Genome-Wide Identification of 2-Oxoglutarate and Fe (II)-Dependent Dioxygenase (2ODD-C) Family Genes and Expression Profiles under Different Abiotic Stresses in Camellia sinensis (L.)

The 2-oxoglutarate and Fe (II)-dependent dioxygenase (2ODD-C) family of 2-oxoglutarate-dependent dioxygenases potentially participates in the biosynthesis of various metabolites under various abiotic stresses. However, there is scarce information on the expression profiles and roles of 2ODD-C genes in Camellia sinensis. We identified 153 Cs2ODD-C genes from C. sinensis, and they were distributed unevenly on 15 chromosomes. According to the phylogenetic tree topology, these genes were divided into 21 groups distinguished by conserved motifs and an intron/exon structure. Gene-duplication analyses revealed that 75 Cs2ODD-C genes were expanded and retained after WGD/segmental and tandem duplications. The expression profiles of Cs2ODD-C genes were explored under methyl jasmonate (MeJA), polyethylene glycol (PEG), and salt (NaCl) stress treatments. The expression analysis showed that 14, 13, and 49 Cs2ODD-C genes displayed the same expression pattern under MeJA and PEG treatments, MeJA and NaCl treatments, and PEG and NaCl treatments, respectively. A further analysis showed that two genes, Cs2ODD-C36 and Cs2ODD-C21, were significantly upregulated and downregulated after MeJA, PEG, and NaCl treatments, indicating that these two genes played positive and negative roles in enhancing the multi-stress tolerance. These results provide candidate genes for the use of genetic engineering technology to modify plants by enhancing multi-stress tolerance to promote phytoremediation efficiency.


Introduction
Plants possess several mechanisms to cope directly with various abiotic stresses, such as extreme temperature, salt, and drought, owing to their sessile nature [1]. One of the important mechanisms for plants under environmental challenges is the regulation of secondary metabolites [2]. Plants can produce more than 200,000 phytochemicals through various metabolic enzymes [3,4]. In particular, oxygenation/hydroxylation reactions are very important for plant growth and development, which are catalyzed by the 2-oxoglutarate-dependent dioxygenase (2OGD) [5,6]. 2OGDs, the second-largest enzyme family in plants, require 2-oxoglutarate (2OG) and molecular oxygen as co-substrates, and ferrous iron Fe(II) as a cofactor to catalyze the oxidation of a substrate with concomitant decarboxylation of 2OG to form succinate and carbon dioxide. 2OGDs were involved in the biosynthesis of many secondary metabolites [6][7][8][9].
Numerous studies have reported that 2ODDs play important roles in the growth and development processes of plants and participate in the regulation of secondary metabolite biosynthesis under various stresses. For example, overexpression of the CrCOMT gene (encoding the caffeic acid O-methyltransferase) from Carex rigescens enhanced salt tolerance in transgenic Arabidopsis. The TaACO1 gene from wheat (Triticum aestivum) also belongs to the 2ODD gene family, which conferred salt sensitivity when overexpressed in transgenic Arabidopsis [13]. The turnover of auxin can be manipulated under salt treatment through inducing the expression of GH3 genes and inhibiting DAO genes [14]. The 2ODD family members AOP and GSL-OH were reported to increase drought and salt tolerance by regulating glucosinolate biosynthesis and metabolism [15]. Moreover, the T-DNA insertional mutations of GSL-OH in Arabidopsis prevented the accumulation of 2-hydroxybut-3-enyl glucosinolate, which decreased the insect resistance of the plants [16]. Overexpression of the DOXC subfamily F3H gene from Camellia sinensis and Lycium chinense in tobacco and Arabidopsis increased their salt and drought tolerance, respectively [17,18]. In addition, overexpression of the SlF3H gene could improve the cold tolerance of Solanum lycopersicum by regulating jasmonic acid accumulation [19]. Ectopic expression of the DOXC subfamily DoFLS1 gene from Dendrobium officinale promoted flavonol accumulation and enhanced tolerance to abiotic stress in Arabidopsis [20]. Overexpression of the DOXC subfamily gene StGA2ox1 in potato improved stress tolerance compared with that of wild-type plants, including salt, drought, and low-temperature tolerance [21].
Tea (C. sinensis) is the oldest natural, nonalcoholic, caffeine-containing beverage and benefits human health due to its wealth of secondary metabolites, including catechins, theanine, polysaccharides, caffeine, and volatiles [22][23][24]. Abiotic stresses considerably affect the yield, quality, and even life of C. sinensis, and such adverse environmental conditions may reduce the performance of the C. sinensis with reduced yield from 65% [25]. Although the functions of 2ODD-C genes have been extensively investigated in multiple model organisms, these enzymes have not been systematically analyzed in C. sinensis. In our current study, a comprehensive analysis of the Cs2ODD-C gene family in C. sinensis was performed for phylogenetic evolution, gene structure, conserved motifs, chromosome location, gene duplication, and expression patterns in different organs and under different abiotic stress conditions. In addition, two candidate genes (Cs2ODD-C36 and Cs2ODD-C21) might play both positive and negative roles in regulating the multi-stress tolerance. Our results provide new insight into the function of Cs2ODD-C genes in C. sinensis and establish a knowledge base for further genetic improvement of C. sinensis. Through this study, we hope to develop a novel molecular basis to improve the multi-stress tolerance of plants and promote the development of phytoremediation technology for multi-stress situations.

Genome-Wide Identification of Cs2ODD-C Gene Family in C. sinensis
To identify the Cs2ODD-C family genes in C. sinensis, the full-length sequence alignments of the DIOX_N (PF14226) and 2OG-FeII_Oxy (PF03171) domains were downloaded from the Pfam database and were used as queries to search the C. sinensis proteome [26]. A total of 153 Cs2ODD-C genes were identified in C. sinensis genome, encoding proteins ranging from 162 aa (Cs2ODD-C129) to 422 aa (Cs2ODD-C104) in length, with an average of 334.4 aa. The molecular weight of Cs2ODD-C family proteins ranged from 17.53 kDa (Cs2ODD-C129) to 47.36 kDa (Cs2ODD104), and the isoelectric points ranged Plants 2023, 12, 1302 3 of 23 from 4.18 (Cs2ODD-C130) to 9.55 (Cs2ODD-C47). The prediction of the subcellular localization of Cs2ODD-C family proteins showed that all proteins were distributed in the cytoplasm (Supplementary Table S1). The detailed information of Cs2ODD-C family genes is provided in Supplementary Table S1, including chromosome location, number of introns, and the gene start and end sites.
Based on the valuable information for genome annotation, 146 of the identified Cs2ODD-C genes were localized on 15 C. sinensis chromosomes, with the remaining 7 genes (Cs2ODD-C1, Cs2ODD-C17, Cs2ODD-C49, Cs2ODD-C70, Cs2ODD-C74, Cs2ODD-C95, and Cs2ODD-C107) unanchored to chromosomes. As shown in Figure 1, the congregate regions and number of Cs2ODD-C genes are irregular across the 15 chromosomes. Chromosome 9 harbored the highest number of Cs2ODD-C genes (n = 18), whereas Chromosome 6 had the smallest number of genes (n = 3). Moreover, the percentage of Cs2ODD-C genes per chromosome varied from 0.09% on Chromosome 6 to 0.65% on Chromosome 15 (Supplementary Table S1). ranging from 162 aa (Cs2ODD-C129) to 422 aa (Cs2ODD-C104) in length, with an average of 334.4 aa. The molecular weight of Cs2ODD-C family proteins ranged from 17.53 kDa (Cs2ODD-C129) to 47.36 kDa (Cs2ODD104), and the isoelectric points ranged from 4.18 (Cs2ODD-C130) to 9.55 (Cs2ODD-C47). The prediction of the subcellular localization of Cs2ODD-C family proteins showed that all proteins were distributed in the cytoplasm (Supplementary Table S1). The detailed information of Cs2ODD-C family genes is provided in Supplementary Table S1, including chromosome location, number of introns, and the gene start and end sites. Based on the valuable information for genome annotation, 146 of the identified Cs2ODD-C genes were localized on 15 C. sinensis chromosomes, with the remaining 7 genes (Cs2ODD-C1, Cs2ODD-C17, Cs2ODD-C49, Cs2ODD-C70, Cs2ODD-C74, Cs2ODD-C95, and Cs2ODD-C107) unanchored to chromosomes. As shown in Figure 1, the congregate regions and number of Cs2ODD-C genes are irregular across the 15 chromosomes. Chromosome 9 harbored the highest number of Cs2ODD-C genes (n = 18), whereas Chromosome 6 had the smallest number of genes (n = 3). Moreover, the percentage of Cs2ODD-C genes per chromosome varied from 0.09% on Chromosome 6 to 0.65% on Chromosome 15 (Supplementary Table S1).  To further explore the relationships among Cs2ODD-C family genes, an unrooted phylogenetic tree in C. sinensis, O. sativa, and Arabidopsis was constructed by PhyML 3.0 software with the maximum-likelihood (ML) method. The Cs2ODD-C family genes clustered into 21 groups (Groups I to XXI) based on the tree topology and the functions of identified AtODD-C genes in Arabidopsis ( Figure 2; Table 1). The number of Cs2ODD-C genes in different groups was different. Group XIV, including 20 Cs2ODD-C genes, was the largest group, followed by Group VI (15 genes), Group VIII (14 genes), and Group IV (13 genes). Group XVIII contained only one Cs2ODD-C gene, making it the smallest group. In addition, Groups IV, VIII, X, XII, XIII, XIV, XVII, XX, and XXI contained more Cs2ODD-C genes in C. sinensis than in Arabidopsis and O. sativa. In addition, two groups (Groups VII and XV) harbored only At2ODD-C or Cs2ODD-C genes without corresponding Os2ODD-C genes in O. sativa, indicating that the number of 2ODD-C genes may have increased in C. sinensis and Arabidopsis or that O. sativa lost these 2ODD-C genes during evolution.

Gene Structure and Conserved Motifs of Cs2ODD-C Family Genes
To further investigate the gene structure diversity and motif composition of Cs2ODD-C genes, the conserved motifs of Cs2ODD-C proteins were analyzed and visualized using the MEME suite. Fifteen putative conserved motifs were identified in Cs2ODD-C proteins (Figures 3a and S1). Motifs 2, 3, and 4 corresponding to the DIOX_N domain, and Motifs 1, 5, and 10 corresponding to the 2OG-Fe II_Oxy domain were shared

Gene Structure and Conserved Motifs of Cs2ODD-C Family Genes
To further investigate the gene structure diversity and motif composition of Cs2ODD-C genes, the conserved motifs of Cs2ODD-C proteins were analyzed and visualized using the MEME suite. Fifteen putative conserved motifs were identified in Cs2ODD-C proteins (Figures 3a and S1). Motifs 2, 3, and 4 corresponding to the DIOX_N domain, and Motifs 1, 5, and 10 corresponding to the 2OG-Fe II_Oxy domain were shared by nearly all Cs2ODD-C proteins. The distributions of some conserved motifs varied among the groups, with some groups having specific conserved motifs. Motif 12 was specifically distributed in all members of Group XVII, whereas Motif 15 was specifically distributed in Groups XII and XIV. The members of Groups I-VI and VII-XI commonly contained Motifs 11 and 14. These results indicated that the specific motifs appearing in different subgroups might be associated with specific functions.
The exon/intron structural patterns of Cs2ODD-C genes are shown in Figure 3b and Supplementary Table S1. The number of exons for the 153 Cs2ODD-C genes varied from 1 to 11. A majority (138 of 153) of the Cs2ODD-C genes have 1-4 introns, and the other 15 Cs2ODD-C genes have 9-11 introns. Cs2ODD-C genes in the same subgroups consistently showed the same numbers and lengths of introns/exons, indicating that they have the same intron/exon organization. For instance, all genes in Group XIII contained only one intron, whereas most members of Groups X-XII possessed two introns, and all members of Group XVII had more than ten introns. Thus, this analysis further verified the topology of the phylogenetic tree of Cs2ODD-C family genes.
showed the same numbers and lengths of introns/exons, indicating that they have same intron/exon organization. For instance, all genes in Group XIII contained only intron, whereas most members of Groups X-XII possessed two introns, and all memb of Group XVII had more than ten introns. Thus, this analysis further verified the topolo of the phylogenetic tree of Cs2ODD-C family genes.

Cis-Regulatory Elements in the Cs2ODD-C Promoter Regions
Cis-regulatory elements are responsible for transcriptional regulation by binding transcription factors. The cis-elements in the promoter regions of Cs2ODD-C genes w analyzed using PlantCARE online software (Supplementary Figure S2). There were functionally annotated cis-elements identified, and these were further classified into th categories: stress-responsive elements (MYB, MBS, AE-box, ABRE, TCA-motif, box and TATA), hormone-responsive elements (RY-element, P-box, GA-motif, TATC-mo ABRE, and Sp1), and light-responsive elements (AT-rich element, I-box, G-box, GA mo AT1-motif, LTR, ATCT-motif, ACE, W-box, and TGA-box). The Cs2ODD-C genes c tained numerous stress-, hormone-, and light-responsive elements, suggesting that th genes might be involved in response to light signaling, plant growth and developme stresses, and hormones. In addition, the promoters of Cs2ODD-C36 and Cs2ODD-C genes contained many stress-related cis-elements, such as CGTCA-motif, TATC-b GARE-motif, ABRE, MBS, P-box, TCA-motif, and GACG-motif, indicating that these t

Cis-Regulatory Elements in the Cs2ODD-C Promoter Regions
Cis-regulatory elements are responsible for transcriptional regulation by binding to transcription factors. The cis-elements in the promoter regions of Cs2ODD-C genes were analyzed using PlantCARE online software (Supplementary Figure S2). There were 23 functionally annotated cis-elements identified, and these were further classified into three categories: stress-responsive elements (MYB, MBS, AE-box, ABRE, TCA-motif, box-III, and TATA), hormone-responsive elements (RY-element, P-box, GA-motif, TATC-motif, ABRE, and Sp1), and light-responsive elements (AT-rich element, I-box, G-box, GA motif, AT1motif, LTR, ATCT-motif, ACE, W-box, and TGA-box). The Cs2ODD-C genes contained numerous stress-, hormone-, and light-responsive elements, suggesting that these genes might be involved in response to light signaling, plant growth and development, stresses, and hormones. In addition, the promoters of Cs2ODD-C36 and Cs2ODD-C21 genes contained many stress-related cis-elements, such as CGTCA-motif, TATC-box, GARE-motif, ABRE, MBS, P-box, TCA-motif, and GACG-motif, indicating that these two genes might play crucial roles in response to various abiotic stresses in C. sinensis (Supplementary Figure S2).

Evolutionary Patterns of the Cs2ODD-C Gene Family
Gene duplication and divergence play important roles in the evolutionary momentum of the genome, and tandem duplication (TD) and whole-genome duplication (WGD) contribute to evolutionary novelty and genome complexity [27]. Among the 153 Cs2ODD-C genes, 36 (23.52%) and 39 (25.49%) were duplicated and retained from WGD/segmental duplication and TD, respectively (Supplementary Figure S3), indicating that these two gene duplication events mainly contribute to the expansion of Cs2ODD-C family genes in C. sinensis.
A collinearity analysis of the Cs2ODD-C gene family in C. sinensis was conducted to further study the underlying evolutionary processes. A total of 33 Cs2ODD-C genes from 23 segmental duplication events were identified in C. sinensis, which accounted for 91.6% of WGD-type Cs2ODD-C genes (Figure 4a; Supplementary Table S2). Cs2ODD-C genes were located within synteny blocks on all chromosomes. In addition, the ratio of non-synonymous to synonymous substitutions (Ka/Ks) is a useful measure of the strength and mode of natural selection acting on protein-coding genes. A Ka/Ks ratio of 1 is indicative of neutral selection (no specific direction), lower than 1 indicates purifying selection, and higher than 1 indicates positive selection [28]. In the present study, the Ka/Ks ratio of 23 Cs2ODD-C gene pairs was less than one, implying that these genes are under negative purifying selection, which maintained the functions of the Cs2ODD-C gene family in C. sinensis (Supplementary Table S2). Moreover, Ks was usually used to estimate the evolutionary dates of genome or gene duplication events. Te WGD/segmental duplicated events in C. sinensis occurred from 0.91 (Ks = 0.0272) to 81.51 mya (Ks = 2.5541).

Expression of Cs2ODD-C Family Genes in Different Tissues of C. sinensis
The expression patterns of Cs2ODD-C genes in different tissues of C. sinensis were evaluated using the previous RNA-seq data from the Tea Plant Information Archive (TPIA) database [29]. Four Cs2ODD-C genes (Cs2ODD-C40, Cs2ODD-C47, Cs2ODD-C53, and Cs2ODD-C58) were not detected in any tissue of C. sinensis. The expression patterns  Tables S3 and S4). A total of 36 orthologous gene pairs of 2ODD-C were identified between C. sinensis and Arabidopsis, whereas there were only 5 orthologous gene pairs of 2ODD-C between C. sinensis and O. sativa identified in this study (Figure 4b,c). The much higher number of orthologous events of Cs2ODD-C-At2ODD-C than that of Cs2ODD-C-Os2ODD-C indicated that C. sinensis is more closely related to Arabidopsis than to O. sativa.

Expression Pattern of Cs2ODD-C Family Genes under MeJA Treatment
The expression patterns of 153 Cs2ODD-C family genes in C. sinensis seedlings that experienced MeJA treatment were evaluated used previous RNA-seq data from the TPIA database ( Figure 6). Under MeJA treatment, 11 Cs2ODD-C genes (Cs2ODD-C49, Cs2ODD-C50, Cs2ODD-C36, Cs2ODD-C27, Cs2ODD-C110, Cs2ODD-C57, Cs2ODD-C8, Cs2ODD-C5, Cs2ODD-C153, Cs2ODD-C125, and Cs2ODD-C18) were upregulated at both 24 h and 48 h, Cs2ODD-C134, Cs2ODD-C145, Cs2ODD-C109, and Cs2ODD-C88) showed downregulated expression at both time points (Figure 6), and this may indicate that these were long-term response genes in C. sinensis under MeJA exposure. Moreover, the expression levels of 47 Cs2ODD-C genes increased to a maximum at 24 h and then sharply decreased at 48 h of MeJA treatment, which may indicate that these were short-term response genes in C. sinensis under MeJA exposure. Both long-term and short-term response genes in C. sinensis might play important roles in the response to MeJA. In addition, 26 Cs2ODD-C genes showed only upregulated expression at 48 h after MeJA treatment; this may indicate that these genes are relatively less responsive to MeJA ( Figure 6). However, there was no significant change in the expression levels of 20 Cs2ODD-C genes between the tissues treated with MeJA and untreated control tissues, indicating that these genes might not be involved in the response to MeJA in C. sinensis ( Figure 6).

Expression Pattern of Cs2ODD-C Family Genes under PEG Treatment
The expression levels of Cs2ODD-C family genes in C. sinensis seedlings under PEG treatment were evaluated using the previous RNA-seq data from the TPIA database ( Figure 7). The expression levels of 28 Cs2ODD-C genes significantly increased at both 24 h and 48 h after PEG treatment, whereas 59 Cs2ODD-C genes showed downregulated expression at both time points. Moreover, the expression levels of 14 Cs2ODD-C genes (Cs2ODD-C106, Cs2ODD-C49, Cs2ODD-C50, Cs2ODD-C66, Cs2ODD-C27, Cs2ODD-C46, Cs2ODD-C4, Cs2ODD-C2, Cs2ODD-C107, Cs2ODD-C110, Cs2ODD-C153, Cs2ODD-C108, Cs2ODD-C136, and Cs2ODD-C42) increased to a maximum at 24 h after PEG treatment and then decreased sharply at 48 h after PEG treatment, which may indicate short-term response genes in C. sinensis under PEG exposure. Both long-term and short-term response genes in C. sinensis might play important roles in the response to PEG. In addition, the expression of 13 Cs2ODD-C genes was only significantly upregulated at 48 h after PEG treatment, suggesting that these genes are relatively less responsive to PEG stress (Figure 7). However, there was no significant difference in the expression levels of 27 Cs2ODD-C genes between the PEG treatment and control groups, thus indicating that these genes might not be associated with drought stress in C. sinensis.

Expression Pattern of Cs2ODD-C Family Genes under MeJA Treatment
The expression patterns of 153 Cs2ODD-C family genes in C. sinensis seedlings that experienced MeJA treatment were evaluated used previous RNA-seq data from the TPIA database ( Figure 6). Under MeJA treatment, 11 Cs2ODD-C genes (Cs2ODD-C49, Cs2ODD-C50, Cs2ODD-C36, Cs2ODD-C27, Cs2ODD-C110, Cs2ODD-C57, Cs2ODD-C8, Cs2ODD- in C. sinensis under MeJA exposure. Both long-term and short-term response genes in C. sinensis might play important roles in the response to MeJA. In addition, 26 Cs2ODD-C genes showed only upregulated expression at 48 h after MeJA treatment; this may indicate that these genes are relatively less responsive to MeJA ( Figure 6). However, there was no significant change in the expression levels of 20 Cs2ODD-C genes between the tissues treated with MeJA and untreated control tissues, indicating that these genes might not be involved in the response to MeJA in C. sinensis ( Figure 6).

Expression Pattern of Cs2ODD-C Family Genes under PEG Treatment
The expression levels of Cs2ODD-C family genes in C. sinensis seedlings under PEG treatment were evaluated using the previous RNA-seq data from the TPIA database (Figure 7). The expression levels of 28 Cs2ODD-C genes significantly increased at both 24 h and 48 h after PEG treatment, whereas 59 Cs2ODD-C genes showed downregulated expression at both time points. Moreover, the expression levels of 14 Cs2ODD-C genes

Expression Pattern of Cs2ODD-C Family Genes under NaCl Stress
Salinity can regulate the growth and development of plants, and salt stress can severely affect plant growth, thus reducing the plant yield. Therefore, we evaluated the expression levels of the 153 Cs2ODD-C family genes in C. sinensis seedlings that experienced NaCl treatment, using the previous RNA-seq data from the TPIA database. We found that the expression of 21 Cs2ODD-C genes was significantly upregulated at both 24 h and 48 h after NaCl treatment, whereas the expression of 58 Cs2ODD-C genes was significantly downregulated at both 24 h and 48 h after NaCl treatment, indicating that these genes may be long-term response genes in C. sinensis under salt stress (Figure 8). Moreover, the expression levels of 20 Cs2ODD-C genes increased to a maximum at 24 h and then decreased sharply at 48 h of NaCl treatment (Figure 8), and this may indicate that these are short-term response genes in C. sinensis under salt stress. In addition, 16 Cs2ODD-C genes were only significantly upregulated 48 h after NaCl treatment, implying that these genes are relatively less responsive to salt stress ( Figure 8). However, the expression levels of 25 Cs2ODD-C genes showed no significant change after NaCl treatment in comparison with those of the controls, thus indicating that these genes might not be involved in the response to the salt stress of C. sinensis (Figure 8).
Cs2ODD-C4, Cs2ODD-C2, Cs2ODD-C107, Cs2ODD-C110, Cs2ODD-C153, Cs2ODD-C108, Cs2ODD-C136, and Cs2ODD-C42) increased to a maximum at 24 h after PEG treatment and then decreased sharply at 48 h after PEG treatment, which may indicate short-term response genes in C. sinensis under PEG exposure. Both long-term and short-term response genes in C. sinensis might play important roles in the response to PEG. In addition, the expression of 13 Cs2ODD-C genes was only significantly upregulated at 48 h after PEG treatment, suggesting that these genes are relatively less responsive to PEG stress ( Figure  7). However, there was no significant difference in the expression levels of 27 Cs2ODD-C genes between the PEG treatment and control groups, thus indicating that these genes might not be associated with drought stress in C. sinensis.  Pairwise comparisons of gene expression levels among MeJA, PEG, and NaCl treatments were performed to further evaluate the expression pattern of Cs2ODD-C genes in C. sinensis. The expression of three Cs2ODD-C genes (Cs2ODD-C125, Cs2ODD-C36, and Cs2ODD-C8) was significantly upregulated at both 24 h and 48 h after MeJA and PEG treatments, whereas four Cs2ODD-C genes (Cs2ODD-C104, Cs2ODD-C109, Cs2ODD-C54, and Cs2ODD-C21) showed downregulated expression at both time points. The expression levels of six Cs2ODD-C genes (Cs2ODD-C2, Cs2ODD-C4, Cs2ODD-C42, Cs2ODD-C46, Cs2ODD-C66, and Cs2ODD-C107) increased to a maximum at 24 h and then decreased at 48 h of both the MeJA and PEG treatments. Only one gene (Cs2ODD-C93) showed upregulated expression at 48 h after both the MeJA and PEG treatments (Figures 6 and 7, and Supplementary Table S5). As shown in Figures 6 and 8, the expression of two Cs2ODD-C genes (Cs2ODD-C27 and Cs2ODD-C36) was upregulated at 24 h and 48 h of MeJA and NaCl treatments, whereas three Cs2ODD-C genes (Cs2ODD-C109, Cs2ODD-C143, and Cs2ODD-C21) showed downregulated expression at both time points in both treatments.

Expression Pattern of Cs2ODD-C Family Genes under NaCl Stress
The expression levels of four Cs2ODD-C genes (Cs2ODD-C30, Cs2ODD-C4, Cs2ODD-C72, and Cs2ODD-C95) increased to a maximum at 24 h and then decreased at 48 h of MeJA and NaCl treatments. Furthermore, four Cs2ODD-C genes (Cs2ODD-C116, Cs2ODD-C117, Cs2ODD-C67, and Cs2ODD-C52) were only significantly upregulated at 48 h of MeJA and NaCl treatments (Figures 6 and 8; Supplementary Table S6). Under the PEG and NaCl treatments, nine Cs2ODD-C genes (Cs2ODD-C117, Cs2ODD-C 140, Cs2ODD-C35, Cs2ODD-C36, Cs2ODD-C39, Cs2ODD-C44, Cs2ODD-C121, and Cs2ODD-C6) showed significantly upregulated expression at 24 h and 48 h, while 37 Cs2ODD-C genes showed downregulated expression at both time points. The expression levels of two Cs2ODD-C genes (Cs2ODD-C110 and Cs2ODD-C48) increased to a maximum at 24 h and then decreased at 48 h of PEG and NaCl treatments (Figures 7 and 8; Supplementary Table S7). It is worth noting that one Cs2ODD-C gene (Cs2ODD-C36) showed upregulated expression at both 24 h and 48 h for the MeJA, PEG, and NaCl treatments, whereas the Cs2ODD-C gene (Cs2ODD-C21) was downregulated at both time points in all three treatments (Figures 6-8 Salinity can regulate the growth and development of plants, and salt stress can severely affect plant growth, thus reducing the plant yield. Therefore, we evaluated the expression levels of the 153 Cs2ODD-C family genes in C. sinensis seedlings that experienced NaCl treatment, using the previous RNA-seq data from the TPIA database. We found that the expression of 21 Cs2ODD-C genes was significantly upregulated at both 24 h and 48 h after NaCl treatment, whereas the expression of 58 Cs2ODD-C genes was significantly downregulated at both 24 h and 48 h after NaCl treatment, indicating that these genes may be long-term response genes in C. sinensis under salt stress (Figure 8). Moreover, the expression levels of 20 Cs2ODD-C genes increased to a maximum at 24 h and then decreased sharply at 48 h of NaCl treatment (Figure 8), and this may indicate that these are short-term response genes in C. sinensis under salt stress. In addition, 16 Cs2ODD-C genes were only significantly upregulated 48 h after NaCl treatment, implying that these genes are relatively less responsive to salt stress ( Figure 8). However, the expression levels of 25 Cs2ODD-C genes showed no significant change after NaCl treatment in comparison with those of the controls, thus indicating that these genes might not be involved in the response to the salt stress of C. sinensis (Figure 8).

Validation of Expression Patterns of 12 Cs2ODD-C Genes under PEG, NaCl, and MeJA Treatments Using qRT-PCR
To validate the reliability of RNA-seq results, twelve Cs2ODD-C genes with high expression after at least two treatments were selected to verify the expression patterns by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) experiments ( Figure 9). Expression comparisons were performed in C. sinensis seedlings treated with PEG, NaCl, and MeJA, and the expression trends in the RT-PCR results were in agreement with the RNA-Seq data.

Discussion
Genes encoding members of the 2OGD superfamily, as the second largest enzyme family in plants, play important roles in growth and development [6,7,30], including proline hydroxylation, the biosynthesis of secondary metabolites, DNA demethylation, and others [16,[31][32][33][34][35]. The ODD-C family of genes, accounting for the majority of 2OGD genes in plants, plays important roles in the biosynthesis or degradation of various secondary metabolites, including hormones, flavonoids, glucosinolate, benzoxazinoid, and monoterpenoid indole alkaloid [6]. However, a systematic characterization of Cs2ODD-C genes in C. sinensis has not yet been performed. In this study, the genome-wide identification and characterization of Cs2ODD-C family genes in C. sinensis were carried out. A total of 153 Cs2ODD-C genes were identified and divided into 21 groups based on phylogeny, gene structure, and protein motif analyses. The number of Cs2ODD-C family genes in C. sinensis was less than in Glycine max (209) and Brassica rapa (154), but more than in Zea mays (75), O. sativa (78), Vitis vinifera (103), Arabidopsis (93), and Fragaria vesca (123). These results showed that the species-specific duplication events contributed to the expansion of the Cs2ODD-C gene family in C. sinensis [36,37].
Gene duplication makes the primary contribution to gene family expansion and genetic novelty. Several patterns of gene duplication, including tandem, proximal, dispersed, and whole-genome duplication (WGD or segmental duplication), contribute differentially to the expansion of specific gene families in plant genomes [38][39][40]. For example, segmental and tandem duplications contributed to the expansion of WRKY and Figure 11. Protein interaction network analysis for the candidate Cs2ODD−Cs involved in response to MeJA, PEG, and NaCl stresses. The online tool STRING was used to predict the network.

Discussion
Genes encoding members of the 2OGD superfamily, as the second largest enzyme family in plants, play important roles in growth and development [6,7,30], including proline hydroxylation, the biosynthesis of secondary metabolites, DNA demethylation, and others [16,[31][32][33][34][35]. The ODD-C family of genes, accounting for the majority of 2OGD genes in plants, plays important roles in the biosynthesis or degradation of various secondary metabolites, including hormones, flavonoids, glucosinolate, benzoxazinoid, and monoterpenoid indole alkaloid [6]. However, a systematic characterization of Cs2ODD-C genes in C. sinensis has not yet been performed. In this study, the genome-wide identification and characterization of Cs2ODD-C family genes in C. sinensis were carried out. A total of 153 Cs2ODD-C genes were identified and divided into 21 groups based on phylogeny, gene structure, and protein motif analyses. The number of Cs2ODD-C family genes in C. sinensis was less than in Glycine max (209) and Brassica rapa (154), but more than in Zea mays (75), O. sativa (78), Vitis vinifera (103), Arabidopsis (93), and Fragaria vesca (123). These results showed that the species-specific duplication events contributed to the expansion of the Cs2ODD-C gene family in C. sinensis [36,37].
Gene duplication makes the primary contribution to gene family expansion and genetic novelty. Several patterns of gene duplication, including tandem, proximal, dispersed, and whole-genome duplication (WGD or segmental duplication), contribute differentially to the expansion of specific gene families in plant genomes [38][39][40]. For example, segmental and tandem duplications contributed to the expansion of WRKY and AP2/ERF transcription factor [41,42]. Transposed duplication was responsible for the proliferation of other important gene families, including MADS-box, F-box, and B3 transcription factors in Brassicales [43]. In the present study, nearly half of Cs2ODD-C family genes in C. sinensis were derived from WGD (or segmental duplication) and tandem duplications, suggesting that these two duplication events played important roles in the expansion of Cs2ODD-C genes in C. sinensis. Gene expansion is accompanied by neofunctionalization and subfunctionalization, as well as new protein-protein interactions and gene-expression patterns. For example, Cs2ODD-C3 and Cs2ODD-C5 were duplicated and retained from WGD. The Cs2ODD-C3 gene was highly expressed in leaves and apical buds, whereas Cs2ODD-C5 showed a high expression level in the roots, flowers, and fruits. Moreover, the expression of Cs2ODD-C5 was downregulated after MeJA treatment, whereas there was no significant difference in the expression level of Cs2ODD-C3 under MeJA stress ( Figure 6).
In plants, the type of cis-acting elements at the 5 regulatory region (promoter) determines the complex regulatory properties of a given gene [44]. Our result showed that the cis-acting elements in the promoters of Cs2ODD-C genes, including light-, stress-, and hormone-responsive elements, are involved in light, stress, and hormone responses, and this was consistent with previous studies [45][46][47]. Zhu et al. (2020) identified the cis-acting elements in the promoters of key carotenoid pathway genes from Citrus species, which were classified into light-, stress-, and hormone-responsive elements [48].
The members of 2ODD-C family are involved in biosynthesis of secondary metabolites, including hormones (auxin, GA, jasmonic acid, salicylic acid, and ethylene) [33][34][35], flavonoids [23], benzylisoquinoline alkaloids [7], glucosinolates [16,31], tropane alkaloids [49], monoterpene indole alkaloids [50], benzoxazinoids [7], coumarins [7], mugineic acid [7], and steroidal glycoalkaloids [12]. These secondary metabolites directly or indirectly respond to abiotic stress. Our results showed that 64.05% (98), 74.5% (114), and 75.16% (115) of Cs2ODD-C family genes in C. sinensis showed differential expression patterns under MeJA, PEG, and NaCl treatment, respectively, suggesting that the Cs2ODD-C family genes played essential roles in regulating various abiotic stresses. Moreover, paired comparison analysis further showed that 14, 13, and 49 Cs2ODD-C genes displayed the same expression pattern in the MeJA vs. PEG, MeJA vs. NaCl, and PEG vs. NaCl comparisons, implying that these genes might play important roles under these two abiotic stresses, respectively. In addition, two Cs2ODD-C genes, Cs2ODD-C36 and Cs2ODD-C21, were up-and downregulated after MeJA, PEG, and NaCl treatments, indicating that these two genes played both positive and negative roles in enhancing the tolerance to abiotic stress. Functional annotation revealed that the orthologous genes of Cs2ODD-C36 and Cs2ODD-C21 in O. sativa and Arabidopsis were IDS3 (Os07g07410) and GA3ox, respectively. A previous study revealed that the overexpression of GA3ox reduced stress tolerance in Arabidopsis [51,52]. Stress-induced DELLA accumulation reduced the bioactive GA content by inhibiting the expression level of GA3ox, increased the activities of reactive oxygen species (ROS) detoxification enzymes (catalases and Cu/Zn-superoxide dismutases) and reduced the ROS accumulation in plants [53,54]. In addition, previous studies reported that iron-containing enzymes (superoxide dismutase, catalase, and glutathione peroxidase) were involved in the detoxification of ROS [55,56], and iron deficiency is believed to be dependent on the type and quantity of mugineic acid [57]. A functional annotation showed that the orthologous genes of Cs2ODD-C36 in O. sativa was IDS3 (Os07g07410) encoding 2 -deoxymugineic-acid 2 -dioxygenase, which was involved in the formation of mugineic acids [58]. Thus, we could propose potential molecular mechanisms for the underlying roles of Cs2ODD-C genes in response to various abiotic stresses. One explanation is that stress-induced DELLA accumulation in C. sinensis reduces the bioactive GA content by decreasing the expression levels of Cs2ODD-C21 genes, thus enhancing stress tolerance. Another explanation is that the Cs2ODD-C36 gene involved in the biosynthesis of mugineic acids plays important roles in enhancing abiotic stress tolerance in C. sinensis by improving the absorption of iron to enhance the activity of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) ( Figure 12). improving the absorption of iron to enhance the activity of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) ( Figure 12).

Conclusions
In the present study, 153 Cs2ODD-C genes were identified in the C. sinensis genome and were classified into 21 groups based on the sequence similarity and phylogenetic relationships. The conserved domain, gene structure, and evolutionary relationships of Cs2ODD-C genes were also established and analyzed. Investigation of cis-regulatory elements of Cs2ODD-C genes indicated that many Cs2ODD-C genes are involved in regulating abiotic stress tolerance. A comprehensive analysis revealed that two candidate genes, namely Cs2ODD-C36 and Cs2ODD-C21, may be involved in both positively and negatively regulating the multi-stress tolerance. The above results could provide a basis for the functional characterization of Cs2ODD-C genes and also provide candidate genes for the future improvement of leaf colorization in C. sinensis.

Identification and Classification of Cs2ODD-C Family Genes in C. sinensis
The genome sequence and corresponding annotations of the C. sinensis were obtained from the TPIA database (http://tpia.teaplant.org, accessed on 15 February 2022). An HMM file was constructed through the full alignment files of the DIOX_N (PF14226) and 2OG-FeII_Oxy (PF03171) domains, using the hmmbuild program with the HMMER3 software package [59]. The C. sinensis protein databases were then used to conduct HMM searches. Short proteins (<100 amino acids) and the redundant sequences mapped to a similar location on the same chromosome were removed from all candidate genes in the chromosomal localizations. Finally, the core domains (DIOX_N and 2OG-FeII_Oxy) were further used to verify the candidate proteins with Pfam (https://pfam.xfam.org/, accessed on 15 February 2022) and SMART (http://smart.embl-heidelberg.de/, accessed on 15 February 2022) [59,60]. Finally, the identified proteins containing DIOX_N and 2OG-Fe II_Oxy domains were regarded as candidate Cs2ODD-C genes.

Chromosomal Localization, Motif, and Gene Structure Analyses
The physical location and gene structure of Cs2ODD-C genes were acquired in the C. sinensis database, and the isodose distribution of the genes was plotted with MapInspect software (http://mapinspect.software.informer.com/, accessed on 15 February 2022). The conserved motifs in C. sinensis Cs2ODD-C protein sequences were identified with the online program MEME5.0.1 (http://meme.nbcr.net/meme/intro.html, accessed on 15 February 2022) under the following optimized parameters: maximum motif width, 50 bp; minimum motif width, 6 bp; and maximum numbers of different motifs, 20 [61].

Phylogenetic Analysis
The comparative analysis of full-length 2ODD-C protein sequences between C. sinensis and Arabidopsis was conducted with Clustal X2.0 software, using default parameters, which included 93 At2ODD-C and 153 Cs2ODD-C protein sequences [6]. The best-fit model of protein evolution was identified with the Model-Generator program [62]. An unrooted phylogenetic tree was constructed based on maximum likelihood (ML) and best model of JTT and G with 100 bootstraps, using PhyML3.0 software. The phylogenetic tree was visualized by using FIGTREE [63].

Synteny Analysis
Synteny analyses between the Arabidopsis and C. sinensis genomes were performed locally according to the method described by Lee et al. [64]. The potential homologous gene pairs were initially identified across multiple genomes by using BLASTP (E < 1× 10 −5 , top 5 matches). The result of BLASTP was then used as an input file for MCScanX to determine the different type of duplication events (tandem duplication, proximal duplication, WGD/segmental duplication, or dispersed duplication) [65].

Ka/Ks Analysis of Cs2ODD-C Genes
To estimate the evolutionary rates of whole-genome/segmental and tandem-duplicated Cs2ODD-C paralog genes, PAL2NAL software was used to construct a multiple-codon alignment from the corresponding aligned protein sequences. Such codon alignments can be used for synonymous (Ks) and nonsynonymous (Ka) estimation [66]. The Ks and Ka substitutions of Cs2ODD-C genes were calculated using KaKs_Calculator 2.0, which is commonly used to evaluate the pattern of selection [67]. Generally, Ka/Ks > 1 indicates that genes have undergone positive selection, Ka/Ks < 1 indicates that genes have undergone purifying or negative selection, and Ka/Ks = 1 indicates neutral selection [68].

Expression Analysis
The expression patterns of Cs2ODD-C genes in various organs of C. sinensis and under various abiotic stress conditions were obtained from the TPIA database (http:// tpia.teaplant.org, accessed on 15 February 2022). A heat map of the reads per kilobase of transcript per million reads mapped (RPKM) values from RNA-seq data for the relative expression levels of Cs2ODD-C family genes was constructed. The inactive genes were defined as those with RPKM < 2 based on previous reports [69,70]. A gene with RPKM > 2 in at least one organ or stress treatment was regarded as a differentially expressed gene in C. sinensis.

Growth Conditions, Plant Materials Collection, and Stress Treatments
Tea (C. sinensis) seeds were germinated, and the seedlings were planted in a growth chamber (22 • C/18 • C, 14 h photoperiod, sand substrate) for three months. Two-month-old untreated C. sinensis seedlings were collected from the growth chamber and used as control samples. Experimental plants were treated with 10 µM MeJA, 10 mM PEG solution, and 200 mM NaCl, respectively. The treated and untreated samples were collected after 24 h and 48 h treatment, immediately frozen in liquid nitrogen, and stored in a deep freezer at −78 • C until further analysis.

RT-qPCR of Cs2ODD-C Genes
Total RNA was extracted from the tea seedling with TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRA), and the corresponding cDNA was obtained with cDNA Reverse Transcription Kit (TaKaRa). Then qPCR was conducted with SYBR Premix Ex Tag on a DNA Engine Opticon TM 2 system (PCR instrument: Bio-rad T100, manufacturer: Bio-Rad Laboratories Inc, city: State of California, Country: United States). The CsTBP (TATA-box binding protein gene)gene was used as a housekeeping gene to normalize the expression level of target genes in C. sinensis. All primers used in this study are listed in Supplementary Table S10. The reaction conditions of the PCR program were as follows: an initial step at 95 • C for 30 s. After the cycling protocol, melting curves were obtained by increasing the temperature from 60 to 95 • C (0.2 • C −s ) to denature the double-stranded DNA. Then qPCR amplifications were carried out in 96-well plates. The assays were run in an ABI 7500 system, using the SDS v. 1.4 application software (Applied Biosystems, Foster City, CA, USA). The primer efficiency was created based on a five-fold dilution series of cDNA (1:5, 1:25, 1:50, and 1:100), followed by 42 cycles of 95 • C for 15 s, 58 • C for 15 s, 72 • C for 30 s, and 1 s at 80 • C for reading the plate. The expression levels were evaluated by the 2 −∆∆Ct method, with three biological replicates [71]. The amino acid sequences of 12 candidate stress-related Cs2ODD-C proteins were used as the target sequences to construct the tertiary structures with the SWISS-MODEL program (https://swissmodel.expasy.org, accessed on 15 February 2022). The intensive modeling mode was selected for this analysis.

Prediction of Protein Interaction Networks
The protein interaction networks of Cs2ODD-Cs in C. sinensis were predicted using STRING (version 11.0) software (https://www.string-db.org/, accessed on 15 February 2022). The confidence threshold (combined score) was set to 0.5, and "full network" was used as the network type. The protein interaction networks of Cs2ODD-C proteins in C. sinensis were visualized using Cytoscape v3.8.2 software [72].

Statistical Analysis
Significant analyses between two sample groups were calculated using the t-test analysis [73]. All of the expression analyses were conducted for three biological replicates. The average data of three biological replicates were displayed with plus or minus the standard deviation (average ± SD).

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12061302/s1, Figure S1: Sequence logos of Cs2ODD-C proteins. Figure S2: Cis-element analysis of Cs2ODD-C genes from upstream 2000bp sequence to the transcription start site. Figure S3: Proportion of genes originating from diferent replication events. Table S1: The detail information of CsODD genes in C. sinensis. Table S2: Segmentally duplicated Cs2ODD-C gene pairs. Table S3: One-to-one orthologous relationships between C. sinensis and Arabidopsis. Table S4: One-to-one orthologous relationships between C. sinensis and rice.