On August 23, 2025, the research group led by Jie Liu at the Wheat Research Center, China Agricultural University, published an online research article in Plant Communications titled “Unlocking tillering repression through TEOSINTE BRANCHED-B1 mutagenesis enhances wheat grain yield.”
Under laboratory conditions, seeds were subjected to a cold stress treatment. After germination in a Jiupo plant growth chamber, seedlings were transplanted into soil and grown for one week under controlled conditions: 16-hour light / 24 °C and 8-hour dark / 16 °C cycles.
This study introduced single-base mutations into the coding region of the tiller suppression gene TEOSINTE BRANCHED-B1 (TB-B1), significantly increasing the number of spikes per unit area in dense spike wheat varieties and boosting population yield by more than 8 %, providing a novel route and valuable breeding intermediate materials for high-yield wheat molecular breeding.
Using tiller buds collected from field-grown NIL-Heng and NIL-Shi plants, chromatin accessibility and histone modification analyses demonstrated enhanced ATAC-seq signal intensity at the TB-B1 locus in NIL-Heng, with significantly increased levels of H3K27ac (associated with transcriptional activation) and significantly decreased H3K27me3 (associated with transcriptional repression) (Figure 1G). These findings indicate that the r-e-z deletion increases chromatin accessibility in this region and establishes an active chromatin state at the TB-B1 locus, leading to its transcriptional activation.
Figure 1. Large-segment r-e-z deletion alters chromatin state and causes ectopic activation of the adjacent TB-B1 gene.
Figure 2. Identification of transcription factors regulating TB-B1 upstream.
To identify upstream regulators that specifically activate TB-B1 under vernalization, we used the PlantTFDB database (https://planttfdb.gao-lab.org) to identify a NAC (NAM, ATAF1/2, CUC1/2) binding site in the TB-B1 promoter between positions -1171 to -1145 upstream of the start codon, which was absent in the TB-A1 and TB-D1 promoters (Figure 2A). Phylogenetic analysis of NAC family members in wheat identified eight candidate NAC transcription factors (NAC058-D, NAC047-D, NAC074-A, NAC086-D, NAC025-A, NAC042-B, NAC090-B, and NAC007-B). Both yeast one-hybrid (Y1H) and electrophoretic mobility shift assays (EMSA) confirmed that transcription factors such as NAC047-D bind to the TB-B1 promoter region and activate its transcription (Figures 2B-E). These results indicate that NAC047 and related transcription factors act as cold-sensitive upstream regulators that specifically activate TB-B1 expression under vernalization. Transcriptome analysis further identified 1,803 differentially expressed genes (Figures 2F-I).
Figure 3. Mutations in the TB-B1 gene significantly increase tiller number and per-plant yield in r-e-z haplotype wheat.
Considering that activation of TB-B1 reduces tiller number and yield potential in the r-e-z haplotype, large-scale EMS mutagenesis was performed in the NIL-Heng background to screen for TB-B1 functional mutants. Three independent mutant lines were identified: (1) m804, a C→T single nucleotide mutation at position 433 causing a Leu-145-Phe amino acid substitution; (2) m1347, a G→T mutation at position 755 causing an Arg-252-Met substitution; (3) m2294, a G→A mutation at position 811 causing an Ala-271-Thr substitution. Compared to NIL-Heng, these lines showed a significant increase in tiller number and improved other agronomic and yield-related traits (such as spikelets per spike, thousand-grain weight, and grain weight per spike) (Figure 3). The grain yield per plant in these mutants increased by 18.59 %, 18.73 %, and 17.69 %, respectively (Figure 3).
Under standard field plot conditions, m1347 and m2294 were evaluated for field yield traits (Figure 4A). The number of effective spikes per unit area increased by 9.01 % and 12.02 %, respectively (Figures 4B, C). Plot-level grain yield of these lines was elevated by 2.54 %–8.61 % compared to NIL-Heng (Figure 4D), indicating that TB-B1 variation can significantly enhance yield potential in the r-e-z haplotype.
Figure 4. Field yield performance of m1347 and m2294 mutant systems under standard plot conditions.
In summary, this study demonstrates that the r-e-z haplotype deletion negatively regulates tiller number by inducing ectopic activation of the adjacent TB-B1 gene through local chromatin state changes. Introducing missense mutations into TB-B1 disrupts its function, significantly increasing tiller number and crop yield. Thus, combining the r-e-z haplotype deletion with loss-of-function TB-B1 alleles in wheat varieties may enhance yield potential, presenting a promising strategy for high-yield wheat molecular breeding.
Figure 5. Molecular breeding strategy for high-yield wheat based on the r-e-z haplotype and TB-B1 gene mutations.
Author Information
This research was completed under the guidance of Academician Qixin Sun and Professor Zhongfu Ni at the Wheat Research Center, China Agricultural University. Professor Jie Liu served as the corresponding author. PhD candidate Xiangqing Liu, postdoctoral researcher Long Song, and Associate Researcher Xiliu Cheng at the Chinese Academy of Agricultural Sciences are co-first authors. Additional contributors included postdoctoral researcher Haoran Wang and PhD candidate Yiqing Wang from the Wheat Research Center; Researcher Yanming Ma from Xinjiang Academy of Agricultural Sciences; Associate Professor Jinghui Li from Henan University of Science and Technology; Researcher Zefu Lu and Dr. Hongcui Pei from the Chinese Academy of Agricultural Sciences; Associate Researcher Bin Yang and Researcher Mingyi Zhang from Shanxi Agricultural University; and Professor Moussa Benhamed and PhD candidate Qingyi Wang from Université Paris-Saclay. This work was supported by the National Natural Science Foundation of China (32401861, 32372120), the Bio Breeding – National Major Science and Technology Project (2023ZD0402402), the National Key R&D Program of China (2022YFF1003401), and the CNAU 2115 Talent Program.