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Article

Identification and Expression Analysis of C2H2-Type Zinc Finger Protein (C2H2-ZFP) Genes in Bougainvillea in Different Colored Bracts

by
Yushan Wang
1,†,
Yanping Hu
1,2,†,
Wen Liu
1,
Wengang Yu
1,
Jian Wang
1 and
Yang Zhou
1,*
1
Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, School of Tropical Agriculture and Forestry (School of Agricultural and Rural Affairs, School of Rural Revitalization), Hainan University, Haikou 570228, China
2
Key Laboratory of Vegetable Biology of Hainan Province, Hainan Vegetable Breeding Engineering Technology Research Center, The Institute of Vegetables, Hainan Academy of Agricultural Sciences, Haikou 571199, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(6), 659; https://doi.org/10.3390/horticulturae11060659
Submission received: 13 April 2025 / Revised: 3 June 2025 / Accepted: 7 June 2025 / Published: 10 June 2025
(This article belongs to the Special Issue Color Formation and Regulation in Horticultural Plants)
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Abstract

Bougainvillea spp. possesses vibrantly pigmented bracts that exhibit high ornamental value. Zinc finger proteins (ZFPs), one of the most extensive transcription factor families in plants, are implicated in diverse biological functions, including plant morphogenesis, transcriptional regulation, and responses to abiotic stress. Nevertheless, their regulatory roles in bract pigmentation in Bougainvillea remain unexplored. In the present investigation, 105 BbZFP genes were identified from the Bougainvillea genome via bioinformatic analyses and subsequently categorized into five subgroups according to the quantity and arrangement of their structural domains. Analysis of physicochemical characteristics demonstrated that the BbZFP family encompasses both acidic and basic proteins, all of which are hydrophilic and predominantly classified as unstable proteins. Gene structure analysis revealed that the majority of BbZFP genes comprise between one and five– introns. Cis-regulatory element analysis suggested that BbZFP promoter regions harbor multiple elements associated with abiotic stress responses, hormonal regulation, and light responsiveness, implying their possible participation in these physiological processes. Transcriptomic data analysis revealed distinct expression patterns of BbZFP genes among bracts of different colors. A quantitative real-time polymerase chain reaction (RT-qPCR) further confirmed that Bou_68928, Bou_1096, Bou_4400, and Bou_17631 were markedly upregulated in yellow bracts relative to white bracts, suggesting their involvement in flavonoid biosynthesis regulation. Meanwhile, Bou_1096 and Bou_17631 exhibited markedly elevated expression in red-purple bracts compared to white bracts, potentially regulating betacyanin biosynthesis in Bougainvillea. These findings offer candidate genes for molecular breeding strategies aimed at enhancing floral coloration in Bougainvillea. The next step will involve elucidating the functions of these genes in bract coloration.

1. Introduction

Flower color represents one of the most significant traits of ornamental plants, with its expression and regulation subject to modulation by a variety of endogenous and exogenous factors. The diversity in floral pigmentation is largely governed by both the concentration and composition of plant pigments. These pigments are primarily categorized into four main classes: chlorophyll, carotenoids, flavonoids, and betalains [1].
Flavonoids represent a crucial class of secondary metabolites in plants and are extensively distributed as pigments in various ornamental species, determining the orange, red, purple, and blue hues in most plant petals [2]. This group of compounds encompasses a diverse range of structures, including flavones, flavonols, anthocyanidins, proanthocyanidins, flavanones, and isoflavones. Among these, anthocyanidins have been extensively investigated for their pivotal role in regulating floral coloration in plants [2]. Betalains, which are restricted to species within the core Caryophyllales order, are classified into two primary groups: betacyanins and betaxanthins [3,4]. Existing research suggests that betalains and anthocyanins are mutually exclusive within a single plant species, although the molecular mechanisms underpinning this exclusivity remain partially understood, and several explanatory hypotheses have been proposed [4,5,6]. The impact of betalains on floral coloration depends largely on the specific pigment profile of the petal, generating a spectrum of hues ranging from orange to red and other tones [7,8].
Zinc finger proteins (ZFPs) constitute one of the most expansive transcription factor families in plants [9], fulfilling significant roles in a range of biological functions, including plant morphogenesis, transcriptional regulation, and stress adaptation [10]. According to the number and spatial arrangement of cysteine (Cys) and histidine (His) residues involved in zinc ion coordination within their secondary structures, ZFPs have been classified into several subgroups, including C2H2, C2HC, C2HC5, C2C2, C3HC4, C4, C4HC3, and C6 [10,11,12,13]. These proteins are broadly distributed across animals, plants, and microorganisms, with EPF1 from petunia recognized as the first ZFP identified in plants [14]. Evidence suggests that C2H2-type ZFPs exhibit functional diversity, acting not only as DNA-binding transcription factors but also interacting with RNA and various proteins [15]. To date, ZFP gene families have been identified in numerous species, including 176 C2H2-type ZFP genes in Arabidopsis [16], 118 in tobacco, 189 in rice [17], and 129 in cucumber [18]. Based on analyses of both the quantity and classification of C2H2-ZFPs in cucumber [18], soybean [19], and poplar [20], these proteins have been categorized into five principal classes: Q, M, Z, C, and D [18]. Among them, Q-type domains are defined by the plant-specific ‘QALGGH’ sequence and a conserved spacing of ‘X2-C-X2-C-X7-QALGGH-X3-H’; M-type domains are derived from modifications in either the ‘QALGGH’ motif or the spacing between the two Cys and two His residues and are further subdivided into M1–M13 subtypes. Z-type domains are differentiated by variation in the number of amino acids between the second Cys and the first His and are further classified into Z1 and Z2 depending on whether this spacing exceeds 12 residues. The C-type includes 12 residues between the second Cys and first His, whereas D-type domains are distinguished by the absence of the second His in the C2H2 ZFP motif [18]. C2H2-type ZFPs are implicated in mediating plant responses to diverse abiotic stresses, including drought, salinity, cold, oxidative damage, and light stress, in addition to roles in biotic stress defense [21,22]. Moreover, they are involved in several essential developmental processes such as seed germination, floral induction, and leaf senescence [23,24,25,26]. Overexpression of the apple C2H2-type zinc finger protein gene MdZAT5 activates the expression of anthocyanin biosynthesis-related genes in apple callus, thereby positively regulating anthocyanin accumulation [27]. In Satsuma mandarin (Citrus unshiu), the transcriptional cascade mediated by CitZAT4 is initiated by ethylene via CitERF061, linking ethylene signaling to carotenoid metabolism and promoting the development of orange peel color in the fruits [28].
Bougainvillea spp. originates from tropical America and is regarded as a significant ornamental plant in tropical and subtropical regions, while also exhibiting certain medicinal properties [29]. Owing to its vividly colored bracts, Bougainvillea possesses exceptionally high ornamental value [29,30]. As a member of the order of Caryophyllales, its bract pigmentation is predominantly governed by betalains [31,32]. Wu et al. [33] reported that bract coloration in Bougainvillea results from the relative abundance and ratio of betaxanthins and betacyanins, whose coexistence leads to the manifestation of various red hues. By employing transcriptome sequencing and quantitative real-time polymerase chain reaction (RT-qPCR), Wang et al. [30] identified 16 genes—including PAL2, CHS1, ANS, CDOPA5GT, ANR, CHS2, and DOPA—that exhibited significant differential expression among magenta, yellow, white, and cherry-colored bracts, suggesting potential involvement in pigmentation regulation in Bougainvillea. Findings by Kang et al. [32] revealed that the competitive interaction between flavonols and betacyanins represents a primary determinant of red and yellow bract development in Bougainvillea × buttiana ‘Chitra’. Prior research has demonstrated that the C2H2-type ZFP TT1 is one of the key transcription factors involved in the flavonoid biosynthesis pathway [34]. Nevertheless, investigations into C2H2-type ZFPs in Bougainvillea, particularly with respect to their functional roles in floral pigmentation, remain unreported. In the present study, C2H2-type ZFP gene family members were identified from the Bougainvillea genome through bioinformatic approaches and transcriptome data were examined to assess the expression patterns of C2H2 family genes across bracts of different colors. RT-qPCR was subsequently conducted to validate the expression levels of candidate genes. These results contribute to a theoretical framework for molecular breeding strategies targeting bract color traits in Bougainvillea.

2. Materials and Methods

2.1. Identification of C2H2-ZFP Gene Family in Bougainvillea

In order to systematically identify the C2H2-type ZFP genes in Bougainvillea, two complementary approaches were employed. First, the ZFP protein sequences from Arabidopsis thaliana were retrieved from the TAIR database (https://www.arabidopsis.org/index.jsp, assessed on 19 July 2024) [16]. These sequences were used as queries for local Blastp searches against the Bougainvillea genome database hosted by the China National GeneBank (https://www.cngb.org/, accessed on 30 July 2024) (project number: CNP0004115) [35], utilizing TBtools software (v2.310, accessed on 30 July 2024) [36]. Second, the Hidden Markov Model (HMM) profile corresponding to C2H2-ZFP proteins (Pfam ID: PF00096) was retrieved from the UniProt database (https://www.uniprot.org/, assessed on 19 July 2024). This HMM profile was then used as a query to screen the Bougainvillea protein database with a stringent cutoff e-value 1 × 10−10 [18], also performed using TBtools. The overlapping results from the Blastp and HMM analyses were identified. Subsequently, these intersecting sequences were submitted to the PfamScan platform (https://www.ebi.ac.uk/Tools/pfa/pfamscan/, assessed on 20 July 2024) for further domain verification, ultimately yielding the final set of Bougainvillea C2H2-ZFP gene family sequences.
The protein sequences of C2H2-ZFP members from Bougainvillea were submitted to the ExPAsy (https://web.expasy.org/protparam/, assessed on 20 October 2024) [37] and Cell-PLoc 2.0 [38] (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/, assessed on 20 October 2024) platforms to obtain predicted molecular weights, isoelectric points, and subcellular localization data for these C2H2-ZFPs.

2.2. Phylogenetic Analysis and Sequence Alignment of C2H2-ZFPs

The phylogenetic tree of C2H2-ZFP proteins was constructed in MEGA11.0 [39] using the maximum likelihood method. The analysis was performed with default parameters, including the Poisson correction model, pairwise deletion of gaps, and 1000 bootstrap replications. The classification of the ZFP gene family in Bougainvillea was determined based on previously published studies concerning the ZFP gene family in cucumber [18].

2.3. Conserved Domains, Gene Structure, and Cis-Acting Elements Analysis of C2H2-ZFPs

The C2H2-ZFP protein sequences of Bougainvillea were submitted to the MEME platform (http://meme-suite.org/, assessed on 6 August 2024) [40], with the number of motifs specified as 20. The resulting MEME output files were retrieved and visualized using TBtools. Motif comparison results were subsequently uploaded to the WebLogo platform (https://weblogo.berkeley.edu/logo.cgi, assessed on 14 December 2024) for visualization [41]. The CDS sequences and gene structure annotation data for Bougainvillea C2H2-ZFPs were acquired via TBtools, and gene structure visualization was subsequently carried out.
The 2000 bp sequences located upstream of the ATG start codon of C2H2-ZFP genes were extracted using TBtools and designated as promoter sequences. These putative promoter regions were subsequently submitted to the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, assessed on 29 September 2024) online tool [42] for the identification of cis-acting elements.

2.4. Analysis of C2H2-ZFP Gene Expression in Bougainvillea Bracts of Different Colors Using RNA Sequencing (RNA-Seq) Data

The sampled materials included the white variety Bougainvillea spectabilis ‘White Stripe’ (Bou_W), the yellow variety Bougainvillea × buttiana ‘Golden Glow’ (Bou_Y), the red variety Bougainvillea × buttiana ‘Rainbow Orange’ (Bou_R1), the purple varieties Bougainvillea spectabilis ‘Brasiliensis’ (Bou_P1) and Bougainvillea glabra ‘Elizabeth Angus’ (Bou_P2), and canary yellow (Bou_CY) and red (Bou_R2) bracts of Bougainvillea × buttiana ‘Chitra’. The bracts at the flowering stage were collected from three-year-old plant varieties and subjected to transcriptome analysis conducted by Wuhan Metware Biotechnology Co., Ltd. (China). Each sample consisted of three biological replicates, with material pooled from at least five individual plants to ensure representativeness.
Transcriptome data from bracts of Bougainvillea exhibiting various colors were obtained from the SRA database (https://www.ncbi.nlm.nih.gov/sra, assessed on 25 March 2025) (SRP570855). Gene expression levels were calculated and normalized to TPM (transcripts per million) values, as described by previous studies [43]. Differential expression was analyzed using DESeq2, with a threshold of FDR < 0.05 and |log2FC| > 1. The Benjamini–Hochberg procedure was applied to correct for multiple testing. The expression data of C2H2-ZFP genes were extracted using their corresponding gene IDs from the normalized TPM values. GO enrichment of the screened genes was conducted using the Metware Cloud, a freely accessible online platform for data analysis (https://cloud.metware.cn, assessed on 20 May 2025). A heatmap representing the differential expression patterns of C2H2-ZFP genes was generated via TBtools. Gene expression clustering across bract samples was subsequently performed using the K-means analysis function in R software (version stats 4.2.0) with default parameters, as available on the Metware Cloud Platform (https://cloud.metware.cn, assessed on 25 March 2025) [44].

2.5. RT-qPCR Validation

RT-qPCR was employed to validate the expression levels of the screened genes. RNA extraction and cDNA synthesis were conducted following the methodology reported in previous research by our laboratory [32]. Reactions were carried out on a QuantStudio 1 Real-Time PCR system (RoChe, Basel, Switzerland) with the ChamQTM Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The 18S gene was selected as the internal reference gene [45], and data analysis was performed using the 2−ΔΔCT method [46]. Three biological replicates were established for the experiment. Primers were designed using Primer 5.0 software, and the corresponding sequences are provided in Table S1. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China).

2.6. Statistical Analysis

Data were analyzed using Excel and visualized using Origin 2024 software (version: OriginPro 10.1.0.178). The results were expressed as the mean ± standard error (SE) of three replicates, and statistical differences were assessed using Student’s t-test. Asterisks (* or **) denote significant differences at p < 0.05 or 0.01, respectively.

3. Results

3.1. Identification and Physicochemical Properties Analysis of C2H2-ZFP Genes in Bougainvillea

Based on the identification of conserved C2H2-ZFP domains and subsequent validation through HMMER and PfamScan analyses, a total of 105 non-redundant C2H2-ZFP genes were identified in the Bougainvillea genome (Table S2). Physicochemical property analysis indicated that BbZFP members comprise between 137 and 1544 amino acids, with molecular weights ranging from 15.55 to 175.39 kDa, isoelectric points spanning from 5.11 to 9.66, and instability indices between 26.44 and 74.25. Among these, 12 proteins exhibited instability indices below 40, thereby classifying them as stable. The grand average of hydropathicity (GRAVY) values for all family members were less than 0, suggesting that the entire gene family encodes hydrophilic proteins. Subcellular localization predictions revealed that Bou_119181 was localized to both the endoplasmic reticulum and nucleus and Bou_93983 was localized to the Golgi apparatus and nucleus, whereas all other members were exclusively localized to the nucleus (Table S2).

3.2. Phylogenetic Analysis of BbZFP Proteins

Based on the phylogenetic relationships of C2H2-ZFP proteins in other plant species [18], the 105 BbZFPs were classified into six subgroups according to their distinct domain compositions: Bb1Mix, Bb2Q, Bb2Mix, Bb3MZD, Bb3Mix, Bb4Mix (Figure 1A, Tables S2 and S3). The results showed that the Bb1Mix group comprised 23 members, each containing a single ZFP domain, among which 21 were identified as Q-type ZFPs and 2 as C-type ZFPs. The Bb2Q group included 23 members, all of which harbored two Q-type ZFP domains. Bb2Mix was characterized as a mixed group containing two ZFP domains, with 12 members assigned to this subgroup. All members of the Bb3MZD group possessed one M1-type zinc finger domain, one Z1-type zinc finger domain, and one D-type domain, comprising a total of 25 BbZFPs. Bb3Mix consisted of 14 members, each with three zinc finger domains. The Bb4Mix subgroup contained four zinc finger domains and included eight BbZFPs (Table S3).

3.3. Analysis of Conserved Motifs of BbZFPs

The analysis results indicated that most BbZFP proteins possess either motif1 or motif3, while the Bb2Q, Bb2Mix, Bb3Mix, and Bb4Mix groups contain multiple motif types—such as motif1, motif3, motif6, and motif8—or repeated instances of motif1 (Figure 1B). Members of the Bb1Mix group exclusively contain motif1, without the presence of motif3, whereas all Bb2Q members harbor motif1, motif3, motif6, and motif10 in a defined arrangement. Bb3MZD group members consistently possess motif1, motif2, motif4, and motif5 arranged in a specific sequence, while the remaining Mix groups exhibit greater variability in motif organization. Both motif1 and motif3 include the plant ZFP domain–conserved sequence ‘QALGGH’ (Figure S1A,B), which demonstrates a high level of conservation. Motif8 and motif16 also align with the structural features of ZFP domains. Notably, motif8 contains the conserved ‘X2-L-X-GH’ sequence characteristic of M7-type zinc finger domains (Figure S1C). Furthermore, motif2, motif14, motif15, motif16, and motif19 display strong evolutionary conservation. It was further observed that genes within the Bb1Mix, Bb2Q, and Bb3MZD groups tend to cluster together, suggesting possible functional similarities among BbZFP proteins in these subgroups.

3.4. Gene Structures Analysis of BbZFPs

Exon–intron structure analysis revealed that the number of introns in BbZFP genes ranged from 0 to 11, with 97 genes containing between 0 and 4 introns (Figure 1C). Within the Bb1Mix group, all members lacked introns except BbZFP83 and BbZFP84, which possessed relatively high intron counts. Most Bb3MZD group members contained between two and five introns, whereas all Bb2Q members were characterized by the complete absence of introns (Figure 1C). BbZFP genes with close phylogenetic relationships exhibited high levels of similarity in both intron number and structural arrangement.

3.5. Cis-Acting Elements in the BbZFP Gene Promoter

The 2000 bp sequence upstream of the ATG codon of BbZFP genes was analyzed as the promoter region to identify cis-acting elements. These elements were categorized into four functional classes: light-responsive elements, hormone-responsive elements, stress-responsive elements, and elements associated with growth and development (Figure 2, Table S4). The hormone-responsive group encompassed five types of elements responsive to auxin, gibberellin, abscisic acid, salicylic acid, and methyl jasmonate. Stress-responsive elements included those associated with anaerobic conditions, low temperatures, drought, defense mechanisms, and general stress stimuli. All BbZFP gene promoters contained at least one hormone-responsive or stress-responsive element, and 92 promoters harbored both (Tables S4 and S5), suggesting the potential involvement of BbZFP genes in hormonal and stress response pathways. Light-responsive elements were identified in the promoter regions of all BbZFP genes (Tables S4 and S6), implying their participation in light-mediated regulatory processes in Bougainvillea. Additionally, elements associated with growth and development were present in nearly all BbZFP promoter sequences (Tables S5 and S6), indicating that the BbZFP gene family may also play roles in broader developmental regulation in Bougainvillea.

3.6. Expression Analysis of BbZFP Genes in Bracts of Different Colors

To investigate the potential involvement of BbZFP genes in bract color formation in Bougainvillea, RNA-seq data were analyzed to examine BbZFP gene expression patterns across varieties with distinct bract colors. Based on the transcriptome data analysis, a total of 61 differentially expressed genes (DEGs) were identified (Tables S7 and S8). GO enrichment analysis demonstrated that all enriched GO terms were classified into three primary categories: biological process (BP), cellular component (CC), and molecular function (MF). Specifically, these categories included fifteen, two, and two significantly enriched terms, respectively (Figure S2, Table S9). Notably, the majority of DEGs were enriched in ‘Cellular process’ and ‘Metabolic process’ under BP, as well as ‘Transcription regulator activity’ under MF.
Among all the DEGs, 27 BbZFP genes were identified as exhibiting significant expression differences between red-purple bract varieties (‘Rainbow Orange’: Bou_R1, and ‘Chitra’: Bou_R2) and purple bract varieties (‘Brasiliensis’: Bou_P1, and ‘Elizabeth Angus’: Bou_P2) in comparison to the white bract variety (‘White Stripe’: Bou_W) (Figure 3A, Table S7). Trend analysis indicated that all DEGs could be clustered into 10 groups according to their expression dynamics. Groups 1 and 3 were found to be upregulated in both red and purple bracts across the examined varieties. Groups 2 and 8 were downregulated in red bracts but demonstrated an initial increase followed by a decrease in expression in purple bracts. Groups 4 and 10 showed consistent downregulation in all color varieties. In contrast, Groups 5, 6, and 7 exhibited marked upregulation in red bracts but only modest or variable expression changes in purple bracts (Figure 3B).
Additionally, 29 BbZFP genes were identified from the transcriptome dataset as exhibiting markedly different expression levels among yellow bract varieties (‘Golden Glow’: Bou_Y), canary yellow bract varieties (‘Chitra’: Bou_CY), and white bract varieties (‘White Stripe’: Bou_W) (Figure 4A, Table S8). Trend analysis classified these DEGs into seven distinct groups according to their expression profiles. Groups 1, 5, and 6 displayed an initial increase in expression followed by a subsequent decrease, whereas Group 4 exhibited an initial decline followed by upregulation. Groups 3 and 7 showed consistent upregulation, while Group 2 demonstrated stable downregulation across all varieties (Figure 4B).

3.7. Validation of Differentially Expressed Genes by RT-qPCR

Based on the results of the heatmap and trend analyses (Figure 3 and Figure 4), genes from Subclasses 6, 7, 8, and 9 in red-purple materials and Subclasses 3 and 7 in yellow materials were selected for RT-qPCR–based expression validation. The results revealed that the RT-qPCR expression patterns were consistent with those obtained from RNA-seq analysis (Figure 5). In red-purple materials, Bou_1096 and Bou_17631 showed significant upregulation (Figure 5A), whereas in yellow bract samples, the expression levels of Bou_68928, Bou_1096, Bou_4400, and Bou_17631 were markedly upregulated compared to those in white bracts (Figure 5B).

4. Discussion

Through the identification of the C2H2-type ZFP family in Bougainvillea, a total of 105 ZFP family genes were detected, a number substantially lower than that reported in A. thaliana (176) [16], rice (189) [17], and maize (211) [47], indicating considerable interspecies variation in the number of C2H2-type ZFP family members. Subcellular localization prediction analysis demonstrated that all C2H2-type ZFPs in Bougainvillea are localized to the nucleus (Table S2), thereby reinforcing their role as transcription factors. Notably, Bou_119181 was additionally identified as being localized to the endoplasmic reticulum, while Bou_93983 was also detected in the Golgi apparatus. In sweet potato, IbZFP17 is localized in the cytoplasm, whereas IbZFP114 is targeted to the chloroplast [48]. Among the 386 ZFPs identified in cotton, 35 members are localized in various organelles, including vacuole, mitochondria, and chloroplast [49]. In citrus, 33.7% of CsZFP proteins are distributed across organelles such as peroxisomes, cytoplasm, and chloroplast [50]. While in apple, a subset of ZFPs is also found in organelles such as the cytoplasm and mitochondria [51]. These findings suggest that ZFPs may play diverse roles in plant growth and development. Gene structure analysis and physicochemical characterization of the BbZFP family were conducted to elucidate the structural and functional diversity of its members. The findings revealed that the BbZFP proteins comprise both acidic and basic types, all of which are hydrophilic. The majority were classified as unstable proteins (instability index value > 40), while only 12 were identified as stable (instability index value < 40) (Table S2). Gene structure analysis revealed limited variation in intron numbers, with most BbZFP genes lacking introns, except for those in the Bb3MZD group, which contained between two and four introns. These results suggest that the BbZFP gene has been conserved throughout the course of evolution.
Conserved motif analysis revealed that all BbZFP proteins contained both motif1 and motif3. These motifs shared the conserved sequence ‘QALGGH’, which is characteristic of plant Q-type C2H2 ZFPs [52]. Mutations within the “QALGGH” sequence have been shown to substantially impair the DNA-binding activity of ZFPs [53]. Alterations in the core sequence of the Q-type domain can give rise to modified Q-type (M-type) ZFPs, which are classified into 13 distinct subtypes [18,54]. Previous studies have demonstrated that Q-type C2H2 ZFPs are broadly involved in plant developmental processes and also serve pivotal functions in plant responses to abiotic stress [55]. Englbrecht et al. [16] categorized the ZFP family in A. thaliana into three groups—A, B, and C—based on the number and spacing of zinc finger domains. In cucumber, Yin et al. [18] proposed a classification system based on domain types, dividing C2H2 ZFPs into five major categories: Q, M, Z, C, and D. In the present study, the BbZFP family was divided into six subfamilies (Table S3). The results indicated that 69 BbZFPs possess one or two Q-type ZFP domains. However, certain members lacked this canonical sequence. For example, members of the Bb3MZD group each contained one M1-type zinc finger domain, one Z1-type domain, and one D-type domain. The sequences within this group were relatively conserved and were all derived from the IDD family, which encodes transcription factors featuring C2H2 ZFP domains [56]. These proteins were accordingly classified into a distinct subgroup, separate from other C2H2-type ZFPs. Previous studies have shown that IDD family members are implicated in a range of biological functions, including morphogenesis, carbohydrate metabolism, cell patterning, hormone signaling, and cell growth, division, and differentiation [57,58,59]. The Bb1Mix, Bb2Mix, and Bb4Mix groups also contained members lacking Q-type zinc finger domains. ZFPs that do not carry the ‘QALGGH’ motif have also been found to be essential for plant development [60]. Collectively, these findings suggest that various ZFP subfamilies contribute to the functional diversity underlying plant growth and development.
Flavonoids are naturally occurring polyphenols in plants and can be categorized into anthocyanins and flavonols based on their structural characteristics [61,62]. Research conducted by Park et al. [63] demonstrated that elevated flavonol content can influence flower pigmentation and root development in tobacco, with higher concentrations resulting in a shift in flower color toward white. The alterations in gene expression levels within the flavonol synthesis pathway, along with the accumulation of flavonols, account for the yellow coloration observed in the cultivar ‘Chitra’ of Bougainvillea [32]. Studies by Koes et al. [34] have confirmed that the C2H2-ZFP protein TT1, a key transcription factor, is involved in the flavonoid biosynthetic pathway, where it interacts with R2R3-MYB and regulates the expression of essential structural genes. Zhao et al. [64] reported that genes associated with the phenylpropanoid and flavonoid biosynthesis pathways are commonly involved in seed coat color formation in Brassica species and identified Bra023223, a gene specifically expressed and encoding the important C2H2-type ZFP TT1 implicated in flavonoid biosynthesis. Soybean GmZFP7 enhances the accumulation of isoflavones by modulating the expression of key synthase genes, GmIFS2 and GmF3H1, in the phenylpropanoid pathway [65]. In golden buckwheat, the FdC2H2-2 gene is induced by jasmonic acid and promotes rutin synthesis and accumulation in vivo by activating the expression of key enzyme genes (FLS, PAL, and F3′5′H) in the rutin biosynthesis pathway [66]. Our results demonstrate that 19 BbZFP genes were upregulated in the yellow-orange group, with notable upregulation trends observed for Bou_68928, Bou_1096, Bou_4400, and Bou_17631 (Figure 5B). These findings suggest that these genes may play a regulatory role in flavonol biosynthesis during the development of yellow bracts in Bougainvillea.
Betalains are exclusively found in plant species belonging to the order Caryophyllales and are classified into two categories: betacyanins and betaxanthins. Current research indicates that betalains and anthocyanins do not coexist within plant tissues [4,67]. Previous studies have suggested that varying compositions of betacyanins and betaxanthins in petals can produce floral coloration ranging from orange to red and other related hues [7,8]. The biosynthesis of betalain pigments in plants utilizes tyrosine as a substrate. Tyrosine is hydroxylated by cytochrome P450 (CYP) to form L-3,4-dihydroxyphenylalanine (L-DOPA). Subsequently, L-DOPA can be catalyzed by L-DOPA oxidase to generate cyclo-DOPA or by 4,5-dihydroxyphenylalanine dioxygenase (DODA) to form betalamic acid [68,69]. Betalamic acid spontaneously combines with amino acids or amines to produce betaxanthin [70]. The synthesis of betacyanin typically follows two pathways: one pathway involves the spontaneous reaction of betalamic acid with cyclo-DOPA to form betanidin, which is then glycosylated by betanidin 5-O-glucosyltransferase (5GT) or betanidin 6-O-glucosyltransferase (6GT) to yield betacyanin; the other pathway involves the conversion of cyclo-DOPA to cDOPA 5-O-glucoside by cDOPA 5-O-glucosyltransferase (cDOPA5GT), followed by its spontaneous reaction with betanidin to form betacyanin [71]. Studies have demonstrated that the synthesis of betacyanin is regulated by transcription factors. For instance, the R2R3-type MYB gene BvMYB1 in sugar beet binds to the promoter of the structural gene BvDODA in the betalain synthesis pathway, upregulating the expression of BvDODA. Overexpression of BvMYB1 in white sugar beet roots regulates betacyanin synthesis, resulting in red root coloration [72]. In pitaya, the HuMYB132 transcription factor positively regulates betalain synthesis by binding to the promoters of HuCYP76AD1-1 and HuDODA1. Silencing HuMYB132 inhibits both the accumulation of betalain and the expression levels of betalain biosynthesis genes in pitaya [73]. Additionally, an RNA-seq study identified a WRKY transcription factor, HpWRKY44, in Hylocereus polyrhizus that binds to the W-box motif in the promoter of the CYP76AD1 gene and may play a role in regulating betalain synthesis [74]. Unlike the transcriptional regulation mechanism of anthocyanins, MYB transcription factors do not form MBW complexes with bHLH and WD40 to regulate the betalain synthesis pathway [72]. However, MYB may form more complex regulatory complexes with other transcription factors to participate in the biosynthesis of betacyanin [75]. In this study, we observed that the expression levels of Bou_1096 and Bou_17631 were significantly higher in red bracts and purple bracts compared to white bracts (Figure 5B). Notably, the expression level in purple bracts was further upregulated relative to that in red bracts. Based on these findings, we hypothesize that these genes may play a role in regulating betacyanin synthesis in Bougainvillea. Analysis of cis-acting elements indicated that the promoters of both Bou_1096 and Bou_17631 genes harbor multiple MYB binding sites (Table S4), implying a potential cooperation with MYB transcription factors in betacyanin synthesis. However, further experimental validation is required to confirm this hypothesis.

5. Conclusions

In this study, a total of 105 BbZFP genes were identified in the genome of Bougainvillea. Their physicochemical properties and genetic characteristics were systematically analyzed. Based on the number of their domains, these genes were classified into six subgroups. Additionally, RNA-seq and RT-qPCR analyses were employed to investigate the expression patterns of BbZFP genes in Bougainvillea bracts of different colors. The results suggest that Bou_68928, Bou_1096, Bou_4400, and Bou_17631 may play roles in regulating flavonoid synthesis, thereby influencing bract yellowing. Furthermore, Bou_1096 and Bou_17631 are likely involved in betacyanin pigment synthesis, affecting the red or purple coloration of Bougainvillea bracts.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11060659/s1, Figure S1: Sequence of motif 1, motif 3, and motif 8; Figure S2: GO enrichment analysis of DEGs; Table S1: Primers used in this study; Table S2: Predicted properties of C2H2-ZFP gene family in Bougainvillea; Table S3: The types and sub-types of C2H2-ZFP domains and their characteristics; Table S4: cis-acting elements in the BbZFP gene promoters; Table S5: Information of cis-acting elements; Table S6: Analysis of cis-elements in the BbZFP genes promoter regions; Table S7: Expression of BbZFP genes in Bougainvillea red bracts (TPM); Table S8: Expression of BbZFP genes in Bougainvillea yellow bracts (TPM); Table S9: GO enrichment analysis of the differentially expressed genes.

Author Contributions

Conceptualization, Y.Z.; formal analysis, Y.W., Y.H., W.L., W.Y., J.W. and Y.Z.; funding acquisition, Y.Z.; investigation, Y.W. and Y.H.; methodology, Y.W. and Y.Z.; supervision, Y.Z.; writing-original draft preparation, Y.W., Y.H. and Y.Z.; writing-review and editing, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Hainan Province Science and Technology Special Fund (ZDYF2024XDNY212).

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Yu, X.; Hu, S.; He, C.; Zhou, J.; Qu, F.; Ai, Z.; Chen, Y.; Ni, D. Chlorophyll metabolism in postharvest tea (Camellia sinensis L.) leaves: Variations in color values, chlorophyll derivatives, and gene expression levels under different withering treatments. J. Agric. Food Chem. 2019, 67, 10624–10636. [Google Scholar] [CrossRef] [PubMed]
  2. Li, X.; Zheng, M.; Gan, Q.; Long, J.; Fan, H.; Wang, X.; Guan, Z. The formation and evolution of flower coloration in Brassica crops. Front. Gene. 2024, 15, 1396875. [Google Scholar] [CrossRef] [PubMed]
  3. Zhao, X.; Zhang, Y.; Long, T.; Wang, S.; Yang, J. Regulation mechanism of plant pigments biosynthesis: Anthocyanins, carotenoids, and betalains. Metabolites 2022, 12, 871. [Google Scholar] [CrossRef] [PubMed]
  4. Gandía-Herrero, F.; García-Carmona, F. Biosynthesis of betalains: Yellow and violet plant pigments. Trends Plant Sci. 2013, 18, 334–343. [Google Scholar] [CrossRef]
  5. Polturak, G.; Aharoni, A. “La Vie en Rose”: Biosynthesis, sources, and applications of betalain pigments. Mol. Plant 2018, 11, 7–22. [Google Scholar] [CrossRef]
  6. Brockington, S.F.; Walker, R.H.; Glover, B.J.; Soltis, P.S.; Soltis, D.E. Complex pigment evolution in the Caryophyllales. New Phytol. 2011, 190, 854–864. [Google Scholar] [CrossRef]
  7. Gandía-Herrero, F.; García-Carmona, F.; Escribano, J. Botany: Floral fluorescence effect. Nature 2005, 437, 334. [Google Scholar] [CrossRef]
  8. Felker, P.; Stintzing, F.C.; Müssig, E.; Leitenberger, M.; Carle, R.; Vogt, T.; Bunch, R. Colour inheritance in cactus pear (Opuntia ficus-indica) fruits. Ann. Appl. Biol. 2008, 152, 307–318. [Google Scholar] [CrossRef]
  9. Takatsuji, H. Zinc-finger proteins: The classical zinc finger emerges in contemporary plant science. Plant Mol. Biol. 1999, 39, 1073–1078. [Google Scholar] [CrossRef]
  10. Laity, J.H.; Lee, B.M.; Wright, P.E. Zinc finger proteins: New insights into structural and functional diversity. Curr. Opin. Struct. Biol. 2001, 11, 39–46. [Google Scholar] [CrossRef]
  11. Han, G.; Lu, C.; Guo, J.; Qiao, Z.; Sui, N.; Qiu, N.; Wang, B. C2H2 zinc finger proteins: Master regulators of abiotic stress responses in plants. Front. Plant Sci. 2020, 11, 298. [Google Scholar] [CrossRef] [PubMed]
  12. Kielbowicz-Matuk, A. Involvement of plant C2H2-type zinc finger transcription factors in stress responses. Plant Sci. 2012, 185–186, 78–85. [Google Scholar] [CrossRef] [PubMed]
  13. Lyu, T.; Cao, J. Cys2/His2 zinc-finger proteins in transcriptional regulation of flower development. Int. J. Mol. Sci. 2018, 19, 2589. [Google Scholar] [CrossRef]
  14. Takatsuji, H.; Mori, M.; Benfey, P.N.; Ren, L.; Chua, N.H. Characterization of a zinc finger DNA-binding protein expressed specifically in Petunia petals and seedlings. EMBO J. 1992, 11, 241–249. [Google Scholar] [CrossRef] [PubMed]
  15. Gamsjaeger, R.; Liew, C.K.; Loughlin, F.E.; Crossley, M.; Mackay, J.P. Sticky Fingers: Zinc-fingers as protein-recognition motifs. Trends Biochem. Sci. 2007, 32, 63–70. [Google Scholar] [CrossRef]
  16. Englbrecht, C.C.; Schoof, H.; Böhm, S. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome. BMC Genom. 2004, 5, 39. [Google Scholar] [CrossRef]
  17. Agarwal, P.; Arora, R.; Ray, S.; Singh, A.K.; Singh, V.P.; Takatsuji, H.; Kapoor, S.; Tyagi, A.K. Genome-wide identification of C2H2 zinc-finger gene family in rice and their phylogeny and expression analysis. Plant Mol. Biol. 2007, 65, 467–485. [Google Scholar] [CrossRef]
  18. Yin, J.; Wang, L.; Zhao, J.; Li, Y.; Huang, R.; Jiang, X.; Zhou, X.; Zhu, X.; He, Y.; He, Y.; et al. Genome-wide characterization of the C2H2 zinc-finger genes in Cucumis sativus and functional analyses of four CsZFPs in response to stresses. BMC Plant Biol. 2020, 20, 359. [Google Scholar] [CrossRef]
  19. Liu, Q.; Wang, Z.; Xu, X.; Zhang, H.; Li, C. Genome-wide analysis of C2H2 zinc-finger family transcription factors and their responses to abiotic stresses in poplar (Populus trichocarpa). PLoS ONE 2015, 10, e0134753. [Google Scholar] [CrossRef]
  20. Yuan, S.; Li, X.; Li, R.; Wang, L.; Zhang, C.; Chen, L.; Hao, Q.; Zhang, X.; Chen, H.; Shan, Z.; et al. Genome-wide identification and classification of soybean C2H2 zinc finger proteins and their expression analysis in legume-Rhizobium symbiosis. Front. Microbiol. 2018, 9, 126. [Google Scholar] [CrossRef]
  21. Lu, Y.; Wang, K.; Ngea, G.L.N.; Godana, E.A.; Ackah, M.; Dhanasekaran, S.; Zhang, Y.; Su, Y.; Yang, Q.; Zhang, H. Recent advances in the multifaceted functions of Cys2/His2-type zinc finger proteins in plant growth, development, and stress responses. J. Exp. Bot. 2024, 75, 5501–5520. [Google Scholar] [CrossRef] [PubMed]
  22. Wang, J.Y.; Wang, J.P.; Yang, H.F. Identification and functional characterization of the NAC gene promoter from Populus euphratica. Planta 2016, 244, 417–427. [Google Scholar] [CrossRef] [PubMed]
  23. Han, G.; Li, Y.; Qiao, Z.; Wang, C.; Zhao, Y.; Guo, J.; Chen, M.; Wang, B. Advances in the regulation of epidermal cell development by C2H2 zinc finger proteins in plants. Front. Plant Sci. 2021, 12, 754512. [Google Scholar] [CrossRef] [PubMed]
  24. Klupczyńska, E.A.; Pawłowski, T.A. Regulation of seed dormancy and germination mechanisms in a changing environment. Int. J. Mol. Sci. 2021, 22, 1357. [Google Scholar] [CrossRef]
  25. Liu, Y.; Khan, A.R.; Gan, Y. C2H2 zinc finger proteins response to abiotic stress in plants. Int. J. Mol. Sci. 2022, 23, 2730. [Google Scholar] [CrossRef]
  26. Xu, W.; Zhu, W.; Yang, L.; Liang, W.; Li, H.; Yang, L.; Chen, M.; Luo, Z.; Huang, G.; Duan, L.; et al. SMALL REPRODUCTIVE ORGANS, a SUPERMAN-like transcription factor, regulates stamen and pistil growth in rice. New Phytol. 2022, 233, 1701–1718. [Google Scholar] [CrossRef]
  27. Wang, D.-R.; Yang, K.; Wang, X.; Lin, X.-L.; Rui, L.; Liu, H.-F.; Liu, D.-D.; You, C.-X. Overexpression of MdZAT5, an C2H2-type zinc finger protein, regulates anthocyanin accumulation and salt stress response in apple calli and Arabidopsis. Int. J. Mol. Sci. 2022, 23, 1897. [Google Scholar] [CrossRef]
  28. Sun, Q.; He, Z.; Ye, J.; Wei, R.; Feng, D.; Zhang, Y.; Chai, L.; Cheng, Y.; Xu, Q.; Deng, X. A novel C2H2-type zinc-finger transcription factor, CitZAT4, regulates ethylene-induced orange coloration in Satsuma mandarin flavedo (Citrus unshiu Marc.). J. Int. Plant Biol. 2024, 67, 294–310. [Google Scholar] [CrossRef]
  29. Saleem, H.; Usman, A.; Mahomoodally, M.F.; Ahemad, N. Bougainvillea glabra (choisy): A comprehensive review on botany, traditional uses, phytochemistry, pharmacology and toxicity. J. Ethnopharmacol. 2021, 266, 113356. [Google Scholar] [CrossRef]
  30. Wang, F.; Yao, G.; Li, J.; Zhu, W.; Li, Z.; Sun, Z.; Xin, P. Mining and expression analysis of color related genes in Bougainvillea glabra bracts based on transcriptome sequencing. Sci. Rep. 2024, 14, 24491. [Google Scholar] [CrossRef]
  31. Tanaka, Y.; Sasaki, N.; Ohmiya, A. Biosynthesis of plant pigments: Anthocyanins, betalains and carotenoids. Plant J. 2008, 54, 733–749. [Google Scholar] [CrossRef] [PubMed]
  32. Kang, Y.; Li, Y.; Zhang, T.; Wang, P.; Liu, W.; Zhang, Z.; Yu, W.; Wang, J.; Wang, J.; Zhou, Y. Integrated metabolome, full-length sequencing, and transcriptome analyses unveil the molecular mechanisms of color formation of the canary yellow and red bracts of Bougainvillea × buttiana ‘Chitra’. Plant J. 2023, 116, 1441–1461. [Google Scholar] [CrossRef] [PubMed]
  33. Wu, Q.; Fu, X.; Chen, Z.; Wang, H.; Wang, J.; Zhu, Z.; Zhu, G. Composition, color stability and antioxidant properties of betalain-based extracts from bracts of Bougainvillea. Molecules 2022, 27, 5120. [Google Scholar] [CrossRef] [PubMed]
  34. Koes, R.; Verweij, W.; Quattrocchio, F. Flavonoids: A colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci. 2005, 10, 236–242. [Google Scholar] [CrossRef]
  35. Lan, L.; Zhao, H.; Xu, S.; Kan, S.; Zhang, X.; Liu, W.; Liao, X.; Tembrock, L.R.; Ren, Y.; Reeve, W.; et al. A high-quality Bougainvillea genome provides new insights into evolutionary history and pigment biosynthetic pathways in the Caryophyllales. Hortic. Res. 2023, 10, uhad124. [Google Scholar] [CrossRef]
  36. 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]
  37. Gasteiger, E.; Hoogland, C.; Gattiker, A.; Duvaud, S.; Wilkins, M.R.; Appel, R.D.; Bairoch, A. Protein identification and analysis tools on the Expasy Server. In The Proteomics Protocols Handbook; Walker, J.M., Ed.; Humana Press: Totowa, NJ, USA, 2005; pp. 571–607. [Google Scholar]
  38. Chou, K.C.; Hong-Bin Shen, H.B. Cell-PLoc 2.0: An improved package of web-servers for predicting subcellular localization of proteins in various organisms. Nat. Sci. 2010, 2, 1090–1103. [Google Scholar] [CrossRef]
  39. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef]
  40. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, W202–W208. [Google Scholar] [CrossRef]
  41. Crooks, G.E.; Hon, G.; Chandonia, J.M.; Brenner, S.E. WebLogo: A sequence logo generator. Genome Res. 2004, 14, 1188–1190. [Google Scholar] [CrossRef]
  42. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef] [PubMed]
  43. Corchete, L.A.; Rojas, E.A.; Alonso-Lopez, D.; Rivas, J.D.L.; Gutierrez, N.C.; Burguillo, F.J. Systematic comparison and assessment of RNA-seq procedures for gene expression quantitative analysis. Sci. Rep. 2020, 10, 19737. [Google Scholar] [CrossRef] [PubMed]
  44. Yang, C.; Shen, S.; Zhou, S.; Li, Y.; Mao, Y.; Zhou, J.; Shi, Y.; An, L.; Zhou, Q.; Peng, W.; et al. Rice metabolic regulatory network spanning the entire life cycle. Mol. Plant 2022, 15, 258–275. [Google Scholar] [CrossRef] [PubMed]
  45. Sun, R.; Liu, S.; Gao, J. Cloning and expression analysis of flavanone 3-hydroxylase gene from Bougainvillea spectabilis. Biotechnol. Bull. 2022, 38, 122–128. (In Chinese) [Google Scholar]
  46. Cao, Y.; Liu, S.; Sun, R.; Chen, H.; Xie, Z.; Huang, L.; Chen, Y. Cloning and expression analysis of 4,5-DOPA dioxygenase gene from Bougainvillea glabra Choisy. Mol. Plant Breed. 2018, 16, 54–60. (In Chinese) [Google Scholar]
  47. Wei, K.; Pan, S.; Li, Y. Functional characterization of maize C2H2 zinc-finger gene family. Plant Mol. Biol. Rep. 2016, 34, 761–776. [Google Scholar] [CrossRef]
  48. Du, T.; Zhou, Y.; Qin, Z.; Li, A.; Wang, Q.; Li, Z.; Hou, F.; Zhang, L. Genome-wide identification of the C2H2 zinc finger gene family and expression analysis under salt stress in sweet potato. Front. Plant Sci. 2023, 14, 1301848. [Google Scholar] [CrossRef]
  49. Salih, H.; Odongo, M.R.; Gong, W.; He, S.; Du, X. Genome-wide analysis of cotton C2H2-zinc finger transcription factor family and their expression analysis during fiber development. BMC Plant Biol. 2019, 19, 400. [Google Scholar] [CrossRef]
  50. Jia, H.H.; Xu, Y.T.; Yin, Z.J.; Qing, M.; Xie, K.D.; Guo, W.W.; Wu, X.M. Genome-wide identification of the C2H2-Zinc finger gene family and functional validation of CsZFP7 in citrus nucellar embryogenesis. Plant Reprod. 2023, 36, 287–300. [Google Scholar] [CrossRef]
  51. Liu, K.; Hou, Q.; Yu, R.; Deng, H.; Shen, L.; Wang, Q.; Wen, X. Genome-wide analysis of C2H2 zinc finger family and their response to abiotic stresses in apple. Gene 2024, 904, 148164. [Google Scholar] [CrossRef]
  52. Liu, Z.; Coulter, J.A.; Li, Y.; Zhang, X.; Meng, J.; Zhang, J.; Liu, Y. Genome-wide identification and analysis of the Q-type C2H2 gene family in potato (Solanum tuberosum L.). Int. J. Biol. Macromol. 2020, 153, 327–340. [Google Scholar] [CrossRef] [PubMed]
  53. Muthamilarasan, M.; Bonthala, V.S.; Mishra, A.K.; Khandelwal, R.; Khan, Y.; Roy, R.; Prasad, M. C2H2 type of zinc finger transcription factors in foxtail millet define response to abiotic stresses. Func. Integr. Genomics 2014, 14, 531–543. [Google Scholar] [CrossRef]
  54. Jiao, Z.; Wang, L.; Du, H.; Wang, Y.; Wang, W.; Liu, J.; Huang, J.; Huang, W.; Ge, L. Genome-wide study of C2H2 zinc finger gene family in Medicago truncatula. BMC Plant Biol. 2020, 20, 401. [Google Scholar] [CrossRef]
  55. Liu, H.; Liu, Y.; Liu, F.; Zeng, L.; Xu, Y.; Jin, Q.; Wang, Y. Genome-wide identification of the Q-type C2H2 zinc finger protein gene family and expression analysis under abiotic stress in lotus (Nelumbo nucifera G.). BMC Genom. 2024, 25, 648. [Google Scholar] [CrossRef]
  56. Colasanti, J.; Tremblay, R.; Wong, A.Y.; Coneva, V.; Kozaki, A.; Mable, B.K. The maize INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein family in higher plants. BMC Genom. 2006, 7, 158. [Google Scholar] [CrossRef]
  57. Kumar, M.; Le, D.T.; Hwang, S.; Seo, P.J.; Kim, H.U. Role of the INDETERMINATE DOMAIN genes in plants. Int. J. Mol. Sci. 2019, 20, 2286. [Google Scholar] [CrossRef]
  58. Coelho, C.P.; Huang, P.; Lee, D.Y.; Brutnell, T.P. Making roots, shoots, and seeds: IDD gene family diversification in plants. Trends Plant Sci. 2018, 23, 66–78. [Google Scholar] [CrossRef]
  59. Wu, X.; Tang, D.; Li, M.; Wang, K.; Cheng, Z. Loose plant architecture1, an INDETERMINATE DOMAIN protein involved in shoot gravitropism, regulates plant architecture in rice. Plant Physiol. 2013, 161, 317–329. [Google Scholar] [CrossRef]
  60. Lyu, T.; Liu, W.; Hu, Z.; Xiang, X.; Liu, T.; Xiong, X.; Cao, J. Molecular characterization and expression analysis reveal the roles of Cys/His zinc-finger transcription factors during flower development of Brassica rapa subsp. chinensis. Plant Mol. Biol. 2020, 102, 123–141. [Google Scholar] [CrossRef]
  61. Liu, Q.; Liaquat, F.; He, Y.; Munis, M.F.H.; Zhang, C. Functional annotation of a full-length transcriptome and identification of genes associated with flower development in Rhododendron simsii (Ericaceae). Plants 2021, 10, 649. [Google Scholar] [CrossRef]
  62. Du, H.; Lai, L.; Wang, F.; Sun, W.; Zhang, L.; Li, X.; Wang, L.; Jiang, L.; Zheng, Y. Characterisation of flower colouration in 30 Rhododendron species via anthocyanin and flavonol identification and quantitative traits. Plant Biol. 2018, 20, 121–129. [Google Scholar] [CrossRef] [PubMed]
  63. Park, S.; Kim, D.H.; Yang, J.H.; Lee, J.Y.; Lim, S.H. Increased flavonol levels in Tobacco expressing AcFLS affect flower color and root growth. Int. J. Mol. Sci. 2020, 21, 1011. [Google Scholar] [CrossRef]
  64. Zhao, H.; Basu, U.; Kebede, B.; Qu, C.; Li, J.; Rahman, H. Fine mapping of the major QTL for seed coat color in Brassica rapa var. Yellow Sarson by use of NIL populations and transcriptome sequencing for identification of the candidate genes. PLoS ONE 2019, 14, e0209982. [Google Scholar] [CrossRef]
  65. Feng, Y.; Zhang, S.; Li, J.; Pei, R.; Tian, L.; Qi, J.; Azam, M.; Agyenim-Boateng, K.G.; Shaibu, A.S.; Liu, Y.; et al. Dual-function C2H2-type zinc-finger transcription factor GmZFP7 contributes to isoflavone accumulation in soybean. New Phytol. 2023, 237, 1794–1809. [Google Scholar] [CrossRef]
  66. Long, O.; Lu, X.; Shi, Y.; Li, G.; Zhang, K.; Wang, J.; Zhou, M. Analysis of C2H2-ZFP family genes and functional characterization of FdC2H2-2 in rutin synthesis and accumulation in golden buckwheat. J. Plant Genet. Res. 2023, 24, 1161–1173. (In Chinese) [Google Scholar]
  67. Zhao, L.; Li, Y.; Li, Y.; Chen, W.; Yao, J.; Fang, S.; Lv, Y.; Zhang, Y.; Zhu, S. Systematical characterization of the cotton Di19 gene family and the role of GhDi19-3 and GhDi19-4 as two negative regulators in response to salt stress. Antioxidants 2022, 11, 2225. [Google Scholar] [CrossRef]
  68. DeLoache, W.C.; Russ, Z.N.; Narcross, L.; Gonzales, A.M.; Martin, V.J.; Dueber, J.E. An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nat. Chem. Biol. 2015, 11, 465–471. [Google Scholar] [CrossRef]
  69. Chang, Y.C.; Chiu, Y.C.; Tsao, N.W.; Chou, Y.L.; Tan, C.M.; Chiang, Y.H.; Liao, P.C.; Lee, Y.C.; Hsieh, L.C.; Wang, S.Y.; et al. Elucidation of the core betalain biosynthesis pathway in Amaranthus tricolor. Sci. Rep. 2021, 11, 6086. [Google Scholar] [CrossRef]
  70. Hou, Y.; Liu, X.; Li, S.; Zhang, X.; Yu, S.; Zhao, G.R. Metabolic engineering of Escherichia coli for de novo production of betaxanthins. J. Agric. Food Chem. 2020, 68, 8370–8380. [Google Scholar] [CrossRef]
  71. Feng, Y.; Yan, X.; Guo, F.; Wang, S.; Liu, Z.; Long, W. Identification, expression analysis of quinoa betalain biosynthesis genes and their role in seed germination and cold stress. Plant Signal. Behav. 2023, 18, 2250891. [Google Scholar] [CrossRef]
  72. Hatlestad, G.J.; Akhavan, N.A.; Sunnadeniya, R.M.; Elam, L.; Cargile, S.; Hembd, A.; Gonzalez, A.; McGrath, J.M.; Lloyd, A.M. The beet Y locus encodes an anthocyanin MYB-like protein that activates the betalain red pigment pathway. Nat. Genet. 2015, 47, 92–96. [Google Scholar] [CrossRef] [PubMed]
  73. Xie, F.; Chen, C.; Chen, J.; Chen, J.; Hua, Q.; Shah, K.; Zhang, Z.; Zhao, J.; Hu, G.; Chen, J.; et al. Betalain biosynthesis in red pulp pitaya is regulated via HuMYB132: A R-R type MYB transcription factor. BMC Plant Biol. 2023, 23, 28. [Google Scholar] [CrossRef] [PubMed]
  74. Cheng, M.N.; Huang, Z.J.; Hua, Q.Z.; Shan, W.; Kuang, J.F.; Lu, W.J.; Qin, Y.H.; Chen, J.Y. The WRKY transcription factor HpWRKY44 regulates CytP450-like1 expression in red pitaya fruit (Hylocereus polyrhizus). Hortic. Res. 2017, 4, 17039. [Google Scholar] [CrossRef]
  75. Timoneda, A.; Feng, T.; Sheehan, H.; Walker-Hale, N.; Pucker, B.; Lopez-Nieves, S.; Guo, R.; Brockington, S. The evolution of betalain biosynthesis in Caryophyllales. New Phytol. 2019, 224, 71–85. [Google Scholar] [CrossRef]
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Figure 1. Phylogenetic relationships, motifs, and gene structures of BbZFP family members. (A) A phylogenetic tree of 105 BbZFP proteins was constructed using the maximum likelihood method. (B) Conserved motifs of BbZFP proteins. Different motifs are represented by various colored boxes. (C) Gene structures of BbZFP genes. Exon(s) and intron(s) are represented by green boxes and black lines, respectively. The phylogenetic tree, conserved motifs, and gene structures were predicted using TBtools.
Figure 1. Phylogenetic relationships, motifs, and gene structures of BbZFP family members. (A) A phylogenetic tree of 105 BbZFP proteins was constructed using the maximum likelihood method. (B) Conserved motifs of BbZFP proteins. Different motifs are represented by various colored boxes. (C) Gene structures of BbZFP genes. Exon(s) and intron(s) are represented by green boxes and black lines, respectively. The phylogenetic tree, conserved motifs, and gene structures were predicted using TBtools.
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Figure 2. The number of BbZFP promoters containing various cis-acting elements. Different colors represent different cis-element types. The numbers above the columns represent the number of promoters.
Figure 2. The number of BbZFP promoters containing various cis-acting elements. Different colors represent different cis-element types. The numbers above the columns represent the number of promoters.
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Figure 3. Expression analysis of BbZFP genes in red-purple bracts based on RNA-seq. (A) Hierarchical clustering of BbZFP genes expression profiles across different colored bracts. The different colors correspond to the log2-transformed fold change with blue and red indicating down- and up-regulation, respectively. (B) K-means analysis of BbZFP genes shown in (A). (C) Materials used in (A). Bou_R1: ‘Rainbow Orange’; Bou_R2: ‘Chitra’ red bract; Bou_P1: ‘Brasiliensis’; Bou_P2: ‘Elizabeth Angus’; Bou_W: ‘White Stripe’.
Figure 3. Expression analysis of BbZFP genes in red-purple bracts based on RNA-seq. (A) Hierarchical clustering of BbZFP genes expression profiles across different colored bracts. The different colors correspond to the log2-transformed fold change with blue and red indicating down- and up-regulation, respectively. (B) K-means analysis of BbZFP genes shown in (A). (C) Materials used in (A). Bou_R1: ‘Rainbow Orange’; Bou_R2: ‘Chitra’ red bract; Bou_P1: ‘Brasiliensis’; Bou_P2: ‘Elizabeth Angus’; Bou_W: ‘White Stripe’.
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Figure 4. Expression analysis of BbZFP genes in yellow bracts based on RNA-seq. (A) Hierarchical clustering of BbZFP genes expression profiles across different colored bracts. The different colors correspond to the log2-transformed fold change with blue and red indicating down- and upregulation, respectively. (B) K-means analysis of BbZFP genes shown in (A). (C) Materials used in (A). Bou_Y: ‘Golden Glow’; Bou_CY: ‘Chitra’canary yellow bract; Bou_W: ‘White Stripe’.
Figure 4. Expression analysis of BbZFP genes in yellow bracts based on RNA-seq. (A) Hierarchical clustering of BbZFP genes expression profiles across different colored bracts. The different colors correspond to the log2-transformed fold change with blue and red indicating down- and upregulation, respectively. (B) K-means analysis of BbZFP genes shown in (A). (C) Materials used in (A). Bou_Y: ‘Golden Glow’; Bou_CY: ‘Chitra’canary yellow bract; Bou_W: ‘White Stripe’.
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Figure 5. RT-qPCR validation of the differentially expressed genes. (A) RT-qPCR validation of the differentially expressed genes in the red-purple group. (B) RT-qPCR validation of the differentially expressed genes in the yellow group. The Y axis on the left represents the values of RT-qPCR, while the Y axis on the right represents the values of RNA-seq. Error bars indicated the standard deviation of three independent replicates. Asterisks (** or ***) indicate a significant difference at p < 0.01 or p < 0.001, respectively. Bou_W: ‘White Stripe’; Bou_Y: ‘Golden Glow’; Bou_CY: ‘Chitra’canary yellow bract; Bou_R2: ‘Chitra’ red bract; Bou_P2: ‘Elizabeth Angus’.
Figure 5. RT-qPCR validation of the differentially expressed genes. (A) RT-qPCR validation of the differentially expressed genes in the red-purple group. (B) RT-qPCR validation of the differentially expressed genes in the yellow group. The Y axis on the left represents the values of RT-qPCR, while the Y axis on the right represents the values of RNA-seq. Error bars indicated the standard deviation of three independent replicates. Asterisks (** or ***) indicate a significant difference at p < 0.01 or p < 0.001, respectively. Bou_W: ‘White Stripe’; Bou_Y: ‘Golden Glow’; Bou_CY: ‘Chitra’canary yellow bract; Bou_R2: ‘Chitra’ red bract; Bou_P2: ‘Elizabeth Angus’.
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Wang, Y.; Hu, Y.; Liu, W.; Yu, W.; Wang, J.; Zhou, Y. Identification and Expression Analysis of C2H2-Type Zinc Finger Protein (C2H2-ZFP) Genes in Bougainvillea in Different Colored Bracts. Horticulturae 2025, 11, 659. https://doi.org/10.3390/horticulturae11060659

AMA Style

Wang Y, Hu Y, Liu W, Yu W, Wang J, Zhou Y. Identification and Expression Analysis of C2H2-Type Zinc Finger Protein (C2H2-ZFP) Genes in Bougainvillea in Different Colored Bracts. Horticulturae. 2025; 11(6):659. https://doi.org/10.3390/horticulturae11060659

Chicago/Turabian Style

Wang, Yushan, Yanping Hu, Wen Liu, Wengang Yu, Jian Wang, and Yang Zhou. 2025. "Identification and Expression Analysis of C2H2-Type Zinc Finger Protein (C2H2-ZFP) Genes in Bougainvillea in Different Colored Bracts" Horticulturae 11, no. 6: 659. https://doi.org/10.3390/horticulturae11060659

APA Style

Wang, Y., Hu, Y., Liu, W., Yu, W., Wang, J., & Zhou, Y. (2025). Identification and Expression Analysis of C2H2-Type Zinc Finger Protein (C2H2-ZFP) Genes in Bougainvillea in Different Colored Bracts. Horticulturae, 11(6), 659. https://doi.org/10.3390/horticulturae11060659

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