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Article

Whole-Genome Analysis of ZF-HD Genes among Three Dendrobium Species and Expression Patterns in Dendrobium chrysotoxum

by
Xin He
1,2,
Xuewei Zhao
1,2,
Qinyao Zheng
2,
Meng-Meng Zhang
1,2,
Ye Huang
2,
Zhong-Jian Liu
1,2,* and
Siren Lan
1,2,*
1
College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization, College of Landscape Architecture and Art, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(6), 610; https://doi.org/10.3390/horticulturae10060610
Submission received: 20 May 2024 / Revised: 4 June 2024 / Accepted: 4 June 2024 / Published: 8 June 2024
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Abstract

:
ZF-HD transcription factors, which are unique to land plants, are involved in the regulation of abiotic stress response and related signaling pathways, and play a crucial role in plant growth and development. Dendrobium is one of the largest genera of orchids, with a high ornamental and ecological value. However, the specific functions of the ZF-HDs in Dendrobium remain unknown. In this study, we identified a total of 53 ZF-HDs from D. chrysotoxum (17), D. catenatum (23), and D. huoshanense (13), and analyzed their physicochemical properties, phylogenetic relationships, chromosomal locations, protein structures, conserved motifs, and expression patterns. The phylogenetic relationships revealed that 53 ZF-HDs were classified into six subfamilies (ZHDI–V and MIF), and all ZF-HD proteins contained motif 1 and motif 4 conserved domains, while a minority of these proteins had exons. The analysis of cis-elements in the promoters of ZF-HDs from three Dendrobium species showed that growth- and development-related elements were the most prevalent, followed by hormone response and abiotic stress response elements. Through collinearity analysis, 14 DchZF-HDs were found to be collinear with DhuZF-HDs, and 12 DchZF-HDs were found to be collinear with DcaZF-HDs. Furthermore, RT-qPCR analysis revealed that DchZF-HDs play a regulatory role in the development of lateral organs during the flowering process. The results indicated that DchZHD2 plays a role in the unpigmented bud stage, while DchMIF8 and DchZHD16 play significant roles during the pigmented bud and initial bloom stages. Hence, this study provides a crucial basis for further exploring ZF-HDs functions in regulating the floral organs of orchids.

1. Introduction

Initially discovered as potential modulators of the C4 phosphoenolpyruvate carboxylase gene, known as PEP-Case, ZF-HD proteins have garnered attention within C4 species [1]. Zinc finger-homeodomain (ZF-HD) transcription factors have two main domains: the C2H2-type ZF domain situated at the N-terminal, and the HD domain located at the C-terminal [2]. According to the different conserved domains, the ZF-HD gene family can be classified into ZHD and MIF subfamilies [3,4], where the MIF subfamily exclusively consists of the ZF domain [5]. ZF-HD transcription factors have a significant impact on regulating seed germination [6], flower development [7], and stress reactions [8].
Recently, bioinformatics has been conducted on the ZF-HD transcription factors of many plants, revealing their expression patterns in various aspects of growth and development, such as Arabidopsis thaliana [2], Oryza sativa [9], Brassica rapa [10], and Vitis vinifera [11]. ZF-HD transcription factors have been shown to be important for the early stages of flower development [12]. The 14 ZF-HDs detected in A. thaliana were all expressed in the flower, and 6 AtZF-HDs did not produce any noticeable floral phenotypes when expressed individually, indicating that AtZF-HDs probably perform convergent functions in controlling the development of flowers, thereby hinting at their potential overlapping roles in floral regulation [13]. CsZF-HD5 is highly expressed in Camellia sinensis flower tissues, suggesting that it plays a key role in flower development [14]. In the presence of abiotic stress, ZF-HD genes play a pivotal regulatory role. Research on the O. sativa ZF-HD gene family has shown that the overexpression of OsZHD1 affects the development of O. sativa leaves [15], and the four OsZHDs bound by OsDREBIB promoter can be induced by drought stress and low temperatures in O. sativa [16]. Moreover, the expression of ZF-HD transcription factors in Triticum aestivum and Brassica rapa was significantly upregulated under abiotic stress [10,17].
Orchidaceae rank among the largest and most geographically diverse groups within the realm of flowering plants, boasting an excess of 28,000 species [18]. Dendrobium is a large genus in the family Orchidaceae, with significant ornamental and medicinal values [19]. Flowers, which are considered an important ornamental trait, have been widely studied in Dendrobium [20]. As sequencing technology continues to advance, numerous Dendrobium genomes have been documented, including those of D. chrysotoxum [21], D. nobile [22], D. huoshanense [23], and D. catenatum [24]. These data have laid a solid foundation for exploring the key biological characteristics of Dendrobium. Previous studies have found that a variety of transcription factors occupy a crucial position in the floral development, growth, and adaptation to adversity of Dendrobium [25,26]. Despite extensive research on the ZF-HD gene family’s role in other plants, there is still a scarcity of reports focusing on Dendrobium.
In this study, we analyzed ZF-HDs in D. chrysotoxum, D. huoshanense, and D. catenatum, encompassing physicochemical properties, gene structure, phylogenetic tree construction, collinearity analysis, protein structure, and cis-elements. Specifically, the study focused on the regulatory role of the ZF-HD transcription factors on the flower of D. chrysotoxum. Our research provides new insights on the molecular mechanisms and morphological diversity of Dendrobium flower organ development. The results of this study may also contribute to broadening our understanding of the role of ZF-HD transcription factors in regulating flower organ development.

2. Results

2.1. Identification and Analysis of ZF-HD Proteins

ZF-HD proteins from three Dendrobiums were initially obtained using Blast and HMMER searches. A total of 17 DchZF-HDs, 23 DcaZF-HDs, and 13 DhuZF-HDs were named consecutively from top to bottom utilizing their chromosomal locations. The amino acids (AAs) of ZF-HDs varied from 85 aa (DchZHD9, DcaZHD8) to 858 aa (DhuZHD4), with an average length of 250 aa. The grand average of hydrophilic values (GRAVY) of the ZF-HDs ranged from −0.977 (DhuZHD9) to −0.293 (DhuZHD4), and the whole ZF-HDs showed GRAVY values less than 0, suggesting that all ZF-HD proteins were hydrophilic. The theoretical isoelectric point (pI) ranged from 5.36 (DhuZHD6) to 10.17 (DhuZHD9). The isoelectric points (pI) of 4 ZF-HDs were less than 7.0, which was acidic, and the isoelectric points of the remaining 49 ZF-HDs were more than 7.0, which was alkaline. The molecular weight (MW) of 53 ZF-HDs ranged from 9.38 kDa (DchZHD15) to 92.47 kDa (DhuZHD4), with an average MW of 27.58 kDa. The aliphatic index (AI) ranged from 36.08 (DcaMIF7) to 80.65 (DhuZHD4). The instability index (II) ranged from 46.94 (DhuZHD5) to 84.44 (DcaZHD1), indicating that most ZF-HD proteins are unstable. In addition, subcellular localization prediction revealed that the ZF-HD proteins of the three Dendrobiums were predominantly localized to the nucleus, pointing to a possible alike with most transcription factors (Table 1). The protein sequence of ZF-HDs from three Dendrobiums are shown in Table S1.

2.2. Phylogeny and Classification of ZF-HDs

We used 70 ZF-HD protein sequences from D. chrysotoxum, D. huoshanense, D. catenatum, and A. thaliana, to construct the neighbor-joining (NJ) phylogenetic tree. Based on the AtZF-HDs classification, the 53 Dendrobium ZF-HDs were divided into ZHD and MIF subfamilies, including 17 DchZF-HDs, 13 DhuZF-HDs, and 23 DcaZF-HDs. The ZHD subfamilies could be divided into ZHDI, ZHDII, ZHDIII, ZHDIV, and ZHDV subfamilies. The ZHDI subfamily had the most members (17), while the ZHDV subfamily had the fewest members (5) (Figure 1).

2.3. ZF-HD Structure and Motif Analysis

The MEME online tool was utilized to predict the conserved motifs of the ZF-HD proteins. The findings exhibited that the bulk of conserved motifs were located in the C-terminal domain, and the designated sequence of motifs began with 6, continued with 9, 1, 4, 10, 7, 5, 2, 3, and concluded with 8 (Figure 2B). In the ZF-HD domain, motif 1 and motif 4 were present in all ZF-HD proteins, which may be active regions for performing functions (Figure 2D). Conserved motifs from the same subfamily were similar, motif 5 was observed only in the ZHDI subfamily, and motif 10 only existed in the ZHDII subfamily. In addition, to delve deeper into the characteristics of ZF-HD genes, an intricate analysis of their intron–exon structure was executed, and the resulting structures were visualized by TBtools. Most of the 70 ZF-HDs did not contain introns, accounting for 71.7%, although a limited number of genes contained one to five introns (Figure 2C). A total of 13.2% of ZF-HDs had two exons and one intron.

2.4. Chromosomal Localization, Collinearity Analysis, and Promoter Analysis of ZF-HDs

In total, 17 DchZF-HDs and 13 DhuZF-HDs were distributed on chromosomes, and 23 DcaZF-HDs were localized to the unanchored scaffold. Seventeen DchZF-HDs were located on ten chromosomes, with Chr10 containing the greatest number of DchZF-HDs (5) (Figure 3A). Thirteen DhuZF-HDs were located on nine chromosomes, with Chr3 containing the greatest number of DchZF-HDs (3) (Figure 3B). In addition, Chr2 and Chr12 contained two DhuZF-HDs; Chr5, Chr7, Chr9, Chr10, Chr14, and Chr17 all contained only one DhuZF-HD. Twenty-three DcaZF-HDs were distributed on 17 scaffolds, with NW_021319500.1 containing the greatest number of DcaZF-HDs, specifically six genes in total (Figure 3C). All three Dendrobium species had gene tandem repeats, with D. chrysotoxum and D. huoshanense exhibiting two pairs of gene tandem repeats each. D. catenatum had six gene tandem repeats on NW_021319500.1 and a pair of gene tandem repeats on NW_021319360.1.
Collinearity analyses of ZF-HDs in three Dendrobium species were conducted. More collinear gene pairs were observed between the genomes of D. chrysotoxum and D. huoshanense than between those of D. chrysotoxum and D. catenatum. In total, 14 DchZF-HDs were collinear with DhuZF-HDs, and 12 DchZF-HDs were collinear with DcaZF-HDs. These results suggest that ZF-HD genes in D. chrysotoxum are more closely related to DhuZF-HDs than DcaZF-HDs (Figure 4).
To investigate the underlying functions of ZF-HDs in three Dendrobium species, an examination of cis-elements within the promoter regions of ZF-HDs was conducted using PlantCARE. DchZF-HDs contained 872 cis-elements, DcaZF-HDs harbored 1142 cis-elements, and DhuZF-HDs exhibited 785 cis-elements. A further analysis showed that, within the 17 DchZF-HDs, light-responsive elements accounted for 25.8%, growth and development-related elements made up 37.4%, plant hormone-responsive elements comprised 18.6%, and abiotic stress-responsive elements constituted 18.2% (Table S2). Notably, the AT~TATA-box element was the most abundant, with a total of 15 elements in DchZHD17. Among the 13 DhuZF-HDs, light-responsive elements accounted for 17.6%, growth and development-related elements for 35.9%, plant hormone-responsive elements for 24.3%, and abiotic stress-responsive elements for 17.6%. The AT~TATA element appeared most frequently, with a total of 62 elements in DhuZHD9. Among the 23 DcaZF-HDs, light-responsive elements constituted 21.2%, growth- and development-related elements 30.3%, plant hormone-responsive elements 24.4%, and abiotic stress-responsive elements 24.1%. The AT~TATA-box element was also the most numerous, with a total of 15 elements in DcaZHD10 (Figure 5). To sum up, cis-elements related to plant growth response were the most frequently occurring in Dendrobium, followed by light-responsive elements, phytohormone-related elements, and abiotic stress-related elements, indicating that ZF-HDs are particularly important in regulating plant growth and development. In addition, the ZF-HDs of three Dendrobium species contain a large amount of MeJA-responsive elements, which is a hormone involved in plant signaling. Previous studies have shown that MeJA maybe be used against salt stress, drought stress, low temperature stress, heavy metal stress, and toxicities of other elements [28]. Therefore, ZF-HDs may play an important role in influencing stress tolerance in Dendrobium species. We also found meristem expression-related elements in three Dendrobium species, indicating that ZF-HDs are important for meristem development.

2.5. Prediction of Protein Structure

An analysis of ZF-HD proteins in three Dendrobium species revealed random coil as the main secondary structure, followed by α-helix, extended strand, and β-turn (Table S3). Using SWISS-MODEL for homology modeling, the tertiary structures of ZF-HD proteins from D. chrysotoxum (Figure S1), D. catenatum (Figure S2), and D. huoshanense (Figure S3) were predicted. The homology between the three Dendrobium species and the modeling templates exceeded 70%, indicating a strong structural similarity and high model reliability. Among SWISS-MODEL metrics, GMQE correlated positively with 3D model quality [29]. D. chrysotoxum and D. catenatum GMQE values exceeded 0.5, indicating good modeling, while four ZF-HD proteins in D. huoshanense had scores below 0.5 (DhuZHD4, DhuZHD6, DhuMIF7, and DhuZHD9), indicating high variability (Table S4).

2.6. Expression Pattern of ZF-HDs in D. chrysotoxum

Based on FPKM values, we drew a heatmap of ZF-HDs (Figure 6). The results showed that DchZHD2 was highly expressed in the S1 stage, especially in the sepal. Conversely, DchMIF8 and DchZHD16 were highly expressed in the S2 and S3 stages, with DchMIF8 exhibiting the highest expression in the sepal in the S2 stage and DchZHD16 having the highest expression in the gynostemium in the S3 stage. DchZHD6 is almost non-expressive, indicating that it may have lost its regulation of floral organ development. In addition, the study found that genes from the same subfamily have similar expression patterns. Consistent expression patterns were observed in DchMIF8 and DchMIF9 belonging to the MIF subfamily, as well as DchZHD1 and DchZHD2 belonging to the ZHDII subfamily. Overall, most DchZF-HDs were generally highly expressed in the S1 stage, while their expression decreased in the later stages of floral organ development. The temporal and spatial specificity exhibited by these ZF-HDs may be the key to regulating the growth and development of various floral organs of D. chrysotoxum at different stages.

2.7. qRT-PCR Analysis of ZF-HDs in D. chrysotoxum

The results revealed that the expression levels of the MIF subfamily genes were higher than those of the ZHD subfamily genes. To deeply understand the expression patterns of the two subfamilies in different flower organs at various developmental stages, DchZHD2, DchMIF8, and DchZHD16 were selected for qRT-PCR analysis (Figure 7). DchZHD2 exhibited expression across all flower organs except gynostemium, peaking in the lips during stage S1, which may have a significant promoting effect on early flower organ development. Meanwhile, DchMIF8 showed the highest expression in the gynostemium during S1, and DchZHD16 showed the highest expression in the petal during S3. The expression of DchZHD16, which may be involved in promoting the morphogenesis of D. chrysotoxum, showed an upregulated trend. DchZHD2 is significantly expressed in the early stages of flower organ development and subsequently exhibits a downward trend with flower organ development. In contrast, as flowers develop, the expression of genes DchMIF8 and DchZHD16 shows an overall upward trend.

3. Discussion

Transcription factors (TFs) are crucial proteins that play a regulatory role in various biological processes during plant growth, including flowering, fruiting, and fruit ripening [7,30,31]. As a transcriptional regulator, zinc finger-homeodomain (ZF-HD) has received increasing attention due to its pivotal roles in modulating plant growth, development, and responses to various biotic and abiotic stresses. Research has shown that ZF-HD transcription factors have been identified in various plants such as A. thaliana (14) [13], Malus domestica (19) [32], Pisum sativum (18) [33], Gossypium hirsutum (49), Gossypium barbadense (50), Gossypium arboretum (22), and Gossypium raimondii (32) [34]. Although ZF-HDs have been discovered in numerous plants, there remains a paucity of research regarding their specific functions within orchids. In our study, 17 ZF-HDs in D. chrysotoxum, 23 ZF-HDs in D. catenatum, and 13 ZF-HDs in D. huoshanense were identified, which represent a smaller number of genes compared to some of the aforementioned plants. The phenomenon indicates that these three Dendrobiums may have experienced genetic losses during the process of evolution.
ZF-HDs are typically classified into two major subfamilies, ZHD and MIF, based on their phylogenetic tree. Similarly, the ZF-HDs in three Dendrobium species also followed this classification. The ZHD subfamily includes I–V subfamilies, which is similar to the classification of A. thaliana and Solanum lycopersicum [5,7]. The number of ZF-HDs in the MIF subfamily was similar, but there was a significant difference in the number of ZF-HDs in the ZHD subfamily (15 DchZHDs, 11 DcaZHDs, and 21 DhuZHDs). Previous investigations have indicated that the distribution pattern of motifs within subfamilies is intricately linked to their functional attributes. We conducted an analysis of the conserved motifs present within ZF-HD family proteins. Notably, the conserved motifs exhibited significant differences between the ZHD and MIF subfamilies. However, within the same subfamilies, we observed similar conserved motifs, indicating a degree of conservation within each subfamily despite the overall variability [6,35]. Research has shown that genes with introns exhibit greater length and more diverse functions and have advantages in species evolution or recombination [14,32,36]. However, 71.7% of ZF-HDs in this study lacked introns (Figure 2C). This phenomenon is similar to earlier reports on conserved gene characteristics. Some studies have found that the number of introns is also related to the plant’s adaptability to the environment, indicating that plant genes with fewer introns exhibit greater adaptability to the external environment and often respond quickly to stress [37]. Intron-free genes belong to large families that regulate physiological and biochemical processes and contribute to growth and development processes [38]. Therefore, many ZF-HD members in the three Dendrobiums may be able to respond quickly to environmental changes.
The process of gene duplication had a significant impact on the distinctive and varied characteristics of plants, emerging as a dominant force driving the expansion of gene families. Gene duplication generally includes fragment replication and tandem replication, which is the key to promoting biological evolution [39]. Tandem duplications were discovered in three Dendrobium species. The expansion of tandemly duplicated gene families and the increase in gene dosage may have enhanced the genetic diversity and adaptability of Dendrobium in response to environmental changes [40]. The variation in the number of tandemly duplicated genes among D. chrysotoxum, D. catenatum, and D. huoshanense may be associated with their abilities to adapt to different environmental conditions, such as temperature, soil, and light. Further research will reveal the specific functions of these duplicated genes and their impacts on the biological characteristics of Dendrobium, providing new insights into plant evolution and adaptation mechanisms.
Investigating promoters is imperative for enhancing our fundamental comprehension of gene regulation [41]. We have identified a series of functional cis-elements in the ZF-HD promoter of three Dendrobium species, encompassing diverse functional categories such as factors responsive to light, phytohormones, stress, as well as components pertaining to plant growth and development. Many growth responsive cis-elements were found in the upstream 2000 bp promoter region of 53 ZF-HDs, accounting for 37.39% (326/872) of the total DchZF-HDs, 35.92% (282/785) of the DhuZF-HDs, and 30.30% (346/1142) of the DcaZF-HDs. Among them, AT~TATA-box elements responsive to growth are the most abundant among the three Dendrobium species, with 15 in DchZHD17, 62 in DhuZHD9, and 15 in DcaZHD10 (Figure 5). ZF-HDs in three Dendrobium species harbor many MeJA-responsive elements, indicating their potential significance in enhancing stress tolerance. In addition, we identified meristem expression-related elements, indicating that ZF-HDs are important for meristem development. The differences in the number of these elements may reflect their different roles and regulatory mechanisms in the growth and development of different Dendrobium species.
Exploring the genes associated with flower morphology significantly contributes to advancing our comprehension of functional genes and the evolutionary dynamics linked to orchid flower development [42]. Presently, investigations into orchid flower organs predominantly concentrate on elucidating the intricacies of the flowering process, floral morphology, color, and fragrance [43]. SPL [44], B-Box [45], and MADS-box gene families [46] have all been recognized for their involvement in the developmental processes of flowers. In addition, the ZF-HD gene family also plays a role in flower morphogenesis. Research showed that AtZHD5 has an impact on both floral structure and leaf formation [13]. Similarly, Wang et al. [10] identified heightened expression levels in most BraZF-HDs within flowers, emphasizing their pivotal role in the regulation of flowering processes in Brassica pekinensis. Our work was based on transcriptome expression levels of D. chrysotoxum ZF-HDs in five flower organs of three stages. The findings revealed that members of the ZF-HD gene family exhibit varying transcript abundances at different stages within the flower buds, indicating that these genes possess distinct expression patterns and functions during the development of floral organs. There was a notable elevation in the expression levels of DchMIF8, DchMIF9, and DchZHD16 during the latter stages of flower organ development (Figure 6). The expression of DchZHD2 decreased with the development of flower organs, while the expression of genes DchMIF8 and DchZHD16 generally showed an upward trend. This observation aligns with the identification of numerous ZF-HDs associated with flowering in Malus pumila [47]. The qRT-PCR analysis of key genes showed that the relative expression level of DchZHD2 was highest in the lip during the S1 stage, while the relative expression levels of DchMIF8 and DchZHD16 peaked in the petal during the S3 stage. During early flower development, DchZHD2 expression was prominent but declined with flower organ development. Conversely, the expression of DchMIF8 and DchZHD16 was on the rise as with flower organ development. This suggests that different genes may play distinct regulatory roles at various stages of flower organ development, potentially involved in processes such as flower morphogenesis, flower opening, or other processes related to floral development. In brief, the growth and development of three Dendrobiums and the flower organ development of D. chrysotoxum may be regulated by members of the ZF-HD gene family.

4. Material and Methods

4.1. Experimental Materials and Data Sources

For this study, plant material sourced from wild-type plants cultivated in a greenhouse at Fujian Agriculture and Forestry University (26°05′ N, 119°13′ E) was utilized. Various parts of D. chrysotoxum flowers, specifically sepals (Se), petals (Pe), gynostemium (Gy), ovary (Ov), and lips (Lip), were collected across the unpigmented bud stage (S1), pigmented bud stage (S2), and initial bloom stage (S3). Subsequently, the collected samples were placed in liquid nitrogen for rapid freezing, followed by storage in a −80 °C degrees freezer for future use.
We accessed the National Center for Biotechnology Information database (NCBI, https://www.ncbi.nlm.nih.gov/, accessed on 18 September 2023) to acquire the genomic sequences and annotation files pertaining exclusively to three Dendrobiums. The BioProject accession number for D. chrysotoxum is PRJNA664445 (release date: 10 September 2021, accessed on 18 September 2023) [48], for D. catenatum it is PRJNA262478 (release date: 26 February 2016, accessed on 18 September 2023) [49], and for D. huoshanense it is PRJNA597621 (release date: 12 January 2021, accessed on 18 September 2023) [21]. Moreover, the protein sequence files corresponding to ZF-HD in A. thaliana were obtained from the Plant Transcription Factor Database (PlantTFDB, http://planttfdb.gao-lab.org/index.php?sp=Ath, accessed on 18 September 2023).

4.2. Identification and Physicochemical Properties of ZF-HD Genes

Using the protein sequence of A.thaliana ZF-HD proteins as a probe, local blast was performed using TBtools v1.120 software [50], and the obtained sequence was reverse-blasted using the Blast tool in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 September 2023). To further identify ZF-HDs in the three Dendrobiums, we used the Hidden Markov Model (HMM) configuration profiles of ZF-HD (PF04770) from Pfam 34.0 database for analysis. Subsequently, protein sequences that were incomplete or redundant were manually excluded from the dataset. An assessment of protein attributes involving amino acid (aa) composition, isoelectric point (pI), molecular weight (MW), average hydrophilicity (GRAVY), instability index (II), and aliphatic index (AI) was conducted utilizing ExPASy 3.0 (https://www.expasy.org/, accessed on 20 September 2023) [51]. The value of an isoelectric point greater than 7 implied that the protein is alkaline, while a lower value indicates acidic properties [25]. The instability index, which serves as an experimental estimation of the stability of a protein, predicts that a protein is stable if its value is smaller than 40 [52]. And a deeper understanding of the potential subcellular localization of Dendrobium ZF-HD proteins through WoLF PSORT (https://wolfpsort.hgc.jp/, accessed on 21 September 2023) is crucial to elucidating the functional roles of these proteins in the cellular environment [27].

4.3. Phylogenetic Analysis of ZF-HDs

The MEGA7.0 software was employed to introduce 17 ZF-HD protein sequences of A. thaliana (14 AtZHDs and 3 AtMIFs), 17 ZF-HDs of D. chrysotoxum, 23 ZF-HDs of D. catenatum, and 13 ZF-HDs of D. huoshanense. We used the muscle program and default settings for multisequence alignment and the neighbor-joining (NJ) method to construct the phylogenetic tree [53]. The bootstrap method was executed with 500 replicates, setting partial deletion to 50%. The Evolview 3.0 online platform (http://www.evolgenius.info/evolview/#/treeview, accessed on 23 September 2023) beautifies phylogenetic trees, enhancing their clarity and interpretability [54].

4.4. Gene Structures and Conserved Motif Analysis

The NCBI conserved domain database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 23 September 2023) was employed to forecast the conserved domains inherent in Dendrobium ZF-HDs. Moreover, the MEME Suite 5.5.5 online software (https://meme-suite.org/meme/tools/meme/, accessed on 23 September 2023) was utilized to scrutinize the conserved motifs evident in the ZF-HDs of the three Dendrobiums and A. thaliana. The MEME analysis was conducted using default settings, with the adjustment of the maximum number of motifs to ten. The phylogeny analysis was conducted through the utilization of 1000 iterations of the bootstrap approach. Subsequently, the generated output was utilized to create a comprehensive comparative map encompassing the NJ phylogenetic tree (bootstrap method: 500). TBtools was used to integrate and visualize phylogenetic trees, conserved protein motifs, and gene structure.

4.5. Localization and Collinearity Analysis of ZF-HDs on Chromosomes

The genetic sequence and annotation data of D. chrysotoxum, D. catenatum, and D. huoshanense, TBtools were utilized to visualize the chromosomal positions of ZF-HDs within these species. To conduct the collinearity analysis of the ZF-HD gene family among the three Dendrobium species, genomic information for the respective species was retrieved from the NCBI database. The One Step MCScanX-Super Fast and Multiple Synteny Plot plugin in TBtools were used to perform collinearity analysis and visualize the results.

4.6. Analysis of ZF-HD Promoter

To further investigate the transcriptional regulation of Dendrobium ZF-HDs, TBtools extracted the upstream 2000 bp sequence of the ZF-HDs’ promoter. Subsequently, PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 18 October 2023) was utilized to perform the analysis of the promoter regions of Dendrobium ZF-HDs, to predict the occurrence of cis-elements. The identification of these elements provided a reference basis for the potential transcriptional regulatory networks governing Dendrobium ZF-HDs expression. The resulting data were processed and organized using Excel, to facilitate further scientific exploration.

4.7. Protein Structure Prediction

The SWISS-MODEL software (https://swissmodel.expasy.org/interactive, accessed on 26 October 2023) is widely recognized in the field of bioinformatics for its accuracy and reliability in protein structure prediction [55]. By utilizing SWISS-MODEL, we were able to generate a three-dimensional representation of the Dendrobium ZF-HD proteins. Additionally, we employed the SOPMA program (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html, accessed on 26 October 2023) [56] for predicting the secondary structure of these proteins. SOPMA is a well-established method that helped us to predict the presence of various secondary structural elements, such as alpha-helices and beta-sheets, within the protein sequence. Through combining the results from both SWISS-MODEL and SOPMA, we obtained a comprehensive understanding of the structural characteristics of orchid ZF-HD proteins, which is crucial for further elucidating their functions in the cellular environment.

4.8. Analysis of Expression and RT-qPCR

We utilized the FastPure Plant Total RNA Isolation Kit (for polysaccharide- and polyphenol-rich tissues) (Vazyme Biotech Co., Ltd., Nanjing, China) to extract RNA from flower organs at different flowering stages. Leveraging the RSEM v1.2.8 tool, we accurately calculated the gene expression levels for each individual sample, thereby obtaining Fragments Per Kilobase of transcript per Million Fragments (FPKM) values. These computed FPKM values were then employed to generate a comprehensive heatmap using TBtools, visualizing gene expression levels.
Utilizing the PrimerScript® RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China), which was accompanied by gDNA Eraser, we performed reverse transcription. This methodology was employed to eliminate any potential genomic DNA contamination and enhance the process of cDNA synthesis. Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix was used for the qRT-PCR analysis on an ABI 7500 Real-Time System that was from Yeasen Biotechnology (Shanghai) Co., Ltd., Shanghai, China. In this study, a 96-well plate with a 20 μL reaction system in each well was utilized for the experimental setup. Primers for candidate and internal reference genes for RT-qPCR were designed using Primer Premier 5.0 software. A reference gene, Maker75111, was selected for use. Using the 2−∆∆CT method and Graphpad prism 7.0 software, we evaluated the relative expression levels of the target genes.

5. Conclusions

For this study, we identified 17 DchZF-HDs, 23 DcaZF-HDs, and 13 DhuZF-HDs and classified them into ZHD (ZHDI–V) and MIF subfamilies based on their phylogenetic relationships. Members of the same subfamily had similar gene structures and conserved domains. Analyses of phylogenetics, gene structure, motif composition, collinearity, chromosomal localization, and cis-elements in the three Dendrobium species were carried out. In addition, the results of our expression and qRT-PCR analyses indicated that DchMIF8 and DchZHD16 play a significant role in the S2/S3 stage, and changes in the expression level of DchZHD2 may affect the development of floral organs in the S1 stage. In summary, this study provides a reference for researchers to better understand how ZF-HDs are involved in regulating floral organ development and determination.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10060610/s1. Figure S1. Prediction of the Tertiary Structure of D. chrysotoxum; Figure S2. Prediction of the Tertiary Structure of D. catenatum; Figure S3. Prediction of the Tertiary Structure of D. huoshanense. Table S1. Characteristics of ZF-HD proteins from three Dendrobium orchids; Table S2. Statistics of Cis-acting Element Quantity; Table S3. Secondary structure of ZF-HDs proteins in three Dendrobiums; Table S4. Detailed information of the predicted three-dimensional (3D) of ZF-HDs proteins in three Dendrobiums; Table S5. The FPKM values of ZF-HDs in D. chrysotoxum; Table S6. qRT-PCR primers.

Author Contributions

X.H. finalized the manuscript and integrated all other authors’ comments; X.Z. and Q.Z. provided the data; M.-M.Z. and Y.H. analyzed the data; S.L. and Z.-J.L. conceived the study, coordinated with all coauthors, and supervised the whole project. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by scientific funding from the Technical Services for Introduction and Domestication of Orchids in Sanjiangkou Botanical Garden, Fuzhou (KH240047A).

Data Availability Statement

The original data presented in the study are openly available in the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, accessed on 18 September 2023) genome database. The BioProject number for D. chrysotoxum is PRJNA664445, for D. catenatum PRJNA262478, and for D. huoshanense PRJNA597621.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic tree generated for the 70 ZF-HDs retrieved from D. chrysotoxum, D. catenatum, D. huoshanense, and A. thaliana.
Figure 1. Phylogenetic tree generated for the 70 ZF-HDs retrieved from D. chrysotoxum, D. catenatum, D. huoshanense, and A. thaliana.
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Figure 2. The motif and gene structure of ZF-HDs in D. chrysotoxum, D. catenatum, D. huoshanense, and A. thaliana. (A) Phylogenetic tree of 70 ZF-HDs constructed using MEGA7.0. (B) The conserved motif of ZF-HD proteins. (C) The ZF-HDs structure. (D) The sequence information for motif 1 and motif 4 (* indicates highly conserved cysteine residues).
Figure 2. The motif and gene structure of ZF-HDs in D. chrysotoxum, D. catenatum, D. huoshanense, and A. thaliana. (A) Phylogenetic tree of 70 ZF-HDs constructed using MEGA7.0. (B) The conserved motif of ZF-HD proteins. (C) The ZF-HDs structure. (D) The sequence information for motif 1 and motif 4 (* indicates highly conserved cysteine residues).
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Figure 3. The ZF-HDs distribution on the chromosomes of three Dendrobiums. (A) D. chrysotoxum, (B) D. catenatum, and (C) D. huoshanense. Black designates the name of chromosomes, and red is employed to indicate the name of ZF-HDs.
Figure 3. The ZF-HDs distribution on the chromosomes of three Dendrobiums. (A) D. chrysotoxum, (B) D. catenatum, and (C) D. huoshanense. Black designates the name of chromosomes, and red is employed to indicate the name of ZF-HDs.
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Figure 4. Collinearity analysis of ZF-HDs in three Dendrobiums.
Figure 4. Collinearity analysis of ZF-HDs in three Dendrobiums.
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Figure 5. Analysis of cis-element components of the promoter of ZF-HDs across three Dendrobiums. (A) D. chrysotoxum, (B) D. huoshanense, and (C) D. catenatum. The left segment represents the count of cis-elements in ZF-HDs, while the right segment provides the statistical analysis of different categories of ZF-HDs. The types and numbers of ZF-HDs are listed in Table S2.
Figure 5. Analysis of cis-element components of the promoter of ZF-HDs across three Dendrobiums. (A) D. chrysotoxum, (B) D. huoshanense, and (C) D. catenatum. The left segment represents the count of cis-elements in ZF-HDs, while the right segment provides the statistical analysis of different categories of ZF-HDs. The types and numbers of ZF-HDs are listed in Table S2.
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Figure 6. The expression levels of 17 ZF-HD genes during various developmental periods (S1: unpigmented bud stage; S2: pigmented bud stage; S3: initial bloom stage) and in distinct components (Pe: petals; Se: sepals; Ov: ovary, Lip: lip; Gy: gynostemium) of D. chrysotoxum. The FPKM values of ZF-HDs observed in D. chrysotoxum are detailed in Table S5.
Figure 6. The expression levels of 17 ZF-HD genes during various developmental periods (S1: unpigmented bud stage; S2: pigmented bud stage; S3: initial bloom stage) and in distinct components (Pe: petals; Se: sepals; Ov: ovary, Lip: lip; Gy: gynostemium) of D. chrysotoxum. The FPKM values of ZF-HDs observed in D. chrysotoxum are detailed in Table S5.
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Figure 7. Real-time reverse transcription quantitative PCR (RT–qPCR) validation of DchZF-HDs. S1 represents the unpigmented bud stage, S2 represents the pigmented bud stage, and S3 represents the initial bloom stage. The Y-axis signifies the relative expression values (2−ΔΔCT). Additionally, the red asterisk serves as an indicator of the significance level of the P-value in the respective test (** p < 0.01, **** p < 0.0001). Primers and RT-qPCR analysis of DchZF-HDs are shown in Table S6.
Figure 7. Real-time reverse transcription quantitative PCR (RT–qPCR) validation of DchZF-HDs. S1 represents the unpigmented bud stage, S2 represents the pigmented bud stage, and S3 represents the initial bloom stage. The Y-axis signifies the relative expression values (2−ΔΔCT). Additionally, the red asterisk serves as an indicator of the significance level of the P-value in the respective test (** p < 0.01, **** p < 0.0001). Primers and RT-qPCR analysis of DchZF-HDs are shown in Table S6.
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Table 1. The physicochemical properties of ZF-HD proteins from three Dendrobium species.
Table 1. The physicochemical properties of ZF-HD proteins from three Dendrobium species.
NameGene IDAA 1GRAVY 2pI 3Mw 4 (kDa)AI 5II 6Subcellular Localization 7
DchZHD1Maker81525216−0.593 8.73 23.70 57.55 60.85 Nucleus.
DchZHD2Maker28129216−0.593 8.73 23.70 57.55 60.85 Nucleus.
DchZHD3Maker69363217−0.767 8.96 24.50 57.60 83.88 Nucleus.
DchZHD4Maker78792314−0.787 8.25 34.50 51.56 66.90 Nucleus.
DchZHD5Maker102972221−0.871 8.68 25.19 48.55 69.86 Nucleus.
DchZHD6Maker82648297−0.666 6.37 32.51 59.19 53.96 Nucleus.
DchZHD7Maker74870286−0.598 9.07 31.28 59.34 57.57 Nucleus.
DchMIF8Maker100805126−0.917 9.04 13.68 39.60 67.20 Mitochondrial.
DchMIF9Maker57367129−0.788 7.61 14.24 46.20 64.53 Mitochondrial.
DchZHD10Maker92643249−0.691 8.99 27.41 60.00 61.99 Nucleus.
DchZHD11Maker54517450−0.471 8.87 49.31 67.62 48.02 Cytoplasmic.
DchZHD12Maker53183249−0.623 8.99 27.72 59.16 61.03 Nucleus.
DchZHD13Maker57691232−0.570 9.57 25.57 70.13 57.09 Nucleus.
DchZHD14Maker77088198−0.622 9.24 22.43 60.10 70.26 Cytoplasmic.
DchZHD15Maker6122885−0.326 8.70 9.38 55.18 60.76 Chloroplast.
DchZHD16Maker61267293−0.670 7.63 31.52 60.00 57.07 Nucleus.
DchZHD17Maker58363273−0.666 7.05 28.88 49.52 49.81 Nucleus.
DhuZHD1Dhu000007045198−0.648 9.12 22.64 56.16 72.97 Nucleus.
DhuZHD2Dhu000019223198−0.648 9.12 22.64 56.16 72.97 Nucleus.
DhuZHD3Dhu000028169146−0.770 9.65 17.02 46.16 81.27 Nucleus.
DhuZHD4Dhu00002429185813.510 9.64 92.47 80.65 47.66 Cytoplasmic.
DhuZHD5Dhu000009472280−0.386 5.48 30.32 73.86 46.94 Nucleus.
DhuZHD6Dhu000013154390−0.381 5.36 41.72 66.54 48.07 Cytoplasmic.
DhuMIF7Dhu000014526260−0.955 5.39 28.60 45.12 68.88 Nucleus.
DhuZHD8Dhu000009060250−0.638 8.79 27.01 57.80 54.54 Nucleus.
DhuZHD9Dhu000025178626−0.977 10.17 70.34 57.68 65.98 Nucleus.
DhuZHD10Dhu000014745216−0.584 8.73 23.75 58.01 62.13 Nucleus.
DhuZHD11Dhu000004498216−0.581 8.73 23.74 58.01 62.27 Nucleus.
DhuZHD12Dhu000014369125−0.942 9.04 13.54 37.60 70.65 Mitochondrial.
DhuZHD13Dhu000016048219−0.762 8.54 24.84 52.10 59.08 Nucleus.
DcaZHD1rna-XM_020822518.2218−0.855 8.77 24.54 56.01 84.44 Nucleus.
DcaZHD2rna-XM_020823161.2263−0.719 8.92 29.55 58.56 66.36 Nucleus.
DcaZHD3rna-XM_020825047.1222−0.725 8.55 25.09 50.99 59.33 Nucleus.
DcaMIF4rna-XM_020824991.2129−0.736 7.60 14.16 49.22 65.82 Mitochondrial.
DcaZHD5rna-XM_020824023.2216−0.595 8.73 23.72 57.13 62.13 Nucleus.
DcaZHD6rna-XM_020838304.2249−0.646 8.92 26.89 57.67 50.52 Nucleus.
DcaMIF7rna-XM_020825579.2125−0.962 9.04 13.51 36.08 70.65 Mitochondrial.
DcaZHD8rna-XM_020842089.185−0.420 8.10 9.68 52.82 51.73 Chloroplast.
DcaZHD9rna-XM_020842002.2293−0.683 7.64 31.54 57.99 56.76 Nucleus.
DcaZHD10rna-XM_020831316.1200−0.684 8.17 21.60 45.00 67.27 Nucleus.
DcaZHD11rna-XM_020837938.2249−0.650 8.99 27.69 57.99 59.00 Nucleus.
DcaZHD12rna-XM_020837921.2249−0.650 8.99 27.69 57.99 59.00 Nucleus.
DcaZHD13rna-XM_020837895.2249−0.650 8.99 27.69 57.99 59.00 Nucleus.
DcaZHD14rna-XM_020837890.2249−0.650 8.99 27.69 57.99 59.00 Nucleus.
DcaZHD15rna-XM_028698070.1249−0.650 8.99 27.69 57.99 59.00 Nucleus.
DcaZHD16rna-XM_020837900.2249−0.650 8.99 27.69 57.99 59.00 Nucleus.
DcaZHD17rna-XM_020825507.2258−0.748 9.15 28.94 58.29 63.93 Nucleus.
DcaZHD18rna-XM_020846562.2199−0.611 9.24 22.74 54.42 74.46 Nucleus.
DcaZHD19rna-XM_020841238.2285−0.604 9.07 31.21 60.56 58.07 Nucleus.
DcaZHD20rna-XM_020831512.2264−0.762 6.59 28.25 47.01 59.03 Nucleus.
DcaZHD21rna-XM_020848621.2273−0.675 7.05 28.91 49.16 49.50 Nucleus.
DcaZHD22rna-XM_020845498.2312−0.747 8.53 34.04 52.56 65.46 Nucleus.
DcaZHD23rna-XM_020823387.2147−0.784 9.79 17.17 46.53 81.65 Nucleus.
1 AA, amino acid; 2 GRAVY, grand average of hydrophobicity; 3 pI, theoretical isoelectric point; 4 Mw, molecular weight; 5 AI, aliphatic index; 6 II, instability index; 7 subcellular localization predicted by WoLF PSORT [27].
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MDPI and ACS Style

He, X.; Zhao, X.; Zheng, Q.; Zhang, M.-M.; Huang, Y.; Liu, Z.-J.; Lan, S. Whole-Genome Analysis of ZF-HD Genes among Three Dendrobium Species and Expression Patterns in Dendrobium chrysotoxum. Horticulturae 2024, 10, 610. https://doi.org/10.3390/horticulturae10060610

AMA Style

He X, Zhao X, Zheng Q, Zhang M-M, Huang Y, Liu Z-J, Lan S. Whole-Genome Analysis of ZF-HD Genes among Three Dendrobium Species and Expression Patterns in Dendrobium chrysotoxum. Horticulturae. 2024; 10(6):610. https://doi.org/10.3390/horticulturae10060610

Chicago/Turabian Style

He, Xin, Xuewei Zhao, Qinyao Zheng, Meng-Meng Zhang, Ye Huang, Zhong-Jian Liu, and Siren Lan. 2024. "Whole-Genome Analysis of ZF-HD Genes among Three Dendrobium Species and Expression Patterns in Dendrobium chrysotoxum" Horticulturae 10, no. 6: 610. https://doi.org/10.3390/horticulturae10060610

APA Style

He, X., Zhao, X., Zheng, Q., Zhang, M. -M., Huang, Y., Liu, Z. -J., & Lan, S. (2024). Whole-Genome Analysis of ZF-HD Genes among Three Dendrobium Species and Expression Patterns in Dendrobium chrysotoxum. Horticulturae, 10(6), 610. https://doi.org/10.3390/horticulturae10060610

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