Extra Early-Flowering (exe) Mutants in Einkorn Wheat Generated by Heavy-Ion Beam Irradiation
Abstract Four extra early-heading mutants, named extra early-flowering1 (exe1), exe2, exe3, and exe4, were identified in diploid einkorn wheat (Triticum monococcum L.) following heavy-ion beam mutagenesis. Based on their phenotypes in the field, the four exe mutants were classified into two groups: Type I (moderately extra early-heading type; exe1 and exe3) and Type II (extremely extra early-heading type; exe2 and exe4). Analysis of VERNALIZATION 1 (VRN1), a flowering promoter gene, showed that it was more highly expressed at earlier stages of vegetative growth in Type II mutants than in Type I mutants. Our analyses indicate that the difference in earliness between Type I and Type II mutants is associated with differences in the expression level of VRN1.
Improving our understanding of the molecular mechanisms of flowering, the phase transition from vegetative to reproductive growth associated with heading time, is one of the most important goals for wheat breeding at the present time. In bread wheat (Triticum aestivum L.), heading time is genetically determined by three characteristics: vernalization requirement; photoperiod sensitivity; and narrow-sense earliness (earliness per se). Three genes have been identified to determine the requirement of vernalization, namely VERNALIZATION 1 (VRN1), VRN2 and VRN3.
VRN1 encodes an APETALA1/FRUITFULL-like MADS-box transcription factor that is up-regulated by vernalization (Yan et al. 2003; Murai et al. 2003; Trevaskis et al. 2003; Danyluk et al. 2003). Recent studies revealed that expression of VRN1 is epigenetically suppressed in seedlings at an earlier stage of the vegetative growth phase, and that the repressive histone state is modified by the vernalization signal, leading to the up-regulation of VRN1 (Oliver et al. 2009; Diallo et al. 2012). The level of VRN1 transcription gradually increases during the seedling growth stage without the need for further vernalization (Murai et al. 2003; Kitagawa et al. 2012), suggesting that the epigenetic status of VRN1 is also modified by aging. Furthermore, VRN1 shows a diurnal expression pattern that is affected by daylight, with a long photoperiod producing up-regulation of its expression level (Shimada et al. 2009). In summary, these observations indicate that the VRN1 expression is also controlled by autonomous and photoperiodic pathways, as well as the vernalization pathway.
The VRN2 locus consists of two linked ZCCT genes, ZCCT1 and ZCCT2, which encode a protein with a zinc finger motif and a CCT domain (Yan et al. 2004). Natural variations have been identified in the VRN2 locus. Simultaneous deletion or non-functional mutations of these two ZCCT genes result in a plant showing the spring habit (Distelfeld et al. 2009), indicating that VRN2 is a flowering repressor gene. A high level of VRN2 expression is observed in seedlings at the 1-leaf stage, while expression is down-regulated by vernalization and aging; by contrast, VRN1 shows the opposite pattern with low expression in seedlings and up-regulated expression after vernalization (Shimada et al. 2009). It has also been reported that VRN2 shows a diurnal expression pattern and that a long photoperiod up-regulates its expression level (Dubcovsky et al. 2006; Trevaskis et al. 2006), suggesting that the VRN2 expression is affected by photoperiod as well as vernalization.
VRN3 encodes a Raf kinase inhibitor-like protein with a high similarity to the Arabidopsis FLOWERING LOCUS T (FT) protein, which is a florigen (Yan et al. 2006). Transgenic wheat plants overexpressing VRN3 show an extra early-flowering phenotype without the need for vernalization (Yan et al. 2006; Shimada et al. 2009), indicating that VRN3 is a strong flowering promoter. Under long day conditions, VRN3 shows a diurnal expression pattern; however, expression is very low under short day conditions (Shimada et al. 2009; Kitagawa et al. 2012).
Based on data from expression, transgenic and mutant analyses, we developed a gene network model for the interaction of VRN1, VRN2 and VRN3 in leaves (Shimada et al. 2009). In this model, VRN1 is upstream of VRN3 and activates VRN3 expression under long day conditions. Thus, VRN1 is proposed to play a role as an integrator of the vernalization and photoperiodic signals. Trevaskis (2010) put forward an alternative gene network model for VRN1, VRN2 and VRN3; this model was based on the results of investigations using barley. This alternative model postulates that VRN1 and VRN3 mutually up-regulate each other: VRN1 first activates VRN3 expression, and then VRN3 further activates VRN1. The model was referred to as “the flowering model for temperate cereals” in a review paper on flowering in plants (Andres and Coupland 2012). More recently, a third model was suggested by Chen and Dubcovsky (2012). This model proposes that VRN1 is activated by VRN3 and then suppresses VRN2 expression. In this model, VRN1 is not essential for flowering; this conclusion was drawn from an analysis of a VRN1 mutant line. However, it is not certain that the mutant line is a true VRN1 knock-out, because its genotypic alteration is a point mutation and VRN1 mRNA is transcribed.
To obtain more information about the flowering mechanism in wheat, we are developing a large-scale panel of mutants induced by heavy-ion beam mutagenesis; these mutants are being systematically screened for effects on flowering time (Murai et al. 2013). Heavy-ion beam irradiation is effective at producing gene deletion mutants (null mutations) (Kazama et al. 2011, 2013). Here we describe four newly identified extra early-flowering mutant lines in diploid einkorn wheat, which have been named extra early-flowering 1 (exe1), exe2, exe3, and exe4.