: Protein sequence of CCOs from L. barbarum, N. tabacum, A. thaliana, S. lycopersicum, Crocus sativus, and L. ruthenicum; Supplemental Table S4: Sequences Alignment of CCD4-1s and their promoters from L. barbarum and L. ruthenicum; Supplemental Table S5 Binding site prediction results of CCD4-1s promoters.

Author Contributions

Conceptualization, J.C. (Jinhuan Chen); methodology, W.L.; formal analysis, W.L. and J.C. (Jiahang Che); investigation, W.L.; data curation, J.C. (Jinhuan Chen), W.L., J.C. (Jiahang Che) and Q.L.; writing—original draft preparation, W.L.; writing—review and editing, J.C. (Jinhuan Chen), C.W. and G.D.; visualization, W.L.; project administration, J.C. (Jinhuan Chen); funding acquisition, J.C. (Jinhuan Chen) and Q.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Research and Development Program of Ningxia, grant number 2021BEF02005; the Key Research and Development Program of Ningxia, grant number 2022BBF01001-02, and National College Students’ Science and Technology Innovation Project: X202210022069.

Data Availability Statement

The datasets supporting the results presented in this manuscript are included within the article (and its Supplementary Materials). Raw data of L. barbarum and L. ruthenicum fruit developmental process sequenced libraries are available in the NCBI SRA database under number PRJNA483521 [https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA483521], accessed on 20 April 2022. Raw data of stress responsive transcriptomes of L. ruthenicum are available in the NCBI SRA database under number PRJNA285517 [https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA285517], accessed on 20 April 2022.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Hitchcock, C.L. A Monographic Study of the Genus Lycium of the Western Hemisphere. Ann. Mo. Bot. Gard. 1932, 19, 179. [Google Scholar] [CrossRef]
  2. Nee, M. Flora of China, Vol. 17, Verbenaceae Through Solanaceae. Brittonia 1996, 48, 611. [Google Scholar] [CrossRef]
  3. Yu, Z.; Xia, M.; Lan, J.; Yang, L.; Wang, Z.; Wang, R.; Tao, H.; Shi, Y. A Comprehensive Review on the Ethnobotany, Phytochemistry, Pharmacology and Quality Control of the Genus Lycium in China. Food Funct. 2023, 14, 2998–3025. [Google Scholar] [CrossRef] [PubMed]
  4. Zhang, Q.; Chen, W.; Zhao, J.; Xi, W. Functional Constituents and Antioxidant Activities of Eight Chinese Native Goji Genotypes. Food Chem. 2016, 200, 230–236. [Google Scholar] [CrossRef]
  5. Avalos, J.; Carmen Limón, M. Biological Roles of Fungal Carotenoids. Curr. Genet. 2015, 61, 309–324. [Google Scholar] [CrossRef]
  6. Sun, T.; Rao, S.; Zhou, X.; Li, L. Plant Carotenoids: Recent Advances and Future Perspectives. Mol. Hortic. 2022, 2, 3. [Google Scholar] [CrossRef]
  7. Maoka, T. Carotenoids as Natural Functional Pigments. J. Nat. Med. 2020, 74, 1–16. [Google Scholar] [CrossRef]
  8. Kim, Y.; Hwang, I.; Jung, H.-J.; Park, J.-I.; Kang, J.-G.; Nou, I.-S. Genome-Wide Classification and Abiotic Stress-Responsive Expression Profiling of Carotenoid Oxygenase Genes in Brassica Rapa and Brassica Oleracea. J. Plant Growth Regul. 2016, 35, 202–214. [Google Scholar] [CrossRef]
  9. Lin, J.; Massonnet, M.; Cantu, D. The Genetic Basis of Grape and Wine Aroma. Hortic. Res. 2019, 6, 81. [Google Scholar] [CrossRef]
  10. Yuan, H.; Zhang, J.; Nageswaran, D.; Li, L. Carotenoid Metabolism and Regulation in Horticultural Crops. Hortic. Res. 2015, 2, 15036. [Google Scholar] [CrossRef]
  11. Lim, J.; Lim, C.W.; Lee, S.C. Core Components of Abscisic Acid Signaling and Their Post-Translational Modification. Front. Plant Sci. 2022, 13, 895698. [Google Scholar] [CrossRef]
  12. Waadt, R.; Seller, C.A.; Hsu, P.-K.; Takahashi, Y.; Munemasa, S.; Schroeder, J.I. Plant Hormone Regulation of Abiotic Stress Responses. Nat. Rev. Mol. Cell Biol. 2022, 23, 680–694. [Google Scholar] [CrossRef] [PubMed]
  13. Rehman, N.U.; Li, X.; Zeng, P.; Guo, S.; Jan, S.; Liu, Y.; Huang, Y.; Xie, Q. Harmony but Not Uniformity: Role of Strigolactone in Plants. Biomolecules 2021, 11, 1616. [Google Scholar] [CrossRef] [PubMed]
  14. Yoneyama, K.; Brewer, P.B. Strigolactones, How Are They Synthesized to Regulate Plant Growth and Development? Curr. Opin. Plant Biol. 2021, 63, 102072. [Google Scholar] [CrossRef] [PubMed]
  15. Liang, N.; Yao, M.-D.; Wang, Y.; Liu, J.; Feng, L.; Wang, Z.-M.; Li, X.-Y.; Xiao, W.-H.; Yuan, Y.-J. Cs CCD2 Access Tunnel Design for a Broader Substrate Profile in Crocetin Production. J. Agric. Food Chem. 2021, 69, 11626–11636. [Google Scholar] [CrossRef]
  16. Ilg, A.; Yu, Q.; Schaub, P.; Beyer, P.; Al-Babili, S. Overexpression of the Rice Carotenoid Cleavage Dioxygenase 1 Gene in Golden Rice Endosperm Suggests Apocarotenoids as Substrates in Planta. Planta 2010, 232, 691–699. [Google Scholar] [CrossRef]
  17. Auldridge, M.E.; Block, A.; Vogel, J.T.; Dabney-Smith, C.; Mila, I.; Bouzayen, M.; Magallanes-Lundback, M.; DellaPenna, D.; McCarty, D.R.; Klee, H.J. Characterization of Three Members of the Arabidopsis Carotenoid Cleavage Dioxygenase Family Demonstrates the Divergent Roles of This Multifunctional Enzyme Family. Plant J. 2006, 45, 982–993. [Google Scholar] [CrossRef] [PubMed]
  18. Cheng, L.; Huang, N.; Jiang, S.; Li, K.; Zhuang, Z.; Wang, Q.; Lu, S. Cloning and Functional Characterization of Two Carotenoid Cleavage Dioxygenases for Ionone Biosynthesis in Chili Pepper (Capsicum annuum L.) Fruits. Sci. Hortic. 2021, 288, 110368. [Google Scholar] [CrossRef]
  19. Mathieu, S.; Terrier, N.; Procureur, J.; Bigey, F.; Günata, Z. A Carotenoid Cleavage Dioxygenase from Vitis vinifera L.: Functional Characterization and Expression during Grape Berry Development in Relation to C13-Norisoprenoid Accumulation. J. Exp. Bot. 2005, 56, 2721–2731. [Google Scholar] [CrossRef]
  20. Meng, N.; Yan, G.-L.; Zhang, D.; Li, X.-Y.; Duan, C.-Q.; Pan, Q.-H. Characterization of Two Vitis Vinifera Carotenoid Cleavage Dioxygenases by Heterologous Expression in Saccharomyces Cerevisiae. Mol. Biol. Rep. 2019, 46, 6311–6323. [Google Scholar] [CrossRef]
  21. Rodrigo, M.J.; Alquézar, B.; Alós, E.; Medina, V.; Carmona, L.; Bruno, M.; Al-Babili, S.; Zacarías, L. A Novel Carotenoid Cleavage Activity Involved in the Biosynthesis of Citrus Fruit-Specific Apocarotenoid Pigments. J. Exp. Bot. 2013, 64, 4461–4478. [Google Scholar] [CrossRef] [PubMed]
  22. Ren, C.; Guo, Y.; Kong, J.; Lecourieux, F.; Dai, Z.; Li, S.; Liang, Z. Knockout of VvCCD8 Gene in Grapevine Affects Shoot Branching. BMC Plant Biol. 2020, 20, 47. [Google Scholar] [CrossRef]
  23. Cutler, A.J.; Krochko, J.E. Formation and Breakdown of ABA. Trends Plant Sci. 1999, 4, 472–478. [Google Scholar] [CrossRef] [PubMed]
  24. Martínez-Andújar, C.; Martínez-Pérez, A.; Ferrández-Ayela, A.; Albacete, A.; Martínez-Melgarejo, P.A.; Dodd, I.C.; Thompson, A.J.; Pérez-Pérez, J.M.; Pérez-Alfocea, F. Impact of Overexpression of 9-Cis-Epoxycarotenoid Dioxygenase on Growth and Gene Expression under Salinity Stress. Plant Sci. 2020, 295, 110268. [Google Scholar] [CrossRef] [PubMed]
  25. González-Villagra, J.; Rodrigues-Salvador, A.; Nunes-Nesi, A.; Cohen, J.D.; Reyes-Díaz, M.M. Age-Related Mechanism and Its Relationship with Secondary Metabolism and Abscisic Acid in Aristotelia Chilensis Plants Subjected to Drought Stress. Plant Physiol. Biochem. 2018, 124, 136–145. [Google Scholar] [CrossRef] [PubMed]
  26. Xia, H.; Wang, X.; Zhou, Y.; Su, W.; Jiang, L.; Deng, H.; Li, M.; Zhuang, Q.; Xie, Y.; Liang, D. Biochemical and Molecular Factors Governing Flesh-Color Development in Two Yellow-Fleshed Kiwifruit Cultivars. Sci. Hortic. 2021, 280, 109929. [Google Scholar] [CrossRef]
  27. Noronha, H.; Silva, A.; Silva, T.; Frusciante, S.; Diretto, G.; Gerós, H. VviRafS5 Is a Raffinose Synthase Involved in Cold Acclimation in Grapevine Woody Tissues. Front. Plant Sci. 2022, 12, 754537. [Google Scholar] [CrossRef]
  28. Cai, X.; Jiang, Z.; Tang, L.; Zhang, S.; Li, X.; Wang, H.; Liu, C.; Chi, J.; Zhang, X.; Zhang, J. Genome-Wide Characterization of Carotenoid Oxygenase Gene Family in Three Cotton Species and Functional Identification of GaNCED3 in Drought and Salt Stress. J. Appl. Genet. 2021, 62, 527–543. [Google Scholar] [CrossRef]
  29. Huang, Y.; Guo, Y.; Liu, Y.; Zhang, F.; Wang, Z.; Wang, H.; Wang, F.; Li, D.; Mao, D.; Luan, S.; et al. 9-Cis-Epoxycarotenoid Dioxygenase 3 Regulates Plant Growth and Enhances Multi-Abiotic Stress Tolerance in Rice. Front. Plant Sci. 2018, 9, 162. [Google Scholar] [CrossRef]
  30. Toscano, S.; Trivellini, A.; Cocetta, G.; Bulgari, R.; Francini, A.; Romano, D.; Ferrante, A. Effect of Preharvest Abiotic Stresses on the Accumulation of Bioactive Compounds in Horticultural Produce. Front. Plant Sci. 2019, 10, 1212. [Google Scholar] [CrossRef]
  31. da Silva Oliveira, C.E.; Zoz, T.; de Castro Seron, C.; Boleta, E.H.M.; de Lima, B.H.; Souza, L.R.R.; Pedrinho, D.R.; Matias, R.; dos Santos Lopes, C.; de Oliveira Neto, S.S.; et al. Can Saline Irrigation Improve the Quality of Tomato Fruits? Agron. J. 2022, 114, 900–914. [Google Scholar] [CrossRef]
  32. Zhou, R.; Yu, X.; Li, X.; Mendanha dos Santos, T.; Rosenqvist, E.; Ottosen, C.-O. Combined High Light and Heat Stress Induced Complex Response in Tomato with Better Leaf Cooling after Heat Priming. Plant Physiol. Biochem. 2020, 151, 1–9. [Google Scholar] [CrossRef] [PubMed]
  33. Zhang, R.-R.; Wang, Y.-H.; Li, T.; Tan, G.-F.; Tao, J.-P.; Su, X.-J.; Xu, Z.-S.; Tian, Y.-S.; Xiong, A.-S. Effects of Simulated Drought Stress on Carotenoid Contents and Expression of Related Genes in Carrot Taproots. Protoplasma 2021, 258, 379–390. [Google Scholar] [CrossRef]
  34. Zhao, Y.-H.; Deng, Y.-J.; Wang, Y.-H.; Lou, Y.-R.; He, L.-F.; Liu, H.; Li, T.; Yan, Z.-M.; Zhuang, J.; Xiong, A.-S. Changes in Carotenoid Concentration and Expression of Carotenoid Biosynthesis Genes in Daucus Carota Taproots in Response to Increased Salinity. Horticulturae 2022, 8, 650. [Google Scholar] [CrossRef]
  35. Islam, T.; Yu, X.; Badwal, T.S.; Xu, B. Comparative Studies on Phenolic Profiles, Antioxidant Capacities and Carotenoid Contents of Red Goji Berry (Lycium barbarum) and Black Goji Berry (Lycium ruthenicum). Chem. Cent. J. 2017, 11, 59. [Google Scholar] [CrossRef] [PubMed]
  36. Cao, Y.-L.; Li, Y.; Fan, Y.-F.; Li, Z.; Yoshida, K.; Wang, J.-Y.; Ma, X.-K.; Wang, N.; Mitsuda, N.; Kotake, T.; et al. Wolfberry Genomes and the Evolution of Lycium (Solanaceae). Commun. Biol. 2021, 4, 671. [Google Scholar] [CrossRef] [PubMed]
  37. Chen, H.; Zuo, X.; Shao, H.; Fan, S.; Ma, J.; Zhang, D.; Zhao, C.; Yan, X.; Liu, X.; Han, M. Genome-Wide Analysis of Carotenoid Cleavage Oxygenase Genes and Their Responses to Various Phytohormones and Abiotic Stresses in Apple (Malus domestica). Plant Physiol. Biochem. 2018, 123, 81–93. [Google Scholar] [CrossRef]
  38. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  39. Letunic, I.; Khedkar, S.; Bork, P. SMART: Recent Updates, New Developments and Status in 2020. Nucleic Acids Res. 2021, 49, D458–D460. [Google Scholar] [CrossRef]
  40. Letunic, I.; Bork, P. 20 Years of the SMART Protein Domain Annotation Resource. Nucleic Acids Res. 2018, 46, D493–D496. [Google Scholar] [CrossRef]
  41. Lu, S.; Wang, J.; Chitsaz, F.; Derbyshire, M.K.; Geer, R.C.; Gonzales, N.R.; Gwadz, M.; Hurwitz, D.I.; Marchler, G.H.; Song, J.S.; et al. CDD/SPARCLE: The Conserved Domain Database in 2020. Nucleic Acids Res. 2020, 48, D265–D268. [Google Scholar] [CrossRef] [PubMed]
  42. The UniProt Consortium; Bateman, A.; Martin, M.-J.; Orchard, S.; Magrane, M.; Agivetova, R.; Ahmad, S.; Alpi, E.; Bowler-Barnett, E.H.; Britto, R.; et al. UniProt: The Universal Protein Knowledgebase in 2021. Nucleic Acids Res. 2021, 49, D480–D489. [Google Scholar] [CrossRef]
  43. Berardini, T.Z.; Reiser, L.; Li, D.; Mezheritsky, Y.; Muller, R.; Strait, E.; Huala, E. The Arabidopsis Information Resource: Making and Mining the “Gold Standard” Annotated Reference Plant Genome: Tair: Making and Mining the “Gold Standard” Plant Genome. Genesis 2015, 53, 474–485. [Google Scholar] [CrossRef] [PubMed]
  44. Wilkins, M.R.; Gasteiger, E.; Bairoch, A.; Sanchez, J.-C.; Williams, K.L.; Appel, R.D.; Hochstrasser, D.F. Protein Identification and Analysis Tools in the ExPASy Server. In 2-D Proteome Analysis Protocols; Humana Press: Totowa, NJ, USA, 1998; Volume 112, pp. 531–552. ISBN 978-1-59259-584-6. [Google Scholar]
  45. Wang, C.; Dong, Y.; Zhu, L.; Wang, L.; Yan, L.; Wang, M.; Zhu, Q.; Nan, X.; Li, Y.; Li, J. Comparative Transcriptome Analysis of Two Contrasting Wolfberry Genotypes during Fruit Development and Ripening and Characterization of the LrMYB1 Transcription Factor That Regulates Flavonoid Biosynthesis. BMC Genom. 2020, 21, 295. [Google Scholar] [CrossRef]
  46. Bailey, T.L.; Johnson, J.; Grant, C.E.; Noble, W.S. The MEME Suite. Nucleic Acids Res. 2015, 43, W39–W49. [Google Scholar] [CrossRef] [PubMed]
  47. Hou, X.; Rivers, J.; León, P.; McQuinn, R.P.; Pogson, B.J. Synthesis and Function of Apocarotenoid Signals in Plants. Trends Plant Sci. 2016, 21, 792–803. [Google Scholar] [CrossRef]
  48. Othman, R.; Mohd Zaifuddin, F.A.; Hassan, N.M. Carotenoid Biosynthesis Regulatory Mechanisms in Plants. J. Oleo Sci. 2014, 63, 753–760. [Google Scholar] [CrossRef]
  49. Floss, D.S.; Walter, M.H. Role of Carotenoid Cleavage Dioxygenase 1 (CCD1) in Apocarotenoid Biogenesis Revisited. Plant Signal. Behav. 2009, 4, 172–175. [Google Scholar] [CrossRef]
  50. Zhao, J.; Li, J.; Zhang, J.; Chen, D.; Zhang, H.; Liu, C.; Qin, G. Genome-Wide Identification and Expression Analysis of the Carotenoid Cleavage Oxygenase Gene Family in Five Rosaceae Species. Plant Mol. Biol. Rep. 2021, 39, 739–751. [Google Scholar] [CrossRef]
  51. Frusciante, S.; Diretto, G.; Bruno, M.; Ferrante, P.; Pietrella, M.; Prado-Cabrero, A.; Rubio-Moraga, A.; Beyer, P.; Gomez-Gomez, L.; Al-Babili, S.; et al. Novel Carotenoid Cleavage Dioxygenase Catalyzes the First Dedicated Step in Saffron Crocin Biosynthesis. Proc. Natl. Acad. Sci. USA 2014, 111, 12246–12251. [Google Scholar] [CrossRef]
  52. Yao, Y.; Jia, L.; Cheng, Y.; Ruan, M.; Ye, Q.; Wang, R.; Yao, Z.; Zhou, G.; Liu, J.; Yu, J.; et al. Evolutionary Origin of the Carotenoid Cleavage Oxygenase Family in Plants and Expression of Pepper Genes in Response to Abiotic Stresses. Front. Plant Sci. 2022, 12, 792832. [Google Scholar] [CrossRef] [PubMed]
  53. Yue, X.-Q.; Zhang, Y.; Yang, C.-K.; Li, J.-G.; Rui, X.; Ding, F.; Hu, F.-C.; Wang, X.-H.; Ma, W.-Q.; Zhou, K.-B. Genome-Wide Identification and Expression Analysis of Carotenoid Cleavage Oxygenase Genes in Litchi (Litchi Chinensis Sonn.). BMC Plant Biol. 2022, 22, 394. [Google Scholar] [CrossRef] [PubMed]
  54. Zhou, Q.; Li, Q.; Li, P.; Zhang, S.; Liu, C.; Jin, J.; Cao, P.; Yang, Y. Carotenoid Cleavage Dioxygenases: Identification, Expression, and Evolutionary Analysis of This Gene Family in Tobacco. IJMS 2019, 20, 5796. [Google Scholar] [CrossRef]
  55. Dobeš, C.H.; Mitchell-Olds, T.; Koch, M.A. Extensive Chloroplast Haplotype Variation Indicates Pleistocene Hybridization and Radiation of North American Arabis Drummondii, A. × Divaricarpa, and A. Holboellii (Brassicaceae): Pleistocene HYBRIDIZATION OF ARABIS SPECIES. Mol. Ecol. 2004, 13, 349–370. [Google Scholar] [CrossRef] [PubMed]
  56. Koch, M.A.; Dobeš, C.; Kiefer, C.; Schmickl, R.; Klimeš, L.; Lysak, M.A. Supernetwork Identifies Multiple Events of Plastid TrnF(GAA) Pseudogene Evolution in the Brassicaceae. Mol. Biol. Evol. 2007, 24, 63–73. [Google Scholar] [CrossRef]
  57. Koch, M.A.; Dobeš, C.; Matschinger, M.; Bleeker, W.; Vogel, J.; Kiefer, M.; Mitchell-Olds, T. Evolution of the TrnF(GAA) Gene in Arabidopsis Relatives and the Brassicaceae Family: Monophyletic Origin and Subsequent Diversification of a Plastidic Pseudogene. Mol. Biol. Evol. 2005, 22, 1032–1043. [Google Scholar] [CrossRef]
  58. Harrison, P.M. Digging for Dead Genes: An Analysis of the Characteristics of the Pseudogene Population in the Caenorhabditis Elegans Genome. Nucleic Acids Res. 2001, 29, 818–830. [Google Scholar] [CrossRef]
  59. Lätari, K.; Wüst, F.; Hübner, M.; Schaub, P.; Beisel, K.G.; Matsubara, S.; Beyer, P.; Welsch, R. Tissue-Specific Apocarotenoid Glycosylation Contributes to Carotenoid Homeostasis in Arabidopsis Leaves. Plant Physiol. 2015, 168, 1550–1562. [Google Scholar] [CrossRef]
  60. González-Pérez, S.; Gutiérrez, J.; García-García, F.; Osuna, D.; Dopazo, J.; Lorenzo, Ó.; Revuelta, J.L.; Arellano, J.B. Early Transcriptional Defense Responses in Arabidopsis Cell Suspension Culture under High-Light Conditions. Plant Physiol. 2011, 156, 1439–1456. [Google Scholar] [CrossRef]
  61. Ramel, F.; Mialoundama, A.S.; Havaux, M. Nonenzymic Carotenoid Oxidation and Photooxidative Stress Signalling in Plants. J. Exp. Bot. 2013, 64, 799–805. [Google Scholar] [CrossRef]
  62. Llewellyn, C.A.; Airs, R.L.; Farnham, G.; Greig, C. Synthesis, Regulation and Degradation of Carotenoids Under Low Level UV-B Radiation in the Filamentous Cyanobacterium Chlorogloeopsis Fritschii PCC 6912. Front. Microbiol. 2020, 11, 163. [Google Scholar] [CrossRef] [PubMed]
  63. Karppinen, K.; Zoratti, L.; Sarala, M.; Carvalho, E.; Hirsimäki, J.; Mentula, H.; Martens, S.; Häggman, H.; Jaakola, L. Carotenoid Metabolism during Bilberry (Vaccinium myrtillus L.) Fruit Development under Different Light Conditions Is Regulated by Biosynthesis and Degradation. BMC Plant Biol. 2016, 16, 95. [Google Scholar] [CrossRef] [PubMed]
  64. Ge, S.; He, L.; Jin, L.; Xia, X.; Li, L.; Ahammed, G.J.; Qi, Z.; Yu, J.; Zhou, Y. Light-dependent Activation of HY5 Promotes Mycorrhizal Symbiosis in Tomato by Systemically Regulating Strigolactone Biosynthesis. New Phytol. 2022, 233, 1900–1914. [Google Scholar] [CrossRef] [PubMed]
  65. Qin, X.; Qin, B.; He, W.; Chen, Y.; Yin, Y.; Cao, Y.; An, W.; Mu, Z.; Qin, K. Metabolomic and Transcriptomic Analyses of Lycium barbarum L. under Heat Stress. Sustainability 2022, 14, 12617. [Google Scholar] [CrossRef]
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Figure 1. Phylogenetic tree of CCO protein family from L. barbarum, N. tabacum, A. thaliana, S. lycopersicum, Crocus sativus, and L. ruthenicum. A maximum likelihood (ML) phylogenetic tree was constructed by Tbtools with Auto model and 5000 ultrafast bootstraps. Similar names of CCO gene were distinguished by different colors of background and edges. Same species were exhibited in same color of stripes. Stars indicate CCOs identified in L. barbarum. Detailed information of all CCO proteins is listed in Supplemental Table S3.
Figure 1. Phylogenetic tree of CCO protein family from L. barbarum, N. tabacum, A. thaliana, S. lycopersicum, Crocus sativus, and L. ruthenicum. A maximum likelihood (ML) phylogenetic tree was constructed by Tbtools with Auto model and 5000 ultrafast bootstraps. Similar names of CCO gene were distinguished by different colors of background and edges. Same species were exhibited in same color of stripes. Stars indicate CCOs identified in L. barbarum. Detailed information of all CCO proteins is listed in Supplemental Table S3.
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Figure 2. LbCCOs gene structure and cis-acting elements analysis with ML phylogenetic tree of LbCCO proteins. (A) Ten motifs were found in the LbCCOs whose sequence logos are shown in (B) of LbCCO proteins analyzed by Tbtools. (C) Conserved domain of LbCCOs. The portion without RPE65 domain of each protein was indicated in color gray. (D) CDS and intron organization of LbCCOs. There is an intron between the two CDS of each gene.
Figure 2. LbCCOs gene structure and cis-acting elements analysis with ML phylogenetic tree of LbCCO proteins. (A) Ten motifs were found in the LbCCOs whose sequence logos are shown in (B) of LbCCO proteins analyzed by Tbtools. (C) Conserved domain of LbCCOs. The portion without RPE65 domain of each protein was indicated in color gray. (D) CDS and intron organization of LbCCOs. There is an intron between the two CDS of each gene.
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Figure 3. Prediction of cis-acting elements. (A) Prediction of cis-acting elements in the 2000 bp upstream (from the ATG) sequences of LbCCO promoters. The portion without cis-acting elements of each promoter was indicated in color gray. (B) Clustered heatmap of the number of different elements in each of hormone-responsive and environment-responsive. Darker redness indicates a larger number of specific elements in CCOs.
Figure 3. Prediction of cis-acting elements. (A) Prediction of cis-acting elements in the 2000 bp upstream (from the ATG) sequences of LbCCO promoters. The portion without cis-acting elements of each promoter was indicated in color gray. (B) Clustered heatmap of the number of different elements in each of hormone-responsive and environment-responsive. Darker redness indicates a larger number of specific elements in CCOs.
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Figure 4. LbCCOs location and collinearity with chromosomes of A. thaliana and S. lycopersicum. (A) 12 LbCCOs located to five chromosomes. The scale on the outer circle is in megabases (Mb). Inner and mid-side circles show the gene density by color or column height. The connections indicate gene tandem. (B) Collinearity of chromosomes of L. barbarum with those of A. thaliana and S. lycopersicum. The connections indicate the relationships with colinear genes. The labelled LbCCOs are colinear with the CCOs linked by the darker lines in the two other species. The rest unlabeled LbCCOs were identified as non-colinear genes of AtCCO or SlCCO.
Figure 4. LbCCOs location and collinearity with chromosomes of A. thaliana and S. lycopersicum. (A) 12 LbCCOs located to five chromosomes. The scale on the outer circle is in megabases (Mb). Inner and mid-side circles show the gene density by color or column height. The connections indicate gene tandem. (B) Collinearity of chromosomes of L. barbarum with those of A. thaliana and S. lycopersicum. The connections indicate the relationships with colinear genes. The labelled LbCCOs are colinear with the CCOs linked by the darker lines in the two other species. The rest unlabeled LbCCOs were identified as non-colinear genes of AtCCO or SlCCO.
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Figure 5. Fruit color changing pattern (A) and CCO expression pattern clustered heatmap (B–E); (B) clustered heat map of CCOs expression pattern of L. barbarum (Lb) and L. ruthenicum (Lr) fruit at green stage (G), turning color stage (T), and mature stage (M); (C) clustered CCOs express pattern at stamens (XR), pistils(CR), green fruits(GF), mature fruits(MF), leaves on current year branches(NL), leaves on last year branches(OL), stem tip (S) of L. barbarum (Lb) and L. ruthenicum (Lr); (D) clustered heat map of LrCCOs expression pattern at leaves of L. ruthenicum in salt and saline-alkali stress; (E) clustered heat map of LbCCOs expression pattern at green and red fruits of L. barbarum plant in Yinchuan (NX) and Nomhon (QH). The circle color and size, which were based on scaled TPM values, were used to illustrate the expression abundance, the numbers in circle of legend are scaled TPM values while the numbers in the circle of the panel are TPM values (no number is displayed for TPM value < 2). The vertical lines and groupings on the right side of the panel are clusters of genes with different expression profiles.
Figure 5. Fruit color changing pattern (A) and CCO expression pattern clustered heatmap (B–E); (B) clustered heat map of CCOs expression pattern of L. barbarum (Lb) and L. ruthenicum (Lr) fruit at green stage (G), turning color stage (T), and mature stage (M); (C) clustered CCOs express pattern at stamens (XR), pistils(CR), green fruits(GF), mature fruits(MF), leaves on current year branches(NL), leaves on last year branches(OL), stem tip (S) of L. barbarum (Lb) and L. ruthenicum (Lr); (D) clustered heat map of LrCCOs expression pattern at leaves of L. ruthenicum in salt and saline-alkali stress; (E) clustered heat map of LbCCOs expression pattern at green and red fruits of L. barbarum plant in Yinchuan (NX) and Nomhon (QH). The circle color and size, which were based on scaled TPM values, were used to illustrate the expression abundance, the numbers in circle of legend are scaled TPM values while the numbers in the circle of the panel are TPM values (no number is displayed for TPM value < 2). The vertical lines and groupings on the right side of the panel are clusters of genes with different expression profiles.
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Figure 6. Subcellular localization of LbCCD4-1 and LrCCD4-1 protein. LbCCD4-1-EGFP and LrCCD4-1 fusion proteins driven by the 35S promoter were transiently expressed in tobacco epidermal cells. Images were observed by laser confocal microscopy. Scale bar = 50 µm.
Figure 6. Subcellular localization of LbCCD4-1 and LrCCD4-1 protein. LbCCD4-1-EGFP and LrCCD4-1 fusion proteins driven by the 35S promoter were transiently expressed in tobacco epidermal cells. Images were observed by laser confocal microscopy. Scale bar = 50 µm.
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Table
Table 1. Predicted physiochemical properties of LbCCO proteins.
Table 1. Predicted physiochemical properties of LbCCO proteins.
Gene SymbolGene IDProtein Length (aa)MW (kDa)pIInstability IndexAliphatic IndexGRAVYSubcellular Localization
LbCCD1Lba06g0113391873.08.1131.3579.72−0.250Cytoplasm
LbCCD3-1Lba04g01414713102.96.4735.6578.16−0.278Cytoplasm
LbCCD3-2Lba06g0001664067.56.1935.1889.67−0.189Cytoplasm
LbCCD4-1Lba04g0094559973.06.3839.8379.12−0.250Chloroplast
LbCCD4-2Lba09g02158110562.28.5536.8181.80−0.179Chloroplast
LbCCD4-3Lba09g02160539123.46.1636.7379.78−0.233Chloroplast
LbCCD7Lba06g02264643102.96.2741.1577.60−0.372Cytoplasm
LbCCD8Lba04g0142155662.26.3729.6082.07−0.343Cytoplasm
LbNCED1Lba11g0056260760.06.3138.7675.80−0.403Chloroplast
LbNCED2Lba09g0172455772.66.2041.9482.03−0.364Cytoplasm
LbNCED6-1Lba05g00348587123.46.4843.9084.48−0.330Chloroplast
LbNCED6-2Lba05g0035050660.05.5438.8384.33−0.303Cytoplasm
Table
Table 2. Numbers of predicted TFBS in CCD4-1s.
Table 2. Numbers of predicted TFBS in CCD4-1s.
TFBSLbCCD4-1proLrCCD4-1pro
AP21112
ARF11
ARR-B55
B369
BBR-BPC1213
BES122
bHLH88
bZIP77
C2H21414
C3H22
CAMTA22
CPP33
Dof4240
EIL22
ERF2626
G2-like1010
GATA33
GRAS33
HD-ZIP44
LBD66
MIKC_MADS2325
MYB1515
MYB_related22
NAC3537
Nin-like22
SBP22
TCP88
Trihelix11
VOZ11
WRKY66
YABBY11
SUM265272
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Li, W.; Che, J.; Lian, Q.; Wang, C.; Dai, G.; Chen, J. Identification and Expression Pattern of the Carotenoid Cleavage Oxygenase Gene Family in Lycium Suggest CCOs Respond to Abiotic Stress and Promote Carotenoids Degradation. Forests 2023, 14, 983. https://doi.org/10.3390/f14050983

AMA Style

Li W, Che J, Lian Q, Wang C, Dai G, Chen J. Identification and Expression Pattern of the Carotenoid Cleavage Oxygenase Gene Family in Lycium Suggest CCOs Respond to Abiotic Stress and Promote Carotenoids Degradation. Forests. 2023; 14(5):983. https://doi.org/10.3390/f14050983

Chicago/Turabian Style

Li, Weinan, Jiahang Che, Qile Lian, Cuiping Wang, Guoli Dai, and Jinhuan Chen. 2023. "Identification and Expression Pattern of the Carotenoid Cleavage Oxygenase Gene Family in Lycium Suggest CCOs Respond to Abiotic Stress and Promote Carotenoids Degradation" Forests 14, no. 5: 983. https://doi.org/10.3390/f14050983

APA Style

Li, W., Che, J., Lian, Q., Wang, C., Dai, G., & Chen, J. (2023). Identification and Expression Pattern of the Carotenoid Cleavage Oxygenase Gene Family in Lycium Suggest CCOs Respond to Abiotic Stress and Promote Carotenoids Degradation. Forests, 14(5), 983. https://doi.org/10.3390/f14050983

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