Genome-wide identification and characterization of TCP gene family in Dendrobium nobile and their role in perianth development

Abstract

TCP is a widely distributed, essential plant transcription factor that regulates plant growth and development. An in-depth study of TCP genes in Dendrobium nobile, a crucial parent in genetic breeding and an excellent model material to explore perianth development in Dendrobium, has not been conducted. We identified 23 DnTCP genes unevenly distributed across 19 chromosomes and classified them as Class I PCF (12 members), Class II: CIN (10 members), and CYC/TB1 (1 member) based on the conserved domain and phylogenetic analysis. Most DnTCPs in the same subclade had similar gene and motif structures. Segmental duplication was the predominant duplication event for TCP genes, and no tandem duplication was observed. Seven genes in the CIN subclade had potential miR319 and -159 target sites. Cis-acting element analysis showed that most DnTCP genes contained many developmental stress-, light-, and phytohormone-responsive elements in their promoter regions. Distinct expression patterns were observed among the 23 DnTCP genes, suggesting that these genes have diverse regulatory roles at different stages of perianth development or in different organs. For instance, DnTCP4 and DnTCP18 play a role in early perianth development, and DnTCP5 and DnTCP10 are significantly expressed during late perianth development. DnTCP17, 20, 21, and 22 are the most likely to be involved in perianth and leaf development. DnTCP11 was significantly expressed in the gynandrium. Specially, MADS-specific binding sites were present in most DnTCP genes putative promoters, and two Class I DnTCPs were in the nucleus and interacted with each other or with the MADS-box. The interactions between TCP and the MADS-box have been described for the first time in orchids, which broadens our understanding of the regulatory network of TCP involved in perianth development in orchids.

Keywords: Dendrobium nobile; TCP gene family; expression pattern; perianth development; protein interaction.

Figure 1

Chromosome localization, gene duplication, and collinearity analysis of DnTCP genes. (A) The figure shows the distribution of DnTCP genes from D. nobile across 19 chromosomes. The scale on the left indicates the chromosome length. The scale is in megabases, Mb. (B) Genomic locations and segmental duplication of the DnTCP genes. The red lines link paralogous TCP genes. (C) Collinearity of the TCP genes between D. nobile and A thaliana, O. sativa, and D. chrysotoxum. The blue lines indicate the TCP gene pairs.

Figure 2

Multiple sequence alignment, the motifs, and the gene structures of DnTCPs. (A) The sequence alignment of TCP domains from D. nobile. The conserved regions (basic helix–loop–helix) the structure has been marked. (B) The sequence logo of TCP conserved regions. (C) Multiple sequence alignment of an R domain in TCP members. The conserved amino acids are in black.

Figure 3

The phylogenetic analysis and miRNAs-binding site recognition of DnTCP genes. (A) The phylogenetic analysis of TCPs and among D. nobile (blue block), A. thaliana (green circle), O. sativa (orange circle), C. goeringii (gray circle), P. equestris (violet circle), and D.catenatum (yellow circle). The unrooted phylogenetic tree was constructed using the neighbor-joining (NJ) method implemented in the MEGA X software with 1000 bootstrap replicates. The brownish-red, pink, and red ring indicate PCF, CYC/TB1, and CIN subclade, respectively. (B) TCP family members and distribution pattern of D. nobile, Arabidopsis, O. sativa, C. goeringii, P. equestris, and D.catenatum. (C) Analysis of the putative target sites for miR319 and miR159 in DnTCP mRNAs. Mismatches were represented by yellow.

Figure 4

Conserved motif and gene structure of the DnTCP genes. Phylogenetic tree of 23 DnTCP proteins in the left; The conserved motifs of the DnTCP genes in the middle and each colored box represents a motif of the protein; exon–intron structures of DnTCP proteins in the right. Blue squares indicate CDS and black lines indicate introns.

Figure 5

Cis-acting regulatory elements and MADS-box binding sites analysis of DnTCP genes. (A) Number of each cis-acting element in the promoter region of DnTCP genes. Based on the functional annotation, the cis-acting elements were classified into three main types: plant development and stress physiology, light-responsive, and phytohormone-responsive. (B) MADS-box binding sites (CArG-boxes) within the putative promoters of DnTCP genes of D. nobile. The different colors of rectangle represent indicate the different CArG-box variants, respectively.

Figure 6

Heat map clustering of codons RSCU of DnTCP genes.

Figure 7

Gene ontology (GO) annotation and prediction of interaction network among DnTCPs. (A) GO annotations of DnTCPs. The red, green, and blue bars represent GO terms of biological process, cellular component, and molecular function, respectively. (B) Analysis of interaction network among DnTCPs. The circle, octagon, and diamond represent PCF, CIN, and CYC/TB1 subclade, respectively. Different color blocks represent the number of interaction proteins. The black line represents the combined score of two interaction proteins, and a higher score corresponds to a thicker line.

Figure 8

Heat map of the expression patterns of 23 DnTCP genes. The expression profile data by qRT-PCR. Expression values are log2-transformed. The expression levels are represented by the color bar.

Figure 9

Spatio−temporal expression analysis of DnTCP genes at different perianth developmental periods or different organs of D. nobile. Relative values of gene expression were normalized to the expression of 18S rRNA. Statistically significant differences were conducted using the one-way ANOVA test and indicated with *(P<0.05), **(P<0.01), ***(P<0.001), and ****(P<0.001). Error bars mean SD values.

Figure 10

Subcellular localization analysis of DnTCP9 and DnTCP18 in Nicotiana benthamiana. Localization results of 35S:: GFP empty plasmid as the control. Co-localization of 35S:: DnTCP9-GFP or DnTCP18-GFP with nucleus marker DAPI and endoplasmic reticulum marker ER-mCherry. Scale bar = 20 µm.

Figure 11

Yeast-two-hybrid analysis of protein–protein interactions among DnTCP proteins or DnTCP proteins and DnSEP3-like. AD-T/BD-53 and AD-T/BD-Lam are the positive and negative controls, respectively.