Safflower CtFT genes orchestrating flowering time and flavonoid biosynthesis

Abstract

Background: Safflower thrives in dry environments but faces difficulties with flowering in wet and rainy summers. Flavonoids play a role in flower development and can potentially alleviate these challenges. Furthermore, the FLOWERING LOCUS T (FT) family of phosphatidylethanolamine-binding protein (PEBP) genes play a crucial role in the photoperiodic flowering pathway. However, their direct impact on flowering and flavonoid biosynthesis under different light duration is elusive.

Results: Utilizing the genome sequencing of Safflower (Jihong NO.1), the current study identifies three specific genes (CtFT1, CtFT2, and CtFT3) that exhibit upregulation in response to long-day conditions. The overexpression of CtFT2, displayed an early, whereas CtFT1 and CtFT3 late flowering phenotype in Arabidopsis thaliana. Interestingly, the transient overexpression of CtFT1 in safflower leaves caused early flowering, while overexpressing CtFT2 and CtFT3 led to late flowering. Additionally, overexpressing CtFT3 in Arabidopsis and CtFT1, CtFT2, and CtFT3 in safflower leaves, significantly increased flavonoid synthesis.

Conclusions: These findings showed that overexpression of CtFT genes could affect the flowering time and significantly increase the flavonoid content of safflower. The function of CtFT gene is different in safflower and Arabidopsis. This study provides valuable insights into the role of CtFT genes in flower formation and flavonoid synthesis in safflower, which may help in improving safflower breeding quality and its adaptability to diverse environmental conditions.

Keywords: CtFTs; Flavonoid biosynthesis; Flower formation; Safflower.

Fig. 1

Chromosomal localization, Phylogenetic analysis and conserved topology of PEBP family in safflower. A the chromosome wise distribution of CtPEBP genes in safflower. The blue vertical lines indicate different safflower chromosome (Chr) and the number of chromosomes is indicated at the left side of each chromosome. The left scale bar used for locating genes was demonstrated in Mb. B Phylogenetic analysis of PEBP family members from safflower, Arabidopsis, rice and soybean. The five-pointed star represents safflower PEBP family members. C The gene structure and conserved protein motif analysis of PEBP family genes in safflower. The blue colored boxes represent untranslated regions (UTR) and red colored boxes represents coding sequences (CDS). The connections between UTR and CDS lines represent introns. The scale at the bottom is drawn to facilitate the comparison of the relative lengths of various genes and proteins. D Multiple sequence alignment of CtFT and their homologs and identification of conserved domains of PEBP family

Fig. 2

Expression patterns of CtFT genes in safflower under contrasting light conditions Expression pattern of safflower CtFT genes (A) under short-day conditions (8 h light / 16 h darkness). B under long-day conditions (16 h light / 8 h darkness)

Fig. 3

Phenotypic analysis of CtFTs transgenic Arabidopsis compared to WT and ft mutants. A Representative T3 transgenic Arabidopsis lines alongside WT (Col-0) and ft mutants were cultivated under long-day (LD) conditions and photographed simultaneously five weeks after planting. B Time of flowering (days). C The number of rosette leaves at the time of blossoming, measured from plants with four leaves of uniform size at transplantation. (D) The length of the main stem at the onset of flowering in Arabidopsis. Each dataset is based on 20 biological replicates

Fig. 4

Differential regulation of flowering time in safflower after transient overexpression of CtFT genes. A Prior to injection of pGreenII62-SK-CtFTs and empty vectors. B After 20 days of injection with pGreenII62-SK-CtFTs and empty vector, illustrating variation in flowering time

Fig. 5

Correlation of CtFT genes and flavonoid accumulation in Arabidopsis. A, C, E: Gene expression and total flavonoid content analysis in WT, ft mutant, and two OE transgenic lines of CtFT1 CtFT2 and CtFT3 genes. B, D, F: Relative expression level of key genes involved in flavonoid biosynthesis pathway in WT, ft mutant, and two OE transgenic lines of CtFT1, CtFT2 and CtFT3 genes. Each value represents three biological repeats, and three technical repeats. The error line represents the standard deviation

Fig. 6

Transient overexpression of CtFT genes expression of FT gene, content of total flavonoids and expression of key enzyme genes in flavonoid biosynthesis pathway in safflower and empty vector control. A, C, E: relative expression and total flavonoid content of CtFT genes in instantaneous overexpression of safflower and injection empty vector safflower. B, D, F: relative expression of key enzyme genes of flavonoid biosynthesis pathway in instantaneous overexpression of safflower and injection empty vector safflower. Each value represents three biological repeats, and each creature repeats three technical repeats. The error line represents the standard deviation of the repetition of three organisms