Genome-Wide Identification of TCP Gene Family in Dendrobium and Their Expression Patterns in Dendrobium chrysotoxum

The TCP gene family are plant-specific transcription factors that play important roles in plant growth and development. Dendrobium chrysotoxum, D. nobile, and D. huoshanense are orchids with a high ornamental value, but few studies have investigated the specific functions of TCPs in Dendrobium flower development. In this study, we used these three Dendrobium species to analyze TCPs, examining their physicochemical properties, phylogenetic relationships, gene structures, and expression profiles. A total of 50 TCPs were identified across three Dendrobium species; they were divided into two clades—Class-I (PCF subfamily) and Class-II (CIN and CYC/TB1 subfamilies)—based on their phylogenetic relationships. Our sequence logo analysis showed that almost all Dendrobium TCPs contain a conserved TCP domain, as well as the existence of fewer exons, and the cis-regulatory elements of the TCPs were mostly related to light response. In addition, our transcriptomic data and qRT-PCR results showed that DchTCP2 and DchTCP13 had a significant impact on lateral organs. Moreover, changes in the expression level of DchTCP4 suggested its important role in the phenotypic variation of floral organs. Therefore, this study provides a significant reference for the further exploration of TCP gene functions in the regulation of different floral organs in Dendrobium orchids.


Introduction
In recent years, many studies have shown that TCPs are widely involved in plant growth and development, including seed germination [1,2], flower development [3][4][5], leaf development [3,6], gametophyte development [7][8][9], and lateral branching [10].Previous analyses have shown that TCP is a member of the RHH superfamily, but the crystal structures of the Class-I TCP domain from AtTCP15 and the Class-II TCP domain from AtTCP10 in complex with different double-stranded DNA (dsDNA) reveal that the TCP domain is a distinct DNA-binding motif, and the homodimeric TCP domains adopt a unique three-site recognition mode [11,12].Based on the differences in the TCP domains, the TCP gene family can be divided into two subfamilies: Class-I (PCF or TCP-P) and Class-II (TCP-C) [13].Class-I has a conserved four-amino-acid deletion in the TCP domain.Class-II is further subdivided into two clades: the CINCINATA(CIN) clade and the CYC/TB1 clade [14].
The distinct roles that TCPs play in plant growth and development are the subject of ongoing investigations.For instance, Fragaria × ananassa TCP11 interacts with MYB or bHLH gene family transcription factors to positively regulate the synthesis genes of proanthocyanidins and flavonoids [15].TCP TFs also play a role in plant abiotic stress alleviation [16].The CUC genes, which controls the morphogenesis of the shoot lateral organs and regulates the formation of the shoot meristem, is of critical importance to plant development [17].Moreover, TCP TFs have been extensively studied for their roles in the process of flowering and fruit development.For example, CYC/TB1 genes plays an important role in forming the symmetry of flowers, and CIN genes affects petal development.The deletion of TCP5 in mutant A. thaliana causes wider petals than that of wild-type A. thaliana [18].Therefore, it is necessary to explore the specific functions of TCPs in orchids.
As the largest family of monocotyledons, Orchidaceae account for 10% of total angiosperms worldwide.Therefore, they are ideal for studying biodiversity and evolution.Orchids have specialized flower organs, mainly including three sepals, two petals, one highly specialized lip, and a gynoecium [19].Dendrobium, the second largest genus of Orchidaceae, contains approximately 1450 species, the majority of which are characterized by a fleshy stem with abundant polysaccharides and grow in diverse habitats [20].TCPs have been widely reported in model plants and main crops; some studies have also explored the roles of Dendrobium TCPs in response to phytohormones by treating samples with plant hormones like methyl jasmonate (MeJA), abscisic acid (ABA), and salicylic acid(SA) [21].Furthermore, some have conducted research on plant habits and photosynthesis pathways, investigating the diversity of biological functions of TCPs in Dendrobium species [22].However, no studies have explored the regulating functions of TCPs in different flower development stages among Dendrobium species.With the development of sequencing technology, some orchid genomes have been reported, such as D. chrysotoxum [23], D. nobile [24], D. huoshanense [25], Dendrobium officinale [26], and Phalaenopsis aphrodite [27], and this provides a basis for orchid TCP gene family analysis.
In this study, a gene structure analysis, phylogenetic tree construction, collinearity analysis, and a cis-acting element analysis of the TCP gene family in three Dendrobium species were carried out.Specially, the expression patterns of five different floral parts in three different development stages (unpigmented bud, pigmented bud, and half-opened flower) of D. chrysotoxum were analyzed for the first time.Our results provide new insights into the molecular mechanisms of floral organ development and morphological diversity in Dendrobium.Exploring the regulation function of the TCP gene family in the flower organ of different development stages of Dendrobium is of great significance for the breeding and development of orchid resources.This study's results may also help to broaden our understanding of the roles of TCPs in regulating flower development and floral organ determination.

Identification and Physicochemical Properties of the TCPs
In this study, we identified the TCPs of three Dendrobium species based on the results of Blast and HMMER.A total of 15, 23, and 12 TCPs were identified in D. chrysotoxum, D. nobile, and D. huoshanense, respectively.In total, 50 TCPs were named DchTCP1-15, DnoTCP1-23, and DhuTCP1-12 according to their distribution order on chromosomes.Furthermore, the EXPASY online tool was used to analyze the physicochemical properties of Dendrobium.The deduced protein length of TCPs varied considerably in the number of amino acids (AA), ranging from 106 aa (DhuTCP9) to 786 aa (DnoTCP15, DhuTCP3), and the average length was 355aa.The theoretical isoelectric point (pI) ranged from 5.62 (DhuTCP9) to 9.86 (DchTCP1).The isoelectric points of 15 TCP proteins were less than 7.0, which was acidic, and the isoelectric points of the remaining 35 TCPs were more than 7.0, which was alkaline.The molecular weight (MW) of 50 TCPs in Dendrobium ranged from 11.41 (DhuTCP9) to 88.54 kDa (DhuTCP3), with an average MW of 38.84 kDa.The deducted grand average of hydrophilic values (GRAVY) of the TCPs ranged from −0.792 to −0.118, and the whole TCPs showed GRAVY values less than 0, suggesting that all the TCPs were hydrophilic.The aliphatic index (AI) ranged from 58.63 (DchTCP12) to 84.73 (DnoTCP6), and the instability index (II) ranged from 38.49 (DchTCP11) to 74.13 (DchTCP8).The physicochemical properties of 50 TCPs in three Dendrobium species are listed in Table S1.

Phylogenetic Analysis of TCPs
To analyze the evolutionary relationship of TCPs in Dendrobium, a phylogenetic tree consisting of TCPs was constructed from four species, including 24 AthTCPs, 15 DchTCPs, 23 DnoTCPs, and 12 DhuTCPs.A total of 74 TCPs were divided into two clades: Class-I (PCF genes) and Class-II (CYC/TB1 genes and CIN genes) (Figure 1).There were more members in Class-II than Class-I, and in Class-I, while there were more members of the CIN subfamily than there were in the CYC/TB1 subfamily, the number of Class-II members in D. huoshanense was similar to the other two Dendrobium species, but there were significant differences in Class-I (a total of 15 members; 3 DchPCFs, 11 DnoPCFs, and 1 DhuPCF).
to -0.118, and the whole TCPs showed GRAVY values less than 0, suggesting that all the TCPs were hydrophilic.The aliphatic index (AI) ranged from 58.63 (DchTCP12) to 84.73 (DnoTCP6), and the instability index (II) ranged from 38.49 (DchTCP11) to 74.13 (DchTCP8).The physicochemical properties of 50 TCPs in three Dendrobium species are listed in Table S1.

Phylogenetic Analysis of TCPs
To analyze the evolutionary relationship of TCPs in Dendrobium, a phylogenetic tree consisting of TCPs was constructed from four species, including 24 AthTCPs, 15 DchTCPs, 23 DnoTCPs, and 12 DhuTCPs.A total of 74 TCPs were divided into two clades: Class-I (PCF genes) and Class-II (CYC/TB1 genes and CIN genes) (Figure 1).There were more members in Class-II than Class-I, and in Class-I, while there were more members of the CIN subfamily than there were in the CYC/TB1 subfamily, the number of Class-II members in D. huoshanense was similar to the other two Dendrobium species, but there were significant differences in Class-I (a total of 15 members; 3 DchPCFs, 11 DnoPCFs, and 1 DhuPCF).huoshanense.The phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA7.0 software and was divided into two classes (three subfamilies) according to the classification of AtTCPs.

Gene Structure and Motif Analysis of TCPs
The conserved protein motifs of the 50 TCP proteins in Dendrobium were predicted using the MEME online tool, and 10 motifs were set as the upper bound.The results showed that most conserved motifs existed in the C-terminal domain, and the order was Motif2, Motif1, Motif4, Motif8, Motif5, Motif6, Motif3, Motif7, Motif10, Motif9 (Figure 2A).The phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA7.0 software and was divided into two classes (three subfamilies) according to the classification of AtTCPs.

Gene Structure and Motif Analysis of TCPs
The conserved protein motifs of the 50 TCP proteins in Dendrobium were predicted using the MEME online tool, and 10 motifs were set as the upper bound.The results showed that most conserved motifs existed in the C-terminal domain, and the order was Motif2, Motif1, Motif4, Motif8, Motif5, Motif6, Motif3, Motif7, Motif10, Motif9 (Figure 2A).Motif1 was the most conserved, Motif 6 existed only in CIN, and Motif7 was unique to Class-II.Almost all PCF proteins included Motif1 and Motif3; most CIN proteins included Motif2, Motif1, Motif4, and Motif5, and the CYC/TB1 proteins included Motif1, Motif2, and Motif7.This suggests that Class-II proteins contained more Motifs than Class-I proteins.To obtain the sequence logo of the TCP domains in Dendrobium, we conducted multiple sequence alignments.The results showed that the TCP domain encoded by Motif1 was highly conserved (Figure 2C).
of exons in the Class-I genes was less than that in the Class-II genes, and Dendrobium e hibited fewer exon numbers than other orchids [28].Due to differing intron lengths, t lengths of the genes among the CIN lineage were different.For example, the length of t encoded proteins of DchTCP13 and DchTCP14 were similar (324aa and 323aa), but t gene lengths were about 1.2 kb and 12 kb.Therefore, it is speculated that the CIN linea might play an important regulatory role in Dendrobium's growth, development, a adaptability to the external environment at the levels of gene copy number and gene stru ture.

Chromosomal Localization of TCPs
Based on the relevant genome annotation files, the gene location on the chromosom of D. chrysotoxum, D. nobile, and D. huoshanense were examined.DchTCP15 was localiz to the unanchored scaffold, and the remaining fourteen DchTCPs were distributed on ni chromosomes.In addition, a pair of gene tandem repeats existed on Chr14 (DchTC DchTCP10) (Figure 3A).A total of 23 DnoTCPs were distributed on 13 chromosomes of nobile (Figure 3B).Three chromosomes had three genes, four chromosomes had two gen and the remaining six chromosomes only contained one gene.Moreover, the distributi of DnoTCPs was similar at the top, middle, and bottom of the chromosomes.Twelve Dh TCPs were distributed on nine chromosomes of D. huoshanense (Figure 3C).Only two Dh TCPs (DhuTCP1, DhuTCP8) were localized on the bottom of chromosomes, and a pair gene tandem repeats existed on Chr16.To further study the characteristics of TCPs in Dendrobium, we analyzed the intronexon structure.The results showed that all 50 TCPs had introns that ranged from zero to two, and exons ranged from one to three (Figure 2B).Up to 80% of TCPs had no introns.The longest intron appeared at DchTCP1, followed by DchTCP14.In addition, the number of exons in the Class-I genes was less than that in the Class-II genes, and Dendrobium exhibited fewer exon numbers than other orchids [28].Due to differing intron lengths, the lengths of the genes among the CIN lineage were different.For example, the length of the encoded proteins of DchTCP13 and DchTCP14 were similar (324aa and 323aa), but the gene lengths were about 1.2 kb and 12 kb.Therefore, it is speculated that the CIN lineage might play an important regulatory role in Dendrobium's growth, development, and adaptability to the external environment at the levels of gene copy number and gene structure.

Chromosomal Localization of TCPs
Based on the relevant genome annotation files, the gene location on the chromosomes of D. chrysotoxum, D. nobile, and D. huoshanense were examined.DchTCP15 was localized to the unanchored scaffold, and the remaining fourteen DchTCPs were distributed on nine chromosomes.In addition, a pair of gene tandem repeats existed on Chr14 (DchTCP9, DchTCP10) (Figure 3A).A total of 23 DnoTCPs were distributed on 13 chromosomes of D. nobile (Figure 3B).Three chromosomes had three genes, four chromosomes had two genes, and the remaining six chromosomes only contained one gene.Moreover, the distribution of DnoTCPs was similar at the top, middle, and bottom of the chromosomes.Twelve DhuTCPs were distributed on nine chromosomes of D. huoshanense (Figure 3C).Only two DhuTCPs (DhuTCP1, DhuTCP8) were localized on the bottom of chromosomes, and a pair of gene tandem repeats existed on Chr16.

Cis-Acting Regulatory Elements Analysis of TCPs
To explore the regulatory function of Dendrobium, the 2000 bp promoter regions of the DchTCPs were retrieved to identify putative cis-regulatory elements.In total, 321, 499, and 226 promoter elements were identified in D. chrysotoxum (Figure 4A), D. nobile (Figure 4B), and D. huoshanense, respectively (Figure 4C).The functions included phytohormone responsiveness, stress responsiveness, plant growth and development responsiveness,

Cis-Acting Regulatory Elements Analysis of TCPs
To explore the regulatory function of Dendrobium, the 2000 bp promoter regions of the DchTCPs were retrieved to identify putative cis-regulatory elements.In total, 321, 499, and 226 promoter elements were identified in D. chrysotoxum (Figure 4A), D. nobile (Figure 4B), and D. huoshanense, respectively (Figure 4C).The functions included phytohormone responsiveness, stress responsiveness, plant growth and development responsiveness, and light response.Cis-acting regulatory elements related to light response were the most frequently occurring elements in Dendrobium, followed by MeJA-responsive and ABA-responsive elements, thereby indicating that the regulation of TCP functions by light is particularly im-portant during plant growth and development.However, the wound-responsive element was only found in DhuTCP12, and the auxin-responsive element was only observed in DhuTCP1 (Figure 4C).The top three Dendrobium TCPs with the highest number of cis-acting regulatory elements were as follows: DnoTCP7 (34), DnoTCP8 (33), and DhuTCP12 (32).We also discovered meristem expression elements in three Dendrobium species, indicating that TCPs play an important role in meristem tissue.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 6 and light response.Cis-acting regulatory elements related to light response were the m frequently occurring elements in Dendrobium, followed by MeJA-responsive and ABA sponsive elements, thereby indicating that the regulation of TCP functions by light is ticularly important during plant growth and development.However, the wound-res sive element was only found in DhuTCP12, and the auxin-responsive element was observed in DhuTCP1 (Figure 4C).The top three Dendrobium TCPs with the highest n ber of cis-acting regulatory elements were as follows: DnoTCP7 (34), DnoTCP8 (33), DhuTCP12 (32).We also discovered meristem expression elements in three Dendro species, indicating that TCPs play an important role in meristem tissue.S2.

Collinearity Analysis of TCPs
Six pairs of segmentally duplicated genes occurred in D. chrysotoxum genome (Figure 5A).Significantly, a pair of tandemly repeated genes (DchTCP9 and DchTCP10) was observed on Chr14.The D. nobile genome showed four pairs of segmentally duplicated genes (Figure 5B).Interestingly, one pair of segmentally duplicated genes was present in the same chromosome (CM039726.1).However, only two pairs of segmentally duplicated genes were present in the D. huoshanense genome (Figure 5C), which also contained a pair of tandemly repeated genes (DhuTCP6 and DhuTCP7) on Chr16.S2.

Collinearity Analysis of TCPs
Six pairs of segmentally duplicated genes occurred in D. chrysotoxum genome (Figure 5A).Significantly, a pair of tandemly repeated genes (DchTCP9 and DchTCP10) was observed on Chr14.The D. nobile genome showed four pairs of segmentally duplicated genes (Figure 5B).Interestingly, one pair of segmentally duplicated genes was present in the same chromosome (CM039726.1).However, only two pairs of segmentally duplicated genes were present in the D. huoshanense genome (Figure 5C), which also contained a pair of tandemly repeated genes (DhuTCP6 and DhuTCP7) on Chr16.

Expression Patterns of TCPs in D. chrysotoxum
To further understand the expression patterns of the TCPs, we drew a heatmap of the TCPs in three different parts of D. chrysotoxum at different developmental stages (Figure 6).We found that DchTCP12 was not detected in the transcriptome of D. chrysotoxum.DchTCP1, DchTCP2, and DchTCP13 showed similar expression patterns during three stages, all belonging to the CIN subfamily.DchTCP6, 7, 10, 11, and 14 exhibited high expression in all three stages, but no expression was observed in the ovary in S1.DchTCP4 represented high expression levels in the petals and lips in S1.However, DchTCP3 showed high expression only in the ovary and sepal in S1.DchTCP5 belonging to CYC/TB1 subfamily only showed high expression in the gynostemium in S1.Interestingly, DchTCP8 and DchTCP15 pertaining to the PCF subfamily were mainly expressed in S3.The expression levels of DchTCP15 were higher than DchTCP8, while DchTCP15 exhibited high expression patterns in the ovary in S1.

Expression Patterns of TCPs in D. chrysotoxum
To further understand the expression patterns of the TCPs, we drew a heatmap of the TCPs in three different parts of D. chrysotoxum at different developmental stages (Figure 6).We found that DchTCP12 was not detected in the transcriptome of D. chrysotoxum.DchTCP1, DchTCP2, and DchTCP13 showed similar expression patterns during three stages, all belonging to the CIN subfamily.DchTCP6, 7, 10, 11, and 14 exhibited high expression in all three stages, but no expression was observed in the ovary in S1.DchTCP4 represented high expression levels in the petals and lips in S1.However, DchTCP3 showed high expression only in the ovary and sepal in S1.DchTCP5 belonging to CYC/TB1 subfamily only showed high expression in the gynostemium in S1.Interestingly, DchTCP8 and DchTCP15 pertaining to the PCF subfamily were mainly expressed in S3.The expression levels of DchTCP15 were higher than DchTCP8, while DchTCP15 exhibited high expression patterns in the ovary in S1.

The qRT-PCR Analysis of TCPs in D. chrysotoxum
The results regarding the expression patterns showed that the expression levels of Class-II genes in the five flower parts were generally higher than that of the Class-I genes.To further understand the expression patterns of the two classes in different flower parts through the different stages of flower development, DchTCP2 (Class-I), DchTCP4 (Class-II), and DchTCP13 (Class-I) were selected for qRT-PCR analysis.The results showed a relatively significant correlation between the expression trend of the selected genes and the expression level of the transcriptome data (Figure 7).DchTCP2 and DchTCP13 were expressed in all flower parts except the ovary in S1, and the expression levels in S1 were significantly higher than those in S2 and S3.In addition, DchTCP4 represented no expression in the sepal in S1; the high expression in the sepal in S2 increased, and downregulation was observed in the sepal in S3.The highest expression of DchTCP4 was observed in the petal in S1, though this gradually declined in S2 and S3.S3.

The qRT-PCR Analysis of TCPs in D. chrysotoxum
The results regarding the expression patterns showed that the expression levels of Class-II genes in the five flower parts were generally higher than that of the Class-I genes.
To further understand the expression patterns of the two classes in different flower parts through the different stages of flower development, DchTCP2 (Class-I), DchTCP4 (Class-II), and DchTCP13 (Class-I) were selected for qRT-PCR analysis.The results showed a relatively significant correlation between the expression trend of the selected genes and the expression level of the transcriptome data (Figure 7).DchTCP2 and DchTCP13 were expressed in all flower parts except the ovary in S1, the expression levels in S1 were significantly higher than those in S2 and S3.In addition, DchTCP4 represented no expression in the sepal in S1; the high expression in the sepal in S2 increased, and downregulation was observed in the sepal in S3.The highest expression of DchTCP4 was observed in the petal in S1, though this gradually declined in S2 and S3.S3.S3.

The qRT-PCR Analysis of TCPs in D. chrysotoxum
The results regarding the expression patterns showed that the expression levels of Class-II genes in the five flower parts were generally higher than that of the Class-I genes.
To further understand the expression patterns of the two classes in different flower parts through the different stages of flower development, DchTCP2 (Class-I), DchTCP4 (Class-II), and DchTCP13 (Class-I) were selected for qRT-PCR analysis.The results showed a relatively significant correlation between the expression trend of the selected genes and the expression level of the transcriptome data (Figure 7).DchTCP2 and DchTCP13 were expressed in all flower parts except the ovary in S1, and the expression levels in S1 were significantly higher than those in S2 and S3.In addition, DchTCP4 represented no expression in the sepal in S1; the high expression in the sepal in S2 increased, and downregulation was observed in the sepal in S3.The highest expression of DchTCP4 was observed in the petal in S1, though this gradually declined in S2 and S3.S4.Y-axis represents relative expression values  S4.

Discussion
Orchidaceae is one of the most precious wild plant resources in the world, and the Dendrobium genus is particularly important due to its high ornamental value and medicinal value [29].TCP proteins are plant-specific TFs that play a critical role in regulating plant growth and development.Although TCPs have been found in plants such as Z. mays, O. sativa, and A. majus, insufficient research has been conducted on their specific roles in orchids [30][31][32].In this study, we identified fifty TCPs in three Dendrobium species.Our study's results showed that the number of TCPs in D. chrysotoxum (15) and D. huoshanense (12) were similar to C. goeringii (14) [33] but less than Phalaenopsis equestris (23) [34] and D. catenatum (25) [35].D. chrytosoxum and D. huoshanense may have suffered genetic loss during evolution.This indicates that genome size, chromosome numbers, and gene duplication are potentially responsible for the differences.
The TCP gene family was divided into two classes including three subfamilies (PCF, CIN, and CYC/TB1) based on a phylogenetic tree that pertained to A. thaliana, D. chrysotoxum, D. nobile, and D. huoshanense.The phylogenetic analysis indicated that only AtTCP16 was not found to have a similar homolog.The number of TCPs in Class-II was similar among the three Dendrobium species, but there was a significant difference in the number of TCPs in Class-I (3 DchPCFs, 11 DnoPCFs, and 1 DhuPCF).The number of amino acids in the TCPs of three Dendrobium species demonstrated a significant difference, ranging from 106 aa (DhuTCP9) to 786 aa (DnoTCP15, DhuTCP3).These results showed the potential complexity of the origin and evolution of the Dendrobium TCP gene family, suggesting a strong relationship with biological functional diversity [36].
Our gene structure analysis indicated that the three Dendrobium species highly similar exon-intron structures.Almost all reported orchids have long introns, but only three long introns were observed in this study.Interestingly, they were all displayed in D. chrysotoxum.C. goeringii only has four long introns [33].The genes with introns only account for 20% of all 50 TCPs in Dendrobium, possibly due to intron loss during gene structure evolution [37].Despite not providing any benefit in recombination nor species evolution, intron-free genes frequently react quickly to stress [38].Furthermore, we analyzed the conserved motifs in three Dendrobium species.The Class-II genes containing Motif1, 2, and 4 were more conservative than the Class-I genes.Motif 6 only showed in CIN subfamilies, suggesting that CIN genes may have particular biological functions.Through the Motif logo in three Dendrobium species and the above analysis, it can be concluded that the TCP domain of Dendrobium is relatively conserved in the plant evolutionary process.
Whole genome duplication (WGD) [39][40][41] is considered an important factor in genome evolution and gene family formation and expansion [42,43].D. chrysotoxum, D. nobile, and D. huoshanense have experienced at least two whole-genome duplication events [21][22][23].Therefore, the TCP number and distribution in the three Dendrobium species have some differences.There was a pair of tandemly repeated genes on Chr14 in D. chrysotoxum and Chr16 in D. huoshanense, respectively.The collinearity analysis results showed six pairs of segmentally duplicated genes in D. chrysotoxum, four pairs of segmentally duplicated genes in D. nobile, and only two pairs of segmentally duplicated genes in D. huoshanense.Overall, these findings indicate that whole-genome duplication plays a role in the expansion of the TCP gene family of Dendrobium.
The prediction of promoter elements at the transcriptional level provides a more thorough understanding of gene regulation [44].The Cis-acting elements for a series of functional types were identified in the TCPs of three Dendrobium species, including light-responsive, phytohormone-responsive, stress-responsive, and plant growth and development elements.In this study, we found that the light-responsive elements were the most frequently occurring elements in the 2000 bp upstream sequences from Dendrobium, accounting for 41.3% of all cis-acting elements.Light affects multiple developmental processes, including seed germination, shade avoidance, and photoperiod responses [45].So, our analysis indicated that TCPs may play a role in these developmental processes.The cis-acting elements of phytohormone accounted for 16.1% of all elements, mainly including MeJA.MeJA is a vital cellular regulator against biotic and abiotic stresses in different development processes [46].Therefore, it is speculated that TCPs may impact the stress resistance of Dendrobium.The proportion of biotic stresses and growth and development elements were 16.0% and 26.6%, respectively.More cis-acting elements were related to meristem expression in the last type.The identification of TCP1 in the regulation brassinosteroid (BR) biosynthesis suggested that TCP1 may cause the uneven distribution of BRs in floral meristems [47].In the inflorescence shoot apex and seed, DELLA proteins release TCPs to simulate cell division in the root apical meristem, thereby separately stimulating shoot elongation and seed germination [48,49].Based on the above discussion, we can infer that Dendrobium TCPs is actively involved in meristem development.
It has been reported that TCP transcription factors are involved in plant development processes such as gametophyte development, lateral branching, seed germination, flower development, and leaf development [1][2][3][4][5][6][7][8][9][10].Through expression analysis, we found that, among all of the detected DchTCPs, the expression profiles of the Class-II genes were more active than that of the Class-I genes.Moreover, the DNA sequences recognized by Class-I or Class-II TCPs are not mutually exclusive, suggesting that they may share the same target genes and that they function together in regulating gene expression [50].It would be interesting to determine if Class-I and Class-II TCPs interact with the same target gene in Dendrobium.Moreover, the expression levels of TCPs from the CYC/TB1 subfamily were lower than those from the CIN subfamily, and DchTCP12 from the CYC/TB1 subfamily was not detected in the transcriptome.DchTCP4 and DchTCP15 from the PCF subfamily showed active expression levels in the gynostemium and ovary in the unpigmented bud stage, further confirming the findings of a previous study that reported that PCFs in the TCP gene family regulate seed germination and gametophyte development [51].Interestingly, DchTCP2 and DchTCP13 participated in all parts in S1 except the ovary, and their expression levels in S1 were much higher than those of the other two stages.Based on these results, we speculate that DchCINs may be involved in the color transformation process of flower buds.In addition, these two genes were expressed in both sepal and petal in the S1 and S2, which is consistent with the function of CINs participating in the development of lateral organs [52,53].It has been reported that the TCP gene BRC1 interacts with flowering genes and modulates floral activity in the axillary buds [54].Furthermore, the decrease in five Class-II TCPs may be related to the delayed flowering phenotype of Arabidopsis mutants [55].We speculate that DchTCP2 and DchTCP13 may exert an important effect on the regulation of florescence.Previous studies have found that the downregulation of AtTCP14, 15 from the PCF subfamily may affect the development of outer whorls and gynoecia [56].Combining the downregulation of DchTCP4 during three stages, we propose that DchTCP4 belonging to PCF subfamily may have a major effect on the phenotype of floral organs.

Plant Materials
The plant materials were collected from the National Orchid Germplasm Resources of Fujian Agriculture and Forestry University, Fuzhou, China.We used five different parts of D. chrysotoxum (petal, sepal, lip, ovary, and gynostemium) at three developmental stages, namely the unpigmented bud stage (S1), the pigmented bud stage (S2), and the half-opened flower stage (S3).All samples were frozen at −80 • C.

Identification and Physicochemical Properties of the TCP Gene Family
In order to identify potential TCPs in Dendrobium, 24 A. thaliana TCP proteins were used as a probe in a local BLASTp search (built-in TBtools v 1.120) [26].The Hidden Markov Model (HMM) profile of the TCP conserved domain (PF03634) from the online database (http://pfam-legacy.xfam.org/,accessed on 13 June 2023) was used to further identify TCPs in Dendrobium using the Simple HMM Search in TBtools.The results of BLASTp and HMMER were combined to manually remove incomplete and redundant protein sequences, and uncertain genes were uploaded to the NCBI (http://blast.ncbi.nlm.njh.gov/,accessed on 13 June 2023) website prior to a BLASTp search.The online software ExPASy (http://www.expasy.org/,accessed on 13 June 2023) was used to analyze the amino acid (AA) content, isoelectric points (pIs), molecular weights (MWs), hydrophilic large averages (GRAVY), instability index (II), and fat index (AI) of the proteins [27].

Phylogenetic Analysis of TCPs
The 24 TCP protein sequences of A. thalian (AthTCP), 23 TCP protein sequences of D. nobile (DnoTCP), 12 TCP protein sequences of D. huoshanense (DhuTCP), and 15 TCP protein sequences of D. chrysotoxum (DchTCP) were introduced into the MEGA7.0software.The alignment sequences selected that were using the ClustalW program for multisequence alignment to assess the similarities between proteins, Gap Opening and Gap Extend, were 15 and 6.66, respectively; the DNA Weight Matrix selection was the IUB; other values were kept as the default values.Then, the neighbor-joining (NJ) method was carried out using 500 replications of the bootstrap method to construct a phylogenetic tree consisting of a total of 50 sequences.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches.In addition, we performed the bootstrap method through using 500 replicates (partial deletion with 50%).The online software Evloview3.0(https://evolgenius.info//evolview-v2/# mytrees/tree/ATDc%20TCP, accessed on 15 June 2023) was used to improve and beautify the phylogenetic tree.

Conserved Motifs and Gene Structure Analysis of TCPs
The conserved domains of the TCPs were analyzed using the NCBI conserved domain database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi,accessed on 13 June 2023).For conserved motif prediction, all TCP protein sequences of Dendrobium were submitted to the Multiple Em for Motif Elicitation (MEME) online tool (http://memesuite.org/meme/tools/meme,accessed on 13 June 2023) for identification.We set the maximum number of motifs at ten, and the other parameters were kept at the defaults.Finally, the phylogenetic tree, conserved motifs, and gene structures of the Dendrobium TCPs were visualized using TBtools.

Collinearity Analysis and Chromosomal Localization of TCPs
Using the Gene Location Visualize tool from the GTF/GFF programs in TBtools with the annotation files for Dendrobium, we obtained the chromosomal locations of the Dendrobium TCPs.For collinearity analysis, the genome data of three Dendrobium species were compared to themselves via the use of the One Step MCScanx program in TBtools.Then, we used the One Step MCScanx program and the Advance Circos to visualize the duplication patterns in three Dendrobium species.

Promoter Element Analysis of TCP Genes
We used TBtools to obtain a 2000 bp gene sequence upstream of the promoter codon from the genome of three Dendrobium species to identify putative cis elements in the promotor [26].PlantCARE online software (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 June 2023) was used to analyze cis-regulatory elements in the promoter region of the TCPs in Dendrobium.Finally, the data were processed and appropriately sorted using Excel 2016 software before being visualized using TBtools v2.003.

Expression Analysis and RT-qPCR
Transcriptome sequencing and library construction were completed by Bgi Genomics Co., Ltd.(Shenzhen, China).Bowtie2 2.2.9 was used to compare the clean reads to the genomic sequence and RSEMv1.2.8 software was used to calculate the gene expression level of each sample.Gene expression levels were measured as fragments per kilobase of transcript per million fragments (FPKM) values.The data were visualized using TBtools v2.003, and a heat map was generated for D. chrysotoxum.
Total RNA was extracted from the plant materials using a FastPure Plant Total RNA Isolation Kit (for polysaccharide-and polyphenol-rich tissues) (Vazyme Biotech Co., Ltd., Nanjing, China).A reverse Transcript Kit PrimerScript ®® RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) was used for reverse transcription to remove the contaminated genomic DNA and generate cDNA.Primer Premier 5 software was used to design primers, and Maker75111 was selected as the reference gene based on the transcriptome data (Table S4).Hieff UNICON ® Universal Blue qPCR SYBR Green Master Mix was used for a qRT-PCR analysis on an ABI 7500 Real-Time System.The experimental setup utilized 96-well plates with a 20 µL reaction system in each well, and three biological replicates of the study were performed.The RT-qPCR conditions were 2 min at 95 • C in the holding stage and 40 cycles of 10 s at 95 • C and 30 s at 60 • C in the cycling stage.The relative expression levels of the target genes were calculated using the 2 −∆∆CT method for the experimental data and normalized using GraphPad Prism 7.0 [57].

Figure 1 .
Figure 1.Phylogenetic tree of 74 TCP proteins from A. thaliana, D. chrysotoxum, D. nobile, D.huoshanense.The phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA7.0 software and was divided into two classes (three subfamilies) according to the classification of AtTCPs.

Figure 1 .
Figure 1.Phylogenetic tree of 74 TCP proteins from A. thaliana, D. chrysotoxum, D. nobile, D. huoshanense.The phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA7.0 software and was divided into two classes (three subfamilies) according to the classification of AtTCPs.

Figure 2 .
Figure 2. Conserved motifs, gene structure, and conserved domains of TCPs.(A) Phylogenetic t and conserved motifs of the TCPs of D. chrysotoxum, D. nobile, and D. huoshanense.(B) Gene structu of the TCPs of D. chrysotoxum, D. nobile, and D. huoshanense.(C) Sequence logo of Motif1 of D. chr otoxum, D. nobile, and D. huoshanense, which encodes the TCP domain.

Figure 2 .
Figure 2. Conserved motifs, gene structure, and conserved domains of TCPs.(A) Phylogenetic tree and conserved motifs of the TCPs of D. chrysotoxum, D. nobile, and D. huoshanense.(B) Gene structure of the TCPs of D. chrysotoxum, D. nobile, and D. huoshanense.(C) Sequence logo of Motif1 of D. chrysotoxum, D. nobile, and D. huoshanense, which encodes the TCP domain.

Figure 3 .
Figure 3. Chromosome distribution in three Dendrobium species.(A) Chromosome distribution in D. chrysotoxum.(B) Chromosome distribution in D. nobile.(C) Chromosome distribution in D. huoshanense.The yellow letters represent chromosome names, while the red letters represent gene names.

Figure 3 .
Figure 3. Chromosome distribution in three Dendrobium species.(A) Chromosome distribution in D. chrysotoxum.(B) Chromosome distribution in D. nobile.(C) Chromosome distribution in D. huoshanense.The yellow letters represent chromosome names, while the red letters represent gene names.

Figure 4 .
Figure 4. Regulatory elements in the promotor region of three Dendrobium species.(A) The cisacting elements of D. chrysotoxum.(B) The cis-acting elements of D. nobile.(C) The cis-acting elements of D. huoshanense.Raw data are listed in TableS2.

Figure 4 .
Figure 4. Regulatory elements in the promotor region of three Dendrobium species.(A) The cisacting elements of D. chrysotoxum.(B) The cis-acting elements of D. nobile.(C) The cis-acting elements of D. huoshanense.Raw data are listed in TableS2.

Figure 7 .
Figure 7. Real-time fluorescence quantitative expression analysis on DchTCPs at the different stages of flower development.Raw data are listed in Table S4.Y-axis represents relative expression values

Figure 7 .
Figure 7. Real-time fluorescence quantitative expression analysis on DchTCPs at the different stages of flower development.Raw data are listed in TableS4.Y-axis represents relative expression values

Figure 7 .
Figure 7. Real-time fluorescence quantitative expression analysis on DchTCPs at the different stages of flower development.Raw data are listed in Table S4.Y-axis represents relative expression values (2−∆∆CT).The red asterisk indicates the P value in the significance test (* p < 0.05, ** p < 0.01, **** p < 0.0001).Primers and RT-qPCR analyses of TCP genes are shown in TableS4.