Next Article in Journal
Exploration of the Conformational Scenario for α-, β-, and γ-Cyclodextrins in Dry and Wet Conditions, from Monomers to Crystal Structures: A Quantum-Mechanical Study
Previous Article in Journal
Gas-Phase vs. Grain-Surface Formation of Interstellar Complex Organic Molecules: A Comprehensive Quantum-Chemical Study
Previous Article in Special Issue
Heat Stress Alleviation by Exogenous Calcium in the Orchid Dendrobium nobile Lindl: A Biochemical and Transcriptomic Analysis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 23
10.3390/ijms242316825
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Identification, Expression, and Molecular Characterization of the CONSTANS-like Gene Family in Seven Orchid Species

by
Yonglu Wei
,
Jianpeng Jin
,
Zengyu Lin
,
Chuqiao Lu
,
Jie Gao
,
Jie Li
,
Qi Xie
,
Wei Zhu
,
Genfa Zhu
and
Fengxi Yang
*
Guangdong Key Laboratory of Ornamental Plant Germplasm Innovation and Utilization, Environmental Horticulture Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(23), 16825; https://doi.org/10.3390/ijms242316825
Submission received: 7 November 2023 / Revised: 23 November 2023 / Accepted: 25 November 2023 / Published: 27 November 2023
(This article belongs to the Special Issue Orchid Biochemistry 3.0)
Browse Figures
Review Reports Versions Notes

Abstract

:
The orchid is one of the most distinctive and highly valued flowering plants. Nevertheless, the CONSTANS-like (COL) gene family plays significant roles in the control of flowering, and its functions in Orchidaceae have been minimally explored. This research identified 68 potential COL genes within seven orchids’ complete genome, divided into three groups (groups I, II, and III) via a phylogenetic tree. The modeled three-dimensional structure and the conserved domains exhibited a high degree of similarity among the orchid COL proteins. The selection pressure analysis showed that all orchid COLs suffered a strong purifying selection. Furthermore, the orchid COL genes exhibited functional and structural heterogeneity in terms of collinearity, gene structure, cis-acting elements within their promoters, and expression patterns. Moreover, we identified 50 genes in orchids with a homology to those involved in the COL transcriptional regulatory network in Arabidopsis. Additionally, the first overexpression of CsiCOL05 and CsiCOL09 in Cymbidium sinense protoplasts suggests that they may antagonize the regulation of flowering time and gynostemium development. Our study will undoubtedly provide new resources, ideas, and values for the modern breeding of orchids and other plants.

1. Introduction

The orchid family (Orchidaceae) is one of the most species-rich plant families. There are approximately 30,000 orchid species with colorful and aromatic flowers that are appreciated worldwide [1,2,3]. In addition to its economic importance, the orchid offers ecological, ornamental, medicinal, aesthetic, and cultural value [4,5,6]. Molecular phylogenetic analyses suggest that the orchid family consists of five mono-phyletic subfamilies: Apostasioideae, Vanilloideae, Cypripedioideae, Orchidoideae, and Epidendroideae [2,7]. The diverse Vanilla genus, which belongs to the Vanilloideae family, has extensive usage in various industries, such as food, pharmaceuticals, cosmetics, beverages, and traditional crafts [8,9,10]. Epidendroideae, with approximately 21,160 species, is the largest of the five orchid subfamilies [11]. Due to their diversity, many species from several genera are very prominent industrial commodities. For instance, Dendrobium is a valuable traditional herb with high commercial worth [12]. Cymbidium, on the other hand, has been grown in China for thousands of years and is renowned as the ‘King of Fragrance’ [13].
The induction of flowering is a key step leading to proper flower development in the orchid, and several functional pathways, including photoperiod, vernalization, environmental temperature, phytohormones, and autonomous flowering pathways, have been identified to control flowering induction [14,15,16,17,18,19]. CONSTANS-LIKE (COL) genes, phosphatidyl ethanolamine-binding protein (PEBP) genes, and several members of the MADS-box gene family in these pathways have been suggested to play a key role in flowering time regulation [20,21,22]. However, due to several major technical obstacles, including the long vegetative phase, the low efficiency of the genetic transformation systems, and the time-consuming process of tissue culture, the molecular mechanism has yet to be elucidated [23].
CONSTANS, a member of the zinc fingered transcription factor family, is an important regulator of plant responses to the photoperiod, playing a key role in the regulation of flowering [24]. The COL family genes contain two conserved elements: the N-terminal BBX (B-box) domain, which consists of four cysteines with a specific structure of (C-X2-C-X16-C-X2-C); and the C-terminal CCT (CONSTANS, CO-like, and TIMING of CAB1) domain [25,26]. Since the discovery of the first COL gene in Arabidopsis [27], numerous members of the COL gene family have been identified in various plant species, including 12 in grapevine (Vitis vinifera) [28], 13 in lotus (Nelumbo nucifera) [29], 14 in Populus [30], 15 in petunia (Petunia axillaris) [31], 16 in rice (Oryza sativa) [32], 17 in Arabidopsis [20], 19 in maize (Zea mays) [33], 22 in sunflower (Helianthus annuus) [34], 25 in banana (Musa acuminata) [35], 26 in soybean (Glycine max) [36], and 42 in cotton (Gossypium hirsutum) [37].
The sequence of the orchid genome provides genetic resources for gene functional studies, and the study of the orchid flowering-time genes can therefore provide essential information for the further modification of orchid varieties to increase yield. Previously, there was limited information on the functions of genes involved in flowering time regulation in orchids [5,6]. Here, we identified seven COL genes and investigated their properties, such as chromosome location, gene organization, cis-acting elements, protein–protein interactions (PPIs), and gene expression pattern. In addition, we were surprised to find that the CsiCOL genes in C. sinense may be related to the development of the gynostemium, which is one of the typical characteristics of orchids that distinguishes them from other plants [38]. First, the overexpression of CsiCOL05 and CsiCOL09 in C. sinense protoplasts confirms the regulation of the flowering time and flower development genes. Our results provide useful information for characterizing COL gene functions in orchids and other plants.

2. Results

2.1. Basic Characterization of COL Genes in Orchidaceae

In the present study, the genomes of five Epidendroideae (Cymbidium sinense; Csi, Dendrobium catenatum; Dca, Dendrobium chrysotoxum; Dch, Dendrobium huoshanense; and Dhu, Phalaenopsis equestri; Peq); one Apostasioideae (Apostasia shenzhenica; Ash); and one Vanilloideae (Vanilla planifolia; Vpl). Orchidaceae species were thoroughly scanned for gene identification. Finally, a total of 68 putative COL genes were identified in all seven orchid families by the HMM program and subsequently verified by the Pfam and blastp databases, with all COL genes containing both B-box and CCT domains. To distinguish the sixty-eight genes, we named them CsiCOL1 to CsiCOL10, DcaCOL01 to DcaCOL09, DchCOL01 to DchCOL10, DhuCOL01 to DhuCOL08, PeqCOL01 to PeqCOL07, AshCOL01 to AshCOL10, and VplCOL01 to VplCOL14, according to their physical location on the chromosomes. Detailed information is provided in Supplementary Table S1. The DhuCOL genes were dispersed on six chromosomes of D. huoshanense: one in chromosome 1 (DhuCOL05), 3 (DhuCOL04), 14 (DhuCOL03), and 18 (DhuCOL06); and two in chromosome 7 (DhuCOL07 and DhuCOL08) and 17 (DhuCOL01 and CsiCOL02), respectively. The VplCOL genes were scattered on nine chromosomes and one contig of the V. planifolia genome by one in chromosome 3 (VplCOL12), 4 (VplCOL11), 7 (VplCOL08), 8 (VplCOL07), 9 (VplCOL06), and JADCNL010000338 (VplCOL01); and two in chromosome 2 (VplCOL13 and VplCOL14), 5 (VplCOL09 and VplCOL10), 13 (VplCOL04 and VplCOL05), and 14 (VplCOL02 and VplCOL03), respectively. The DchCOL genes were scattered on nine chromosomes and one contig of the D. chrysotoxum genome by one in chromosome 7 (DchCOL06), 11 (DchCOL03), 16 (DchCOL02), 18 (DchCOL09), and unchr_scaffold_742 (DchCOL01); and two in chromosome 9 (DchCOL07 and DchCOL08) and 12 (DchCOL04 and DchCOL05), respectively. The CsiCOL genes were distributed on seven chromosomes and one contig of the C. sinense genome, with one in chromosome 3 (CsiCOL04), 7 (CsiCOL10), 8 (CsiCOL09), 15 (CsiCOL07), 20 (CsiCOL03), and contig4269 (CsiCOL08); and two in chromosome 6 (CsiCOL05 and CsiCOL06) and 9 (CsiCOL01 and CsiCOL02), respectively.
The amino acid sequences of seven orchid COL proteins range from 163 (Ash-COL01) to 499 (DhuCOL01) in length, and the molecular weights are between 18.43 kDa (AshCOL4) and 54.68 kDa (DhuCOL01). The minimum and maximum isoelectric points are 4.86 (VplCOL13) and 9.36 (VplCOL02), respectively, among the orchid COL genes. The protein instability index analysis showed that, except for AshCOL01 (39.52), DcaCOL01 (29.5), and VplCOL06 (36.52), all seven Orchidaceae COL members belong to unstable proteins (instability index > 40) [39]. The prediction results of the subcellular location showed that the COL genes of Orchidaceae are localized in the nucleus, mitochondria, and several other locations.

2.2. Phylogenetic Analysis of COL Genes

In order to understand the evolutionary relationships of COL family genes in Orchidaceae, we constructed an unrooted tree (Figure 1), using 101 COL proteins from A. thaliana (17), O. sativa (16), A. shenzhenica (10), D. catenatum (9), D. huoshanense (8), V. planifolia (14), D. chrysotoxum (10), C. sinense (10), and P. equestri (7). All of the COL proteins were verified by the Pfam and Blastp databases as containing both the B-box and the CCT domains. In Arabidopsis, COLs are divided into three groups according to their sequence alignment, with group I COLs containing two B boxes, group II COLs containing one normal and one divergent B box, and group III COLs containing only one B box domain [20,32]. Each group contained at least one COL protein from the nine different plant species, whereas P. equestri was not expected in III. The distribution of Orchidaceae COL proteins was twenty-eight (AshCOL02-AshCOL04, AshCOL07, AshCOL09, DcaCOL02, DcaCOL09, DhuCOL02, DhuCOL03, DhuCOL06, DhuCOL07, VplCOL01, VplCOL04-VplCOL06, VplCOL08, VplCOL13, DchCOL03, DchCOL05, DchCOL07, DchCOL09, CsiCOL01, CsiCOL04, CsiCOL05, CsiCOL09, and PeqCOL02-PeqCOL04) in group I; twenty-eight (Ash-COL01, AshCOL06, AshCOL08, AshCOL10, DcaCOL01, DcaCOL03, DcaCOL04, DcaCOL06, DcaCOL07, DhuCOL01, DhuCOL08, VplCOL02, VplCOL03, VplCOL09, VplCOL10-VplCOL12, DchCOL04, DchCOL08, DchCOL10, CsiCOL02, CsiCOL03, CsiCOL06, CsiCOL08, PeqCOL01, and PeqCOL05-PeqCOL07) in group II; and twelve (AshCOL05, DcaCOL05, DcaCOL08, DhuCOL04, DhuCOL05, VplCOL07, VplCOL14, DchCOL01, DchCOL02, DchCOL06, CsiCOL07, and CsiCOL10) in group III.

2.3. Sequence Structure Analysis of COL Members

Sequence structure information was predicted using MEME. Fifteen types of conserved motifs were represented by numbers from 1 to 10 (Figure 2A and Supplementary Table S2). All Orchidaceae COL members contain two conserved domains, one or two N-terminal B-boxes (motif 2 and motif 5), and one CCT domain (motif 1) near the C-terminus. The number of COL motifs ranged from two (VplCOL01) to seven (CsiCOL06). Even though the majority of the orchid COL proteins contained these six conserved protein domains, the motifs of each sub-clade are still different from each other. For instance, motifs 6 and 8 were present only in class II. An orchid VP motif (motif 9) alongside the CCT domain, which is important for binding to the COP1 gene to regulate light signaling cascades [40]. The conserved motif was identified in the group I COL members in Orchidaceae. It has also been detected in A. thaliana [26] and cucumber [41].
The intron–exon structure was analyzed (Figure 2B) to gain further insight into the characteristics of orchid COL genes. The results showed that the orchid COL family is composed of one to six exons. Although the gene structure is similar in each subgroup, orchid COL genes have a high degree of variation in intron length and exon number. Although most of the orchid COL genes in group II have longer introns than the other subgroups, the gene structure of VplCOL13 showed a significant difference in the length of the introns (Figure 2B), which may be a peculiar feature of orchids.

2.4. Chromosomal Location, Collinearity and Evolutionary Analysis of Orchid COL Genes

According to the annotation file of the four orchid species, the densities of the 300 kb inheritance interval genes were obtained and then further transformed into a gradient colored heatmap on the orchid chromosome or scaffold. The Tbtools program was used to visualize the chromosomal locations of the COL genes [42] in the chromosomal genome of the four orchids. The 39 COL genes are distributed unevenly and widely over the 28 chromosomes (Figure 3 and Supplementary Table S1). There are 9, 9, 8, and 13 COL genes in the C. sinense, D. chrysotoxum, D. huoshanense, and V. planifolia genomes, respectively (Figure 3A–D). Interestingly, only one or two of the COL genes were observed on the same chromosome in four species of Orchidaceae; otherwise, each COL gene in group III of C. sinense, D. chrysotoxum, and D. huoshanense was located on an independent chromosome. However, four chromosomes (Chr3, 4, 5, and 14) were exclusively divided into six COLs belonging to group II in the genome of V. planifolia.
A collinearity analysis was performed between C. sinense and five plant species (Supplementary Table S5), including one dicot (A. thaliana) and four monocots (D. huoshanense, D. chrysotoxum, V. planifolia, and O. sativa). The number of COL collinearity gene pairs between C. sinense and A. thaliana, D. huoshanense, D. chrysotoxum, V. planifolia, and O. sativa are 1, 7, 8, 8, and 2, respectively (Supplementary Table S5). The comparative genome analysis revealed more conserved collinear blocks (Figure 4), and the collinear analysis with orchids revealed multiple-to-one phenomena. For example, CsiCOL1 has a collinearity relationship with both VplCOL06 and VplCOL08. However, relatively few collinearity gene pairs were detected between C. sinense and the model plant A. thaliana or O. sativa. With more collinearity gene pairs of the COL genes (one on one), the collinearity analysis between C. sinense and the other three Orchidaceae showed that COL genes in groups I, II, and III were involved in the formation of collinearity gene pairs; however, only group I and II COL members in C. sinense produced collinearity with those in A. thaliana and O. sativa, indicating that the COL sequences in group I and II are relatively conserved in the evolutionary history.
For the analysis of the selection pressure, we calculated the ratio of the non-synonymous substitutions per non-synonymous site (Ka) to the synonymous substitutions per synonymous site (Ks) of 40 gene pairs that were selected on the basis of sequence similarity. The results showed that the Ka/Ks ratios of all COL genes were <1, with most values < 0.4, indicating that all orchid COLs have undergone strong purifying selection (Supplementary Table S6) [43].

2.5. Cis-Acting Elements in the Promoters of Orchid COL Genes

Cis-acting elements in the promoters of orchid COL genes: In the promoter regions of Orchidaceae COL genes (Figure 5 and Table S7). the composition of cis-acting elements was detected. We detected 941 light-responsive evolved elements, including G-box, GT1 motif, GATA motif, ACE, and 3-AF1 binding sites. These elements indicate that COL genes can be used as light sensors in flowering plants [44]. Among them, 70 and 209 promoter regions were found to contain GT1 motif and G-box cis-acting elements, respectively. Phytohormone-responsive elements, mainly correlated with GA (GARE motif), MeJA (CGTCA motif), and auxin (AuxRR core), were also detected in the promoter regions. Moreover, the developmental element CAT-box was found at three promoter regions, and its specific function is associated with meristematic expression. A circadian element was found in 3 (AshCOL01, AshCOL06, and AshCOL09) in A. shenzhenica, 2 (CsiCOL01 and CsiCOL03) in C. sinense, 2 (DcaCOL05 and DcaCOL09) in D. catenatum, 1 (DhuCOL07) in D. huoshanense, and 1 (VplCOL06) in V. planifolia, corresponding to the circadian expression pattern of COL genes [45,46]. The possible functions and expression patterns of COL genes are related to the composition of cis-acting elements.

2.6. Expression Profiles of Orchid COL Genes in Different Tissues and Differentially Expressed CsiCOL Genes during ABA Treatment

We analyzed the expression based on seven Orchidaceae transcriptome data in different tissues, including roots, stems, leaves, flowers, fruits, and seeds, to shed light on the potential functions of orchid COL genes during plant development. The COL genes showed different patterns of expression in the different tissues (Figure 6). For example, VplCOL08 was highly expressed in all tissues, while VplCOL01 and VplCOL09 were lowly expressed. All genes were highly expressed in specific tissues. This suggests that they may have critical functions in these tissues. For example, CsiCOL4 and CsiCOL6 showed a high level of expression in flowers but a low level of expression in leaves. Interestingly, CsiCOL genes showed tissue-specific expression in individual floral organs of different flower varieties (Figure 7). For example, CsiCOL09 was more highly expressed in the column, while CsiCOL01 showed complementary expression patterns, suggesting that the two genes play opposite roles in column formation; similar patterns occur in regard to the Arabidopsis flowering time, which is regulated by CO and COL09 genes [47]. In addition, CsiCOL05 was highly expressed in all cultivars, indicating an essential function in flower development. Under ABA treatment, the expression of CsiCOL04 and CsiCOL10 in the leaves was highly reduced (Supplementary Figure S1).

2.7. Protein Structure Prediction

The tertiary structure of most orchid COLs is highly conserved, characterized by four β-sheet and four α-helices; the N-terminus had an anti-parallel β-sheet formed by β1 and β2. The α1 helix aligned in parallel with the β-sheet. A C-terminal short α2 helix was present vertically against α1, forming the compact structure (Figure 8). The mirrored structure with a similar arrangement was connected by a loop structure. Except for 3 COLs (VplCOL02, VplCOL03, and CsiCOL04), which showed two β-sheets and three α-helices, 16 COLs (AshCOL02, AshCOL05, AshCOL08, DcaCOL05, DcaCOL08, DhuCOL04-07, VplCOL14, DchCOL01, DchCOL02, DchCOL05-07, CsiCOL07, and CsiCOL10) showed two β-sheets and two α-helices, DchCOL09 had two β-sheets and one α-helices, 2 COLs (VplCOL05 and VplCOL06) had two α-helices, and 3 COLs (AshCOL07, VplCOL02, and PeqCOL04) exhibited only two β-sheets. Each strand in the asymmetric unit interacts with symmetrically related molecules on both sides to form an extended linear head-to-tail oligomeric configuration, resulting in an enhanced performance in transcriptional regulation [48]. Secondary structure prediction revealed that all orchid COL proteins are composed of an α-helix (Hh), extended strands (Ee), β-turns (Tt), and random coils (Cc), which, on average, account for 31.07%, 10.65%, 3.14%, and 55.15% of the protein structure, respectively (Supplementary Table S8).

2.8. Correlation Analysis between CsiCOLs and Regulation of Co-Activity-Related Transcription Factors

To determine the potential transcriptional regulation mechanism of CsiCOL, the cor-relation analysis between the expression of the CsiCOLs and CO-activated transcription factor was performed (Figure 9). The results indicated that three groups of CsiCOL members showed a significant correlation with SHAGGY-like kinase 12 (SK12: Mol003238, Mol012561, and Mol003091) and PSEUDO-RESPONSE REGULATOR (PRR: Mol011035 and Mol029571) transcription factors, suggesting conserved functions of the genes within each cluster, such as, in plant growth, stress-related metabolic regulation [49], and the circadian clock [50]. JUMANJI 28 (JMJ28: Mol011862) was significantly related to CsiCOLs in group I and group II. FLOWERING BHLH (FBH: Mol010847) was significantly correlated with CsiCOLs only in group I, while FK506-binding protein (FKBP12: Mol015806) was significantly correlated with CsiCOLs only in group III. Additionally, three genes were only significantly associated with CsiCOLs in group II, that is, GIGANTEA (GI: Mol017612), TARGET OF AT (TOE: Mol008285), and FLAVIN-BINDING KELCHREPEAT F-BOX1 (FKF1: Mol013985), respectively. This also indicates that the functions of CsiCOL genes diversified during evolution.

2.9. Exploration of Subcellular Localization and Regulatory Analysis by Transient Overexpression of CsiCOLs in Cymbidium Protoplasts

Protoplast transformation is particularly important for studying gene function in crop species, which often have unique genetic traits that are not present in model plants [51,52]. To elucidate the influence of CsiCOLs on the temporal association of flowering and floral morphogenesis (Figure 10A), we initiated a transient overexpression experiment, using CsiCOL05 and CsiCOL09 in C. sinense protoplasts. Subcellular localization studies reveal a nuclear disposition of CsiCOL05. In contrast, the subcellular location of CsiCOL09 is observed in both the nucleus and cell membrane (Figure 10B). These results, derived from subcellular localization assessments, suggest that different COLs genes may have different functions in orchids. We selected ten candidate genes: four FLOWERING LOCUST (FT), TERMINAL FLOWER 1 (TFL1), APETALA1 (AP1), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), LEAFY (LFY), and AGAMOUS1 (AG1), based on gene ontology and the extant literature, which were then validated using quantitative real-time polymerase chain reaction (qRT-PCR) (Supplementary Table S4). The FT/TFL1 genes form the phosphatidylethanolamine binding protein (PEBP) family, which has been implicated in activating flowering downstream of COL [21,53,54]. Previous studies have implicated AP1, SOC1, LFY, and AG1 in the regulation of flowering duration and floral patterning in orchids [6,55,56,57]. Notably, CsiCOL05 had a significantly higher expression; meanwhile, for CsiCOL09, the expression level was much lower in gynostemium (Figure 10A). The relative expression level of four genes (CsiFT, CsiAP1, CsiSOC1, and CsiLFY) was significantly upregulated compared to the control, and the expression level of two genes (CsiTFL1 and CsiAG1) was significantly downregulated in CsiCOL05-OE (Figure 10C). In contrast, the opposite result was observed for CsiCOL09-OE (Figure 10D). In summary, our results demonstrated that CsiCOL05 and CsiCOL09 have similar functions but different regulatory modes.

3. Discussion

Although the COL gene family has been reported in many plants, it has rarely been reported in Orchidaceae. The orchid is a mainstay of the worldwide floriculture trade and a popular ornamental crop [58,59], so the completion of the genomes of orchids provided an opportunity to study the COL gene family in Orchidaceae species. A comparison of COL gene families in seven orchids showed that they all have the typical conserved domains of the BBX and CCT domains [26]. The occurrence of B-box domains in orchid species may suggest that COLs, which respond to biotic [60] and abiotic stresses [61] in angiosperms, may control similar functions in Orchidaceae. The second motif of the CCT domain is important in mediating protein–protein interactions, such as Nuclear Factor-Y (NFY) [62], CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) [63], and TOPLESS (TPL) [64]. Therefore, variations in CCT domains could potentially explain the significant diversity in flowering time observed across Orchidaceae [65].
In this study, the COL gene family was systematically compared and analyzed. Seven orchids, Arabidopsis, and rice were used to construct a phylogenetic tree. However, not all of the COL proteins that are clustered in group I have two B-box domains. For example, CsiCOL04 and CsiCOL09 were classified in group I but contain only one B-box domain. In this study, the phylogenetic tree-based classification results are not exactly the same as in Arabidopsis [20,32]. The phylogenetic relationship of the orchid COL genes in accordance with that of the Orchidaceae genome indicates the conservation nature and necessity of the COL genes in orchids [66]. During plant evolution, gene duplication events played a critical role in expanding gene families [67]. There were only two pairs in D. huoshanense, which was much less than in V. planifolia (8). The significant proliferation of orchid COL duplicates in V. planifolia may be attributed to the protracted flowering duration through natural selection. The collinearity analysis revealed that segregation was key to COL gene family expansion. As the first study on COL gene families in orchids, our study serves as a useful data resource for future comparative and functional genome studies on COL gene families.
Among the hundreds of flowering time genes that have been described to date [68]. COL is a key regulator of photoperiod flowering [69]. COL binds the conserved TGTG (N2-3) ATG motif in the FT promoter region, thereby activating FT/TFL to regulate the flowering time [70,71]. However, FT and TFL1 have contrasting effects on both the flowering time and floral pattern [72]. Until now, only a few studies have defined the COL gene family in Orchidaceae species. The overexpression of PaCOL1 in Arabidopsis exhibited earlier flowering under short-day (SD) conditions [73]. Two genes from cymbidium homology with CsiCOL05, CsCOL1, and CeCOL were overexpressed in Arabidopsis, resulting in early flowering and increased levels of AtFT expression under long-day (LD) conditions [74]. Similarly, the ectopic expression of Orchid FT/TFL from Oncidium Gower Ramsey and Dendrobium in Arabidopsis promoted or inhibited the flowering time and loss of inflorescence indeterminacy [75,76]. The basic leucine-zipper (bZIP) transcription factor FLOWERING LOCUS D (FD) plays a key role in the opposing interaction of FT and TFL1, which is dependent on competitive binding partners. This interaction triggers the transition from vegetative to reproductive growth by activating genes related to inflorescence and floral meristem identity, such as SOC1, AP1, and LFY [21,77,78]. Simultaneously, AG1 was found to be linked to the formation of the column in orchids [57,79]. Combined with the changes of related genes after the overexpression of CsiCOL05 and CsiCOL09 in protoplasts, we speculated over the antagonistic effects of CsiCOL05 and CsiCOL09 on the regulation of the flowering time and gynostemium development in C. sinense (Figure 11).
In addition, in grapevine, VvCOL1 has been implicated in regulating the induction and maintenance of bud dormancy [80]. MaCOL1 was reported to be associated with stress tolerance and abiotic fruit ripening in the banana [81]. TaCol-B5, orthologous to COL5, modifies spike architecture and enhances grain yield in wheat [82]. The participation of ABA is seen in the regulation of plant growth and plant development, and it helps ensure yield stability [83]. In pak choi, the floral transition can be expedited, as ABA directly stimulates BrCO transcription through the involvement of BrABF3 (abscisic acid-responsive transcription factors, ABF) [84]. Upon ABA treatment, CsiCOL5 exhibited heightened expression throughout all tissues, underscoring its crucial involvement in the overall growth and development of C. sinense.
With the application of cell engineering and gene editing technologies in orchid molecular breeding [85,86], we anticipate the identification of a growing number of valuable functional genes. Orchid COLs, serving as key functional genes governing abiotic stress responses, flowering time regulation, and flower development within the Orchidaceae family, represent a valuable resource for enhancing orchid breeding. For instance, the Cymbidium gynostemium-like perianth variety will lose its floral scent [56,87], while the regulation of gynostemium development may be influenced by CsiCOLs. Moreover, a majority of orchids produce exceedingly minuscule seeds, often described as ‘dust-like’ [88,89]. Intriguingly, recent research has uncovered that the COL gene has the potential to govern seed size in plants [90,91]. This is not only essential for advancing the creation of new varieties with desirable traits through molecular breeding methods but also for comprehending the consequences of gene duplication and loss in the evolution of Orchidaceae and other plant species.

4. Materials and Methods

4.1. Basic Characterization of COL Genes in Orchidaceae

All COL proteins were identified as containing both B-box and CCT domains [20]. All COL genes in the genomes of C. sinense, P. equestris, D. catenatum, D. chrysotoxum, D. huoshanense, V. planifolia, and A. shenzhenica were identified using the Hidden Markov Model (HMM) program and related Pfam accessions (B-box and CCT domains corresponding to PF00643.19 and PF06203.9). All selected COL proteins were further identified using the Pfam database (http://pfam.xfam.org/, (accessed on 26 December 2022)) and Blastp in NCBI (https://www.ncbi.nlm.nih.gov/, (accessed on 30 December 2022)). The conserved domains B-box and CCT were confirmed. In order to distinguish the COL genes, we named them on the basis of their physical location on the chromosomes in the orchid’s genome. Basic information on the number of amino acids, molecular weight, theoretical isoelectric point (pI), and instability index (with a value < 40 considered as stable) was obtained using the ProtParam tool (https://web.expasy.org/protparam/, (accessed on 18 January 2023)). Then, the subcellular location information was obtained using the online tool BUSCA (http://busca.biocomp.unibo.it, (accessed on 22 January 2023)). All the basic information of the COL genes can be found in Supplementary Table S1.

4.2. Phylogenetic Tree Construction

To explore the phylogenetic relationships and taxonomy of COL genes, the phylogenetic tree was constructed using COL proteins from seven orchids, Arabidopsis, and rice. The AthCOL and OsaCOL protein sequences come from the TAIR database (http://www.arabidopsis.org/, (accessed on 15 December 2022)) and Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/, (accessed on 15 December 2022)). Sequence alignments were performed using the most accurate algorithm, JTT + I + G4, with 1000 cycles of iterative refinement, using MAFFT v.7.475 [92]. The phylogenetic tree was reconstructed from 1000 replicates of the ultrafast bootstrap ML tree, using IQ-TREE v1.6.12 [93], with the selection of the best-fitting model by ModelFinder [94].

4.3. Chromosomal Location, Comparative Genome Collinearity Analysis, and Selective Pressure

Chromosomal location, comparative genome collinearity analysis, and selective pressure. The collinearity relationship of COL genes in C. sinense and the other five plant species (D. huoshanense, D. chrysotoxum, V. planifolia, A. thaliana and O. sativa) was identified using MCScanX [95]. The corresponding COL genes were mapped to chromosomes based on physical location from the orchid genome database, the TAIR database, and the rice genome annotation project. Gene colinear relationships were visualized using Tbtools v1.125 and Circos software v2.0 [96]. KaKs_Calculator 3.0 software [97] was then used to calculate the synonymous substitution rate (Ka) and nonsynonymous substitution rate (Ks) for gene pairs.

4.4. Conserved Motifs of Orchidaceae COL Genes

Basic information, such as the physical location, amino acid sequence, and nucleotide sequence, of orchid COL genes was obtained from seven Orchidaceae genomes. Conserved motifs were identified using the MEME online site (http://meme-suite.org/tools/meme, (accessed on 11 February 2023)) with the following parameters: maximum motifs number, 10; minimum and maximum widths, 6 and 50. The basic information about the sequence of the motifs is listed in Supplementary Table S2.

4.5. Analysis of the Cis-Acting Elements

For the analysis of cis-acting elements in their promoter region, the upstream sequences (2000 bp) of the orchid COL genes were collected. This analysis was carried out with PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, (accessed on 19 February 2023)) [98], and the results were exported with Tbtools [42].

4.6. Growth Conditions of C. sinense, Sample Collection, and Abscisic Acid (ABA) Treatments

C. sinense ‘Qi Hei’ is a famous traditional cultivar in China. For the ABA treatment, we used 2-year-old C. sinense ‘Qi Hei’ in flowerpots sprayed weekly with 100 μM ABA for one month, with deionized water treatment as control groups. Each biological replicate was a mixture of three plants. All the plants in this study were grown in the greenhouse of the Institute of Environmental Horticulture of the Guangdong Academy of Agricultural Sciences (Guangzhou, China). The greenhouse was maintained at 25 ± 1 °C and 80% humidity. Roots, stems, leaves, flowers, and fruit tissues were collected from the cultivated plantlets. All the samples were collected and immediately frozen in liquid nitrogen and then stored at −80 °C until they were used for further analysis.

4.7. Protein Tertiary Structure Prediction

SWISS-MODEL (https://swissmodel.expasy.org/interactive, (accessed on 25 February 2023)) [99] was used to draw and visualize the protein tertiary structure prediction of orchid COLs. The tertiary structure is rainbow-colored to represent order from the N-terminus to C-terminus. The program SOPMA (https://npsa-prabi.ibcp.fr/, (accessed on 25 February 2023)) [100] was used to predict the secondary structure.

4.8. Correlation Analysis between CsiCOLs and Transcription Factors

To predict the regulation of CsiCOLs activity, transcription factors whose function has been verified in Arabidopsis [101] were used as query sequences to identify the homologous genes of C. sinense, including TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATINGCELLFACTOR (TCP) (AtTCP2, AtTCP3, AtTCP4, AtTCP10 and AtTCP24), PHYTOCHROME AND FLOWERING TIME1 (PFT1), GI, JMJ28, FBH1, FBH2, FBH3, FBH4, CYCLING DOF FACTOR (CDF) (CDF1, CDF2, CDF3, CDF4, CDF5 and CDF6), TOE1, TOE2, TOE3, CRYPTOCHROME (CRY) (CRY1 and CRY2), PHYTOCHROME-DEPENDENT LATE-FLOWERING (PHL), FKF1, PHYTOCHROME (PHY), PHYA, PHYB, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1), TIMING OF CAB EXPRESSION 1 (TOC1), PRR (PRR5, PRR7 and PRR9), CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), SUPPRESSOR OF PHYTOCHROME A (SPA) (SPA1, SPA3 and SPA4), SK12, FKBP12, and EARLY FLOWERING 3 (ELF3) (Supplementary Table S3). In addition, the ggcor function in R language 4.2 was used to visualize the correlation heat map.

4.9. C. sinense Leaf Protoplast Transformation and Subcellular Localization

Protoplast isolation and transfection from the leaf bases of C. sinense were carried out based on previous protocols [102,103]. The vectors used were generated by cloning the coding sequences (CDSs) of CsiCOL05 and CsCOL09 into the PAN580-GFP vector. The aforementioned vectors containing pAN580-GFP, pAN580-CsiCOL05-GFP, and pAN580-CsCOL09-GFP were then introduced into Escherichia coli DH5α competent cells (Tiangen, Beijing, China) according to the manufacturer’s guidelines. This step was followed by large-scale bacterial propagation and extraction of plasmid DNA, using the Endo-Free Plasmid Maxi Kit (Omega Biotek, Norcross, GA, USA). The resulting plasmid DNA was then concentrated and adjusted to various concentrations, up to 2.0 μg/μL, before being introduced into orchid protoplasts. After incubation at 23 °C in a light-free environment for 16 h, the fluorescence emitted by the GFP or GFP-protein fusions could be observed using an LSM710 confocal laser scanning microscope. For nuclear visualization, the transfected protoplasts were stained with 50 μg/mL DAPI (Sigma-Aldrich Chemie, Steinheim, Germany) at 37 °C for 10 min. Excitation of DAPI signals was performed with a blue diode laser, using a 405 nm excitation line with a 485 nm long-pass barrier filter.

4.10. RNA Extraction and qRT-PCR Analysis

The transfectants (5 × 105) were collected independently in three biological replicates and stored at −80 °C immediately after being frozen in liquid nitrogen. Total RNA was extracted from these samples, using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then, using 1 μg RNA, a 20 μL cDNA system was synthesized according to the guidelines of the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Otsu, Japan). qRT-PCR was performed on the Bio-Rad iCycler Real-Time PCR Detection System (Hercules, CA, USA). This was performed with three biological replicates, using TaKaRa SYBR Premix Ex Taq™ (Tli RNaseH Plus, TaKaRa, Otsu, Japan). The total reaction system consisted of 20 μL containing 10 μL of SYBR Premix (2×), 1 μL cDNA, 1 μL each of sense and antisense primer (10 μM), and 7 μL of ddH2O. The qRT-PCR program was structured as follows: an initial predenaturation phase at 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 56 °C for 30 s, and a final extension phase at 72 °C for 30 s. We used Primer Premier 5.0 for primer pair design, and the NCBI Blast program was used to determine the specificity of all primers (see Supplementary Table S4). The β-actin gene (Mol013347) in C. sinense was used as an internal reference. Relative expression levels were calculated using the 2−ΔΔCT method [104].

5. Conclusions

Within the scope of this study, a total of 68 COL genes were successfully identified across seven distinct orchid species, and their phylogenetic tree, gene structure, chromosome localization, collinearity, gene replication, selective pressure, cis-acting elements, expression patterns, and protein structure were characterized. Furthermore, we delved into the regulation of co-activity-associated transcription factors within C. sinense. Remarkably, we conducted an exploration of subcellular localization and performed regulatory analyses through the transient overexpression of two CsiCOLs in Cymbidium protoplasts. We plotted potential regulatory patterns concerning their impact on flowering time and flower development. Our findings offer valuable insights and a conceptual foundation for exploring the unique functions of orchid growth and development, which carry significant implications for contemporary orchid breeding practices.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms242316825/s1.

Author Contributions

Conceptualization, G.Z. and F.Y.; methodology, Y.W.; software, C.L.; validation, Z.L., W.Z. and J.L.; formal analysis, Q.X.; investigation, J.J. and Y.W; resources, G.Z.; data curation, Y.W.; writing—original draft preparation, Y.W.; writing—review and editing, F.Y.; visualization, J.G.; supervision, Y.W.; project administration, G.Z.; funding acquisition, G.Z. and F.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the 2022 Provincial Rural Revitalization Strategy Special Fund Seed Industry Revitalization Project (2022-NBA-00-015), National Key RD Program (2018YFD1000400 and 2019YFD1001003), Innovation Team of Modern Agriculture Industry Technology System in Guangdong Province (2023KJ121), and Guangdong Academy of Agricultural Sciences Discipline Team Construction Project (202127TD, XT202212, R2020PY-JX018, and BZ202006).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Fay, M.F.; Chase, M.W. Orchid biology: From Linnaeus via Darwin to the 21st century. Preface. Ann. Bot. 2009, 104, 359–364. [Google Scholar] [CrossRef] [PubMed]
  2. Chase, M.W.; Cameron, K.M.; Freudenstein, J.V.; Pridgeon, A.M.; Salazar, G.; van den Berg, C.; Schuiteman, A. An updated classification of Orchidaceae. Bot. J. Linn. Soc. 2015, 177, 151–174. [Google Scholar] [CrossRef]
  3. Kindlmann, P.; Kull, T.; McCormick, M. The Distribution and Diversity of Orchids. Diversity 2023, 15, 810. [Google Scholar] [CrossRef]
  4. Mérillon, J.-M.; Kodja, H. Orchids Phytochemistry, Biology and Horticulture; Springer: Berlin/Heidelberg, Germany, 2019. [Google Scholar] [CrossRef]
  5. Zhang, D.; Zhao, X.; Li, Y.; Ke, S.; Yin, W.; Lan, S.; Liu, Z. Advances and prospects of orchid research and industrialization. Hortic. Res. 2022, 9, uhac220. [Google Scholar] [CrossRef] [PubMed]
  6. Li, Y.; Zhang, B.; Yu, H. Molecular genetic insights into orchid reproductive development. J. Exp. Bot. 2022, 73, 1841–1852. [Google Scholar] [CrossRef]
  7. Hsiao, Y.Y.; Fu, C.H.; Ho, S.Y.; Li, C.I.; Chen, Y.Y.; Wu, W.L.; Wang, J.S.; Zhang, D.Y.; Hu, W.Q.; Yu, X.; et al. OrchidBase 4.0: A database for orchid genomics and molecular biology. BMC Plant Biol. 2021, 21, 371. [Google Scholar] [CrossRef]
  8. Liaqat, F.; Xu, L.; Khazi, M.I.; Ali, S.; Rahman, M.U.; Zhu, D. Extraction, purification, and applications of vanillin: A review of recent advances and challenges. Ind. Crop. Prod. 2023, 204, 117372. [Google Scholar] [CrossRef]
  9. Das, P.; Chandra, T.; Negi, A.; Jaiswal, S.; Iquebal, M.A.; Rai, A.; Kumar, D. A comprehensive review on genomic resources in medicinally and industrially important major spices for future breeding programs: Status, utility and challenges. Curr. Res. Food Sci. 2023, 7, 100579. [Google Scholar] [CrossRef]
  10. Hammer, K.; Khoshbakht, K. A domestication assessment of the big five plant families. Genet. Resour. Crop Evol. 2015, 62, 665–689. [Google Scholar] [CrossRef]
  11. Freudenstein, J.V.; Chase, M.W. Phylogenetic relationships in Epidendroideae (Orchidaceae), one of the great flowering plant radiations: Progressive specialization and diversification. Ann. Bot. 2015, 115, 665–681. [Google Scholar] [CrossRef] [PubMed]
  12. Wu, W.; Lin, Y.; Farag, M.A.; Li, Z.; Shao, P. Dendrobium as a new natural source of bioactive for the prevention and treatment of digestive tract diseases: A comprehensive review with future perspectives. Phytomedicine 2023, 114, 154784. [Google Scholar] [CrossRef] [PubMed]
  13. Hew, C.S.; Wong, Y.S. Chinese Cymbidium Orchid: History of Chinese Cymbidium; World Scientific: Singapore, 2023. [Google Scholar] [CrossRef]
  14. Boss, P.K.; Bastow, R.M.; Mylne, J.S.; Dean, C. Multiple pathways in the decision to flower: Enabling, promoting, and resetting. Plant Cell 2004, 16 (Suppl. S1), S18–S31. [Google Scholar] [CrossRef] [PubMed]
  15. Baurle, I.; Dean, C. The timing of developmental transitions in plants. Cell 2006, 125, 655–664. [Google Scholar] [CrossRef] [PubMed]
  16. Wickland, D.P.; Hanzawa, Y. The FLOWERING LOCUS T/TERMINAL FLOWER 1 Gene Family: Functional Evolution and Molecular Mechanisms. Mol. Plant 2015, 8, 983–997. [Google Scholar] [CrossRef] [PubMed]
  17. Xu, S.; Chong, K. Remembering winter through vernalisation. Nat. Plants 2018, 4, 997–1009. [Google Scholar] [CrossRef]
  18. Taylor, C.M.; Kamphuis, L.G.; Zhang, W.; Garg, G.; Berger, J.D.; Mousavi-Derazmahalleh, M.; Bayer, P.E.; Edwards, D.; Singh, K.B.; Cowling, W.A.; et al. INDEL variation in the regulatory region of the major flowering time gene LanFTc1 is associated with vernalization response and flowering time in narrow-leafed lupin (Lupinus angustifolius L.). Plant Cell Environ. 2019, 42, 174–187. [Google Scholar] [CrossRef] [PubMed]
  19. Zhang, W.; Yuan, J.; Cheng, T.; Tang, M.J.; Sun, K.; Song, S.L.; Xu, F.J.; Dai, C.C. Flowering-mediated root-fungus symbiosis loss is related to jasmonate-dependent root soluble sugar deprivation. Plant Cell Environ. 2019, 42, 3208–3226. [Google Scholar] [CrossRef] [PubMed]
  20. Robson, F.; Costa, M.M.; Hepworth, S.R.; Vizir, I.; Pineiro, M.; Reeves, P.H.; Putterill, J.; Coupland, G. Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants. Plant J. 2001, 28, 619–631. [Google Scholar] [CrossRef]
  21. Jin, S.; Nasim, Z.; Susila, H.; Ahn, J.H. Evolution and functional diversification of FLOWERING LOCUS T/TERMINAL FLOWER 1 family genes in plants. Semin. Cell Dev. Biol. 2021, 109, 20–30. [Google Scholar] [CrossRef]
  22. Nam, J.; dePamphilis, C.W.; Ma, H.; Nei, M. Antiquity and evolution of the MADS-box gene family controlling flower development in plants. Mol. Biol. Evol. 2003, 20, 1435–1447. [Google Scholar] [CrossRef]
  23. Huang, J.; BolañosVillegas, P.; Chen, F. Regulation of Flowering in Orchids. In The Orchid Genome; Chen, F., Chin, S., Eds.; Springer International Publishing: Cham, Switzerland, 2021; pp. 73–94. [Google Scholar] [CrossRef]
  24. Imaizumi, T.; Schultz, T.F.; Harmon, F.G.; Ho, L.A.; Kay, S.A. FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis. Science 2005, 309, 293–297. [Google Scholar] [CrossRef] [PubMed]
  25. Khanna, R.; Kronmiller, B.; Maszle, D.R.; Coupland, G.; Holm, M.; Mizuno, T.; Wu, S.H. The Arabidopsis B-box zinc finger family. Plant Cell 2009, 21, 3416–3420. [Google Scholar] [CrossRef] [PubMed]
  26. Gangappa, S.N.; Botto, J.F. The BBX family of plant transcription factors. Trends Plant Sci. 2014, 19, 460–470. [Google Scholar] [CrossRef] [PubMed]
  27. Putterill, J.; Robson, F.; Lee, K.; Simon, R.; Coupland, G. The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors. Cell 1995, 80, 847–857. [Google Scholar] [CrossRef] [PubMed]
  28. Wang, L.; Xue, J.; Dai, W.; Tang, Y.; Gong, P.; Wang, Y.; Zhang, C. Genome-wide Identification, Phylogenetic Analysis, and Expression Profiling of CONSTANS-like (COL) Genes in Vitis vinifera. J. Plant Growth Regul. 2019, 38, 631–643. [Google Scholar] [CrossRef]
  29. Cao, D.; Lin, Z.; Huang, L.; Damaris, R.N.; Li, M.; Yang, P. A CONSTANS-LIKE gene of Nelumbo nucifera could promote potato tuberization. Planta 2021, 253, 65. [Google Scholar] [CrossRef]
  30. Li, J.; Gao, K.; Yang, X.; Khan, W.U.; Guo, B.; Guo, T.; An, X. Identification and characterization of the CONSTANS-like gene family and its expression profiling under light treatment in Populus. Int. J. Biol. Macromol. 2020, 161, 999–1010. [Google Scholar] [CrossRef] [PubMed]
  31. Khatun, K.; Debnath, S.; Robin, A.H.K.; Wai, A.H.; Nath, U.K.; Lee, D.J.; Kim, C.K.; Chung, M.Y. Genome-wide identification, genomic organization, and expression profiling of the CONSTANS-like (COL) gene family in petunia under multiple stresses. BMC Genom. 2021, 22, 727. [Google Scholar] [CrossRef] [PubMed]
  32. Griffiths, S.; Dunford, R.P.; Coupland, G.; Laurie, D.A. The evolution of CONSTANS-like gene families in barley, rice, and Arabidopsis. Plant Physiol. 2003, 131, 1855–1867. [Google Scholar] [CrossRef]
  33. Song, N.; Xu, Z.; Wang, J.; Qin, Q.; Jiang, H.; Si, W.; Li, X. Genome-wide analysis of maize CONSTANS-LIKE gene family and expression profiling under light/dark and abscisic acid treatment. Gene 2018, 673, 1–11. [Google Scholar] [CrossRef]
  34. Niu, T.; Wang, X.; Abbas, M.; Shen, J.; Liu, R.; Wang, Z.; Liu, A. Expansion of CONSTANS-like genes in sunflower confers putative neofunctionalization in the adaptation to abiotic stresses. Ind. Crops Prod. 2022, 176, 114400. [Google Scholar] [CrossRef]
  35. Chaurasia, A.K.; Patil, H.B.; Azeez, A.; Subramaniam, V.R.; Krishna, B.; Sane, A.P.; Sane, P.V. Molecular characterization of CONSTANS-Like (COL) genes in banana (Musa acuminata L. AAA Group, cv. Grand Nain). Physiol. Mol. Biol. Plants 2016, 22, 1–15. [Google Scholar] [CrossRef]
  36. Song, X.; Duan, W.; Huang, Z.; Liu, G.; Wu, P.; Liu, T.; Li, Y.; Hou, X. Comprehensive analysis of the flowering genes in Chinese cabbage and examination of evolutionary pattern of CO-like genes in plant kingdom. Sci. Rep. 2015, 5, 14631. [Google Scholar] [CrossRef] [PubMed]
  37. Cai, D.; Liu, H.; Sang, N.; Huang, X. Identification and characterization of CONSTANS-like (COL) gene family in upland cotton (Gossypium hirsutum L.). PLoS ONE 2017, 12, e0179038. [Google Scholar] [CrossRef] [PubMed]
  38. Perez-Escobar, O.A.; Bogarín, D.; Przelomska, N.A.; Ackerman, J.D.; Balbuena, J.A.; Bellot, S.; Bühlmann, R.P.; Cabrera, B.; Cano, J.A.; Charitonidou, M. The Origin and Speciation of Orchids. bioRxiv 2023. bioRxiv:2023.09.10.556973. [Google Scholar] [CrossRef]
  39. Gamage, D.G.; Gunaratne, A.; Periyannan, G.R.; Russell, T.G. Applicability of Instability Index for In vitro Protein Stability Prediction. Protein Pept. Lett. 2019, 26, 339–347. [Google Scholar] [CrossRef] [PubMed]
  40. Lau, K.; Podolec, R.; Chappuis, R.; Ulm, R.; Hothorn, M. Plant photoreceptors and their signaling components compete for COP1 binding via VP peptide motifs. EMBO J. 2019, 38, e102140. [Google Scholar] [CrossRef]
  41. Tian, Z.; Qin, X.; Wang, H.; Li, J.; Chen, J. Genome-Wide Identification and Expression Analyses of CONSTANS-Like Family Genes in Cucumber (Cucumis sativus L.). J. Plant Growth Regul. 2022, 41, 1627–1641. [Google Scholar] [CrossRef]
  42. 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] [PubMed]
  43. Zhang, Z.; Li, J.; Zhao, X.; Wang, J.; Wong, G.K.; Yu, J. KaKs_Calculator: Calculating Ka and Ks Through Model Selection and Model Averaging. Genom. Proteom. Bioinform. 2006, 4, 259–263. [Google Scholar] [CrossRef]
  44. Simon, S.; Ruhl, M.; de Montaigu, A.; Wotzel, S.; Coupland, G. Evolution of CONSTANS Regulation and Function after Gene Duplication Produced a Photoperiodic Flowering Switch in the Brassicaceae. Mol. Biol. Evol. 2015, 32, 2284–2301. [Google Scholar] [CrossRef] [PubMed]
  45. Campoli, C.; Drosse, B.; Searle, I.; Coupland, G.; von Korff, M. Functional characterisation of HvCO1, the barley (Hordeum vulgare) flowering time ortholog of CONSTANS. Plant J. 2012, 69, 868–880. [Google Scholar] [CrossRef] [PubMed]
  46. Kikuchi, R.; Kawahigashi, H.; Oshima, M.; Ando, T.; Handa, H. The differential expression of HvCO9, a member of the CONSTANS-like gene family, contributes to the control of flowering under short-day conditions in barley. J. Exp. Bot. 2012, 63, 773–784. [Google Scholar] [CrossRef]
  47. Cheng, X.F.; Wang, Z.Y. Overexpression of COL9, a CONSTANS-LIKE gene, delays flowering by reducing expression of CO and FT in Arabidopsis thaliana. Plant J. 2005, 43, 758–768. [Google Scholar] [CrossRef]
  48. Zeng, X.; Lv, X.; Liu, R.; He, H.; Liang, S.; Chen, L.; Zhang, F.; Chen, L.; He, Y.; Du, J. Molecular basis of CONSTANS oligomerization in FLOWERING LOCUS T activation. J. Integr. Plant Biol. 2022, 64, 731–740. [Google Scholar] [CrossRef] [PubMed]
  49. Li, C.; Zhang, B.; Yu, H. GSK3s: Nodes of multilayer regulation of plant development and stress responses. Trends Plant Sci. 2021, 26, 1286–1300. [Google Scholar] [CrossRef] [PubMed]
  50. Hayama, R.; Sarid-Krebs, L.; Richter, R.; Fernandez, V.; Jang, S.; Coupland, G. PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length. EMBO J. 2017, 36, 904–918. [Google Scholar] [CrossRef]
  51. Xu, Y.; Li, R.; Luo, H.; Wang, Z.; Li, M.W.; Lam, H.M.; Huang, C. Protoplasts: Small cells with big roles in plant biology. Trends Plant Sci. 2022, 27, 828–829. [Google Scholar] [CrossRef] [PubMed]
  52. Chen, Z.; Debernardi, J.M.; Dubcovsky, J.; Gallavotti, A. Recent advances in crop transformation technologies. Nat. Plants 2022, 8, 1343–1351. [Google Scholar] [CrossRef] [PubMed]
  53. Su, C.; Wang, Y.; Yu, Y.; He, Y.; Wang, L. Coordinative regulation of plants growth and development by light and circadian clock. aBIOTECH 2021, 2, 176–189. [Google Scholar] [CrossRef]
  54. Wang, S.-L.; An, H.R.; Tong, C.-G.; Jang, S. Flowering and flowering genes: From model plants to orchids. Hortic. Environ. Biotechnol. 2021, 62, 135–148. [Google Scholar] [CrossRef]
  55. Teo, Z.W.N.; Zhou, W.; Shen, L. Dissecting the Function of MADS-Box Transcription Factors in Orchid Reproductive Development. Front. Plant Sci. 2019, 10, 1474. [Google Scholar] [CrossRef] [PubMed]
  56. Yang, F.X.; Gao, J.; Wei, Y.L.; Ren, R.; Zhang, G.Q.; Lu, C.Q.; Jin, J.P.; Ai, Y.; Wang, Y.Q.; Chen, L.J.; et al. The genome of Cymbidium sinense revealed the evolution of orchid traits. Plant Biotechnol. J. 2021, 19, 2501–2516. [Google Scholar] [CrossRef]
  57. Su, S.; Shao, X.; Zhu, C.; Xu, J.; Tang, Y.; Luo, D.; Huang, X. An AGAMOUS-like factor is associated with the origin of two domesticated varieties in Cymbidium sinense (Orchidaceae). Hortic. Res.-Engl. 2018, 5, 48. [Google Scholar] [CrossRef]
  58. Chugh, S.; Guha, S.; Rao, I.U. Micropropagation of orchids: A review on the potential of different explants. Sci. Hortic. 2009, 122, 507–520. [Google Scholar] [CrossRef]
  59. Chao, Y.T.; Yen, S.H.; Yeh, J.H.; Chen, W.C.; Shih, M.C. Orchidstra 2.0-A Transcriptomics Resource for the Orchid Family. Plant Cell Physiol. 2017, 58, e9. [Google Scholar] [CrossRef] [PubMed]
  60. Libault, M.; Wan, J.; Czechowski, T.; Udvardi, M.; Stacey, G. Identification of 118 Arabidopsis transcription factor and 30 ubiquitin-ligase genes responding to chitin, a plant-defense elicitor. Mol. Plant-Microbe Interact. 2007, 20, 900–911. [Google Scholar] [CrossRef] [PubMed]
  61. Shalmani, A.; Jing, X.Q.; Shi, Y.; Muhammad, I.; Zhou, M.R.; Wei, X.Y.; Chen, Q.Q.; Li, W.Q.; Liu, W.T.; Chen, K.M. Characterization of B-BOX gene family and their expression profiles under hormonal, abiotic and metal stresses in Poaceae plants. BMC Genom. 2019, 20, 27. [Google Scholar] [CrossRef]
  62. Gnesutta, N.; Kumimoto, R.W.; Swain, S.; Chiara, M.; Siriwardana, C.; Horner, D.S.; Holt, B.F., 3rd; Mantovani, R. CONSTANS Imparts DNA Sequence Specificity to the Histone Fold NF-YB/NF-YC Dimer. Plant Cell 2017, 29, 1516–1532. [Google Scholar] [CrossRef]
  63. Ordoñez-Herrera, N.; Trimborn, L.; Menje, M.; Henschel, M.; Robers, L.; Kaufholdt, D.; Hansch, R.; Adrian, J.; Ponnu, J.; Hoecker, U. The Transcription Factor COL12 Is a Substrate of the COP1/SPA E3 Ligase and Regulates Flowering Time and Plant Architecture. Plant Physiol. 2018, 176, 1327–1340. [Google Scholar] [CrossRef]
  64. Plant, A.R.; Larrieu, A.; Causier, B. Repressor for hire! The vital roles of TOPLESS-mediated transcriptional repression in plants. New Phytol. 2021, 231, 963–973. [Google Scholar] [CrossRef] [PubMed]
  65. Fjellheim, S.; Young, D.A.; Paliocha, M.; Johnsen, S.S.; Schubert, M.; Preston, J.C. Major niche transitions in Pooideae correlate with variation in photoperiodic flowering and evolution of CCT domain genes. J. Exp. Bot. 2022, 73, 4079–4093. [Google Scholar] [CrossRef] [PubMed]
  66. Zhang, G.; Hu, Y.; Huang, M.Z.; Huang, W.C.; Liu, D.K.; Zhang, D.; Hu, H.; Downing, J.L.; Liu, Z.J.; Ma, H. Comprehensive phylogenetic analyses of Orchidaceae using nuclear genes and evolutionary insights into epiphytism. J. Integr. Plant Biol. 2023, 65, 1204–1225. [Google Scholar] [CrossRef] [PubMed]
  67. Harris, R.M.; Hofmann, H.A. Seeing is believing: Dynamic evolution of gene families. Proc. Natl. Acad. Sci. USA 2015, 112, 1252–1253. [Google Scholar] [CrossRef]
  68. Kinoshita, A.; Richter, R. Genetic and molecular basis of floral induction in Arabidopsis thaliana. J. Exp. Bot. 2020, 71, 2490–2504. [Google Scholar] [CrossRef] [PubMed]
  69. Osnato, M.; Cota, I.; Nebhnani, P.; Cereijo, U.; Pelaz, S. Photoperiod Control of Plant Growth: Flowering Time Genes Beyond Flowering. Front. Plant Sci. 2021, 12, 805635. [Google Scholar] [CrossRef] [PubMed]
  70. Cao, J.; Yuan, J.; Zhang, Y.; Chen, C.; Zhang, B.; Shi, X.; Niu, R.; Lin, F. Multi-layered roles of BBX proteins in plant growth and development. Stress Biol. 2023, 3, 1. [Google Scholar] [CrossRef]
  71. Tiwari, S.B.; Shen, Y.; Chang, H.; Hou, Y.; Harris, A.; Ma, S.F.; McPartland, M.; Hymus, G.J.; Adam, L.; Marion, C.; et al. The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis-element. New Phytol. 2010, 187, 57–66. [Google Scholar] [CrossRef]
  72. Zhu, Y.; Klasfeld, S.; Wagner, D. Molecular regulation of plant developmental transitions and plant architecture via PEPB family proteins: An update on mechanism of action. J. Exp. Bot. 2021, 72, 2301–2311. [Google Scholar] [CrossRef]
  73. Ke, Y. The Study of Phalaenopsis CONSTANS-like Gene Regulatory Networks; National Central University: Taoyuan City, Taiwan, 2020. [Google Scholar]
  74. Zhang, J.; Zhao, X.; Tian, R.; Zeng, S.; Wu, K.; Teixeira da Silva, J.A.; Duan, J. Molecular Cloning and Functional Analysis of Three CONSTANS-Like Genes from Chinese Cymbidium. J. Plant Growth Regul. 2020, 39, 1061–1074. [Google Scholar] [CrossRef]
  75. Hou, C.; Yang, C. Functional Analysis of FT and TFL1 Orthologs from Orchid (Oncidium Gower Ramsey) that Regulate the Vegetative to Reproductive Transition. Plant Cell Physiol. 2009, 50, 1544–1557. [Google Scholar] [CrossRef] [PubMed]
  76. Li, Y.; Zhang, B.; Wang, Y.; Gong, X.; Yu, H. DOTFL1 affects the floral transition in orchid Dendrobium Chao Praya Smile. Plant Physiol. 2021, 186, 2021–2036. [Google Scholar] [CrossRef]
  77. Zhu, Y.; Klasfeld, S.; Jeong, C.W.; Jin, R.; Goto, K.; Yamaguchi, N.; Wagner, D. TERMINAL FLOWER 1-FD complex target genes and competition with FLOWERING LOCUS T. Nat. Commun. 2020, 11, 5118. [Google Scholar] [CrossRef]
  78. Bratzel, F.; Turck, F. Molecular memories in the regulation of seasonal flowering: From competence to cessation. Genome Biol. 2015, 16, 1–14. [Google Scholar] [CrossRef] [PubMed]
  79. Li, J.; Wang, L.; Chen, X.; Zeng, L.; Su, Y.; Liu, Z. Characterization of Two AGAMOUS-like Genes and Their Promoters from the Cymbidium faberi (Orchidaceae). Plants 2023, 12, 2740. [Google Scholar] [CrossRef] [PubMed]
  80. Almada, R.; Cabrera, N.; Casaretto, J.A.; Ruiz-Lara, S.; Gonzalez Villanueva, E. VvCO and VvCOL1, two CONSTANS homologous genes, are regulated during flower induction and dormancy in grapevine buds. Plant Cell Rep. 2009, 28, 1193–1203. [Google Scholar] [CrossRef] [PubMed]
  81. Chen, J.; Chen, J.Y.; Wang, J.N.; Kuang, J.F.; Shan, W.; Lu, W.J. Molecular characterization and expression profiles of MaCOL1, a CONSTANS-like gene in banana fruit. Gene 2012, 496, 110–117. [Google Scholar] [CrossRef]
  82. Zhang, X.; Jia, H.; Li, T.; Wu, J.; Nagarajan, R.; Lei, L.; Powers, C.; Kan, C.C.; Hua, W.; Liu, Z.; et al. TaCol-B5 modifies spike architecture and enhances grain yield in wheat. Science 2022, 376, 180–183. [Google Scholar] [CrossRef]
  83. Kishor, P.B.K.; Tiozon, R.N.; Fernie, A.R.; Sreenivasulu, N. Abscisic acid and its role in the modulation of plant growth, development, and yield stability. Trends Plant Sci. 2022, 27, 1283–1295. [Google Scholar] [CrossRef]
  84. Zhang, C.; Zhou, Q.; Liu, W.; Wu, X.; Li, Z.; Xu, Y.; Li, Y.; Imaizumi, T.; Hou, X.; Liu, T. BrABF3 promotes flowering through the direct activation of CONSTANS transcription in pak choi. Plant J. 2022, 111, 134–148. [Google Scholar] [CrossRef]
  85. Sevilleno, S.; Cabahug-Braza, R.; An, H.; Lim, K.; Hwang, Y. The role of cytogenetic tools in orchid breeding. Korean J. Agric. Sci. 2023, 50, 193–206. [Google Scholar] [CrossRef]
  86. Balilashaki, K.; Dehghanian, Z.; Gougerdchi, V.; Kavusi, E.; Feizi, F.; Tang, X.; Vahedi, M.; Hossain, M.M. Progress and Prospect of Orchid Breeding: An Overview. In Advances in Orchid Biology, Biotechnology and Omics; Springer: Berlin/Heidelberg, Germany, 2023; pp. 261–283. [Google Scholar] [CrossRef]
  87. Ramya, M.; Lee, S.Y.; An, H.R.; Park, P.M.; Kim, N.S.; Park, P.H. MYB1 transcription factor regulation through floral scent in Cymbidium cultivar ‘Sael Bit’. Phytochem. Lett. 2019, 32, 181–187. [Google Scholar] [CrossRef]
  88. Yam, T.W.; Nair, H.; Hew, C.S.; Arditti, J. History-Seeds: Orchid Seeds and their Germination: An Historical Account. In Orchid Biology: Reviews and Perspectives, VIII; Springer: Berlin/Heidelberg, Germany, 2002; pp. 387–504. [Google Scholar] [CrossRef]
  89. Lee, Y.-I.; Yeung, E.C. The orchid seed coat: A developmental and functional perspective. Bot. Stud. 2023, 64, 1–15. [Google Scholar] [CrossRef] [PubMed]
  90. Yu, B.; He, X.; Tang, Y.; Chen, Z.; Zhou, L.; Li, X.; Zhang, C.; Huang, X.; Yang, Y.; Zhang, W. Photoperiod controls plant seed size in a CONSTANS-dependent manner. Nat. Plants 2023, 9, 343–354. [Google Scholar] [CrossRef]
  91. Achary, R.K.; Majee, M. CONSTANS, a key-player connecting day length to seed size. Trends Plant Sci. 2023, 28, 975–977. [Google Scholar] [CrossRef]
  92. Katoh, K.; Standley, D.M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 2013, 30, 772–780. [Google Scholar] [CrossRef]
  93. Nguyen, L.T.; Schmidt, H.A.; von Haeseler, A.; Minh, B.Q. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 2015, 32, 268–274. [Google Scholar] [CrossRef] [PubMed]
  94. Kalyaanamoorthy, S.; Minh, B.Q.; Wong, T.K.F.; von Haeseler, A.; Jermiin, L.S. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods 2017, 14, 587–589. [Google Scholar] [CrossRef]
  95. Wang, Y.; Tang, H.; Debarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.H.; Jin, H.; Marler, B.; Guo, H.; et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef]
  96. Krzywinski, M.; Schein, J.; Birol, I.; Connors, J.; Gascoyne, R.; Horsman, D.; Jones, S.J.; Marra, M.A. Circos: An information aesthetic for comparative genomics. Genome Res. 2009, 19, 1639–1645. [Google Scholar] [CrossRef]
  97. Zhang, Z. KaKs_calculator 3.0: Calculating selective pressure on coding and non-coding sequences. Genom. Proteom. Bioinform. 2022, 20, 536–540. [Google Scholar] [CrossRef] [PubMed]
  98. Lescot, M.; Dehais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouze, 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]
  99. Schwede, T.; Kopp, J.; Guex, N.; Peitsch, M.C. SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res. 2003, 31, 3381–3385. [Google Scholar] [CrossRef] [PubMed]
  100. Geourjon, C.; Deléage, G. SOPMA: Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput. Appl. Biosci. 1995, 11, 681–684. [Google Scholar] [CrossRef] [PubMed]
  101. Takagi, H.; Hempton, A.K.; Imaizumi, T. Photoperiodic flowering in Arabidopsis: Multilayered regulatory mechanisms of CONSTANS and the florigen FLOWERING LOCUS T. Plant Commun. 2023, 4, 100552. [Google Scholar] [CrossRef] [PubMed]
  102. Ren, R.; Gao, J.; Lu, C.; Wei, Y.; Jin, J.; Wong, S.M.; Zhu, G.; Yang, F. Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids. Int. J. Mol. Sci. 2020, 21, 2264. [Google Scholar] [CrossRef]
  103. Ren, R.; Gao, J.; Yin, D.; Li, K.; Lu, C.; Ahmad, S.; Wei, Y.; Jin, J.; Zhu, G.; Yang, F. Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops. Front. Plant Sci. 2021, 12, 626015. [Google Scholar] [CrossRef]
  104. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
Ijms 24 16825 g001
Figure 1. Molecular phylogenetic analysis of 101 COL proteins from Arabidopsis. thaliana, Oryza. sativa, A. shenzhenica, D. catenatum, D. huoshanense, V. planifolia, D. chrysotoxum, C. sinense, and P. equestri. Branches are colored according to the species color scheme on the bottom right.
Figure 1. Molecular phylogenetic analysis of 101 COL proteins from Arabidopsis. thaliana, Oryza. sativa, A. shenzhenica, D. catenatum, D. huoshanense, V. planifolia, D. chrysotoxum, C. sinense, and P. equestri. Branches are colored according to the species color scheme on the bottom right.
Ijms 24 16825 g001
Ijms 24 16825 g002
Figure 2. The conserved motifs and gene structure of the COL gene family in seven orchid species. (A) The conserved motifs of the COL gene family. (B) The gene structure of the COL gene family.
Figure 2. The conserved motifs and gene structure of the COL gene family in seven orchid species. (A) The conserved motifs of the COL gene family. (B) The gene structure of the COL gene family.
Ijms 24 16825 g002
Ijms 24 16825 g003
Figure 3. Chromosomal distribution of the COL genes in four orchids. Chromosome (Chr) names in yellow are on the left, and gene names are on the right. The scale on the left is in megabases (Mb). Gradient colors from red (high) to blue (low) indicate gene density in heat maps on orchid chromosomes by setting the estimated inheritance interval to 300 kb. Red indicates high gene density, and blue indicates low gene density. (A) C. sinense. (B) D. chrysotoxum. (C) D. huoshanense. (D) V. planifolia.
Figure 3. Chromosomal distribution of the COL genes in four orchids. Chromosome (Chr) names in yellow are on the left, and gene names are on the right. The scale on the left is in megabases (Mb). Gradient colors from red (high) to blue (low) indicate gene density in heat maps on orchid chromosomes by setting the estimated inheritance interval to 300 kb. Red indicates high gene density, and blue indicates low gene density. (A) C. sinense. (B) D. chrysotoxum. (C) D. huoshanense. (D) V. planifolia.
Ijms 24 16825 g003
Ijms 24 16825 g004
Figure 4. Chromosomal distributions of COL genes and schematic interchromosomal relationships. (A–D) Synteny analysis of four orchid genomes identifies segmental duplication pairs of COL subfamilies. Dark orchid and red colored lines indicate homologous pairs of COL genes and their corresponding chromosomal locations, respectively. (E) Intragenomic synteny between C. sinense (20 chromosomes) and each of rice (12 chromosomes), D. chrysotoxum (19 chromosomes), D. huoshanense (19 chromosomes), and V. planifolia (14 chromosomes).
Figure 4. Chromosomal distributions of COL genes and schematic interchromosomal relationships. (A–D) Synteny analysis of four orchid genomes identifies segmental duplication pairs of COL subfamilies. Dark orchid and red colored lines indicate homologous pairs of COL genes and their corresponding chromosomal locations, respectively. (E) Intragenomic synteny between C. sinense (20 chromosomes) and each of rice (12 chromosomes), D. chrysotoxum (19 chromosomes), D. huoshanense (19 chromosomes), and V. planifolia (14 chromosomes).
Ijms 24 16825 g004
Ijms 24 16825 g005
Figure 5. Cis-acting elements in the promoter regions of COL genes. Elements with similar regulatory functions are displayed in the same color. The number of each type of element is shown on the right.
Figure 5. Cis-acting elements in the promoter regions of COL genes. Elements with similar regulatory functions are displayed in the same color. The number of each type of element is shown on the right.
Ijms 24 16825 g005
Ijms 24 16825 g006
Figure 6. The expression profile of COL genes among different tissues in seven orchids. (A) A. shenzhenica and (B) C. sinense. Floral bud0, dormant lateral buds; floral bud1, 1−5 mm floral bud; floral bud2, 6−10 mm floral bud; floral bud3, 11−15 mm floral bud; floral bud4, 16−20 mm floral bud; floral bud5, blooming flower. (C) D. catenatum, (D) D. chrysotoxum, (E) D. huoshanense, (F) P. equestri, and (G) V. planifolia.
Figure 6. The expression profile of COL genes among different tissues in seven orchids. (A) A. shenzhenica and (B) C. sinense. Floral bud0, dormant lateral buds; floral bud1, 1−5 mm floral bud; floral bud2, 6−10 mm floral bud; floral bud3, 11−15 mm floral bud; floral bud4, 16−20 mm floral bud; floral bud5, blooming flower. (C) D. catenatum, (D) D. chrysotoxum, (E) D. huoshanense, (F) P. equestri, and (G) V. planifolia.
Ijms 24 16825 g006
Ijms 24 16825 g007
Figure 7. Gene expression patterns of CsiCOL genes in individual floral organs of different flower varieties. (A) Wild type (WT), gynostemium-like perianth variety (GPV), multi-perianth variety (MPV), labellum-like perianth variety (LaPV), and null-lip variety (NLV); 1 to 4 represent the individual floral organ sepal, petal, labellum, and column, respectively. (B) Cartoon heat map of tissue-specific expression of CsiCOL genes in C. sinense floral organs.
Figure 7. Gene expression patterns of CsiCOL genes in individual floral organs of different flower varieties. (A) Wild type (WT), gynostemium-like perianth variety (GPV), multi-perianth variety (MPV), labellum-like perianth variety (LaPV), and null-lip variety (NLV); 1 to 4 represent the individual floral organ sepal, petal, labellum, and column, respectively. (B) Cartoon heat map of tissue-specific expression of CsiCOL genes in C. sinense floral organs.
Ijms 24 16825 g007
Ijms 24 16825 g008
Figure 8. Protein tertiary structure of 68 COL genes from seven species of Orchidaceae. The tertiary structures are colored in rainbow order, representing the N-to-C terminuses.
Figure 8. Protein tertiary structure of 68 COL genes from seven species of Orchidaceae. The tertiary structures are colored in rainbow order, representing the N-to-C terminuses.
Ijms 24 16825 g008
Ijms 24 16825 g009
Figure 9. Representative regulatory network for CsiCOLs and CO regulatory genes. (A) Correlations of expression patterns between CsiCOLs and other transcription factors. (B) A model for CsiCOLs and CO regulators identified by their sequence homology to Arabidopsis in C. sinense.
Figure 9. Representative regulatory network for CsiCOLs and CO regulatory genes. (A) Correlations of expression patterns between CsiCOLs and other transcription factors. (B) A model for CsiCOLs and CO regulators identified by their sequence homology to Arabidopsis in C. sinense.
Ijms 24 16825 g009
Ijms 24 16825 g010
Figure 10. Subcellular localization and expression patterns of genes involved in flower development in response to the induction of CsiCOLs. (A) RT-qPCR analysis of CsiCOL05 and CsiCOl09 genes at different floral organs of standard and GPV variety C. sinense. (B) Subcellular localization of CsiCOL05 and CsiCOL09 protein in C. sinense protoplasts. Bar = 10 μm. (C,D) Relative expression levels of CsiFT, CsiTFL1, CsiAP1, CsiSOC1, CsiLFY, and CsiAG1 genes in response to CsiCOL05 and CsiCOL09 induction by qRT-PCR, respectively. Significant differences analyzed between the CsiCOLs-OE and the WT (pAN580-GFP vector) by Student’s t-test, using R (*** p  <  0.001, ** p  <  0.01, and * p  <  0.05).
Figure 10. Subcellular localization and expression patterns of genes involved in flower development in response to the induction of CsiCOLs. (A) RT-qPCR analysis of CsiCOL05 and CsiCOl09 genes at different floral organs of standard and GPV variety C. sinense. (B) Subcellular localization of CsiCOL05 and CsiCOL09 protein in C. sinense protoplasts. Bar = 10 μm. (C,D) Relative expression levels of CsiFT, CsiTFL1, CsiAP1, CsiSOC1, CsiLFY, and CsiAG1 genes in response to CsiCOL05 and CsiCOL09 induction by qRT-PCR, respectively. Significant differences analyzed between the CsiCOLs-OE and the WT (pAN580-GFP vector) by Student’s t-test, using R (*** p  <  0.001, ** p  <  0.01, and * p  <  0.05).
Ijms 24 16825 g010
Ijms 24 16825 g011
Figure 11. CsiCOL05 and CsiCOL09 schematics of potential regulatory patterns in C. sinense.
Figure 11. CsiCOL05 and CsiCOL09 schematics of potential regulatory patterns in C. sinense.
Ijms 24 16825 g011
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Wei, Y.; Jin, J.; Lin, Z.; Lu, C.; Gao, J.; Li, J.; Xie, Q.; Zhu, W.; Zhu, G.; Yang, F. Genome-Wide Identification, Expression, and Molecular Characterization of the CONSTANS-like Gene Family in Seven Orchid Species. Int. J. Mol. Sci. 2023, 24, 16825. https://doi.org/10.3390/ijms242316825

AMA Style

Wei Y, Jin J, Lin Z, Lu C, Gao J, Li J, Xie Q, Zhu W, Zhu G, Yang F. Genome-Wide Identification, Expression, and Molecular Characterization of the CONSTANS-like Gene Family in Seven Orchid Species. International Journal of Molecular Sciences. 2023; 24(23):16825. https://doi.org/10.3390/ijms242316825

Chicago/Turabian Style

Wei, Yonglu, Jianpeng Jin, Zengyu Lin, Chuqiao Lu, Jie Gao, Jie Li, Qi Xie, Wei Zhu, Genfa Zhu, and Fengxi Yang. 2023. "Genome-Wide Identification, Expression, and Molecular Characterization of the CONSTANS-like Gene Family in Seven Orchid Species" International Journal of Molecular Sciences 24, no. 23: 16825. https://doi.org/10.3390/ijms242316825

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop