Determinate inflorescences have a significant effect on the genetic improvement of rapeseed, so understanding the molecular function underlying the inflorescence trait may be beneficial to oilseed breeding. A previous study found candidate gene BnTFL1 (Terminal Flower 1) for control of the inflorescence trait on Brassica napus chromosome A10 (16,627–16,847 kb). However, little is known about the function of the BnTFL1 gene in B. napus. In this study, we firstly studied the formation of the shoot apical meristem and gene expression in indeterminate and determinate inflorescences; the results showed that the inflorescence architecture and BnA10.TFL1 expression showed significant differences in the shoot apex at the budding stage. Then, two alleles (named BnA10.TFL1 gene from indeterminate and BnA10.tfl1 gene from determinate) were cloned and sequence-analyzed; the results suggest that the open reading frame of the alleles comprises 537 bp, encodes 178 amino acids containing a conserved phosphatidylethanolamine-binding protein (PEBP) domain, and shares high similarity with Arabidopsis thaliana TFL1. To analyze the function of BnA10.TFL1, the BnA10.TFL1 gene was introduced into the determinate A. thaliana tfl1 mutant and B. napus 571 line by complementation experiment. The determinate traits were restored to indeterminate, and expression of BnA10.TFL1 was increased in the indeterminate shoot apex. These results reveal that BnA10.tfl1 is a gene controlling the determinate inflorescence trait. Moreover, the BnA10.TFL1 protein was localized to the nucleus, cytoplasm, and plasma membrane. Collectively, the results of this study help us to understand the molecular mechanism of determinate inflorescences and will provide a reliable research basis for the application of determinate inflorescences in B. napus.

Determinate inflorescence is indeed a pivotal agricultural characteristic in crops, notably impacting the architecture modification of Brassica napus (AACC, 2n = 38). Previous study identified a crucial gene Bnsdt2 that encodes the transcription factor BnaC09.TFL1 (Terminal Flower 1). Here by two alleles were cloned and sequenced from indeterminate 2982 and determinate 4769, respectively, we found that BnaC09.TFL1 harbors two T/C and G/C non-synonymous mutations in exon 1, and contains sixty-six differences in a 1.9 Kb promoter sequence. Subsequently, BnaC09.TFL1 was introduced into B. napus 571 line by genetic complementation and overexpression, transgenic plants 571^CTO lines and 571^TClines were all restored to the indeterminate inflorescence. Interestingly, after BnaC09.TFL1 was knocked out in ‘Westar’, transgenic plants Westar^Tcr lines were mutated to determinate inflorescences. Additionally, a NIL-4769 line was constructed to evaluate the effect of BnaC09.TFL1 on agronomic traits of Brassica napus, the results demonstrated that BnaC09.tfl1 reduced the plant height and increased the branch number and branch thousand grain weight of Brassica napus. Finally, we performed RT-qPCR, GUS staining and subcellular localization experiments to analyze the expression pattern of BnaC09.TFL1, the results showed that the expression of BnaC09.TFL1 at shoot apex of NIL-4769 was higher than that of 4769, GUS activity was detected at apical of Arabidopsis thaliana and BnC09.TFL1-GFP was detected in cell membrane, nucleus and cytoplasm. Our findings provide a firm molecular foundation for the study of rapeseed’s molecular mechanism of determinate inflorescence formation, as well as theoretical guidance for the application of determinate inflorescence in rapeseed breeding.

Brassica rapa, a major oilseed crop in high-altitude areas, is well known for its indeterminate inflorescences. However, this experiment revealed an intriguing anomaly within the plot: a variant displaying a determinate growth habit (520). Determinate inflorescences have been recognized for their role in the genetic enhancement of crops. In this study, a genetic analysis in a determinate genotype (520) and an indeterminate genotype (515) revealed that two independently inherited recessive genes (Brdt1 and Brdt2) are responsible for the determinate trait. BSA-seq and SSR markers were employed to successfully locate the Brdt1 gene, which is localized within an approximate region 72.7 kb between 15,712.9 kb and 15,785.6 kb on A10. A BLAST analysis of these candidate intervals revealed that Bra009508 (BraA10.TFL1) shares homology with the A. thaliana TFL1 gene. Then, BraA10.TFL1 (gene from the indeterminate phenotype) and BraA10.tfl1 (gene from the determinate phenotype) were cloned and sequenced, and the results indicated that the open reading frame of the alleles comprises 537 bp. Using qRT-PCR, it was determined that BraA10.TFL1 expression levels in shoot apexes were significantly higher in NIL-520 compared to 520. To verify the function of BraA10.TFL1, the gene was introduced into the determinate A. thaliana tfl1 mutant, resulting in the restoration of indeterminate traits. These findings demonstrate that BraA10.tfl1 is a gene that controls the determinate inflorescence trait. Overall, the results of this study provide a theoretical foundation for the further investigation of determinate inflorescence.

Rapeseed (Brassica napus L.) with determinate inflorescence (DTI) exhibits desirable traits, including reduced plant height, enhanced lodging resistance, and consistent maturity, making them valuable breeding resources. DTI is modulated by BnaA10.TFL1 and BnaC09.TFL1 (BnaA10/C09.TFL1), which encode the TERMINAL FLOWER 1 protein, a key regulator of flowering time and meristem identity. However, the underlying functional and regulatory mechanisms remain unclear. In this study, we demonstrated that variations in the promoter region of BnaA10/C09.TFL1, rather than the coding region, contributed to the transition from indeterminate inflorescence (IDTI) to DTI in B. napus. Specifically, BnaA10.SEP inhibited BnaA10/C09.TFL1 expression by binding to the GT1-motif in the promoter region of BnaA10/C09.TFL1DTI, contributing to the IDTI phenotype under short-day conditions. Meanwhile, two novel DTI mutants were successfully generated through the simultaneous knockout of BnaA10/C09.TFL1 using the CRISPR/Cas9 system. Furthermore, BnaA10/C09.TFL1 and its homolog BnaA02.FT interacted with BnaA07.14-3-3 instead of directly binding to BnaA08.FD to regulate the development of different inflorescence architectures. Overall, the BnaA10.SEP–BnaA10/C09.TFL1–BnaA07.14-3-3–BnaA08.FD module revealed a new mechanism for DTI formation and a promising strategy for modifying inflorescence architecture traits in B. napus.

The determinate inflorescence trait of Brassica napus L. is associated with various desirable agricultural characteristics. BnTFL1s (BnaA10.TFL1 and BnaC09.TFL1), which encode the transcription factor TERMINAL FLOWER 1 (TFL1), have previously been identified as candidate genes controlling this trait through map-based cloning. However, the mechanism underlying the effects of the BnTFL1 proteins remains unclear. Further, proteins generally interact with each other to fulfill their biological functions. The objective of this study was to construct a cDNA library of the shoot apical meristem (SAM) of B. napus and screen for proteins that interact with BnTFL1s, to better understand its mechanism of action. The recombination efficiency of the yeast two-hybrid (Y2H) library that we constructed was 100%, with insertion fragment lengths ranging from 750 to 2000 bp and a capacity of approximately 1.44 × 10⁷ CFUs (colony-forming units), sufficient for screening protein interactions. Additionally, the bait vector pGBKT7-BnTFL1s was transformed into yeast cells alongside positive and negative controls, demonstrating no toxicity to the yeast cells and no self-activation. This bait was used to screen the SAM cDNA library of B. napus, ultimately identifying two BnTFL1s-interacting proteins: 14-3-3-like protein GF14 omega GRF2. These interactions were verified through one-to-one interaction experiments. This study provides a foundation for further research on the biological functions of the BnTFL1s genes and their regulatory role in inflorescence formation in B. napus, while providing a reference for studying similar mechanisms in other plants.