WPCL-D1, a candidate gene responsible for the early heading phenotype in “E15-1069”
In this study, we mutagenized the Japanese winter wheat cultivar “Kitahonami” with EMS, resulting in the early heading mutant “E15-1069.” Through whole-genome resequencing and bulked segregant analysis based on MutMap (Abe et al. 2012; Sugihara et al. 2022), we detected a nonsense mutation in WPCL-D1 on chromosome 3D in “E15-1069” (Figs. 2 and 3a). This nonsense mutation was located in the Myb domain of WPCL-D1, leading to a truncated protein (Fig. 3b). The evolutionary conservation of the WPCL1 Myb domain in plant species such as A. thaliana, barley, and wheat suggests that WPCL-D1 in “E15-1069” is a loss-of-function allele (Onai and Ishiura 2005; Mizuno et al. 2012; Campoli et al. 2013; Mizuno et al. 2016). In F2 and F3 populations of “Kitahonami” and “E15-1069,” the nonsense mutation in WPCL-D1 was significantly associated with days to heading (Fig. 3d). In A. thaliana, LUX/PCL1 is a circadian clock gene and constitutes the evening complex (EC) along with other clock genes like, ELF3 and ELF4 (Nusinow et al. 2011). The EC regulates plant flowering, growth, and thermomorphogenesis by interacting with other clock components (Huang and Nusinow 2016; Zhang et al. 2021). Mutants of A. thaliana, barley, einkorn wheat, and bread wheat that lack the functional LUX/PCL1 gene exhibited arrhythmic expression patterns of circadian clock genes, resulting in early flowering and heading traits (Helfer et al. 2011; Mizuno et al. 2012; Campoli et al. 2013; Mizuno et al. 2016). Our results in “E15-1069” are consistent with these findings (Figs. 3 and 4). Therefore, the loss-of-function mutation in WPCL-D1 is the most likely cause of the early heading phenotype in the mutant “E15-1069.”
Dosage effect of WPCL1 on gene expression and heading time
The wpcl1 null mutants of einkorn wheat and bread wheat exhibited abnormal expression patterns of circadian clock and photoperiod sensitivity genes (Mizuno et al. 2012; Mizuno et al. 2016). In “E15-1069,” the expression patterns of these genes, including TaTOC1, TaLHY and Ppd-1, deviated from those in “Kitahonami” (Fig. 4). However, these changes in expression patterns do not align with those observed in previous studies of the wpcl1 null mutants. For instance, the expression of TaLHY is reduced at dawn in wpcl1 null mutants (Mizuno et al. 2012; Gawroński et al. 2014; Mizuno et al. 2016); in contrast, “E15-1069” exhibited enhanced TaLHY expression at the same time. Similarly, the bread wheat wpcl1 null mutant and barley hvpcl1 mutant showed high accumulation levels of LUX/PCL1 truncated transcripts near dusk (Campoli et al. 2013; Mizuno et al. 2016). However, the accumulation level of WPCL-D1 transcript was reduced in “E15-1069” at ZT9 (Fig. 4). The plant circadian clock comprises multiple feedback loops (McClung 2011; Bendix et al. 2015). The EC, which includes LUX/PCL1, ELF3, and ELF4, participates in regulating other clock gene expressions (Huang and Nusinow 2016). As “E15-1069” possesses a functional WPCL-A1 allele, disturbance of the feedback loop by wpcl-B1 and wpcl-D1 would be partially compensated by WPCL-A1. However, the altered gene expression pattern in “E15-1069” differs from that in “Kitahonami” and previously reported null mutants. Moreover, differences in gene expression patterns are also observed between “Kitahonami” and “Chihokukomugi”, with slight differences in TaTOC1 gene expression (Fig. 4). These observations suggest that functional homoeologues do not fully compensate for the expression levels of the clock genes, and are also influenced by the gene dosage of functional WPCL1. Additionally, while previous studies using einkorn wheat wpcl1 mutants showed heading 20–30 days earlier than the wild-type (Mizuno et al. 2012; Gawroński et al. 2014), “E15-1069” was headed around 5 days earlier than “Kitahonami.” In addition, the heading date of “Chihokukomugi” was about 4 days later than that of “Kitahonami” (Mizuno et al. 2022). The difference in the extent of early heading between the einkorn mutant and “E15-1069” supports the hypothesis that WPCL1 controls heading time in a dosage-sensitive manner. The accumulation pattern of Ppd-1 transcripts may reflect this suggestion. The transcript accumulation level at the peak of Ppd-1 gene expressions depends on the copy number of functional WPCL1 (Fig. 4). As our hypothesis was made on the basis of an indirect comparison between two different cultivars, further studies are needed to explore the relationship between heading time and the gene dosage effect of circadian clock genes using near-isogenic lines or genome editing lines.
The difference in the days to heading between “Kitahonami” and “Chihokukomugi” coincides with that between “E15-1069” and “Kitahonami.” These results imply that the heading time can be fine-tuned by altering the copy number of functional WPCL1. This feature is advantageous in polyploid wheat improvement because the loss-of-function of WPCL1 in diploid wheat induces substantial changes in heading time. Recently, another circadian clock gene, TaELF-B3, a homoeologue of TaELF3, demonstrated that its deleted allele also leads to earlier heading by approximately four days compared to the wild-type allele (Mizuno et al. 2023). This finding indicated that TaELF-B3 has contributed to the fine-tuning of flowering time in wheat breeding. Both WPCL1 and TaELF3 constitute EC (Alvarez et al. 2023). Thus, manipulating clock genes, particularly EC components, offers the opportunity to create a wide range of flowering times for local adaptation.
Regulation of Ppd-1 gene expression by WPCL1
Wheat Ppd-1 is the homologous gene of A. thaliana PRR7 and PRR3 and primarily regulates photoperiod sensitivity (Beales et al. 2007; Zhang et al. 2016). In A. thaliana, LUX/PCL1 binds to the LUX binding site in the promoter region of PRR9 and represses its expression (Helfer et al. 2011). Therefore, WPCL1 is hypothesized to function as a negative regulator of Ppd-1 by binding to its promoter region and repressing Ppd-1 expression (Mizuno et al. 2016). In the wheat wpcl1 null strain, up-regulation of Ppd-1 was observed only in the photoperiod sensitive allele (Ppd-A1b), and not in the photoperiod insensitive allele (Ppd-D1_Hapl-I) (Mizuno et al. 2016). Photoperiod insensitive alleles, Ppd-A1a and Ppd-D1_Hapl-I, carry 1 kbp and 2 kbp deletions, respectively, both of which have deletions in the 5’ UTR region (Beales et al. 2007; Nishida et al. 2013). These deletions contain LUX binding site motifs (GATACG or GATTCG) discovered in A. thaliana (Helfer et al. 2011). Thus, the photoperiod insensitive allele is assumed to be released from regulation by WPCL1. However, in our study, increased expression levels of Ppd-1 were observed in both the photoperiod-sensitive allele (Ppd-D1_Hapl-II) and the photoperiod insensitive allele (Ppd-A1a). These results suggest that WPCL1 also regulates the expression of Ppd-1 using elements other than the LUX binding site motifs in the deletion region. These additional elements may include the G-box motif or the LUX binding site motif located further upstream of the deletion regions (Helfer et al. 2011; Ezer et al. 2017), or regulation may occur indirectly through other pathways.
Prospects for utilizing the “E15-1069” in breeding
As the mutant “E15-1069” originates from the Japanese elite cultivar “Kitahonami,” it is readily available as a breeding material for fine-tuning heading time. However, it is worth noting that “E15-1069” exhibited a lower yield compared to “Kitahonami” (Table 1). This reduction in grain yield is attributed to a decreased spike number. Since the nonsense mutation of WPCL-D1 only leads to a few days of early heading, the yield decrease likely arises from factors other than a shortened vegetative growth period due to the early transition from the vegetative to reproductive phase. In rice (Oryza sativa L.), a circadian clock gene, OsCCA1, a homologue of TaLHY, negatively regulates tiller number via strigolactone signaling and sugar sensing (Wang et al. 2020). “E15-1069” exhibited higher TaLHY expression at ZT0 than “Kitahonami” (Fig. 4), suggesting that the increased expression of TaLHY in “E15-1069” may have resulted in reduced tillering. Additionally, previous studies have shown that heading/flowering-associated genes like Vrn-1, Ppd-1, and FT influence tiller number in wheat (Kato et al. 2000; Dyck et al. 2004; Dixon et al. 2018; Liu et al. 2019). While the heading/flowering control system appears to be related to tiller number, further research is needed to understand it for more efficient breeding using “E15-1069.”
Exploiting the dosage effect of circadian clock genes such as WPCL1 offers a new breeding material, as described above. Although practical use of these resources requires verification, the combination of clock gene alleles has the potential to create diversity in the genetic control of heading/flowering time. Moreover, these genes enable precise adjustment of heading/flowering time, which was previously challenging by modifying Vrn-1 and Ppd-1. Accurate control of heading/flowering time allows for the expansion of suitable land for wheat cultivation, contributing to increased wheat production.