TaMYB72 directly activates the expression of <i>TaFT</i> to promote heading and enhance grain yield traits in wheat (<i>Triticum aestivum</i> L.)

Abstract

Wheat (Triticum aestivum L.) is a staple food for approximately half the global population. As the population continues to grow, the demand for high-yielding varieties is increasing (Zorb et al., 2018). Higher yields can be achieved by increasing the number and weight of grains produced and by optimizing heading dates to maximize production in local environments (Sakuma and Schnurbusch, 2020). Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing provides great opportunities to precisely create more elite allelic variations, which may promote crop improvement by facilitating the selection of lines with improved yields and desirable heading phenotypes (Gao, 2021). We previously characterized the heading regulatory gene TaMYB72, encoding a MYB transcription factor, and its role in promoting flowering and reducing plant height when transgenically expressed in rice (Oryza sativa L.) (Zhang et al., 2016). In the present study, we have evaluated the effect of using CRISPR/Cas9-based gene editing to insert mutations in TaMYB72 for the enhancement of yield traits in wheat. TaMYB72 was found to have transcriptional activation activity (Figure S1A, B). To determine which part of TaMYB72 mediates this function, we constructed differently truncated variants of the protein. We found that amino acid residues 191–284 were necessary for the transcriptional activation activity, while other regions played other roles in helping TaMYB72 maintain its full activity (Figure S1A, B). Additionally, the three TaMYB72 homeologous genes from the A, B, and D subgenomes of wheat all exhibited a diurnal expression pattern in the leaves, with transcripts accumulating after dawn, reaching a peak after 12 h of light, and decreasing rapidly thereafter (Figure S2). To evaluate the role of TaMYB72 in wheat, we generated knockout mutants of this gene using CRISPR/Cas9. We designed single-guide RNAs (sgRNAs) specifically targeting the first exon of TaMYB72 (Figure 1A), which we then introduced into the pBUE413 vector and transformed into KeNong199 (KN199). Three mutant lines with homozygous or biallelic frameshift mutations at the target sites of TaMYB72 were identified through polymerase chain reaction (PCR) and sequencing analysis and designated KO#1, KO#2, and KO#3. KO#1 has a 609-bp deletion in the A subgenome and 1-bp insertions in the B and D subgenomes, KO#2 has 1-bp insertions in all three subgenomes, and KO#3 has a 14-bp deletion in the A subgenome and 1-bp insertions in the B and D subgenomes at the target sites (Figure 1A). We used these three mutant lines for subsequent phenotypic analyses in the field, along with wild-type (WT; KN199) plants as the negative control. The three mutant lines displayed heading dates about 11 d later (Figure 1B, C) and matured about 6 d later than the WT (Figures 1D, S3). Moreover, we obtained single mutants of only TaMYB72-A, TaMYB72-B, or TaMYB72-D and found that, as expected, these had earlier heading dates than the triple mutants (Figure S4). Functional characterization of TaMYB72 in the regulation of heading date and yield traits in wheat (A) Target sites of single-guide RNAs (sgRNAs) and genotypes of the mutant lines. Red, protospacer-adjacent motif (PAM); blue, sgRNA target sequences; green letters and “+”, nucleotide insertions; green dashed lines and “-”, nucleotide deletions. (B, C) Heading date phenotypes of the wild-type (WT) and TaMYB72 mutant lines generated using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). (D) Plant height phenotypes at maturity. (E) Spike morphologies. (F) Grain numbers per spike. (G, H) Grain lengths and 1,000-grain weights. (I) Relative expression levels of TaFT-7A (TraesCS7A02G115400), TaFT-7B (TraesCS7B02G013100), and TaFT-7D (TraesCS7D02G111600) in the flag leaves of WT and TaMYB72 mutants at the heading stage. Data are means ± SD, n = 3. (J) Diagrams showing the reporters and effectors used in the transient transactivation assays. (K) Results of transient expression assays in Nicotiana benthamiana protoplasts. (L) Effect of TaMYB72 on the expression of the LacZ reporter gene driven by the fragments of target gene promoters in yeast. The pB42AD empty vector and placZi containing the fragments of the target gene promoters were co-transformed as a control. (M) Electrophoretic mobility shift assay (EMSA) of TaMYB72 binding to the TaFT-7B promoter region. Competition for binding was provided using 5×, 10×, and 20× unlabeled probes. Glutathione S-transferase (GST): negative control. (N, O) Enrichment of TaMYB72 on the TaFT-7B promoter, determined using chromatin immunoprecipitation – quantitative polymerase chain reaction (ChIP-qPCR). Throughout, error bars indicate standard deviation; *P < 0.05, **P < 0.01, according to Student's t-test. Scale bars: 20 cm (B, D), 2 cm (E), 1 cm (G). Many heading date genes, such as Ghd7 and FT1, are closely associated with yield in crops (Xue et al., 2008; Chen et al., 2022). Our previous study indicated that the transgenic expression of TaMYB72 in rice significantly reduced its height (Zhang et al., 2016). To evaluate the effect of knocking out this gene on wheat yields, we investigated the agronomic traits of the edited mutants planted in the field. In addition to the expected increase in height, we were surprised to see beneficial changes in some important yield traits in the three mutant lines as compared to the WT, including significant increases in plant height (by approximately 8.09%–17.69%; Figures 1D, S5A), spike length (Figures 1E, S5B), and grain number per spike (Figure 1F). Strikingly, the grain length, weight, and 1,000-grain weight of the three mutant lines were higher than those of the WT (Figures 1G, H, S5C). Taken together, these results show that the loss of TaMYB72 enhances grain yield traits in wheat. Recent studies have shown that FT genes play diverse roles in regulating plant flowering and spikelet development (Chen et al., 2022). To determine whether TaMYB72 regulates the height and yield traits of wheat through the FT-mediated pathway, we first examined the expression of the wheat FT genes TaFT-7A, TaFT-7B, and TaFT-7D in the WT and the mutant lines using reverse transcription – quantitative PCR (RT-qPCR). The expression levels of all three TaFT genes were significantly lower in the TaMYB72 mutant lines than in the WT (Figure 1I). TaFT-7B showed the highest expression among the three homeologous genes; therefore, we selected TaFT-7B as the representative TaFT gene for subsequent analysis. To elucidate whether TaMYB72 activates the transcription of TaFT-7B, we conducted dual-luciferase (LUC) reporter (DLR) assays in Nicotiana benthamiana protoplasts. The LUC gene was fused to the promoter of TaFT-7B to generate reporter constructs, while the effector constructs contained TaMYB72 driven by the CaMV 35S promoter (Figure 1J). The transient expression of TaMYB72 strongly activated the proTaFT-7B::LUC reporters (Figure 1K). Furthermore, yeast one-hybrid (Y1H) assay demonstrated that TaMYB72 can bind to the promoter of TaFT-7B (Figure 1L). A sequence analysis showed that the promoter of TaFT-7B contains cis-regulatory elements targeted by MYB transcription factors (TTGTTT) (Millard et al., 2019). We also conducted an electrophoretic mobility shift assay (EMSA), which revealed that TaMYB72 can effectively and specifically bind to the TaFT-7B promoter sequence (Figure 1M). Additionally, chromatin immunoprecipitation (ChIP)-qPCR assay showed that TaMYB72 proteins were highly enriched at sites containing two MYB-core binding motifs in the -1,059 to -1,064 bp region upstream of the translation start codon of TaFT-7B, confirming the interaction between TaMYB72 and the TaFT-7B promoter (Figure 1N, O). These results indicate that TaMYB72 can directly activate the expression of TaFT to promote heading and improve grain yield traits in wheat. The isolation of key genes associated with yield potential is an effective strategy for meeting the challenges of future crop improvement through genetic engineering (Zeng et al., 2017). Wheat yield is mainly determined by three components: 1,000-grain weight, grain number per spike, and spike number per hectare. The mutation of TaMYB72 through gene editing increased the average grain number per spike and grain weight relative to those of the WT, indicating that TaMYB72 is a potential target gene for increasing grain yield; however, we cannot ignore the fact that the knockout of TaMYB72 delayed wheat heading, making it unsuitable for direct use in breeding. We can therefore explore the pathways through which TaMYB72 regulates the heading date and yield traits to achieve the optimal combination between these two phenotypes in a specific ecological environment. Overall, this study provides new insights into the genetic basis of grain yield traits and the heading date, and also provides a candidate gene with potential breeding value in wheat. We are grateful to Professor Qixin Sun and colleagues from China Agricultural University for kindly providing the CRISPR/Cas9 vector system, and Professor Jiaqiang Sun (ICS-CAAS) for the helpful comments. This research was supported by STI 2030—Major Projects (2023ZD0406802) and the Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSCB-202401). The authors declare no conflict of interest. X.K., X.L., and L.Z. conceived the study and designed the experiments. L.W., Z.X., L.Z., and D.L. performed the experiments. L.Z., Z.X., and L.W. wrote the manuscript. All authors read and approved of its content. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13716/suppinfo Figure S1. Protein structure and transcriptional activity of the TaMYB72 protein in yeast Figure S2. The rhythmic expression patterns of TaMYB72-A, TaMYB72-B and TaMYB72-D under long day (LD) condition Figure S3. The statistics of maturation dates of the wild-type (WT) and TaMYB72 mutant lines generated using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) Figure S4. The phenotypes and genotypes of TaMYB72 aa/bb/dd single and triple mutant lines generated by clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) Figure S5. The phenotype statistics of wild-type (WT) and TaMYB72 mutant lines generated using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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