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Article

Genome-Wide Identification, Characterization, and Expression of TCP Genes Family in Orchardgrass

by
Cheng Wang
†,
Guangyan Feng
†,
Xiaoheng Xu
,
Linkai Huang
,
Gang Nie
,
Dandan Li
and
Xinquan Zhang
*
College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2023, 14(4), 925; https://doi.org/10.3390/genes14040925
Submission received: 29 March 2023 / Revised: 12 April 2023 / Accepted: 13 April 2023 / Published: 16 April 2023
(This article belongs to the Section Plant Genetics and Genomics)
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Abstract

:
Plant-specific TCP transcription factors regulate several plant growth and development processes. Nevertheless, little information is available about the TCP family in orchardgrass (Dactylis glomerata L.). This study identified 22 DgTCP transcription factors in orchardgrass and determined their structure, phylogeny, and expression in different tissues and developmental stages. The phylogenetic tree classified the DgTCP gene family into two main subfamilies, including class I and II supported by the exon–intron structure and conserved motifs. The DgTCP promoter regions contained various cis-elements associated with hormones, growth and development, and stress responses, including MBS (drought inducibility), circadian (circadian rhythms), and TCA-element (salicylic acid responsiveness). Moreover, DgTCP9 possibly regulates tillering and flowering time. Additionally, several stress treatments upregulated DgTCP1, DgTCP2, DgTCP6, DgTCP12, and DgTCP17, indicting their potential effects regarding regulating responses to the respective stress. This research offers a valuable basis for further studies of the TCP gene family in other Gramineae and reveals new ideas for increasing gene utilization.

1. Introduction

The plant-specific TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family was first discovered in 1999 [1]. The acronym TCP comes from four genes in three species: T (TB1 from maize [Zea mays]) [2], C (CYC from snapdragon [Antirrhinum majus]) [3], and P (PCFs from rice [Oryza sativa]) [4]. Members of the TCP family have 59 amino acids and an atypical basic helix–loop–helix (bHLH) motif at the N-terminus called the TCP domain which is responsible for protein–protein interactions, nuclear protein localization, and DNA binding [1,4]. Moreover, the TCP domain classifies these proteins into two groups: class I and II [5]. The clearest distinction between class I and II is the basic region of the TCP domain where class I members lost four amino acids. Class II is divided into CYC/TB1 and CIN subclades [5,6]. Arginine-rich motifs with 18–20 residues (R domain) are usually found in class II [1].
Many TCP proteins are critical in several plant biological processes [5,7], including hormone biosynthesis, signaling transduction, flower development, leaf development and senescence, lateral branching, circadian rhythm, seed germination, defence response, and cell proliferation and differentiation [7,8,9,10,11,12,13,14,15,16,17,18]. For instance, AtTCP14 and AtTCP15 class I proteins regulate leaf morphology and embryonic development during seed germination through the gibberellin (GA) signaling pathway [19,20]. AtTCP20 acts upstream of AtTCP9 to regulate leaf senescence via the jasmonic acid (JA) signal pathway [21]. AtTCP16 is necessary for pollen development in developing microspores [22]. In Arabidopsis thaliana, five miR319a targets in CIN class II gene subgroups (AtTCP2, AtTCP3, AtTCP4, AtTCP10, and AtTCP24) were confirmed important in leaf growth and morphogenesis [9,12]. AtTCP4 is essential for the natural life activity of petals, the multifaceted regulation of JA and auxin (IAA) synthesis, the senescence of ripe leaves, and the age-dependent decomposition of leaf photosynthetic complexes [21,23,24,25,26]. In the CYC/TB1 subclade of class II, LjCYC1 and LjCYC3 (in Lotus japonicus) and AmCYC (in snapdragon) produce a marked effect in floral development [3,27]. Moreover, the A thaliana AtTCP1 and GhCYC2 from Gerbera hybrida control symmetrical petal growth in flowers [28,29]. ZmTB1 in maize, AtBRC1 and AtBRC2 in A thaliana, and OsTB1 in rice modulate branching by negatively regulating axillary bud growth [11,30,31]. Besides, TCPs regulate the circadian clock and plant morphogenetics. TCP2, 3, 11, and 15 combine with the TGGGC (C/T) element to interact with various compositions of the core circadian rhythm, thus mediating the Arabidopsis circadian clock. Additionally, TCP20 and 22 improve the circadian clock [10,32].
Environmental stresses can affect plant growth and development [33], and some TCP family members have been shown to respond to environmental changes. For instance, overexpressing OsTCP19 induces the typical genes from abscisic acid (ABA), methyl jasmonate (Me-JA), ethylene (ET), IAA, cytokinins (CK), and other signaling pathways in rice. These genes reduce reactive oxygen species, the accumulation of fat droplets, and water loss in transgenic plants, thus improving their tolerance to high salt and mannitol treatments [34]. In rice, downregulating OsTCP21 and OsPCF6 enhances tolerance to cold stress by altering the scavenging of reactive oxygen species. Similarly, OsPCF5 and OsPCF8 improve tolerance to cold stress [35,36]. Binding OsPCF2 to the OsNHX1 promoter improves salt and drought tolerance [37]. In A thaliana, TCP20 associates with NLP6/7 to modulate signal transduction and nitrate assimilation [38]. Several TCP genes have also been reported in other species. For example, four miR319 target genes, including AsPCF5, AsPCF6, AsPCF8, and AsTCP14, were downregulated in drought and salt tolerance creeping bentgrass (Agrostis stolonifera) [39,40]. Some TCP genes in common bean (Phaseolus vulgaris) and PeTCP10 in moso bamboo (Phyllostachys edulis) enhance tolerance to salt stress [41,42]. Besides, several GhTCP genes in cotton (Gossypium hirsutum) were upregulated under drought salt and heat stress [17]. Although the TCP gene family plays a major role in regulating plant life processes, there are no reports of their functioning in orchardgrass (D glomerata).
Orchardgrass is a widely cultivated perennial forage grass that is native to central and western Europe, the temperate regions of Asia, and North Africa [43]. D glomerata has a high nutrient content and is among the four major global economic perennial grasses. It establishes fast, recovers quickly after mowing, and has high shade, drought, and barren tolerance [44]. Many valuable genes control orchardgrass development and abiotic stress response. Therefore, this study identified TCPs in orchardgrass through synthetic analysis of the D. glomerata genome (gene structure, conserved motif composition, chromosomal location, and phylogenetic characteristics). Preliminary predictions of DgTCP gene evolution involved the analysis of phylogenetic and gene duplication events and collinearity with other plants. Additionally, TCP expression was assessed in different tissues, developmental stages, and abiotic stresses. The results of this study will be helpful in elucidating orchardgrass adaptation to different environments and may reveal the functions of DgTCPs.

2. Material and Methods

2.1. Identification of Dactylis glomerata TCP Genes

Firstly, the TCP domain (PF03634) HMM profile, which was obtained from the Pfam database (http://pfam.xfam.org/, accessed on 1 March 2022) [45], was the reference for identifying TCP genes from the Dactylis glomerata genome (http://orchardgrassgenome.sicau.edu.cn/, accessed on 20 January 2022) using HMMER 3.0 software (E-value cutoff of 0.01) [46]. Secondly, PFAM was used to further analyze all candidate genes. The confirmed TCPs were aligned with Clustal X2.0 [47], and redundant sequences were discarded. Finally, the physicochemical properties of the DgTCP protein, including protein length, CDS length, isoelectric point, and molecular weight, were determined using ProtParam (http://web.expasy.org/protparam/, accessed on 3 March 2022) [48].

2.2. Phylogenetic Analysis and Classification of DgTCP Genes

The sequences of A thaliana TCP genes were obtained from TAIR (https://www.arabidopsis.org/, accessed on 12 February 2022) [49]. Next, 22 rice and 21 Brachypodium distachyon TCP protein sequences were obtained from Plant TFDB (http://planttfdb.cbi.pku.edu.cn/, accessed on 6 March 2022) [50]. Multiple alignments of the selected TCP sequences were performed using Clustal X2.0 [47]. Based on the neighbor-joining (NJ) method and 1000.0 replicates for bootstrap node support, the phylogenetic tree of orchardgrass, rice, B distachyonwere, and A thaliana were constructed using MEGA7.0 [51] and then beautified via the iTOL website (https://itol.embl.de/itol.cgi/, accessed on 14 April 2022).

2.3. Gene Structure and Motif Analysis

The exon–intron structures of DgTCPs were generated based on available genomic information and coding sequences from the Gene Structure Display Server (GSDS 2.0, http://gsds.cbi.pku.edu.cn/, accessed on 6 May 2022) [52]. The online Multiple Expectation Maximization for Motif Elicitation (MEME) software (http://meme-suite.org/, accessed on 11 May 2022) was used to identify the conserved DgTCP proteins motifs (considering ten maximum motifs and default settings) [53].

2.4. Putative Promoter Cis-Acting Element Analysis

The nucleotide sequences of DgTCPs were acquired from the orchardgrass genome database (http://orchardgrassgenome.sicau.edu.cn/, accessed on 20 January 2022). The 2000 bp region upstream of all DgTCPs was considered the promoter sequence, and the cis-acting promoter elements were appraised via PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 19 June 2022) [54]. The putative cis-acting elements are classified into plant hormone responses, growth and development, and biotic and abiotic stress responses.

2.5. Chromosomal Mapping and Synteny Analysis

The chromosomal position information of each TCP gene was retrieved from orchardgrass genome annotations. MapGene2Chrome (MG2C, http://mg2c.iask.in/mg2c_v2.0/, accessed on 26 June 2022) was used to describe the location of the TCPs on the chromosomes. Next, DgTCP gene duplication was analyzed using MCScanX and default parameters [55]. Using the Dual Synteny Plotter of TBtools, we mapped the TCP gene collinearity between D glomerata, A thaliana, O sativa, Sorghum bicolor, Hordeum vulgare, and B distachyon (https://github.com/CJ-Chen/TBtools, accessed on 21 April 2022) [56]. The genome data of O sativa, S bicolor, B distachyon, and H vulgare were downloaded from the JGI Genome Portal (https://genome.jgi.doe.gov/portal/, accessed on 12 March 2022), and A thaliana was downloaded from TAIR (https://www.arabidopsis.org/, accessed on 12 February 2022).

2.6. Plant Material and Treatments

Seeds of “Baoxing” orchardgrass were grown in a growth chamber at a 22 °C/14 h (day) and 20 °C/10 h (night) cycle. One week after germination, the seedings were irrigated with 1/2 Hogland solution. When the seedlings reached the third to fourth leaf, they were treated with 1/2 Hoagland solution containing 20% polyethylene glycol (PEG6000), 250 mM sodium chloride (NaCl), 100 μM methyl jasmonate (Me-JA), 200 mM sodium bicarbonate (NaHCO3), 100μM abscisic acid (ABA), and 100 μM salicylic acid (SA). The leaves for each treatment were collected separately at 0, 1, 3, 6, 12, and 24 h after treatment, immediately frozen in liquid nitrogen, and then stored at −80 °C for qRT-PCR.

2.7. Expression Profiles of DgTCP Family Members

The expression patterns of TCPs in the root, stem, leaf, spike, and flower were obtained from the orchardgrass genome database (Table S1) [46]. Furthermore, the expression patterns of the floral bud developmental stages before vernalization (BV), vernalization (V), after vernalization (AV), before heading (BH), and heading (H) of the late-flowering variety “Baoxing” and early-flowering variety “Donata” were obtained from the RNA-seq data (Table S2) [57]. The RNA-seq data of the TCP genes in four tissues from varieties D20170203 (low-tillering) and AKZ-NRGR66 7 (high-tillering) were obtained from Xu et al. (Table S3) [58]. The heat maps of the expression patterns were produced using TBtools [56].

2.8. Expression of 14 Selected DgTCP Genes in qRT-PCR

The total RNA of the samples under different treatments was extracted using the Hipure HP plant RNA mini kit (Magen, Guangdong, China). First-strand cDNA was synthesized using the MonScriptTM RTIII ALL-in-One Mix with dsDNase kit (Monad, Suzhou, China) following the manufacturer’s instructions. Primers for the 14 DgTCPs were designed using Primer 5.0 software (Table S4), and qRT-PCR was performed using the MonAmpTM SYBR® green qPCR Mix (Monad) on the Bio-Rad CFX96 instrument. GAPDH was the internal reference gene for normalization [43], and the relative gene expression levels were evaluated by the 2−ΔΔCt method [59]. All qRT-PCR assays were performed with three biological and technical replicates.

3. Results

3.1. Identifying TCP Genes in Orchardgrass

Twenty-two genes were retrieved from the orchardgrass genome and designated as DgTCP1–DgTCP22 based on their chromosomal positioning. The protein sequence length, CDS length, molecular weight, isoelectric point (pI), and gene location of the 22 DgTCPs are captured in Table 1 and Table S5. The smallest protein was 17,433.51(DgTCP1), and the biggest was 47,382.56 kDa (DgTCP16). The pI ranged from 5.09 (DgTCP7) to 9.92 (DgTCP11), and the protein lengths were 165 (DgTCP17) to 454 (DgTCP6) aa.

3.2. Phylogeny and Classification of the DgTCP Proteins

Based on the phylogenetic tree of 22 orchardgrass, 22 rice, 21 B distachyon, and 24 A thaliana, TCP proteins were constructed using the neighbor-joining (NJ) method to clarify the phylogenetic relationships and evolutionary history of the TCP gene family (Figure 1). Two classical subfamilies, class I and class II, were identified from the topology of the NJ tree and A thaliana classification. Eleven DgTCPs belong to class I (PCF or TCP-P), and the other 11 belong to class II (TCP-C) (Figure 1). The class II group is further divided into CYC/TB1 (4 DgTCPs) and CIN subclasses (7 DgTCPs) (Figure 1). Thus, we performed multiple sequence alignments on the TCP domains of all DgTCP members to comprehend the phylogenetic relationships of the DgTCPs. The TCP domain comparison and phylogenetic analysis indicated that orchardgrass TCP proteins have class I (PCF) and class II (CIN and CYC/TB1) groups (Figure 1 and Figure 2). Class I proteins lack four amino acids at their basic domain compared with class II proteins.

3.3. The DgTCP Gene Structure and Protein Motif

The structural characteristics of all DgTCPs were analyzed to comprehend the evolution of the TCP gene family in orchardgrasss (Figure 3b). All class I DgTCP genes, except DgTCP5 and DgTCP22, lack introns. In class II, all CIN genes possess one or two introns, while CYC/TB1 genes lack introns.
Figure 3c shows 10 conserved motifs of the 22 DgTCP proteins which were identified using MEME to reveal the structural characteristics of orchardgrass TCP. The amino acid sequence for each motif (Table S6) shows that the conserved motifs have 6–42 amino acids. All DgTCP protein contain motifs 1 and 2. Moreover, DgTCP proteins from the same subfamilies contain similar motifs. For instance, members of clade PCF contain motif 3, while the clades CIN and CYC/TB1 lack motif 3. All clade CIN members contain motifs 7 and 9, while PCF members contain motifs 5, 6, and 8.
Additionally, some motifs, such as motifs 4 and 10, are shared by two classes. These results indicate that the motifs present only in some subgroups may be associated with unique functions. Nevertheless, the unique functions of these motifs in the plant life cycle have not been identified and need to be explored further.

3.4. Chromosomal Localization, Gene Duplication, and Synteny Analysis

The 22 orchardgrass TCP genes are randomly distributed on 7 chromosomes (Figure 4). Chromosome 4 contained six TCP genes, and chromosomes 3 and 5 had five TCP genes each. Three TCP genes mapped to chromosome 1, but only one mapped to chromosomes 2, 6, and 7.
The duplication event is important for analyzing the evolution and expansion of the gene families. The orchardgrass genome has five pairs of segmental duplicates (Table S7), including DgTCP2/DgTCP14, DgTCP3/DgTCP5, DgTCP4/DgTCP9, DgTCP6/DgTCP12, and DgTCP7/DgTCP22 (Figure 5, linked with red lines).
The five comparative syntenic maps show the evolutionary relationships among TCPs in different species, including A thaliana (dicotyledonous plant), O sativa, S bicolor, B distachyon, and H vulgare (Figure 6). The homologous pairs between D glomerata and the 5 species were 33 (O), 31 (S bicolor), 30 (B distachyon), 23 (H vulgare), and 5 (A thaliana) (Table S8). These results indicate that the TCPs in the monocotyledons are highly conserved and homologous.

3.5. Putative Cis-Acting Elements of Orchardgrass DgTCPs

The cis-elements in promoters are essential for transcriptional regulation and gene function analysis. Therefore, to provide further insight into the gene functions and regulation mechanisms of DgTCP genes, 93 cis-elements possibly involved in phytohormone response, plant growth and development, and stress response were identified to unravel the functions and regulatory mechanisms of DgTCP genes (Table S9). The TATA- and CAAT-box had the most cis-elements among the 22 DgTCPs (Table S9). Interestingly, the AACA-motif, MBSI, HD-Zip 1, circadian, and AuxRR-core only existed in 1 of 22 DgTCPs (Figure 7), indicating their likely unique roles in those genes and, by extension, the regulatory pathways and processes involving those genes.
The promoter regions of one and three DgTCPs are two cis-elements (AACA-motif and GCN4-motif) that participate in endosperm expression. Besides, NON-box and CAT-box were associated with meristem expression plant growth and development. The seed-specific regulatory element (RY element) and zein metabolism regulatory element (O2 site) were identified in six and nine DgTCPs, respectively. In addition, a circadian control element (circadian), a flavonoid biosynthetic regulation element (MBSI), a cell cycle regulation element (MSA-like), and a palisade mesophyll cells regulatory element (HD-Zip 1) were also discovered in the promoter regions of DgTCPs (Figure 7). In several hormone-related cis-elements, the salicylic acid (TCA element), the auxin-responsive element (AuxRR core and TGA element), the gibberellin-responsive element (GARE motif, TATC-box, and P-box), the Me-JA-responsive element (CGTCA motif and TGACG motif), and the ABA-responsive element (ABRE) were found in the promoter region of 9, 9, 15, 19, and 19 DgTCP genes, respectively (Figure 7). In addition, the DgTCP promoters contained several cis-elements that were related to several stresses (drought, anaerobic induction, and low temperature) (Figure 7).

3.6. Expression Profiles of DgTCPs in Different Tissues and Developmental Stages

The expression profiles of 2, 5, and 14 DgTCP genes were the highest in the leaf, spike, and stem, respectively (Figure 8a). Moreover, DgTCP1 and DgTCP9 had higher transcription levels in the flowers, revealing that these genes might be important for the growth of different orchardgrass tissues.
The expression patterns of the early- (Baoxing) and late-flowering (Donata) cultivars were analyzed at five flower bud development stages to identify their potential physiological functions in flowering. In most developmental stages, the expression of DgTCP9 and DgTCP6 was higher in “Baoxing” than “Donata” (Figure 8b). TCP15 expression was similar in “Baoxing” and “Donata” before, during (downregulated), and after vernalization (upregulated). TCP18 was significantly upregulated during vernalization in “Baoxing” but showed no change in “Donata”. However, it was upregulated in “Baoxing” and “Donata” during the late vernalization stage and similar in “Baoxing” and “Donata”. From after vernalization to the heading stage, DgTCP2, DgTCP4, DgTCP16, DgTCP17, and DgTCP21 were significantly upregulated during the before heading stage in “Baoxing” and the heading stage in “Donata”.
Gene expression data were retrieved from four tissues of low- (D20170203) and high-tillering (AKZ-NRGR667) orchardgrass to determine the roles of DgTCPs in regulating growth, and development. All DgTCPs were differentially expressed in the four tissues, contrary to their expression under normal conditions (Figure 9). In tiller buds, over half of the DgTCPs were highly expressed in the low- (D20170203) and high-tillering (AKZ-NRGR667) varieties. The expression of TCP1 was significantly higher in D20170203 than AKZ-NRGR667 in the four tissues. However, the expression of TCP20 was higher in the leaves of AKZ-NRGR667 than in D20170203 but similar in other tissues. The expression of TCP10 was higher in the tiller bud of D20170203 than in AKZ-NRGR667 but similar in other tissues. Interestingly, the expression of TCP9 was higher in the tiller buds of D20170203 than those of AKZ-NRGR667 but lower in the leaves of D20170203. Thus, DgTCP1 and DgTCP10 probably inhibit tiller bud development in the two varieties in a differential pattern, ultimately resulting in different phenotypes.

3.7. The Expression of DgTCP Genes under Six Abiotic Stresses

Figure 10 shows the expressions of 14 DgCTPs in “Baoxing” under six abiotic stresses (drought, salt, alkali, Me-JA, ABA, and SA). Salt stress suppressed the expression of two genes (DgTCP8 and DgTCP18) throughout the salt time points and downregulated eleven genes in the early stages (1–3 h) of salt stress. Drought stress upregulated 13 DgTCPs, and the highest values ranged from 1.13-fold (DgTCP3) to 16.89-fold (DgTCP1). Additionally, six genes (DgTCP2, DgTCP12, DgTCP15, DgTCP16, DgTCP17, and DgTCP18) showed significantly higher expression 1 h after treatment with an alkali solution. In contrast, other DgTCPs were upregulated at 6 h of alkali treatment, while DgTCP8 was suppressed at all time points. Nine DgTCPs were highly induced under ABA treatment, and DgTCP12 displayed the highest expression. Nevertheless, ABA treatment inhibited DgTCP3, DgTCP8, DgTCP9, DgTCP16, and DgTCP19 at all time points. Me-JA treatment upregulated five (DgTCP1, DgTCP2, DgTCP6, DgTCP10, and DgTCP17), one (DgTCP8), and two genes (DgTCP3, DgTCP12), which peaked at 3, 6, and 12 h, respectively. These genes were upregulated ranging from 1.26-fold (DgTCP18) to 20.16-fold (DgTCP12). In contrast, Me-JA showed no observable regulation in four genes (DgTCP4, DgTCP8, DgTCP15, and DgTCP16) but downregulated DgTCP9 and DgTCP19. Furthermore, SA treatment suppressed the expression of DgTCP3, DgTCP9, and DgTCP19 across the time points, with six (DgTCP1, DgTCP2 DgTCP12, DgTCP15, DgTCP16, and DgTCP17) and three genes (DgTCP4, DgTCP6, and DgTCP18) peaking at 6 and 24 h, respectively. Six stress treatments upregulated DgTCP1, DgTCP2, DgTCP6, DgTCP12, and DgTCP17, but DgTCP levels varied under different stresses and time points when combined with the stress expression pattern data.

4. Discussion

The plant-specific TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family are crucial plant-specific transcription factors with various functions in many processes, including hormone biosynthesis, flower development, leaf development, lateral branching, and defence response. To date, the TCP gene family has been identified in many plants, such as switchgrass (Panicum virgatum) [60], maize [61], Arabidopsis, and rice [62]. However, a comprehensive report of the TCP gene family in high-quality forages, such orchardgrass, is lacking.
This research identified 22 TCP genes in the orchardgrass genome [46]. All the corresponding DgTCP proteins have a highly conserved TCP domain (motifs 1 and 2). Thus, DgTCPs possibly have similar DNA-binding and protein–protein interaction patterns [1,4]. Moreover, sequence alignment and phylogenetic analysis revealed that the 22 DgTCPs are divided into two major subclasses (Figure 2 and Figure 3), which is consistent with previous results [5]. Each subclass contained TCP genes from B distachyon, rice, and Arabidopsis. Furthermore, the orchardgrass TCP genes are closely related to the TCPs of rice and B distachyon, indicating that they evolved from a common ancestor as Gramineae. These results show that many TCP genes from the same ancestor possibly experienced different differentiation patterns at different lineages. Moreover, DgTCPs in the same class and subclass had similar exon–intron structures (Figure 3b) and relatively conserved motifs (Figure 3c), further supporting the close evolutionary relationships between DgTCPs.
The number of DgTCPs was higher than those in moso bamboo (16) [63], grapevine (Vitis vinifera) (17) [64], strawberry (Fragaria vesca) (19) [65], and Sorghum (20) [66], In contrast, the number of DgTCPs was lower than the number in A thaliana (24) [62], maize (46) [61], soybean (Glycine max) (54) [67], and tobacco (Nictiana tabacum) (61) [68]. Tandem, segmental, and whole-genome duplication are important sources of the functional diversity and evolution of gene families [69]. Previous research showed that D glomerata experienced whole-genome replication events [46]. This study identified five segmental repeat gene pairs in the DgTCP gene family and no tandem duplication (Figure 5). Segmental duplication was more beneficial for expanding and evolving the D. glomerata TCP gene family. These results are similar to those described in A thaliana and rice, indicating that TCP duplication in plant genomes possibly has a common mechanism [62,70].
Abiotic stresses affect plant growth and development, quality, and yield [33], and TCP genes are broadly involved in the regulatory processes of plant life [71]. Therefore, exploring the potential functions of TCP genes in orchardgrass under different abiotic stresses is necessary. In this study, salt and drought treatments upregulated more than half of the identified DgTCPs, similar to the results from rapeseed (Brassica napus) [72], cotton [17], and switchgrass [60]. Nonetheless, ABA treatment inhibited five DgTCPs at all time points. The ABA signal transduction pathway is significant for stress response [73].
Moreover, TCPs interact with other genes in JA biosynthesis to influence growth, development, and abiotic stress responses. For instance, TCP4 encodes the enzyme that catalyzes a crucial step in JA synthesis by positively regulating the LOX2 gene [9]. Deactivating TCP4 in plants downregulates LOX2, thus reducing JA synthesis and increasing plant sensitivity to stress [9]. The expression patterns of orchardgrass TCP genes were diverse after Me-JA treatment (Figure 10). For example, Me-JA treatment lowered the expression of DgTCP16 of the AtTCP4-like gene, which may be because the promoter region of TCP16 lacks Me-JA-related cis-elements (Figure 7).
In A thaliana, TCP8 and TCP9 combine to the TCP-promoter binding site of the SA biosynthesis gene ICS1, thus enhancing ICS1 expression [74]. Additionally, SA treatment increased the expression of many cis-elements related to SA in the promoter regions of the DgTCP genes (Figure 7), including DgTCP2, DgTCP4, DgTCP12, and DgTCP18 (Figure 10). Therefore, DgTCP genes may play a role in SA transduction. These results suggest that DgTCP genes are essential for plants to cope with abiotic stress.
Gene function can be inferred from the expression profile of that gene [75]. Thus, this research inferred the functions of 22 DgTCPs using their expression patterns in five tissues (Figure 8a). The results showed different expression profiles of the 22 DgTCPs in five tissues, indicating that orchardgrass TCPs might be related to the development of different tissues. The highest expression of several DgTCPs, such as DgTCP3, DgTCP4, DgTCP15, and DgTCP19, occurred in the stem. Several TCP genes are highly expressed in the stem, including 60 in cotton [17], 11 in rapeseed [72], and 9 in soybean [67]. Some DgTCP genes, such as DgTCP1 and DgTCP9, are highly expressed in flowers, indicating that DgTCP genes possibly participate in flowering. As with this study, 16 and 12 highly expressed TCP genes were reported in rapeseed and soybean [67,72]. Moreover, most duplicate gene pairs had the same functions and similar expression patterns, except DgTCP4, DgTCP9, DgTCP7, and DgTCP22, which showed different expression profiles. This diverse expression may be related to differences in the evolution of duplicate genes or upstream regulatory mechanisms, causing functionalization in one of the duplicate genes.
Next, we compared the potential role of TCP genes in regulating flowering time at the five stages of late- and early-flowering orchardgrass (Figure 8b). Flowering is crucial for the growth and development of Gramineae, and variations in flowering time directly affect orchardgrass quality and yield. Moreover, vernalization is a critical way to control flowering time and floral organ development [57]. Thus, the unique expression profiles of DgTCP15 and DgTCP18 at different floral bud developmental stages suggests that these genes may regulate flowering time through the vernalization pathway. For example, A thaliana plants that overexpress AtTCP23 have the late-flowering phenotype [76]. In this study, DgTCP6 was expressed at higher levels in “Donata” than in “Baoxing” from the before vernalization stage to the heading stage of late-flowering “Donata” and early-flowering “Baoxing”. DgTCP6 and AtTCP23 belong to the same branch on the evolutionary tree, indicating that they are homologous genes. This alignment implies that DgTCP6 and AtTCP23 have similar functions. Thus, a high expression of DgTCP6 promotes the late-flowering phenotype in “Donata”. Furthermore, the DgTCP9 gene, which has a similar expression pattern as DgTCP6 (Figure 8b), may also have a similar flowering regulatory function. Moreover, AtTCP4 and AtTCP13, AtTCP7 induce early flowering by directly acting on the AP1 promoter to improve its transcript activation ability and activating the transcription expression of the flowering integration gene SOC1, respectively [77,78]. The three TCPs were grouped with DgTCP16 and DgTCP21, DgTCP17, respectively (Figure 1). Additionally, from the before heading stage to the heading stage, the earlier upregulation of DgTCP16, DgTCP17, and DgTCP21 in “Baoxing” relative to “Donata” indicates that they influence early flowering in “Baoxing” and reflect the functions of AtTCP4 and AtTCP13, AtTCP7 in A thaliana. Altogether, the diverse expression of DgTCPs at the five floral bud stages in the different cultivars indicates their regulatory role in orchardgrass flowering.
Finally, the roles of DgTCP1 in tillering were analyzed in the respective cultivars. Tillering is an important agronomic trait in forage crops as it determines the seed yield and aboveground biomass of forage grasses [79,80]. In this study (Figure 9), the tissue-specific expression patterns of DgTCP1 in the two forage varieties revealed that a high expression of DgTCP1 may suppress tillering. Moreover, OsTCP19, a DgTCP9 homologous gene (Figure 1), negatively regulates rice tillering by inhibiting DLT, which promotes tillering [81]. The unique expression of DgTCP9 in high- and low-tillering materials indicates that DgTCP9 possibly confers low-tillering in “D20170203”. In summary, DgTCPs might be important for tiller development; thus, they require further experimental verification.

5. Conclusions

This study identified 22 DgTCPs from the whole genome of D glomerata. Phylogenetic characteristics divided the 22 DgTCPs into class I and II subfamilies. The study also revealed the protein sequence length, CDS length, pI, and molecular weight of the proteins predicted from the 22 DgTCP genes. Furthermore, we identified many cis-elements in the DgTCP-promoter sequences, revealing a complex regulatory network that possibly controls DgTCP genes. The 22 DgTCP genes contained five pairs of segmental repeat genes distributed on seven chromosomes, indicating that segmental duplication was the primary mechanism for DgTCP gene expansion.
Furthermore, the expression of the DgTCPs under various abiotic stresses at different stages (tiller bud and floral bud) and tissues suggested that many DgTCP genes regulate stress tolerance and development in orchardgrass. Specifically, TCP9 probably regulates flowering time, tiller number, and drought stress in D glomerata. This genome-wide analysis of orchardgrass is significant for identifying new DgTCP genes with novel functions and provides a foundation for breeding high-quality orchardgrass varieties and the functional validation of DgTCP genes in the future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes14040925/s1, Table S1: Expression (FPKM) of DgTCP genes in different tissues. Table S2: Expression (FPKM) of DgTCP genes in different floral bud development stages. Table S3: Expression (FPKM) of DgTCP genes in different development tissues of high- and low-tillering orchardgrass. Table S4: The name and sequence information of primers involved in this study. Table S5: List of the 22 DgTCP genes identified in this study. Table S6: Analysis of conserved motifs of TCP protein in orchardgrass. Table S7: Segmental duplication analysis of TCP genes in orchardgrass genome. Table S8: One-to-one orthologous relationships between orchardgrass and other plants. Table S9: Cis-acting elements of 22 TCP genes in Dactylis glomerata L.

Author Contributions

X.Z. and G.F. conceived and designed the experiments; C.W., G.F., and X.X. performed the experiments; C.W. and X.X. analyzed the data; L.H. and G.N. contributed reagents, materials, and analysis tools; C.W. and G.F. wrote the paper; X.X. and D.L. reviewed and edited the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Forage Breeding Project of Sichuan Province (2021YFYZ0013), National Natural Science Foundation of China, grant number NSFC 32101422, the earmarked fund for Modern Agro-industry Technology Research System (no. CARS-34), the Sichuan Province’s Science Fund for International Cooperation (2022YFH0058), and the Sichuan Province’s Science Fund for Distinguished Young Scholars under grant (2021JDJQ001).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this article and its attached documents. The Dglomerata resources were downloaded from Dactylis glomerata genome database (http://orchardgrassgenome.sicau.edu.cn/, accessed on 20 January 2022). The genome data of the TCP transcription factor gene in O sativa, S bicolor, H vulgare, and B distachyon were downloaded from the Plant TFDB (http://planttfdb.cbi.pku.edu.cn/, accessed on 6 March 2022). The genome data of A thaliana were downloaded from TAIR (https://www.arabidopsis.org/, accessed on 12 February 2022).

Acknowledgments

The authors would like to express their gratitude to Xiaoheng Xu from Sichuan Agricultural University.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. An unrooted phylogenetic tree containing TCP proteins from orchardgrass, rice, A thaliana, and B distachyon. Green shading, CIN subclass; yellow shading, CYC/TB1 subclass; purple shading, PCF subclass. The grey circle, red pentagram, black square, and blue triangle represent the rice, orchardgrass, A thaliana, and B distachyon TCPs, respectively.
Figure 1. An unrooted phylogenetic tree containing TCP proteins from orchardgrass, rice, A thaliana, and B distachyon. Green shading, CIN subclass; yellow shading, CYC/TB1 subclass; purple shading, PCF subclass. The grey circle, red pentagram, black square, and blue triangle represent the rice, orchardgrass, A thaliana, and B distachyon TCPs, respectively.
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Figure 2. The logo and sequence alignment of TCP domains from orchardgrass. The basic helix–loop–helix structure has been marked.
Figure 2. The logo and sequence alignment of TCP domains from orchardgrass. The basic helix–loop–helix structure has been marked.
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Figure 3. The phylogenetic tree of the orchardgrass TCP gene family, the gene structures, and the motifs. (a) The phylogenetic tree of D glomerata TCP proteins (DgTCP). (b) Exon–intron structures of DgTCP proteins. Blue squares indicate UTR, black lines indicate introns, and green squares indicate CDS. (c) The colored squares represent the conserved motifs of the DgTCP proteins.
Figure 3. The phylogenetic tree of the orchardgrass TCP gene family, the gene structures, and the motifs. (a) The phylogenetic tree of D glomerata TCP proteins (DgTCP). (b) Exon–intron structures of DgTCP proteins. Blue squares indicate UTR, black lines indicate introns, and green squares indicate CDS. (c) The colored squares represent the conserved motifs of the DgTCP proteins.
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Figure 4. The chromosome locations of the DgTCP genes. The green bars represent Dglomerata chromosomes.
Figure 4. The chromosome locations of the DgTCP genes. The green bars represent Dglomerata chromosomes.
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Figure 5. Genomic locations and segmental repeats of the DgTCP genes. The red lines represent five segmental duplicates of the 22 DgTCP genes.
Figure 5. Genomic locations and segmental repeats of the DgTCP genes. The red lines represent five segmental duplicates of the 22 DgTCP genes.
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Figure 6. Collinearity of the TCP genes between D glomerata and A thaliana, O sativa, S bicolor, H vulgare, and B distachyon. The red lines in the background represent the TCP gene pairs.
Figure 6. Collinearity of the TCP genes between D glomerata and A thaliana, O sativa, S bicolor, H vulgare, and B distachyon. The red lines in the background represent the TCP gene pairs.
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Figure 7. Cis-elements in the promoter regions of the DgTCP genes. (a) The cis-elements in the DgTCP promoter regions. (b) The number of DgTCPs and corresponding cis-elements (red line) and the total number of cis-elements in the DgTCP gene family (black box). (c) The number of cis-elements (from the promoter regions of each DgTCP) associated with plant growth and development, phytohormone, and stress responses.
Figure 7. Cis-elements in the promoter regions of the DgTCP genes. (a) The cis-elements in the DgTCP promoter regions. (b) The number of DgTCPs and corresponding cis-elements (red line) and the total number of cis-elements in the DgTCP gene family (black box). (c) The number of cis-elements (from the promoter regions of each DgTCP) associated with plant growth and development, phytohormone, and stress responses.
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Figure 8. Expression profiles of 22 DgTCP genes in different orchardgrass tissues and developmental stages. (a) The 22 DgTCPs in different tissues. (b) The 22 DgTCPs in “Baoxing” and “Donata” at 5 developmental stages. Color changes from blue to red represent relatively low or high expression, respectively.
Figure 8. Expression profiles of 22 DgTCP genes in different orchardgrass tissues and developmental stages. (a) The 22 DgTCPs in different tissues. (b) The 22 DgTCPs in “Baoxing” and “Donata” at 5 developmental stages. Color changes from blue to red represent relatively low or high expression, respectively.
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Figure 9. The expression of 22 DgTCP genes in different tissues of D20170203 (low-tillering) and AKZ-NRGR667 (high-tillering). The tissues of D20170203 include the tiller bud, D_LB; the shoot base, D_LS; the root, D_LR; and the leaf, D_LL. The tissues of AKZ-NRGR667 include the tiller bud, A_HB; the shoot base, A_HS; the root, A_HR; and the leaf, A_HL. Color changes from blue to red represent relatively low or high expression, respectively.
Figure 9. The expression of 22 DgTCP genes in different tissues of D20170203 (low-tillering) and AKZ-NRGR667 (high-tillering). The tissues of D20170203 include the tiller bud, D_LB; the shoot base, D_LS; the root, D_LR; and the leaf, D_LL. The tissues of AKZ-NRGR667 include the tiller bud, A_HB; the shoot base, A_HS; the root, A_HR; and the leaf, A_HL. Color changes from blue to red represent relatively low or high expression, respectively.
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Figure 10. The expression of 14 DgTCP genes under drought (PEG), salt (NaCl), alkali (NaHCO3), ABA, Me-JA, and SA treatment by qRT-PCR. The error bars show the standard deviations of the three biological replicates. **, p < 0.01 and *, p < 0.05 show the significance of the differences between the control and treatment groups.
Figure 10. The expression of 14 DgTCP genes under drought (PEG), salt (NaCl), alkali (NaHCO3), ABA, Me-JA, and SA treatment by qRT-PCR. The error bars show the standard deviations of the three biological replicates. **, p < 0.01 and *, p < 0.05 show the significance of the differences between the control and treatment groups.
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Table
Table 1. The 22 TCP genes in orchardgrass.
Table 1. The 22 TCP genes in orchardgrass.
Gene NameGene IDChrProtein Length (aa)Length (bp)Molecular Weight (kDa)Isoelectric Point (pI)StartEnd
DgTCP1DG1C00255.1Chr116549517,433.515.186,629,9066,630,695
DgTCP2DG1C03379.1Chr1418125444,766.779.34146,031,371146,037,761
DgTCP3DG1C04745.1Chr120060021,319.20 9.92194,567,533194,568,868
DgTCP4DG2C01041.1Chr2392117639,526.739.4235,226,59535,228,755
DgTCP5DG3C00669.1Chr319057020,377.039.4120,872,68420,873,306
DgTCP6DG3C02184.1Chr3404121241,896.685.7198,406,79498,409,146
DgTCP7DG3C03958.1Chr328384930,759.216.21165,195,655165,196,687
DgTCP8DG3C05144.1Chr332898433,731.695.09204,201,074204,202,229
DgTCP9DG3C06789.1Chr3384115240,053.438.49251,633,645251,634,796
DgTCP10DG4C00330.1Chr429789131,333.776.5513,226,78613,230,494
DgTCP11DG4C01109.1Chr426880429,570.369.1545,855,85045,859,255
DgTCP12DG4C03206.1Chr4377113139,248.247.99150,217,992150,219,441
DgTCP13DG4C04368.1Chr4335100536,236.479.01187,896,786187,897,790
DgTCP14DG4C05327.1Chr4387116139,895.829.07217,216,581217,221,767
DgTCP15DG4C06102.1Chr4345103535,534.555.80 238,269,030238,270,515
DgTCP16DG5C01593.1Chr5454136247,382.566.4655,126,14655,128,837
DgTCP17DG5C02296.1Chr523871424,165.047.1580,396,97180,398,250
DgTCP18DG5C04172.1Chr526579528,827.166.90 174,582,071174,584,669
DgTCP19DG5C04259.1Chr5426127845,754.546.09176,990,965176,993,974
DgTCP20DG5C05852.1Chr532096033,538.986.05225,545,972225,547,688
DgTCP21DG6C01132.1Chr630290632,382.926.50 31,588,65231,591,023
DgTCP22DG7C01355.1Chr725576527,479.726.1448,396,59948,397,363
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Wang, C.; Feng, G.; Xu, X.; Huang, L.; Nie, G.; Li, D.; Zhang, X. Genome-Wide Identification, Characterization, and Expression of TCP Genes Family in Orchardgrass. Genes 2023, 14, 925. https://doi.org/10.3390/genes14040925

AMA Style

Wang C, Feng G, Xu X, Huang L, Nie G, Li D, Zhang X. Genome-Wide Identification, Characterization, and Expression of TCP Genes Family in Orchardgrass. Genes. 2023; 14(4):925. https://doi.org/10.3390/genes14040925

Chicago/Turabian Style

Wang, Cheng, Guangyan Feng, Xiaoheng Xu, Linkai Huang, Gang Nie, Dandan Li, and Xinquan Zhang. 2023. "Genome-Wide Identification, Characterization, and Expression of TCP Genes Family in Orchardgrass" Genes 14, no. 4: 925. https://doi.org/10.3390/genes14040925

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