An early-flowering einkorn wheat mutant with deletions of PHYTOCLOCK 1/LUX ARRHYTHMO and VERNALIZATION 2 exhibits a high level of VERNALIZATION 1 expression induced by vernalization

Abstract

Using heavy-ion beam mutagenesis of Triticum monococcum strain KU104-1, we identified a mutant that shows extra early-flowering; it was named extra early-flowering 3 (exe3). Here, we carried out expression analyses of clock-related genes, clock downstream genes and photoperiod pathway genes, and found that the clock component gene PHYTOCLOCK 1/LUX ARRHYTHMO (PCL1/LUX) was not expressed in exe3 mutant plants. A PCR analysis of DNA markers indicated that the exe3 mutant had a deletion of wheat PCL1/LUX (WPCL1), and that the WPCL1 deletion was correlated with the mutant phenotype in the segregation line. We confirmed that the original strain KU104-1 carried a mutation that produced a null allele of a flowering repressor gene VERNALIZATION 2 (VRN2). As a result, the exe3 mutant has both WPCL1 and VRN2 loss-of-function mutations. Analysis of plant development in a growth chamber showed that vernalization treatment accelerated flowering time in the exe3 mutant under short day (SD) as well as long day (LD) conditions, and the early-flowering phenotype was correlated with the earlier up-regulation of VRN1. The deletion of WPCL1 affects the SD-specific expression patterns of some clock-related genes, clock downstream genes and photoperiod pathway genes, suggesting that the exe3 mutant causes a disordered SD response. The present study indicates that VRN1 expression is associated with the biological clock and the VRN1 up-regulation is not influenced by the presence or absence of VRN2.

Introduction

The early-flowering or early-heading phenotype in bread wheat (Triticum aestivum) cultivars is important as it can produce an early harvest; this characteristic is particularly beneficial in East Asia as it allows harvesting to occur before the onset of the rainy season. In autumn-sown wheat cultivars grown in central to southwestern Japan, reduced photoperiod sensitivity is the major determinant of earliness (Tanio et al., 2005). The photoperiod response is controlled by three major genes, Photoperiod-A1 (Ppd-A1), Ppd-B1 and Ppd-D1, which are located on the chromosome 2 homoeologs (Scarth and Law, 1984). The barley (Hordeum vulgare) Ppd-1 ortholog, Ppd-H1, has been cloned and identified as a member of the PSEUDO-RESPONSE REGULATOR (PRR) family of Arabidopsis (Turner et al., 2005). PRR proteins contain a receiver-like/pseudo-receiver domain at their N-terminal end and a CCT [CONSTANS (CO), CO-like and TIMING OF CAB EXPRESSION 1 (TOC1)] domain near their C-terminus (Mizuno and Nakamichi, 2005). The CCT motif is a plant-specific and widespread motif, and may be important in regulating the expression of flowering control genes including the CO-like family (Griffiths et al., 2003) and of the vernalization gene of temperate cereals VERNALIZATION 2 (VRN2) (Yan et al., 2004a). In Arabidopsis, the PRR family consists of five members, PRR1/TOC1, PRR3, PRR5, PRR7, and PRR9, and functions as a clock oscillator that is central to circadian rhythms (Mizuno and Nakamichi, 2005). Ppd-H1 shows highest homology to PRR7 among the Arabidopsis PRR genes (Turner et al., 2005), suggesting that Ppd-1 is an ortholog of PRR7 and functions as a clock component. Beales et al. (2007) identified the orthologs of wheat A, B and D genomes, and reported that the photoperiod-insensitive Ppd-D1a allele is associated with a 2089-bp deletion upstream of the coding region. Compared with photoperiod-sensitive Ppd-D1b, Ppd-D1a is misexpressed and this is associated with altered expression of the florigenic gene FLOWERING LOCUS T (FT). Genomic sequence analyses revealed that photoperiod-insensitive Ppd-A1a and Ppd-B1a alleles were associated with a 1085-bp deletion and 308-bp insertion in the 5′ upstream region, respectively (Nishida et al., 2013). Copy number variations (CNVs) are also present at the Ppd-B1a alleles and these cause increased basal gene expression levels (Díaz et al., 2012). The photoperiod-insensitive alleles of Ppd-1 have been used for wheat breeding programs for early-heading varieties in several countries, including Japan (Seki et al., 2011, Seki et al., 2013). In addition, Ppd-1 is associated with yield traits such as spikelet numbers (Worland et al., 1998). Recently, Boden et al. (2015) reported that Ppd-1 plays a key role in inflorescence architecture.

In Arabidopsis, the gene network controlling the circadian clock involves a series of transcriptional and post-transcriptional feedback loops that create gene expression rhythms (reviewed in Bendix et al., 2015; Johansson and Staiger, 2015). Two transcription factors, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), are expressed and active at dawn. During the morning, LHY and CCA1 repress the evening expressing genes, PHYTOCLOCK 1/LUX ARRHYTHMO (PCL1/LUX), EARLY FLOWERING 3 (ELF3) and ELF4. PCL1/LUX, ELF3 and ELF4 are expressed from evening until midnight, and repress the PRR family (PRR5, PRR7 and PRR9). The PRRs then repress LHY and CCA1. Another important negative feedback loop is involved in TIMING OF CAB EXPRESSION 1 (TOC1)/PRR1. TOC1/PRR1 expression is suppressed by LHY and CCA1, and TOC1 represses LHY and CCA1 during the course of the day.

The photoperiod pathway that is mainly composed of FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1), GI (GIGANTEA), CDFs (CYCLING DOF FACTORs), and CO (CONSTANS) is downstream of the circadian clock genes described above (reviewed in Song et al., 2015). During long day (LD) conditions, the expression peaks of the clock-regulated genes FKF1 and GI are in the afternoon. The FKF1 protein absorbs blue light and interacts with GI. The FKF1-GI complex degrades CDF proteins (CDF1, 2, 3, and 5), which are negative regulators of CO. The CO protein induces florigenic FT expression, leading to flowering (Song et al., 2015).

To understand the molecular mechanism of flowering in wheat, we constructed a large-scale mutant panel in diploid einkorn wheat (T. monococcum) using heavy-ion beam mutagenesis (Murai et al., 2013). Einkorn wheat seeds were exposed to a heavy-ion beam and then sown in the field. Selfed seeds from each spike of M₁ plants were used to generate M₂ lines. Every year over the past 15 years, we have obtained approximately 1000 M₂ lines and have built up a mutant panel with 10,000 M₂ lines. This mutant panel is being systematically screened for mutations affecting reproductive growth, and especially for flowering-time mutants. From the large scale mutant panel, we have identified four extra early-flowering mutants, named extra early-flowering1 (exe1), exe2, exe3, and exe4 (Nishiura et al., 2014). The four exe mutants fall into two groups, namely Type I (moderately extra early-flowering type: exe1 and exe3) and Type II (extremely extra early-flowering type: exe2 and exe4). Analysis of plant development in a growth chamber showed that the speed of leaf emergence is accelerated in the exe mutants compared to wild-type (WT) plants. Overall, the speed of leaf emergence is faster in Type II than Type I plants. Type I plants show reduced photoperiodic sensitivity, and Type II plants show a distorted photoperiod response. Analysis of VERNALIZATION 1 (VRN1), a flowering promoter gene, shows that it is more highly expressed in seedlings at early developmental stages in Type II mutants than Type I mutants. These findings indicate that the difference in earliness between Type I and Type II mutants is associated with the level of VRN1 expression. VRN1 is an activator of FT (Shimada et al., 2009), and encodes an APETALA1/FRUITFULL-like (AP1/FUL-like) MADS-box transcription factor that is up-regulated by vernalization (Yan et al., 2003; Murai et al., 2003; Trevaskis et al., 2013; Danyluk et al., 2003).

In this study, we demonstrate that the Type I exe3 mutant has a deletion of a clock component gene PCL1/LUX, abbreviated to Wheat PCL1 (WPCL1), which was previously identified by Mizuno et al. (2012). We also confirm that the original strain KU104-1 carries a natural mutation of a null allele of the VRN2 gene. Therefore, the exe3 mutant has loss-of-function mutations of both WPCL1 and VRN2. The present study indicates that the up-regulation of VRN1 expression after vernalization is induced in the absence of expression in the clock component gene WPCL1; however, VRN1 up-regulation is not influenced by the presence or absence of VRN2.