Characterization of late heading 1, a Heavy-Ion Beam Irradiation-Induced Mutant in Einkorn Wheat (Triticum monococcum) that Suppresses an Early-Flowering Phenotype in Plants with Deletion of PHYTOCLOCK 1

Abstract

The einkorn wheat (Triticum monococcum) strain KU104-2 is an early-flowering line that was generated by X-irradiation of the einkorn wheat strain KU104-1. The early-flowering KU104-2 heads about one month earlier than KU104-1 and has a deletion of the biological-clock-component gene PHYTOCLOCK 1/LUX ARRHYTHMO (PCL1/LUX). In this study, we characterize a newly induced mutant named late-heading 1 (lh1) that was generated by heavy-ion beam irradiation of KU104-2. This new mutant shows partial suppression of the early-flowering phenotype of KU104-2. Under field conditions, lh1 plants head about one week later than KU104-2; lh1 mutants show no other significant differences in agricultural traits such as plant height, spike length, and spikelet number per spike compared with KU104-2. Analysis of plant development in a growth chamber showed that lh1 mutants exhibit a late-flowering phenotype under short-day (SD) conditions, but not under long-day (LD) conditions. This late-flowering phenotype under SD conditions is associated with the down-regulation of the flowering promoter gene VERNALIZATION 1 (VRN1) in leaves of plants at the 5^th-leaf stage of growth, i.e., at the late vegetative phase. Our findings suggest that the expression level of VRN1 in the vegetative phase under short-day conditions is important for the determination of flowering time in wheat.

Flowering time is associated with maturing time and is one of the most important agricultural traits in cereal crops such as wheat (Triticum aestivum) and barley (Hordeum vulgare). It is largely controlled by two environment-related characteristics, namely, photoperiod response and vernalization requirement. Photoperiod response is a key character for the determination of earliness in East Asia that allows harvesting to occur before the onset of the rainy season (Tanio et al. 2005). The photoperiod response in cereal crops such as wheat and barley is mainly controlled by the gene Photoperiod 1 (Ppd1), which is a member of the PSEUDO-RESPONSE REGULATOR (PRR) gene family first identified in Arabidopsis (Turner et al. 2005). Vernalization requirement prevents plants from transiting to the reproductive phase before winter and thereby avoids reproductive organs being damaged by cold temperatures. Therefore, the vernalization requirement is an important adaptive trait for long-day plants that germinate in autumn. Three loci have been found to control vernalization, namely, VERNALIZATION 1 (VRN1), VRN2, and VRN3 (Distelfeld et al. 2009). VRN1 encodes an APETALA1/FRUITFULL-like (AP1/FUL-like) MADS-box transcription factor that is up-regulated by vernalization (Danyluk et al. 2003, Murai et al. 2003, Trevaskis et al. 2003, Yan et al. 2003). The expression of VRN1 is also up-regulated by a long photoperiod and by aging (Murai et al. 2003, Nishiura et al. 2014), indicating that its expression is associated with the flowering phenotype. In contrast to VRN1, VRN2 functions as a flowering suppressor and, when present as a recessive allele, controls vernalization non-requirement (spring habit). The VRN2 locus consists of two linked genes, ZCCT1 and ZCCT2, which encode a protein with a zinc finger motif and a CCT domain (Yan et al. 2004). A high level of VRN2 expression is observed in seedlings at early development, while expression is down-regulated by vernalization and aging; this is the opposite pattern of VRN1 (Shimada et al. 2009). VRN3 encodes a Raf kinase inhibitor-like protein with high similarity to the Arabidopsis FLOWERING LOCUS T (FT) protein, which is a florigen (Yan et al. 2006). Therefore, VRN3 is also called FT1 in barley (Hemming et al. 2008, Sasani et al. 2009) and Wheat FT (WFT) in wheat (Shimada et al. 2009). 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 flowering promoter (a florigen gene) in temperate crops. In a previous study, we demonstrated that the VRN1 protein binds to the promoter region of the WFT gene, suggesting that the expression of WFT is directly controlled by VRN1 (Tanaka et al. 2018).

The einkorn early-flowering mutant KT3-5 (identical to KU104-2) was induced by X-irradiation of Triticum monococcum strain KT3-1 (identical to KU104-1) and has been examined for heading traits (Shindo and Sasakuma 2001). Map-based cloning identified a clock component gene PHYTOCLOCK 1/LUX ARRHYTHMO (PCL1/LUX) [abbreviated here as Wheat PCL1 (WPCL1)] as a candidate gene for the mutation (Mizuno et al. 2012). It has been reported that KU104-2 shows higher expression of Ppd1 and photoperiod response-related genes (CONSTANS-like genes), resulting in the up-regulation of WFT and induction of early flowering. To better understand the mechanism of the early-flowering phenotype of this mutation, we identified a suppression mutant of early-flowering phenotype in KU104-2, named late-heading 1 (lh1), in a large-scale mutant panel in einkorn wheat strain KU104-2 (Murai et al. 2013); this suppression mutation was induced by heavy-ion beam mutagenesis.

In this study, we first examined agronomic characters, such as heading-time, in lh1mutant plants compared to those of KU104-2 in the field. Then, we performed an analysis of plant development and gene expression patterns of VRN1 and WFT in lh1 and KU104-2 plants using a growth chamber. Our analyses indicate that the late-flowering phenotype in the lh1 mutant is associated with the down-regulation of VRN1 in the leaves during the vegetative phase. Our findings here, together with previous results, indicate that the expression level of VRN1 under short-day conditions is controlled by a biological clock and is important for the determination of flowering time in wheat.

Materials and methods

Plant material

Diploid einkorn wheat (Triticum monococcum) strain KU104-2, the late-heading mutant line lh1 derived from KU104-2, and wild-type strain KU104-1 were used in the experiments. The lh1 mutant was generated by carbon-ion beam (50 Gy and 50 keVμm⁻¹) irradiation and identified in a large-scale mutant panel (Murai et al. 2013); M₈ generation individuals were used in the experiments.

Field experiments

KU104-1, KU104-2, and lh1 mutant plants were grown in an experimental field at Fukui Prefectural University for three seasons, 2013/2014, 2014/2015, and 2015/2016. The heading dates of each line were scored. Spike length, spikelet number per spike, and culm length (total and 1^st to 5^th internode lengths) at the maturation stage were measured on ten shoots and ears of two individual plants of each in season 2015/2016. Inter-line differences were analyzed by ANOVA (analysis of variance), and significant differences between lines were analyzed by LSD (least significant difference) method.

Growth chamber experiments

KU104-1, KU104-2, and lh1 mutant plants were cultivated in a growth chamber under long day (LD: 16 h light/8 h dark) conditions or short day (SD: 10 h light/14 h dark) conditions at 20°C (light intensity ∼100 μE m⁻² s⁻¹). Leaf emergence speed (plastochron), the total number of leaves, and heading times (earliness) were screened in 10 plants of each line. Growth stages were defined by leaf stages: for example, the 4-leaf stage indicates seedlings with four unfolding leaves on the main shoot. Assessment of the leaf stage was performed in each line when all ten individuals had reached the required leaf stage: for example, for the 4-leaf stage, the time for all ten individuals to reach the 4-leaf stage was measured. Flag leaf (final leaf) unfolding time was used as an indicator of the earliness of heading rather than heading time itself.

Gene expression analysis

Diurnal expression of VRN1 and WFT in KU104-2 and lh1 mutant plants at the 5-leaf stage was analyzed in non-vernalized plants grown under LD or SD conditions at 20°C. Leaves were sampled every 6 h. Total RNAs were extracted from leaves using ISOSPIN Plant RNA (Nippon Gene, Japan); cDNAs were synthesized from the total RNAs using TaKaRa PrimeScript^® RT reagent Kit with gDNA Eraser (TaKaRa, Japan). Real-time PCR analyses were performed using a LightCycler 96 (Roche Diagnostics GmbH) with the following gene-specific primer sets: VRN1, TaMADS#11-545 L (5′-GGAGAGGTCACTGCAGGAGGA-3′), and TaMADS#11-698R (5′-GCCGCTGGATGAATGCTG-3′) at an annealing temperature of 65°C; WFT, TaFT-mono3F (5′-GGTACAACTGGTGCCTCGTT-3′) and TaFT-mono3R (5′-GTTGTAGAGCTCGGCGAAGT-3′) at an annealing temperature of 64°C. The relative quantities of the transcripts were determined using a SYBR Green-labelled amplification product from the gene for the Cell Division Control Protein (CDCP) (Paolacci et al. 2009) prepared with the primers CDCP-L (5′-CAAATACGCCATCAGGGAGAACATC-3′) and CDCP-R (5′-CGCTGCCGAAACCACGAGAC-3′) at an annealing temperature of 62°C. The primer sets for VRN1 and WFT were identical to those used in our previous study (Nishiura et al. 2014). Three biological replicates were performed. Each biological sample contained leaves from two individual plants. The significant difference in gene expression levels of lh1 compared with KU104-2 was analyzed by t-test.

Results

Agronomic characters of the lh1 mutant

Heading dates were assessed in the late-heading 1 (lh1) mutant and einkorn wheat strain KU104-1 and KU104-2 in all three seasons in the experimental field (Table 1). KU104-1 is a wild-type strain showing late-flowering, and KU104-2 is an early-flowering mutant derived from KU104-1 that has a deletion of the clock component gene WPCL1 (Mizuno et al. 2012). Under field conditions, KU104-2 exhibited heading approximately one month earlier than KU104-1. The field experiment indicated that the lh1 mutant partially suppressed the early-flowering phenotype of KU104-2 as it headed 6 to 10 days later than KU104-2 (Table 1).

Table 1. Comparison of heading date of late-heading 1 (lh1), KU104-1, and KU104-2 plants.

Season	Heading date			Difference of heading date KU104-2 vs. lh1	Sowing date
	KU104-1	KU104-2	lh1
2013/2014	June 3	May 8	May 14	6 days	Oct. 21, 2013
2014/2015	June 2	May 5	May 15	10 days	Oct. 20, 2014
2015/2016	June 1	May 2	May 8	6 days	Oct. 19, 2015

The agronomic characteristics of the field-grown plants are shown in Table 2. As reported previously (Nishiura et al. 2014), KU104-2 showed significantly smaller spikes with fewer spikelets than KU104-1 plants. Furthermore, KU104-2 plants had a significantly shorter culm length. The reduced culm length is due to the shortening of the 1^st, 4^th, and 5^th internode lengths compared with KU104-2. Interestingly, the 2^nd internode length in KU104-2 plants was significantly larger than in KU104-1 plants. The lh1 mutant had similar agronomic traits to KU104-2 plants, including the longer 2^nd internodes, and there were no significant differences between the two strains (Table 2, Figs. 1A, 1B). Our data indicate that the suppression mutation in lh1 did not affect these agronomic characters.

Table 2. Agronomic characters (means±Standard Deviation) of late-heading 1 (lh1) compared with KU104-1 and KU104-2.

	Spike length (cm)	Spikelet no.	Culm length (cm)	1st internode length (cm)	2nd internode length (cm)	3rd internode length (cm)	4th internode length (cm)	5th internode length (cm)
KU104-1	8.5±0.3b	38±2b	122±2.3b	60.8±2.8b	23.5±1.6a	14.3±1.6	11.8±0.4b	8.2±1.8b
KU104-2	6.9±1.2a	25±6a	108±4.7a	53.5±2.9a	30.7±1.5b	14.8±2.9	7.8±1.7a	3.8±0.8a
lh1	7.3±0.6a	29±2a	103±7.9a	52.3±1.9a	29.3±1.4b	14.3±2.8	7.5±1.6a	3.8±1.3a
Sum of square	17.676	758.5	1598.944	352.444	208.5	92.5	98.944	103.611
p-Value	0.008**	0.000**	0.000**	0.000**	0.000**	0.922 ns	0.000**	0.000**

**: Significantly different at p=0.01 by ANOVA. ns: Not significant. a and b: The same superscript do not differ significantly (p=0.05) from each other when The least significant difference is applied.

Fig. 1. Phenotypic comparison of the lh1 mutant (right) and KU104-2 (left).

(A) Whole plants are grown in the field. (B) One ear taken from a plant grown in the field. (C) Non-vernalized (NV) plants grown in a growth chamber under short-day (SD) conditions. KU104-2 is at the late reproductive phase whereas lh1 mutant plants have just passed phase transition.

Leaf emergence speed (plastochron) in lh1 mutant plants

Leaf emergence timing in KU104-1, KU104-2, and lh1 mutant plants was determined by examining leaf unfolding in seedlings cultivated in a growth chamber under LD or SD conditions (Fig. 2). Under LD conditions, late-flowering KU104-1 plants took about 110 days from sowing to heading, and the 11^th leaf was the flag leaf (Fig. 2A). The production of successive leaves ceased following the initiation of the flag leaf, which could be distinguished from the other leaves by its short blade and the emergence of a spike from its leaf sheath. In contrast, early-flowering KU104-2 took about 70 days for heading (Fig. 2A). KU104-2 displayed a faster plastochron from the 5-leaf stage; the 9^th leaf was the flag leaf, resulting in early-heading compared with KU104-1. The lh1 mutant exhibited a similar plastochron pattern as KU104-2. However, the 8^th leaf was the flag leaf in the mutant, resulting in the heading being about 5 days earlier compared to KU104-2.

Fig. 2. Leaf emergence speed (plastochron) in non-vernalized KU104-1 (green), KU104-2 (blue), and lh1 mutant (orange) plants grown in a growth chamber. The number of days to each leaf stage, after the 1-leaf stage, is indicated; the square dots at the end of the lines indicate flag leaf stages. (A) Days after the 1-leaf stage to flag leaf stage under long-day conditions. (B) Days after the 1-leaf stage to flag leaf stage under short-day conditions. KU104-1 did not reach flag leaf unfolding within 160 days.

Under SD conditions, KU104-1 plants did not unfold the flag leaf until at least 160 days from sowing. In contrast, KU104-2 showed flag leaf unfolding at about 120 days. The lh1 mutant showed a delayed transition from the vegetative to reproductive phases compared with KU104-2 (Fig. 1C), a slower plastochron, and flag leaf unfolding at about 150 days (Fig. 2B). Flag leaf emergence occurred at the 14-leaf and 15-leaf stages in KU104-2 and lh1, respectively. The delay in flag leaf emergence in the lh1 mutant under SD conditions compared to LD conditions indicates that this mutant retained photoperiodic sensitivity, as did KU104-2.

The timing of emergence of the first five leaves was similar in KU104-1, KU104-2, and lh1 mutant plants (Fig. 2); later, during the vegetative phase, there were no differences among the strains. The plastochron appeared to be shorter during the reproductive growth phase (after phase transition) in KU104-2 compared to KU104-1 under both LD and SD conditions. Interestingly, the plastochron of lh1 mutant plants was similar to that of KU104-2 under LD conditions, but not under SD conditions; the plastochron of lh1 mutant plants was slower than that of KU104-2 under SD conditions (Fig. 2B). These results indicate that the mutant gene(s) in lh1 suppresses the effect of the deletion in WPCL1 only under SD conditions.

Diurnal expression patterns of VRN1 and WFT in lh1 mutant

We used real-time PCR analyses to compare the expression patterns of VRN1 and WFT in KU104-2 and lh1 mutant plants (Figs. 3, 4). Here, we found that lh1 mutant plants showed a similar diurnal expression pattern in VRN1 to KU104-2 under LD and SD conditions at the 5-leaf stage of vegetative growth (Fig. 3). However, gene expression levels were different between the two lines: expression levels were significantly lower in lh1 mutant plants than in KU104-2 plants throughout the day. The difference was greater under SD conditions than under LD conditions. WFT showed a similar diurnal expression pattern in KU104-2 and lh1 mutant plants; expression levels in lh1 were significantly lower than in KU104-2 at the dark period (Fig. 4). Drastic down-regulation of WFT was observed in both lines under SD conditions, with lower WFT expression levels in the lh1 mutant (Fig. 4B). These results suggest that the mutant gene in lh1 influences the levels of expression of VRN1 and WFT but not their expression patterns. This finding indicates that the expression levels of these two genes are important for flowering.

Fig. 3. Diurnal expression pattern of VRN1 in non-vernalized KU104-2 (black) and lh1 mutant (red) plants at the 5-leaf stage. The expression level of VRN1 was determined by real-time PCR with the CDCP gene as an internal control for calculating the relative levels of expression. The difference in gene expression level in lh1 compared with KU104-2 was tested by statistical analysis (t-test). Error bars indicate standard error. * and ** indicate significant difference at p=0.05 and p=0.01, respectively. No asterisk means no significant between gene expression levels. (A) Expression pattern under long-day conditions. (B) Expression pattern under short-day conditions.

Fig. 4. Diurnal expression pattern of WFT in non-vernalized KU104-2 (black) and lh1 mutant (red) plants at the 5-leaf stage. The expression level of WFT was determined by real-time PCR with the CDCP gene as an internal control for calculating the relative levels of expression. The difference in gene expression level in lh1 compared with KU104-2 was tested by statistical analysis (t-test). Error bars indicate standard error. * and ** indicate significant difference at p=0.05 and p=0.01, respectively. No asterisk means no significant between gene expression levels. (A) Expression pattern under long-day conditions. (B) Expression pattern under short-day conditions.

Discussion

The X-ray-induced einkorn mutant KU104-2 (KT3-5) exhibited an early-flowering phenotype due to a deletion of a clock-component-gene WPCL1 (Mizuno et al. 2012). As the original strain KU104-1 lacks a VRN2 locus as a natural variation (Nishiura et al. 2014, 2018), KU104-2 doesn’t have both WPCL1 and VRN2. In a previous study, we developed an early-flowering mutant, extra-early flowering 3 (exe3) with a deletion of WPCL1 by heavy-ion beam irradiation of KU104-1 (Nishiura et al. 2014, 2018). Analysis of exe3 and KU104-2 revealed that the loss of function of WPCL1 resulted in early flowering in wheat. The identification of this consequence of modification of WPCL1 means that such mutations can be of value for wheat breeding (Mizuno et al. 2016).

In this study, we identified an einkorn mutant lh1 generated by heavy-ion beam irradiation of KU104-2, that shows partial suppression of the early-flowering phenotype of KU104-2. The mechanism by which this mutation suppresses the phenotype and its potential value for wheat breeding need to be considered. WPCL1 is an ortholog of Arabidopsis clock-related gene PCL1/LUX (Mizuno et al. 2012) that encodes a single-MYB domain GARP transcription factor (Hazen et al. 2005, Onai and Ishiura 2005). PCL1/LUX is an essential factor for the control of the circadian rhythm, and directly regulates the expression of the clock gene PSEUDO-RESPONSE REGULATOR 9 (PRR9) (Helfer et al. 2011). Furthermore, PCL1/LUX forms a protein complex with EARLY FLOWERING 3 (ELF3) and ELF4 to form the ELF4-ELF3-PCL1/LUX complex, the so-called “Evening Complex”. This complex regulates PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PIF5, which control hypocotyl growth under diurnal conditions (Nusinow et al. 2012).

Mutation of PCL1/LUX in Arabidopsis causes early flowering under LD and SD conditions (Hazen et al. 2005). WT Arabidopsis plants display greatly delayed flowering under SD conditions compared with LD conditions; mutant plants show early flowering under SD conditions that are as early as under LD conditions. These phenotypes are very similar to the behavior of exe3 plants (Nishiura et al. 2014). In the Arabidopsis mutant, the LATE ELONGATED HYPOCOTYL (LHY) gene is repressed and TIMING OF CAB2 EXPRESSION 1 (TOC1) is activated under SD conditions (Hazen et al. 2005). Our previous data indicated that WLHY was down-regulated and WTOC1 was up-regulated in the exe3 mutant under SD conditions (Nishiura et al. 2018). The altered gene expression patterns were also observed in KU104-2 (Mizuno et al. 2012). The similarities between phenotypes and gene expression patterns in the Arabidopsis mutant and wheat mutants (exe3 and KU104-2) indicate that PCL1/LUX performs similar functions in the circadian clock systems of both species. Furthermore, we also revealed that the exe3 mutant exhibited a loss of SD-specific expression patterns in genes of the photoperiodic pathway between clock-related genes (WLHY and WTOC1) and CONSTANS-like genes (Nishiura et al. 2018). In a future study, we will examine the detailed gene expression patterns of genes in the photoperiodic pathway, including clock-related genes (WLHY and WTOC1) and CONSTANS-like genes, in the lh1 mutant.

The fact that the exe3 mutant plants did not show SD-specific expression patterns in the photoperiodic pathway between clock-related genes (WLHY and WTOC1) and CONSTANS-like genes (Nishiura et al. 2018) suggests that the exe3 mutant has disordered SD response caused by the deletion of WPCL1. Wheat is a facultative LD plant and SD photoperiods repress flowering; one of the important roles of the circadian clock is SD suppression of flowering in wheat. The present lh1 mutant exhibits a late-heading phenotype under SD conditions, but not under LD conditions. This fact indicates that there is a specific regulation system controlling floral suppression under SD conditions in wheat. This system may be downstream of the biological clock and receive signals from the clock and may work in concert with photoperiodic pathway genes. Future identification of the mutated gene in lh1 will lead to an improved mechanistic understanding of this SD-specific system.

In our previous study of the exe3 mutant, we demonstrated that the early-flowering phenotype was correlated with the level of VRN1 expression (Nishiura et al. 2014). As exe3 lacks VRN2, the regulation of VRN1 expression may be controlled by the biological clock independently of VRN2. The diurnal expression of VRN1 reinforces this idea (Shimada et al. 2009, Nishiura et al. 2014). The disruption of clock function also affects the expression of the florigen gene WFT through VRN1 expression. In this study, the late-flowering phenotype under SD conditions in the lh1 mutant is associated with the down-regulation of VRN1, in leaves at the 5-leaf stage during the late vegetative phase. In conclusion, the present study together with results from earlier studies, suggests that the expression level of VRN1 under short-day conditions is regulated by a biological clock and is a major determinant of earliness in wheat.

Acknowledgements

We are grateful to the National Bioresource Project-Wheat (NBRP-KOMUGI) for providing the WT wheat strain. This work was supported in part by the Grant-in-Aid for Scientific Research from Fukui Prefectural University to K. Murai.

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