Genome-Wide Identification, Characterization and Expression Analysis of TCP Transcription Factors in Petunia

Abstract

The plant-specific TCP transcription factors are well-characterized in both monocots and dicots, which have been implicated in multiple aspects of plant biological processes such as leaf morphogenesis and senescence, lateral branching, flower development and hormone crosstalk. However, no systematic analysis of the petunia TCP gene family has been described. In this work, a total of 66 petunia TCP genes (32 PaTCP genes in P. axillaris and 34 PiTCP genes in P. inflata) were identified. Subsequently, a systematic analysis of 32 PaTCP genes was performed. The phylogenetic analysis combined with structural analysis clearly distinguished the 32 PaTCP proteins into two classes-class Ι and class Ⅱ. Class Ⅱ was further divided into two subclades, namely, the CIN-TCP subclade and the CYC/TB1 subclade. Plenty of cis-acting elements responsible for plant growth and development, phytohormone and/or stress responses were identified in the promoter of PaTCPs. Distinct spatial expression patterns were determined among PaTCP genes, suggesting that these genes may have diverse regulatory roles in plant growth development. Furthermore, differential temporal expression patterns were observed between the large- and small-flowered petunia lines for most PaTCP genes, suggesting that these genes are likely to be related to petal development and/or petal size in petunia. The spatiotemporal expression profiles and promoter analysis of PaTCPs indicated that these genes play important roles in petunia diverse developmental processes that may work via multiple hormone pathways. Moreover, three PaTCP-YFP fusion proteins were detected in nuclei through subcellular localization analysis. This is the first comprehensive analysis of the petunia TCP gene family on a genome-wide scale, which provides the basis for further functional characterization of this gene family in petunia.

Keywords: TCP transcription factors; expression patterns; floral organ size; petal development; petunia.

Figure 1

Phylogenetic relationship of TCP transcription factors from petunia, Arabidopsis and tomato, based on the multiple alignments of the full-length amino acid sequences of TCP proteins from P. axillaris (PaTCP), tomato (SlTCP) and Arabidopsis (AtTCP). The neighbor-joining (NJ) algorithm-based phylogenetic tree was built using MEGA7.0 software with 500 bootstrap replicates. Green, red and blue lines represent the PCF, CYC/TB1 and CIN subclades, respectively.

Figure 2

Multiple sequence alignments of PaTCP proteins and analysis of the putative miR319 binding sites. (a). Alignment of the TCP domain and adjoining sequence for the predicted PaTCP proteins. Overall conserved amino acids were shaded in black. The Basic, Helix I, Loop and Helix II regions are indicated. (b). Alignment of the R-domain of class II PaTCP members. (c). Alignment of putative target areas for PamiR319 (aligned in reverse).

Figure 3

Exon-intron structures of PaTCP genes. The cladogram was produced by MEGA 7.0 software with the NJ method using the full-length sequences of PaTCP proteins. The orange, yellow and blue rectangles were used to cluster the genes into the CIN, CYC/TB1 and PCF subclades, respectively. 5′UTR and 3′UTR were not shown. The exons and introns are represented by a black block and a thin gray line, respectively. The 0, 1 and 2 denote intron phases.

Figure 4

Distribution of the conserved and potential motifs of PaTCP proteins. Each motif is represented by rectangular boxes with different colors and numbered from 1 to 20.

Figure 5

Expression analysis of PaTCP genes by qPCR. Gs, germinating seeds; Co, cotyledons; Sl, young seedlings; R, roots; St, stems; L, leaves; Ab, axillary buds; If, inflorescences; Fb, flower buds (0.5 cm); Fr, fruits. The relative expression level was normalized to the petunia EF1α gene. For each tissue, three biological replicates were used to calculate the mean values ± SD (standard deviation) with the 2^−ΔΔCT method.

Figure 6

Cis-regulatory elements analysis of PaTCP promoters. Based on the functional annotation, the cis-acting elements were categorized into three major classes: plant growth and development, phytohormone responsive or abiotic and biotic stresses-related cis-acting elements (details are shown in Table S3).

Figure 7

Transcriptional profiles of PaTCP genes in the large- and small-flowered lines, ‘L’ and ‘S,’ respectively, during different petal developmental stages: young flower buds (<0.5 cm, S1), extending flower buds (when flower buds just enclosed by sepals, S2), pre-anthesis (when flower buds extended to full length, S3), semi-open flowers (S4) and fully blooming flowers before the anthers dehisced (S5). The results were normalized to the expression of the petunia EF1α gene. The mean values ± SD (standard deviation) were calculated from three biological replicates with the 2^−ΔΔCT method. The black and dark grey square represented the ‘S’ and ‘L,’ respectively. Asterisks denote statistically significant differences between ‘S’ and ‘L’ at each developmental stage, as determined by Student’s t-test (* p < 0.05, ** p < 0.01).

Figure 8

Subcellular localization of three PaTCP-YFP fusion proteins in tobacco. Empty vector of yellow fluorescence (YFP) was used as control. The co-transformation of the nuclear marker (NF-YA4-mCherry) was used to visualize the nuclei. Merged panel showed the nuclear localization in tobacco epidermis cells. Scale bar, 25 μm.