3.1. Identification and Conserved Domains Analysis of Putative TCP Genes in B. masoniana
The acquisition of the B. masoniana genome sequence offers a valuable resource for comprehensive whole-genome identification (Li et al. 2022). To identify TCP gene family in B. masoniana, we conducted a local BLAST search using Arabidopsis thaliana TCP protein sequences against the B. masoniana genome database. A total of 37 candidate BmaTCPs were selected, and the presence of TCP domains was verified through SMART and Pfam databases. After eliminating redundant sequences manually, we obtained 35 putative TCPs, which were designated as BmaTCP1 to BmaTCP35 based on their sequential accession numbers (Supplementary Data 1).
The involvement of TCP domain has been shown to be crucial in facilitating the dimerization and DNA binding processes of bHLH structures, which are typically comprised of 59 amino acid residues (Yu et al. 2020). The present study successfully identified all TCP structural domains in BmaTCP, which consist of either 55 or 59 amino acid residues and exhibit a bHLH structure (Fig. 1B). In comparison to the BmaTCP II class members, the Class I BmaTCPs members exhibited a lack of four amino acids in the bHLH motif. The loop, helix I, and helix II regions of Class I and Class II BmaTCPs exhibited noticeable differences in their AA sequences; nevertheless, a conserved tandem comprising tryptophan (W) and leucine (L) was detected within helix II. Therefore, based on variations within their TCP domains, BmaTCPs can be categorized into two TCP classes: 17 belong to Class I while 18 BmaTCPs Class II can further be divided into two subcategories (CYC/TB1 and CIN) (Fig. 1A).
3.2. Chromosomal locations and Physical and chemical characteristics Analysis of BmaTCP Genes
A total of 35 putative BmaTCP genes were found to be distributed on chromosomes (Chr) 1 to 15, excluding Chr5 (Fig. 2). Among the identified chromosomes, Chr14 displayed the most putative TCP genes (6 genes), whereas only one TCP was found on Chr2, Chr4, and Chr9.
TCPs in this study display variations in coding sequence (CDS), amino acid sequence, isoelectric point (pI), and molecular weight (MW) (Table 1). BmaTCP32 stands out among the group of 35 BmaTCPs due to its compact size, consisting of only 161 amino acids, while BmaTCP18 represents the largest protein with a length of 478 amino acids. The molecular weight of these proteins ranges from 18.62 kDa (BmaTCP32) to 51.14 kDa (BmaTCP18), and their pI values range from 5.66 (BmaTCP28) to 10.11 (BmaTCP19). Consistent with TCP genes family observed in other species, subcellular localization analysis reveals that BmaTCPs are predominantly localized within the nucleus; however, it should be noted that both BmaTCP4 and BmaTCP22 are predicted to be membrane-bound proteins (Table 1).
3.3. Phylogenetic Investigation and Categorization of Presumed TCP Genes in B. masoniana
To explore the evolutionary connection among the BmaTCP family, an unrooted phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 11.0. This analysis relied on multiple sequence alignments of B. masoniana (35 BmaTCPs), Arabidopsis thaliana (24 AtTCPs), and Oryza sativa (21 OsTCPs). (Fig. 4). All TCP proteins are classified into six subfamilies (Groups A to F). Based on the sequence structure within TCP domain, Groups A, B, and C belong to Class I subclass PCF-type, Group D belongs to Class II subclass CYC/TB1-type, and Groups E and F belong to Class II subclass CIN-type. Comparative analysis of homology and phylogenetic studies between BmaTCPs and genes from other species can facilitate the prediction of potential gene functions (Kapli et al. 2020). Currently, extensive research has been conducted on the role of TCP genes in Arabidopsis, emphasizing the significance of homology analysis between BmaTCPs and AtTCPs for comprehensive exploration and analysis. In Arabidopsis, the co-regulation of leaf margin morphology by AtTCP5 and BEL-like transcription factors (Yu et al. 2020), which are phylogenetically related to BmaTCP1 (Fig. 3), suggests a potential involvement of BmaTCP1 in regulating leaf margin formation. The mRNA of AtTCP4 is regulated by miR319, which functions as one of its targets, thereby playing a pivotal role in leaf morphology and development (Alvarez et al. 2016; Lan et al. 2021). Importantly, it shares sequence homology with BmaTCP3, BmaTCP12, and BmaTCP13, suggesting their potential involvement in the regulation of leaf developmental processes.
3.4. Exon-Intron Organization and Motif Analysis of BmaTCPs in B. masoniana
Based on the genomic and coding sequence analysis of B. masoniana, it was noted that the coding sequences (CDS) exhibited a range in length spanning from 486 to 1437 base pairs. The genes in question possess either zero or one intron. Notably, two-thirds of BmaTCPs exhibit a single exon structure, which is consistent with the gene architecture observed in TCP family members across various species (Ma et al. 2014; Zhou et al. 2016; Wen et al. 2020).
The conserved motifs in BmaTCPs were identified and labeled as Motif 1 to Motif 10 (Fig. 4B). Motif 1, identified in all BmaTCPs, was characterized as a conserved bHLH structure, highlighting its essential contribution to the functionality of BmaTCPs. Class I exclusively contained motifs 2, 4, 5, 8, and 9, while motifs 3,7,6, and10 were specifically observed in Class II. Consequently, BmaTCPs belonging to the same category exhibit conserved motifs, while distinctions are evident between the two categories, implying that BmaTCPs within a specific category may possess analogous functionalities with certain motifs potentially playing vital roles.
3.5. Promotor Cis-Acting Element Analysis of Putative BmaTCP Genes
Cis-regulatory elements present in gene promoters play a pivotal role in governing the expression pattern and localization of genes, thereby contributing significantly to our understanding of transcriptional regulation and gene functionality. In this investigation, we identified multiple cis-regulatory elements associated with hormone response, light response, environmental stress response, growth and development, promoter-related elements, as well as binding sites within the presumed BmaTCP promoters (Fig. 5A). All BmaTCPs exhibited the presence of light-responsive elements. The abundance of light-responsive related elements encompassed Box4, AE-Box, ACE motif, ACA-motif, G-box etc. (Supplementary Data 2). Promoter-related elements were found to be the most prevalent; however, they consisted solely of three types (Box III, CAAT-box, TATA-box).
Among them, a total of 548 putative cis-elements were identified to be involved in the response to plant hormones (such as auxin, methyl jasmonate (MeJA), gibberellins (GA)), environmental stresses (low temperature, drought), and plant growth and development in B. masoniana (Fig. 5B). Except for BmaTCP3 and BmaTCP27, all other BmaTCPs contained anaerobic induction elements, indicating their involvement in gene regulation under hypoxic conditions. Over three-fourths of the BmaTCPs possessed MeJA-responsive elements (CGTCA-motif, TGACG-motif), while only four BmaTCPs lacked abscisic acid-responsive elements (ABRE). Additionally, several hormone-related cis-regulatory elements including auxin response elements (AuxRR core, TGA-element) and gibberellin response elements (P-box, GARE-motif, TATC-box) were identified in the promoters of BmaTCPs. The presence of numerous hormone-responsive elements in the promoter regions of BmaTCPs suggests their significant role in regulating plant growth and development. Almost half of the BmaTCPs contained zein metabolism regulatory element (O2 site) related to maize endosperm protein synthesis regulation among several cis-elements associated with plant growth and development. Furthermore, circadian rhythm regulatory element (circadian), leaf development-related element (HD-Zip1 motif), and seed-specific regulatory element (RY motif) were also discovered within the sequences of BmaTCPs.
3.6. Subcellular Localizations
In order to validate the predicted subcellular localization of BmaTCP, we successfully cloned and fused the full-length coding sequence (CDS) of BmaTCP29 with GFP (with green fluorescent protein) in the pSAT-1403TZ vector for sequencing purposes. Protoplasts from dwarf morning glory leaves were used as material, and the recombinant vector was transiently introduced into protoplasts via PEG-mediated transformation. Subsequently, observation and recording were conducted using fluorescence microscopy. The results of subcellular localization demonstrated exclusive nuclear expression of GFP fused with BmaTCP29, thereby indicating specific nuclear localization and functionality of BmaTCP29 (Fig. 6).
3.7. Expression Patterns of BmaTCPs During Different Leaf Developmental stages
To elucidate the role of BmaTCPs in leaf development, we employed quantitative real-time PCR (qRT-PCR) to investigate the expression profiles of seven BmaTCP genes at different developmental stages (S1-S3) across various leaf regions (Fig. 7). The expression patterns of BmaTCP1 and BmaTCP3 at different developmental stages of the leaf margin are discontinuous. In comparison to other stages, BmaTCP1 exhibits a significantly elevated level of expression during S2 stage. Conversely, BmaTCP3 exhibits relatively low levels of expression during the S2 stage. Compared with other developmental stages, the expression pattern of BmaTCP2 is significantly higher in the S1 stage of leaf margin and subsequently decreases as the leaf matures, indicating its involvement in early leaf margin development. In contrast, BmaTCP12 exhibits high expression specifically in the S3 stage at the leaf margin but shows low expression in the S3 stage at the center of the leaf, suggesting its distinct regulatory role across different regions of the leaf. Furthermore, the promoter of BmaTCP24 contains cis-acting elements associated with meristem expression (Fig. 5) and its expression level is down-regulated during late stages at the leaf margin, implying its potential primary function in cell proliferation processes at this specific region (Fig. 7).