Genome-wide identification of the TCP gene family in Chrysanthemum lavandulifolium and its homologs expression patterns during flower development in different Chrysanthemum species

Abstract

TCP proteins, part of the transcription factors specific to plants, are recognized for their involvement in various aspects of plant growth and development. Nevertheless, a thorough investigation of TCPs in Chrysanthemum lavandulifolium, a prominent ancestral species of cultivated chrysanthemum and an excellent model material for investigating ray floret (RF) and disc floret (DF) development in Chrysanthemum, remains unexplored yet. Herein, a comprehensive study was performed to analyze the genome-wide distribution of TCPs in C. lavandulifolium. In total, 39 TCPs in C. lavandulifolium were identified, showing uneven distribution on 8 chromosomes. Phylogenetic and gene structural analyses revealed that ClTCPs were grouped into classes I and II. The class II genes were subdivided into two subclades, the CIN and CYC/TB1 subclades, with members of each clade having similar conserved motifs and gene structures. Four CIN subclade genes (ClTCP24, ClTCP25, ClTCP26, and ClTCP27) contained the potential miR319 target sites. Promoter analysis revealed that ClTCPs had numerous cis-regulatory elements associated with phytohormone responses, stress responses, and plant growth/development. The expression patterns of ClTCPs during capitulum development and in two different florets were determined using RNA-seq and qRT-PCR. The expression levels of TCPs varied in six development stages of capitula; 25 out of the 36 TCPs genes were specifically expressed in flowers. Additionally, we identified six key ClCYC2 genes, which belong to the class II TCP subclade, with markedly upregulated expression in RFs compared with DFs, and these genes exhibited similar expression patterns in the two florets of Chrysanthemum species. It is speculated that they may be responsible for RFs and DFs development. Subcellular localization and transactivation activity analyses of six candidate genes demonstrated that all of them were localized in the nucleus, while three exhibited self-activation activities. This research provided a better understanding of TCPs in C. lavandulifolium and laid a foundation for unraveling the mechanism by which important TCPs involved in the capitulum development.

Keywords: Chrysanthemum; TCP gene family; capitulum development; expression analysis; ray and disc floret.

Figure 1

Multiple sequence alignments of TCP proteins in C. lavandulifolium and analysis of the potential miR319-targeted sites. (A) TCP domain alignment for the ClTCP proteins. The conserved amino acids were shaded in grey, and the bHLH regions were marked. (B) R domain alignment of the class II subfamily ClTCP members. Multiple sequence alignments were performed with Genedoc. (C) Alignment of potential target sites for miR319 (aligned in reverse). Target sites were taken from the coding sequences of ClTCPs, and the mature miR319 sequence was obtained from miRBase (http://www.mirbase.org/).

Figure 2

Chromosomal positions of TCPs on C. lavandulifolium chromosomes. The scale shows a chromosomal distance of 50 Mb. All 39 ClTCPs were localized to the chromosomes. Segmentally duplicated genes (EVM0004928.1_ClTCP5 and EVM0024248.1_ClTCP6, EVM0001252.1_ClTCP20 and EVM063727.1_ClTCP21) were marked with blue letters. Eleven ClTCPs belong to CYC/TB1 subclade, which are highlighted in red and located on the chromosomes.

Figure 3

Phylogenetic and statistical analyses of TCP members from C. lavandulifolium and Arabidopsis. (A) The tree was built using the Maximum Likelihood (ML) method with 1000 replicates by MEGA X. Multiple sequence alignments of ClTCPs was performed using MAFFT v7. The bootstrap value was indicated by the number above the branches and values less than 50 would not be displayed. Red, green and blue lines indicate the PCF, CIN and CYC/TB1 subclades, respectively. (B) Statistical analysis of TCP members from C. lavandulifolium and A. thaliana.

Figure 4

Conserved motif distributions and gene structure of ClTCPs. (A) The tree was generated based on the sequences of ClTCP proteins. The yellow, purple and green rectangles indicated the clustering of genes into the PCF, CIN and CYC/TB1 subclades, respectively. (B) Conserved motifs in C. lavandulifolium TCP members were identified by MEME tools. Each motif is indicated by a rectangular box of a different color and numbered from 1 to 10. (C) Gene structure analysis of ClTCPs. The coding sequence (CDS), untranslated region (UTRs) and introns are indicated by yellow boxes, green boxes and blank lines, respectively.

Figure 5

Cis-regulator elements analysis of ClTCP promoters. (A) Distribution of cis-regulator elements in the promoter region (2 kb upstream of the transcription start site) of ClTCPs. (B) Statistical analysis on the number and functional type of cis-regulatory elements in all ClTCP promoters in C. lavandulifolium.

Figure 6

Expression patterns of ClTCPs during capitulum development and in two different florets of C. lavandulifolium. Red and blue boxes show high and low expression levels, respectively. Green, black and red color of gene names belong to PCF, CIN, and CYC/TB1, respectively. S1, S2, S5, S6, S9, and S10 indicated the different development stages of capitula (Wen et al., 2022), L indicated reproductive-stage leaf; D1, D2, R1, R2 indicated disc and ray florets at two stages.

Figure 7

Relative expression levels of ClCYC genes in eight different Chrysanthemum samples using qRT-PCR. (A) Capitulum and DFs and RFs of two development stages of C. lavandulifolium (Cl), C. indicum (Ci), C. vesticum (CVW), C. ×morifolium ‘C27’, ‘D91’, ‘G70’, ‘T3’ and C. aromaticum (SN). Bars=1 cm. (B) Expression levels of six ClCYC2 genes in DFs and RFs of the above samples. Data were normalized to the expression data of ClSAND and expressed as the means ± standard error of three biological replicates.

Figure 8

Subcellular localization of six ClCYC2 proteins. Six ClCYC2-GFP fusion recombinants (ClCYC2a-GFP, ClCYC2b-GFP, ClCYC2c-GFP, ClCYC2d-GFP, ClCYC2e-GFP, and ClCYC2f-GFP) and pBI121-GFP as control were transiently expressed in N. benthamiana leaves and observed by a laser scanning confocal microscope 48 hours after injection. Bar=25 µm.

Figure 9

Transactivational analysis of ClCYC2 proteins in Y2HGold strain. The fusion constructs with pGADT7 AD empty, positive constructs (pGBKT7-53) and negative constructs (pGBKT7-lam) were co-transformed into Y2HGold strain and incubated on the SD/-Leu-Trp and SD/-Ade-His-Leu-Trp media.