Comparative Transcriptome Analysis of CCCH Family in Roles of Flower Opening and Abiotic Stress in Osmanthus fragrans

CCCH is a zinc finger family with a typical CCCH-type motif which performs a variety of roles in plant growth and development and responses to environmental stressors. However, the information about this family has not been reported for Osmanthus fragrans. In this study, a total of 66 CCCH predicted genes were identified from the O. fragrans genome, the majority of which had multiple CCCH motifs. The 66 OfCCCHs were found to be unevenly distributed on 21 chromosomes and were clustered into nine groups based on their phylogenetic analysis. In each group, the gene structure and domain makeup were comparatively conserved. The expression profiles of the OfCCCH genes were examined in various tissues, the flower-opening processes, and under various abiotic stresses using transcriptome sequencing and qRT-PCR (quantitative real-time PCR). The results demonstrated the widespread expression of OfCCCHs in various tissues, the differential expression of 22 OfCCCHs during flower-opening stages, and the identification of 4, 5, and 13 OfCCCHs after ABA, salt, and drought stress treatment, respectively. Furthermore, characterization of the representative OfCCCHs (OfCCCH8, 23, 27, and 36) revealed that they were all localized in the nucleus and that the majority of them had transcriptional activation in the yeast system. Our research offers the first thorough examination of the OfCCCH family and lays the groundwork for future investigations regarding the functions of CCCH genes in O. fragrans.


Introduction
Zinc finger proteins are the largest TF (transcription factor) families in eukaryotes and consist of a sequence of one or more cysteine residues with a histidine residue [1,2]. Based on the number and spacing between the conserved cysteines and histidine residues, zinc-finger TFs can be categorized into nine types, including C2H2, CCCH, C3HC4, C4HC3, C2HC5, C2HC, C4, C6, and C8 [3]. Among them, the CCCH zinc-finger TFs as a specific zinc-finger family was first found by Wang et al. in Arabidopsis [2], which contains a typical CCCH-type zinc-finger motif with three conserved cysteines and one histidine residue, accounting for approximately 0.8% of all the zinc finger proteins [4]. In most species, various members of the CCCH zinc-finger family have been identified by genome-wide analysis, including 68 in Arabidopsis [2], 91 in Populus trichocarpa [5], 49 in Dimocarpus longan [6], 88 in wheat [7], and 155 in Brassica napus [8].
Many studies have reported that CCCHs play crucial roles in plant growth and development and adaptive processes. In Arabidopsis, a tandem zinc-finger protein AtTZF3 mediates seed germination under salt treatment as a negative regulator [9]. HUA1 contains CCCH-type zinc finger motifs, of which biological function is involved in stamen/carpel identities and floral determinacy by RNA-binding [10]. Meanwhile, HUA1 also regulates plant size, and hua1 mutant lines are smaller and shorter than WT (wild type) plants [10]. In rice (Oryza sativa L.), CCCH protein SAW1 (swollen anther wall 1) controls anther development by regulation of gibberellin homeostasis [11]. In addition, CCCH type gene IIP4 (ILA1-interacting protein 4) is confirmed in relevant transgenic plants by Zhang et al. [12], and the gene can regulate secondary wall formation and control the mechanical strength of rice. In Cucumis sativus, CsSEF1 (Somatic Embryogenesis Zinc Finger 1) is required for somatic embryogenesis and root development [13]. Furthermore, most research shows that CCCH proteins are involved in hormone signaling and influence plant growth and development. In Arabidopsis, AtTZF4, 5, and 6 are only expressed in seeds, and the three CCCH-type genes involved in ABA-(abscisic acid) and GA-(gibberellic acid) mediate the regulation of seed development [14]. PvCCCH69 is a repressor of leaf senescence as it suppresses the abscisic acid-signaling pathway in switchgrass [15]. In addition, accumulating evidence indicates that a number of CCCH genes participate in plant abiotic stresses and defense responses. For example, the non-tandem CCCH type zinc finger protein IbCCCH18 enhances abiotic stress tolerance in sweet potato (Ipomoea batatas) [16]. The expression of OsTZF5 (CCCH-tandem zinc finger protein 5) increases due to abscisic acid and dehydration stress, and the overexpression of OsTZF5 promotes rice drought tolerance [17]. In Moso bamboo (Phyllostachys edulis), PeCCCH74 enhances drought tolerance and improves the survival rate of Arabidopsis under drought stress treatment [18]. In Switchgrass, plant chilling and freezing tolerance can be significantly improved in the PvCCCH72 transgenic lines through the ABA-mediated pathway [19].
O. fragrans, one of the top ten traditional flowers in China, is an excellent ornamental and practical garden tree that integrates greening, beautification, and fragrance. This ornamental plants have been widely cultivated in Asia due to its unique fragrance, aesthetic and cultural values. However, CCCH genes have not been identified in O. fragrans, and their functions are poorly understood in O. fragrans. In this study, we first performed a genome-wide identification of CCCH family members in the O. fragrans genome. Their chromosomal locations, phylogenetic relationships, gene structural, domain, and CRE (cis-regulatory element) features were then characterized. Finally, the profiles of expression of the CCCHs were explored during flower opening and abiotic stress. In summary, this work provides new insights into the CCCH genes involved in the regulation of flower opening and abiotic stress tolerance, which improves knowledge regarding the diverse functions and features of CCCHs in the breeding of O. fragrans.

Genome-Wide Identification of the OfCCCHs
A total of 66 CCCHs that contained a typical CCCH domain were identified from the O. fragrans genome and were named OfCCCH1-66. The characterizations of the 66 OfCCCH genes are shown in Table S1, including the number of amino acids (length), molecular weights, pI (isoelectric points), and the physical location. The detailed information indicated that the length of the 66 OfCCCH proteins varied and ranged from 127 amino acids (OfCCCH37) to 1195 amino acids (OfCCCH46). In addition, the pI of the 66 OfCCCHs ranged from 4.54 (OfCCCH39) to 9.47 (OfCCCH38). All the OfCCCH genes were mapped on 21 chromosomes, and the highest proportion was located on chromosome 3 (7 members) ( Figure 1).

Phylogenetic Analysis, Gene Duplication, and Synteny Analysis
Based on phylogenetic analysis, the CCCH proteins of Arabidopsis and O. fragrans were divided into nine groups ( Figure 2). A total of 114 of 132 CCCHs fell into one branch and were further divided into two subgroups. The first included 79 CCCHs, and the second consisted of 35 CCCHs. In contrast, the other two branches separated from the previous branch, including 7 and 11 CCCHs, respectively, demonstrating their phylogenic dissociation from the other one. Seventeen segmentally duplicated genes were identified, and no tandem duplicated gene was found. Segmental duplication events were suggested to be the only cause of OfCCCH gene expansion. Remarkably, chromosome 21 had the largest number of segmentally duplicated gene pairs ( Figure 3, Table 1). Among the 17 duplicated gene pairs, all of them evolved under purifying selection (Ka/Ks < 1) ( Table 1). The time of divergence of the OfCCCHs implied that the duplication events occurred 37.57 million years ago (Mya) and continued up until 0.37 Mya, while the most frequent gene duplication events occured 0-10 Mya (Table 1).

Gene Structure, Domain, and CREs Analysis
To better understand the gene structure of OfCCCHs, the intron and exon were identified, and the number of exons ranged from 1 to 24 ( Figure 4). In addition, a total of five types of motifs were found in the OfCCCH family, including ZF-CCCH, ANK, KH, RRM, and WD40 ( Figure 5). Forty-five OfCCCH genes contained one motif, and the rest of the 21 OfCCCH genes possessed two type motifs. For the CREs analysis, a total of 13 CREs were identified in the promoters of 66 OfCCCHs, of which many were in response to hormones such as ABA, MeJA (methyl jasmonate), GA, auxin, and SA (salicylic acid) ( Figure 6). It was determined that 56 of the OfCCCH promoters had ABA-responsive elements, which represented the largest proportion of all OfCCCH promoters, followed by the MeJA-responsive elements (50), the gibberellin-responsive elements (35), the auxin-responsive elements (35), and the salicylic acid-responsive elements (35), but none of these elements were shared by all OfCCCH promoters ( Figure 6).

Phylogenetic Analysis, Gene Duplication, and Synteny Analysis
Based on phylogenetic analysis, the CCCH proteins of Arabidopsis and O. fragrans were divided into nine groups ( Figure 2). A total of 114 of 132 CCCHs fell into one branch and were further divided into two subgroups. The first included 79 CCCHs, and the second consisted of 35 CCCHs. In contrast, the other two branches separated from the previous branch, including 7 and 11 CCCHs, respectively, demonstrating their phylogenic dissociation from the other one. Seventeen segmentally duplicated genes were identified, and no tandem duplicated gene was found. Segmental duplication events were suggested to be the only cause of OfCCCH gene expansion. Remarkably, chromosome 21 had the largest number of segmentally duplicated gene pairs ( Figure 3, Table 1). Among the 17 duplicated gene pairs, all of them evolved under purifying selection (Ka/Ks < 1) ( Table 1). The time of divergence of the OfCCCHs implied that the duplication events occurred 37.57

Transcriptome Analysis of OfCCCH Genes in Different Tissues
Transcriptome analysis was employed to explore the level of transcription of the OfCCCH genes in different tissues (including root, annual branch, perennial branch, young leaf, and mature leaf). As shown in Figure 7, most OfCCCH genes were widely expressed in different tissues; remarkably, high expression of all tissues was observed for OfCCCH6, 20, 25, and 57. Apparent tissue-specific expression was observed for some OfCCCH genes ( Figure 7). For example, OfCCCH3 was predominantly expressed in the annual branch. The expression of five OfCCCHs (OfCCCH7, 32, 35, 36, and 40) was overrepresented in the perennial branch. OfCCCH28 and OfCCCH56 were primarily expressed in the mature leaf.

Transcriptome Analysis of OfCCCH Genes in Different Tissues
Transcriptome analysis was employed to explore the level of transcription of the OfCCCH genes in different tissues (including root, annual branch, perennial branch, young leaf, and mature leaf). As shown in Figure 7, most OfCCCH genes were widely expressed in different tissues; remarkably, high expression of all tissues was observed for OfCCCH6, 20, 25, and 57. Apparent tissue-specific expression was observed for some OfCCCH genes (Figure 7). For example, OfCCCH3 was predominantly expressed in the annual branch. The expression of five OfCCCHs (OfCCCH7, 32, 35, 36, and 40) was overrepresented in the perennial branch. OfCCCH28 and OfCCCH56 were primarily expressed in the mature leaf.

Transcriptome and qRT-PCR Analysis of OfCCCH Genes in Response to Abiotic Stress
The results of the transcriptome showed that a large proportion of OfCCCH genes were differentially expressed in a stress-dependent manner. By comparing the three treatment groups with their control, 4, 5, and 13 DEGs of OfCCCH were found under the ABA, salt, and drought stress treatment, respectively (Table S3). After 12 h ABA stress, three genes (OfCCCH23, 40, and 49) were downregulated, except OfCCCH32 ( Figure 9A). The expression of OfCCCH7, 23, and 32 displayed significant upregulation after salt treatment, while dramatically decreased expression was observed for OfCCCH49 and 64 ( Figure 9A). The expression of OfCCCH8, 17, 18, 28, 39, 45, and 62 was dramatically stimulated, while significant downregulation was observed for six genes (OfCCCH2, 23, 24, 27, 30, and 60) under drought stress treatment ( Figure 9A). The patterns of expression from 0 h to 24 h of the 16 DEGs of OfCCCH were revalidated under different types of abiotic stress using the qRT-PCR method ( Figure 9B). The results showed that all the OfCCCHs showed obvious abiotic stresses response and were consistent with the patterns of expression of transcriptome. In the ABA stress group, OfCCCH23, 32, and 40 were significantly upregulated at 9 h of treatment and then sharply downregulated after 12 h, and the expression of OfCCCH49 displayed a significant downregulation after 3 h ABA stress treatment ( Figure 9B). In the salt group, OfCCCH23 and 32 had a similar pattern of expression. They were all highly expressed at 3 h and 9 h and downregulated at 6 h and 24 h, and OfCCCH49 and 64 were significantly downregulated after 3 h salt stress treatment ( Figure 9B). As shown in Figure 9B, six OfCCCH genes (OfCCCH8, 17, 23, 27, and 28) significantly changed after 3 h of drought stress treatment, while significant upregulation was observed for CCCH39, 45, and 62 after 6 h of drought stress treatment.

Transcriptome and qRT-PCR Analysis of OfCCCH Genes in Response to Abiotic Stress
The results of the transcriptome showed that a large proportion of OfCCCH genes were differentially expressed in a stress-dependent manner. By comparing the three treatment groups with their control, 4, 5, and 13 DEGs of OfCCCH were found under the ABA, salt, and drought stress treatment, respectively (Table S3). After 12 h ABA stress, three genes (OfCCCH23, 40, and 49) were downregulated, except OfCCCH32 ( Figure 9A). The expression of OfCCCH7, 23, and 32 displayed significant upregulation after salt treatment, while dramatically decreased expression was observed for OfCCCH49 and 64 ( Figure 9A). The expression of OfCCCH8, 17,18,28,39,45, and 62 was dramatically stimulated, while significant downregulation was observed for six genes (OfCCCH2, 23,24,27,30, and 60) under drought stress treatment ( Figure 9A). The patterns of expression from 0 h to 24 h of the 16 DEGs of OfCCCH were revalidated under different types of abiotic stress using the qRT-PCR method ( Figure 9B). The results showed that all the OfCCCHs showed obvious abiotic stresses response and were consistent with the patterns of expression of transcriptome. In the ABA stress group, OfCCCH23, 32, and 40 were significantly upregulated at 9 h of treatment and then sharply downregulated after 12 h, and the expression of OfCCCH49 displayed a significant downregulation after 3 h ABA stress treatment ( Figure  9B). In the salt group, OfCCCH23 and 32 had a similar pattern of expression. They were all highly expressed at 3 h and 9 h and downregulated at 6 h and 24 h, and OfCCCH49 and 64 were significantly downregulated after 3 h salt stress treatment ( Figure 9B). As shown in Figure 9B, six OfCCCH genes (OfCCCH8, 17, 23, 27, and 28) significantly changed after 3 h of drought stress treatment, while significant upregulation was observed for CCCH39, 45, and 62 after 6 h of drought stress treatment.

Characterization of the OfCCCH Proteins
To explore the potential function of the OfCCCH genes in the transcriptional regulation system, the representative genes OfCCCH8, 23, 27, and 36 were fused to a C-terminal GFP and expressed in the leaf epidermis of N. benthamiana to detect the subcellular localization. GFP signals were then observed after 36 h. As shown in Figure 10, the GFP fluorescence of the 35S::GFP-OfCCCH8/23/27/36 fusion protein was only detected in the nucleus where it could be involved in regulating transcriptional events. To identify whether these OfCCCH proteins had trans-activation activity, the proteins (OfCCCH8, 23, 27, and 36) were fused in pGBKT7 to form the BD-CCCH vector and then transformed into the AH109 yeast strain. All the yeast cells grew well on the SD/-Trp media. Moreover, OfCCCH8, 27, and 36 grew normally and displayed positive GAL4 activity on X-α-gal-supplemented medium, which suggested that they exhibited transcriptional activation in yeast ( Figure 11). However, OfCCCH23 barely grew on the SD/-T/-A plate, which indicated that OfCCCH23 lacked transactivation activity in yeast and needed to form a complex with other proteins to perform its function of transcriptional activation (Figure 11).

Characterization of the OfCCCH Proteins
To explore the potential function of the OfCCCH genes in the transcriptional regulation system, the representative genes OfCCCH8, 23, 27, and 36 were fused to a C-terminal GFP and expressed in the leaf epidermis of N. benthamiana to detect the subcellular localization. GFP signals were then observed after 36 h. As shown in Figure 10, the GFP fluorescence of the 35S::GFP-OfCCCH8/23/27/36 fusion protein was only detected in the nucleus where it could be involved in regulating transcriptional events. To identify whether these OfCCCH proteins had trans-activation activity, the proteins (OfCCCH8, 23, 27, and 36) were fused in pGBKT7 to form the BD-CCCH vector and then transformed into the AH109 yeast strain. All the yeast cells grew well on the SD/-Trp media. Moreover, OfCCCH8, 27, and 36 grew normally and displayed positive GAL4 activity on X-α-galsupplemented medium, which suggested that they exhibited transcriptional activation in yeast ( Figure 11). However, OfCCCH23 barely grew on the SD/-T/-A plate, which indicated that OfCCCH23 lacked transactivation activity in yeast and needed to form a complex with other proteins to perform its function of transcriptional activation (Figure 11).

Characteristic Analysis of OfCCCH TFs in Osmanthus Fragrans
Zinc-finger TFs, as one of the largest TF families in plants, are critical regulators for multiple biological processes, such as morphogenesis, signal transduction, and environ-

Characteristic Analysis of OfCCCH TFs in Osmanthus Fragrans
Zinc-finger TFs, as one of the largest TF families in plants, are critical regulators for multiple biological processes, such as morphogenesis, signal transduction, and environmental stress responses [5]. Plants have adopted most typical classes of zinc fingers as functional domains in transcription factors whose functions are manifold and reported in many plants, such as Arabidopsis [2], rice [2], and Populus [5]. In this study, a total of 66 CCCH genes were identified in O. fragrans using comprehensive genome-wide analysis (Table S1). Compared with woody plants, the number of OfCCCHs was more than that in Longan [6] and less than that in Populus [5]. Chromosomal mapping of OfCCCHs revealed that the OfCCCHs were not uniformly distributed on 21 chromosomes, of which chromosome 3 harbored the most OfCCCH numbers (Figure 1). Similarity with Arabidopsis, all the OfCCCH genes were clustered in nine clades, according to phylogenetic analysis (Figure 2). Previous studies have shown that segmental duplication is largely responsible for the expansion of Arabidopsis and rice CCCH gene families [2]. Meanwhile, there were 17 OfCCCH gene pairs associated with gene duplication events ( Figure 3, Table 1). We speculated that the multiple nature of segmental duplication might contribute to CCCH family expansion in O. fragrans. For the gene structure and domain analysis, similar distribution patterns of exons were identified in the same branch of phylogenetic tree (Figure 4), implying that these CCCH subfamily genes likely share a common evolutionary origin and similar biological functions. Previous studies have indicated that CCCH proteins have one to six copies of CCCH-type motifs in animals or plants [6,8,20], while we found that one to seven copies of CCCH-type motifs were contained in O. fragrans ( Figure 5). In addition, other four-type motifs were identified in the OfCCCH protein sequences, including ANK, KH, PRM, and WD40 motifs. All of these motifs play important roles in diverse molecular processes [21], splicing and transcriptional regulation [22], growth processes [23], and stress response [24]. Many CREs associated with plant hormones were predicted in the OfCCCH promoters in O. fragrans ( Figure 6). It has been reported that CCCHs are involved in responding to hormone signals to regulate plant development and adaptive ability in Arabidopsis [25] and switchgrass [15]. Therefore, the results suggested that OfCCCH genes might play important roles in the processes of growth and development and stress tolerance by responding of hormone signals in O. fragrans.

OfCCCHs Involved in Flower Opening and Abiotic Stress Tolerance in Osmanthus fragrans
Transcriptome analysis is a valuable tool to uncover gene expression profiles in many biological processes of plants. We investigated the expression profiles of OfCCCH genes in different tissues and found that all the genes were expressed widely in the tissues (Figure 7). Among them, eight OfCCCHs (OfCCCH3, 7, 28, 32, 35, 36, 40, and 56) showed the highest expression in the different tissues of O. fragrans (Figure 7), indicating that these OfCC-CHs might specially regulate the processes of growth and development in these tissues of O. fragrans. In addition, CCCH genes are extensively involved in the regulation of flowering processes in many plants, such as floral development, flowering time, and flower opening. In Arabidopsis, AtKHZ1/2 (CCCH zinc-finger and K-homolog protein) and AtKHZ2 reportedly delay flowering processes [26], while a CCCH Zinc finger protein AaZFP3 from Adonis amurensis can promote flowering process in transgenic A. thaliana [27]. In the process of flower development, HUA1 regulates stamen and carpel formation in A. thaliana [10]. Additionally, DR14 encodes a CCCH zinc finger protein that affects the flower opening process in Ipomoea nil [28]. During the flower opening processes of O. fragrans, a total of 22 OfCCCHs (including 9 upregulation and 13 downregulation) were identified and exhibited differential expression from the S1 to the S2 stages ( Figure 8, Table S2). The results suggested that these DEGs might be involved in the regulation of flower opening. Particularly, OfCCCH3, 16, 22, and 36 upregulated more than 5 times at the S2 stage; therefore, these genes might play an important role in flowering initiation. Moreover, CCCH genes reportedly respond to various abiotic stresses. For example, AtZFP1 (Zinc finger protein 1) improves salt resistance in Arabidopsis [29]. In rice, OsCCCH10 enhances drought tolerance by regulating stress-related genes [30]. In O. fragrans, the differential expression of OfCCCH genes were explored after salt, drought, and ABA treatment, and 5, 13, and 4 DEGs were obtained, respectively ( Figure 9, Table S3). We found these DEGs had ABA responsiveness CREs in their promoters, suggesting that OfCCCHs regulate abiotic tolerance might be associated with the ABA signal in O. fragrans. Remarkably, the gene OfCCCH23 was significantly induced by salt, drought, and ABA at same time. The overlapping responses of OfCCCH genes to multiple abiotic stresses provide potential hub players in O. fragrans acclimation to stressful conditions. Finally, these obtained OfCCCH DEGs play key roles in the regulation of flowering processes and abiotic stresses; therefore, four OfCCCHs were selected, including OfCCCH8, 23, 27, and 36, to further analyze their subcellular localization and transactivation activity (Figures 10 and 11). The characterization of the four proteins provides important information regarding the investigation of their mechanisms related to the regulation of flowering and abiotic tolerance enhancement in O. fragrans.

Plant Materials and Treatments
The materials of O. fragrans 'Yanhong Gui' are preserved in the Osmanthus Germplasm Resource Garden of Zhejiang Agriculture and Forestry University (Hangzhou, China). All the materials were maintained in a growth chamber (DONGNAN INSTRUMENT Co., Ltd., Ningbo, China) with a 12 h/12 h (light/dark) photoperiod, with a relative humidity of 60%. Samples of five tissues, including root, annual branch, perennial branch, young leaf, and mature leaf, were collected. For the flower opening treatment, six year old uniform plants were sent to the same growth chamber with an ambient temperature of 19 • C. Flower buds with similar sizes at the S1 stage (with the outer bud scales unfurled and the inner bud scales still furled) and the S2 stage (with buds that became globular-shaped and visible inside bracts that covered the inflorescences) were collected. For the abiotic stress treatments, 18 branches of O. fragrans were treated with ABA (100 µmol/L), NaCl (200 mmol/L), and mannitol (200 mmol/L) in a growth chamber. In addition, the third or fourth fully expanded leaves from the tip were collected from each plant at 0, 3, 6, 9, 12, and 24 h after treatment. All collected samples were immediately frozen in liquid nitrogen and stored at −80 • C for the subsequent extraction of total RNA and expression profiling. Three biological replicates were established for each treatment.

Genome-Wide Identification of the CCCHs
The genome and protein sequences were obtained from O. fragrans genome database to identify the CCCH gene family in O. fragrans [31]. Information about the OfCCCH structural domain (PF00642, https://pfam.xfam.org/family/PF00642, accessed on 10 June 2022) was obtained from the Pfam protein families database (http://pfam.xfam.org/, accessed on 10 June 2022), and HMMER v3.3.2 (http://hmmer.org/, accessed on 10 June 2022) software was used to search all the protein sequences across the genome with a default e-value and identify genes with the specific conserved domain. Subsequently, the number of domains present in the protein sequences were validated with two online domain analytical tools, including the Batch Web CD-Search of NCBI (https://www.ncbi.nlm.nih.gov, accessed on 10 June 2022) and SMART (http://smart.embl-heidelberg.de/, accessed on 10 June 2022).

Chromosomal Localization, Gene Duplication, and Synteny Analysis
The chromosome information of OfCCCHs was obtained using TBtools software v1.09 with the GFF3 file, which includes the chromosome length and the start and end sites of genes. MG2C software v2.1 (http://mg2c.iask.in/mg2c_v2.1/, accessed on 12 June 2022) was used to map the chromosomes and the gene locations of the OfCCCH gene family. The pattern of duplication pattern of each OfCCCH gene was analyzed using MCScanX v1.0 [32]. The Ks (synonymous) and Ka (non-synonymous) substitution ratios of the gene pairs were assessed using DnaSP v5.0 software [33]. A synteny analysis of OfCCCHs was conducted using the Quick MCScanX Wrapper program in TBtools. The graph of collinearity analysis was drawn using TBtools software.

Phylogenetic Tree Construction and Sequence Analysis of CCCHs
The amino acid sequences of the CCCH proteins of O. fragrans and Arabidopsis thaliana were multiplied and aligned using ClustalX [34] and manually corrected. In addition, the phylogenetic tree was constructed using MEGA 11 software [35] and the neighbor-joining method with a bootstrap of 1000. Subsequently, the phylogenetic tree was created using iTOL.v6 software (https://itol.embl.de/, accessed on 12 June 2022). The structures of OfCCCHs were visualized using GSDS2.0 (http://gsds.gao-lab.org/, accessed on 12 June 2022). The conserved protein motif of each putative OfCCCH family member was analyzed using the MEME program with the motif number to be identified established at 20 and the other parameters set as default. Finally, the information on the molecular weight and the isoelectric points of OfCCCHs were obtained using the online tool EXPASy Compute pI/Mw (https://web.expasy.org/compute_pi/, accessed on 12 June 2022).

Total RNA Extraction and RNA-Sequencing
The total RNA was extracted using an RNAprep pure Kit (TianGen Biotech Co., Ltd., Beijing, China) following the manufacturer's instructions. A 3 µg pool of RNA was used for transcriptome sequencing with three biological replicates using the Illumina Hiseq 2500 platform. Clean reads were obtained by removing reads that contained adapters and poly-N and low-quality reads from the raw data. Unigenes were aligned using BLASTx (e-value < 1 × 10 −5 ) against NR (www.ncbi.nlm.nih.gov/refseq, accessed on 11 January 2022), Swiss-Prot (www.uniprot.org, accessed on 11 January 2022), GO (http://geneontology.org/, accessed on 11 January 2022), and KEGG (https://www. genome.jp/kegg/, accessed on 11 January 2022) databases. FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. Additionally, FPKM (the fragments per kilobase of exon per million mapped reads) was calculated based on the length of the gene and the reads count mapped to this gene. Differential expression between the two conditions was analyzed using the DESeq2 method. Genes with |log2FC| > 1 and an adjusted p-value ≤ 0.05 found using the DESeq2 method were assigned as differentially expressed genes.

Quantitative Real-Time PCR Analysis
The cDNA synthesis was completed using HiScript II Q Select RT Supermix for qPCR (Vazyme Biotech Co., Ltd., Nanjing, China). The qRT-PCR reactions were performed using the LightCycler480II System (Roche, Basel, Switzerland) as described by Yang et al. [36]. The OfACT gene was used as internal normalization for O. fragrans [37]. Triple biological replications were performed for each treatment. The relative level of expression of the target gene was calculated using the 2 −∆∆CT method. The primers were designed using primer 5.0 software, primer sequences are showed in Table S4.

Subcellular Localization of the OfCCCH Proteins
The sequences of ORF (open reading frame) without the termination codon were amplified, and then PCR products were subcloned into the pORE R4 vector (genbank: ay562547.1) as an in-frame C-terminal fusion with GFP (green fluorescent protein). The resulting construct was confirmed via sequencing and transformed into the Agrobacterium tumefaciensstrain strain GV3101 (Weidi Biotechnology Co., Ltd., Shanghai, China). The transformed A. tumefaciens lines were infiltrated into fully expanded leaves of tobacco plants, and each experiment was repeated twice. The GFP signal was investigated using a Zeiss LSM 710 (Carl Zeiss, Jena, Germany) after 36 h. All primer information was showed in the Table S4.

Transcriptional Activation Analysis
A yeast two-hybrid system was used to analyze the transcriptional activation of the OfCCCHs. The full-length sequences of OfCCCH proteins were cloned and fused in the pGBKT7-BD vector. The empty vector was used as a negative control. The vectors were then transformed into the Saccharomyces cerevisiae strain AH109 which contained a MEL1 reporter that encoded α-galactosidase. The expressed MEL1 reporter gene could be detected if the recombinant vector pGBKT7-OfCCCHs was transactivated. SD/-Trp media was used to screen for positive transformants, and the positive clones were further screened on SD/-Trp/-Ade and incubated at 30 • C for 3 days.

Conclusions
In this study, a total of 66 OfCCCH genes were identified in O. fragrans using whole genome analysis. Transcriptome and qRT-PCR analysis results identified 22 differently expressed genes in the flower opening stages, and 5, 13, and 4 differently expressed genes were obtained after salt, drought, and ABA treatment, respectively. The comprehensive analysis of expression patterns demonstrated the importance of OfCCCH genes in the regulation of flower opening and response to abiotic stresses.