Unraveling TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR Transcription Factors in Safflower: A Blueprint for Stress Resilience and Metabolic Regulation

Abstract

Safflower (Carthamus tinctorius L.), a versatile medicinal and economic crop, harbors untapped genetic resources essential for stress resilience and metabolic regulation. The TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors, exclusive to plants, are pivotal in orchestrating growth, development, and stress responses, yet their roles in safflower remain unexplored. Here, we report the comprehensive identification and characterization of 26 safflower TCP genes (CtTCPs), categorized into Class I (PROLIFERATING CELL FACTOR, PCF) and Class II (CINCINNATA and TEOSINTE BRANCHED1/CYCLOIDEA, CIN and CYC/TB1) subfamilies. Comparative phylogenetics, conserved motif, and gene structure analyses revealed a high degree of evolutionary conservation and functional divergence within the gene family. Promoter analyses uncovered light-, hormone-, and stress-responsive cis-elements, underscoring their regulatory potential. Functional insights from qRT-PCR analyses demonstrated dynamic CtTCP expression under abiotic stresses, including abscisic acid (ABA), Methyl Jasmonate (MeJA), Cold, and ultraviolet radiation b (UV-B) treatments. Notably, ABA stress triggered a significant increase in flavonoid accumulation, correlated with the upregulation of key flavonoid biosynthesis genes and select CtTCPs. These findings illuminate the complex regulatory networks underlying safflower's abiotic stress responses and secondary metabolism, offering a molecular framework to enhance crop resilience and metabolic engineering for sustainable agriculture.

Keywords: TCP transcription factors; flavonoid biosynthesis; gene expression profiling; molecular crop improvement; plant regulatory networks; safflower (Carthamus tinctorius L.).

Figure 1

Phylogenetic analysis of TCP genes from Carthamus tinctorius L., Arabidopsis thaliana, and Oryza sativa. The figure displays a neighbor-joining (NJ) phylogenetic tree constructed using the full-length amino acid sequences of TCP genes from safflower (Carthamus tinctorius L., red triangles), Arabidopsis thaliana (green circles), and Oryza sativa (blue stars), generated with MEGAX software (version 10.1.8) and 1000 bootstrap replicates. The TCP gene family is grouped into three clades: PCF (pink), associated with cell cycle regulation; CYC/TB1 (green), involved in axillary bud and leaf development; and CIN (blue), regulating organ size and shape. The 26 CtTCP genes clustered with 24 TCP genes from Arabidopsis thaliana and 21 from Oryza sativa, illustrating both conserved and species-specific functional diversification. This analysis provides insights into TCP gene evolution and specialization, offering a foundation for further functional studies.

Figure 2

Protein motifs, gene structures, and conserved domains of CtTCP genes in safflower. This figure highlights the conserved motifs, gene structures, and conserved domains of the 26 CtTCP genes in safflower (Carthamus tinctorius L.). (a) Conserved motifs: The distribution of 10 conserved motifs is shown as colored blocks, indicating their length and position within the protein sequences. Distinct motif arrangements reflect the functional diversity and evolutionary relationships of CtTCP proteins. (b) Gene structures: The exon–intron organization is illustrated, with yellow boxes representing exons, green boxes for untranslated regions (UTRs), and black lines for introns. The variable exon and intron lengths indicate structural diversity, contributing to functional specialization. (c) Conserved domains: The green and yellow boxes represent subfamily-specific TCP domains, while the pink boxes indicate the core TCP domain. These positions validate the classification and conserved functionality of CtTCP genes. This analysis provides a foundation for understanding the structural and functional roles of CtTCP genes in safflower growth, development, and stress responses.

Figure 3

Cis-acting element analysis of CtTCP gene promoters in safflower. This figure shows cis-acting elements identified within the 2000 bp promoter regions of 26 CtTCP genes in safflower (Carthamus tinctorius L.) using PlantCARE. The phylogenetic tree on the left, constructed via neighbor-joining analysis with bootstrap values at key nodes, illustrates the evolutionary relationships among CtTCP genes. Promoter elements are displayed as colored blocks along the sequences, mapped from the 5′ to 3′ direction. The cis-acting elements are categorized as follows: (1) Hormone-responsive elements linked to gibberellin, abscisic acid, salicylic acid, auxin, and methyl jasmonate (MeJA). (2) Stress-responsive elements associated with drought, low temperature, defense, and anaerobic induction. (3) Growth- and development-related elements, including light responsiveness, circadian control, meristem expression, seed-specific regulation, and cell cycle regulation. (4) Regulatory pathway elements include flavonoid biosynthesis and MYBHv1 binding sites. The diverse distribution of these elements highlights the regulatory complexity of CtTCP genes and their roles in integrating hormonal, stress, and developmental signals, providing a basis for understanding their functions in safflower adaptation and growth.

Figure 4

Expression profiles of CtTCP genes across different organs and developmental stages in safflower. This figure shows the differential expression patterns of CtTCP genes in various organs and developmental stages. (a) Heatmap: The expression levels of 26 CtTCP genes are displayed across roots, stems, leaves, flowers, and seeds at different developmental stages (bud, initial flowering, full bloom, fading flower, and seeds at 10, 20, and 30 days post-anthesis). The color scale (blue to red) indicates low to high expression. Distinct patterns include CtTCP10 and CtTCP24 with high expression in roots, CtTCP7, CtTCP14, and CtTCP15 in stems, CtTCP6 and CtTCP17 in seeds, CtTCP3 and CtTCP12 in leaves, and CtTCP22 and CtTCP20 in flowers at full bloom. (b) qRT-PCR validation: The expression of nine CtTCP genes (CtTCP1, CtTCP3, CtTCP8, CtTCP9, CtTCP10, CtTCP15, CtTCP20, CtTCP22, CtTCP26) in stems, leaves, and flowers was consistent with transcriptome data. Statistical significance is shown by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001). This analysis highlights the diverse roles of CtTCP genes in safflower growth and development.

Figure 5

Expression profiles of CtTCP genes under abiotic stress treatments. The relative expression patterns of nine CtTCP genes were analyzed under four abiotic stress conditions using qRT-PCR, with results presented as mean ± standard deviation (SD). Significant differences compared to the control group (0 h) are indicated by asterisks (ns is not significant, * p < 0.05, ** p < 0.01, *** p < 0.001). (a) ABA (200 μmol): CtTCP1, CtTCP8, CtTCP15, and CtTCP22 were upregulated, peaking at 24 h, while CtTCP9, CtTCP10, and CtTCP20 showed a gradual decline. (b) MeJA (200 μmol): Most genes, including CtTCP1, CtTCP3, CtTCP8, CtTCP9, and CtTCP10, were consistently downregulated across all time points. (c) Cold (4 °C): CtTCP10 and CtTCP26 were downregulated, whereas CtTCP1, CtTCP15, CtTCP20, and CtTCP22 peaked at 9 h, CtTCP3 and CtTCP8 at 12 h, and CtTCP9 at 6 h. (d) UV-B (20,000 lx): CtTCP10, CtTCP20, CtTCP22, and CtTCP26 were downregulated, with modest changes observed in other genes. This analysis underscores the stress-specific expression patterns of CtTCP genes and their potential roles in safflower’s abiotic stress responses.

Figure 6

Effects of ABA treatment on flavonoid content and expression of key flavonoid biosynthetic genes in safflower. (a) Total flavonoid content: Flavonoid content (mg/g) in safflower leaves significantly increased ~2-fold after 24 h of ABA treatment (200 μmol) compared to the control (0 h). (b) Expression of key flavonoid biosynthetic genes: Relative expression levels of CtFLS, CtCHI, CtCHS, CtF3’H, CtF3H, CtDFR, and CtANS were analyzed after 24 h of ABA treatment. CtFLS, CtCHS, CtDFR, and CtANS were significantly upregulated, CtF3’H and CtF3H were downregulated, while CtCHI remained unchanged. Black asterisks denote significant differences compared to the control (* p < 0.05, ** p < 0.01), highlighting ABA’s regulatory role in flavonoid biosynthesis.

Figure 7

Comprehensive Conclusion of CtTCP Gene Roles in Safflower. This figure summarizes the key findings and conclusions of the study on CtTCP gene functions in safflower (Carthamus tinctorius L.). (a) CtTCP Gene Classification: A pie chart depicting the distribution of 26 CtTCP genes into two primary classes: Class I (10 genes, 38.5%) and Class II (16 genes, 61.5%), based on phylogenetic and structural analysis. Class II genes dominate, reflecting their evolutionary and functional diversification. (b) CtTCP Gene Expression Under Abiotic Stresses: A bar chart illustrating the relative fold change in CtTCP gene expression under four abiotic stress treatments: ABA (24-fold), MeJA (18-fold), Cold (12-fold), and UV-B (20-fold). These stressors elicited distinct gene expression responses, highlighting the regulatory roles of CtTCP genes in stress adaptation. (c) Workflow of the Study: A schematic representation of the methodological workflow and major conclusions. Key steps include: (1) CtTCP Gene Identification (n = 26): Comprehensive identification and annotation of CtTCP genes from the safflower genome. (2) Phylogenetic Analysis: Classification of CtTCP genes into Class I and Class II clades, supported by conserved domain analysis. (3) Motif and Domain Analysis: Detailed characterization of conserved motifs and structural domains within CtTCP proteins. (4) Expression Profiling: Analysis of CtTCP gene expression patterns across organs, developmental stages, and under abiotic stresses. (5) Functional Analysis: Validation of gene expression and functional roles in flavonoid biosynthesis and abiotic stress responses. (6) Conclusion: CtTCP genes exhibit multifaceted roles, including tissue-specific functions, regulation of flavonoid biosynthesis, and abiotic stress adaptation. Arrows indicate the progression of the study, emphasizing the integrative approach used to understand CtTCP gene functions. This figure encapsulates the study’s findings, highlighting the significance of CtTCP genes in safflower’s growth, development, and stress resilience.

Figure 8

Hierarchical framework summarizing the roles of CtTCP genes in safflower growth, development, and stress responses. This figure illustrates the multifaceted roles of CtTCP genes in safflower (Carthamus tinctorius L.) based on tissue-specific expression, involvement in flavonoid biosynthesis, and abiotic stress responses. The central node represents the CtTCP gene family, with three major categories branching out: (1) Different Organs: CtTCP genes exhibit differential expression across various organs, including roots, stems, leaves, seeds, and flowers, emphasizing their diverse roles in tissue development and function. (2) Flavonoid Biosynthesis: CtTCP genes regulate key enzymes in the flavonoid biosynthetic pathway, such as CtFLS (Flavonol Synthase), CtCHS (Chalcone Synthase), CtDFR (Dihydroflavonol 4-Reductase), and CtANS (Anthocyanidin Synthase). These enzymes contribute to the biosynthesis of flavonoids, which play critical roles in plant defense and stress resilience. (3) Abiotic Stress Response: CtTCP genes respond to various abiotic stress conditions, including MeJA, ABA, cold, and UV-B. These responses highlight their regulatory role in stress signaling pathways and adaptation mechanisms. Nodes are color-coded to represent categories: gold for the central CtTCP genes, light blue for major functional categories, light green for tissues, light coral for flavonoid-related genes, and orange for abiotic stress responses. The hierarchical arrangement and directed edges depict the relationships and regulatory pathways involving CtTCP genes, providing a comprehensive overview of their functional dynamics in safflower.