Effect of AtSF1 mutation on FT and TSF expression at different temperatures
In a previous study, we found that atsf1-2 mutants exhibited early flowering phenotypes at different temperatures upon downregulation of the expression of SVP, FLM-β, and TEMPRANILLO 2 (Lee et al. 2020). As these important floral repressors affect the transcript levels of FT, SOC1, and TSF, all of which are involved in integrating temperature-dependent flowering signals within the ambient temperature pathway (Lee et al. 2013; Pose et al. 2013), we measured the expression of flowering-promoting genes over a two-day diurnal time course in similar developmental atsf1-2 mutant and wild-type plants grown at 16 and 23°C under LD conditions. Although the expression of FT and TSF was specifically increased in the middle of the night (ZT20) in seven- and eight-day-old atsf1-2 mutant seedlings grown at 23 °C, FT expression at 16 °C showed a statistically significant increase in the morning (ZT0 to 8 or 12), whereas TSF expression increased from night to morning (ZT20 to 8) (Fig. 1a, c). Therefore, phase shifts affected the expression patterns of these two genes. However, SOC1 expression in the atsf1-2 mutants remained unaltered at 16 and 23°C (Fig. 1b). These results suggest that atsf1 mutation leads to early flowering at different temperatures by partially regulating FT and TSF expression.
Effect of genetic interactions among AtSF1, FLM, and FT on temperature-insensitive flowering of atsf1-2 mutants
Because FT acts as a major floral integrator gene under LD conditions (Yoo et al. 2005), we tested the genetic effect of AtSF1, FLM, and FT on flowering time. We first generated double and triple mutants with different combinations of atsf1-2, flm-3, and ft-10 single mutants and measured their flowering times at 16 and 23 °C under LD conditions. Our analysis showed that wild-type plants flowered with a mean total leaf number (TLN) of 27.8 and 14.1 at 16 and 23 °C, respectively. Moreover, the atsf1-2 and flm-3 mutants flowered significantly earlier than the wild-type plants, whereas the ft-10 mutants flowered later than other single mutants and wild-type plants (Fig. 2a), which was consistent with the previous reports (Lee et al. 2013; Lee et al. 2020). The atsf1-2 flm-3 mutants flowered significantly earlier at 16 and 23 °C (10.3 and 9.1 leaves, respectively) than flm-3 single mutants (11.1 and 10.5 leaves at 16 and 23 °C) (Fig. 2a). The atsf1-2 ft-10 mutants (TLN = 35.3 and 17.0 leaves at 16 and 23 °C, respectively) flowered earlier than ft-10 single mutants (TLN = 51.7 and 32.3 leaves) at different temperatures (Fig. 2a). However, flm-3 ft-10 mutants flowered with a TLN of 44.5 and 32.6 leaves at 16 and 23 °C, respectively, which was comparable to that of ft-10 mutants. Furthermore, the atsf1-2 flm-3 ft-10 mutants flowered earlier than the flm-3 ft-10 mutants (40.1 and 28.2 leaves at 16 and 23 °C, respectively) (Fig. 2a). These results indicate that the flm or ft single mutation and flm ft double mutation did not completely suppress the flowering phenotype of atsf1-2 mutants at different temperatures.
To assess the temperature sensitivity of the various mutants, we calculated their LNRs using TLN values at 16 and 23°C. An LNR close to 1.0 indicates that different temperatures have minor effects on flowering. The atsf1-2 or flm-3 single mutants and atsf1-2 flm-3 double mutants had LNR values of approximately 1.0, compared to those of wild-type plants (LNR = 2.0) (Fig. 2b). However, the LNRs of the atsf1-2 ft-10, flm-3 ft-10, and atsf1-2 flm-3 ft-10 mutants were 2.1, 1.4, and 1.4, respectively, indicating that both FLM and FT acted downstream of AtSF1 during temperature response. These results suggest that AtSF1 regulates other flowering time genes, in addition to FLM and FT, to modulate flowering time at different temperatures.
Effect of AtSF1 on LFY expression in the shoot apex regions
Since atsf1-2 ft-10 mutants showed flowered earlier than ft-10 mutants (Fig. 2a) and our previous study have shown that LFY expression was increased at ZT16 in eight-day-old atsf1-2 mutants at 23 °C under LD conditions (Lee et al. 2017), we measured the transcript levels of floral activators LFY and APETALA1 (AP1) over an approximately two-day diurnal time course in the atsf1-2 and wild-type plants grown to similar developmental stages at 16 and 23 °C under LD conditions. RT-qPCR analysis showed that LFY expression levels were increased throughout the time course in the whole seedlings of atsf1-2 mutants grown at 23°C, but not in those grown at 16°C (Fig. 3a). However, the expression of AP1 in atsf1-2 mutants decreased at most ZT points at 23°C, but not at 16°C (Fig. 3b). These results indicate that lesions in AtSF1 affect LFY and AP1 expression significantly in all seedlings at 23°C only.
It has been reported that AtSF1 is strongly expressed in shoot apex regions (Jang et al. 2014); thus, we also examined the expression levels of LFY and AP1 at ZT16 in the shoot apices of 8- and 12-day-old atsf1-2 mutant and wild-type plants grown at 23 and 16°C under LD conditions, respectively. LFY expression was significantly increased in atsf1-2 and atsf1-2 ft-10 mutants at the different temperatures compared to that in the wild-type plants (Fig. 3c). In contrast, AP1 expression in atsf1-2 mutants was lower than that in the wild-type plants at 23 and 16°C (Fig. 3d). Unlike in the whole seedlings of atsf1-2 mutants, LFY and AP1 expression increased and decreased, respectively, in the shoot apex regions of the mutants at different temperatures. This discrepancy suggests that AtSF1 influences flowering by negatively regulating LFY expression in the shoot apex.
Direct binding of FLM to the LFY genomic region
Since it has been established that AtSF1 regulates the AS of FLM pre-mRNA (Lee et al. 2020), we examined whether FLM protein is involved in directly regulating LFY expression. FLM is a type of MADS-box transcription factor, which are DNA-binding proteins that recognise the CArG or vCArG motifs in their target genes (Lee et al. 2013; Pose et al. 2013); thus, we performed ChIP experiments on eight-day-old pFLM:gFLM:GFP flm-3 mutant and wild-type plants at 16 and 23 °C under LD conditions. In these experiments, two regions (upstream promoter region I and part of the first exon and intron region II) and one region (part of the first exon and intron region I) containing CArG or vCArG motifs of the LFY and AP1 loci, respectively, were explored (Fig. 4). Each region lacking the CArG or vCArG motifs of the LFY and AP1 loci was used as a negative control. The FLM-GFP protein was bound to two regions (I and II) in the LFY locus compared to that in the wild-type plants (Fig. 4a), which was consistent with the FLM ChIP-sequencing data (Pose et al. 2013). We observed that the FLM binding to one LFY region I was temperature-dependent, with an approximately 1.8-fold increase in binding at 16 °C compared to that at 23 °C (Fig. 4a). These observations indicate that FLM preferentially binds to the promoter region of LFY at low temperatures. In contrast, FLM binding occurred at both 16 °C and 23 °C in region II (Fig. 4a), suggesting that FLM can bind to the LFY genomic region in a temperature-independent manner. However, differential binding of FLM to the LFY region (NC), a negative control lacking conserved motifs, was not observed at different temperatures. Moreover, no significant FLM enrichment was observed in region I and negative control of the AP1 genomic region (Fig. 4b). Based on the increased LFY expression in atsf1-2 mutants (Fig. 3c) and the direct FLM binding to the LFY genomic locus (Fig. 4a), we conclude that LFY is an in vivo target of FLM and AtSF1 may influence LFY expression through FLM.
Genetic interactions between FLM and LFY
To test the genetic effects of FLM and LFY on flowering time, we measured the flowering times of flm-3, lfy-12, and flm-3 lfy-12 mutants at 16 and 23 °C under LD conditions. The flm-3 lfy-12 mutants flowered significantly later (18.8 and 13.4 leaves, respectively) than flm-3 single mutants at 16 and 23 °C (15.0 and 12.0 leaves, respectively) (Fig. 5a). This indicates that the lfy mutation partially suppressed the early flowering phenotype of flm-3 mutants at different temperatures. The LNRs of flm-3 lfy-12 and flm-3 mutants were 1.4 and 1.3, respectively, compared to those of lfy-12 mutants (LNR = 2.3), indicating that the temperature insensitivity of flm-3 lfy-12 mutants was similar to that of flm-3 mutants (Fig. 5b). In contrast, the LNR of the lfy-12 mutant was 2.3. This result indicates that the temperature sensitivity of lfy-12 mutants was completely suppressed by flm mutation. The results suggest that LFY is another target of the AtSF1-FLM module in addition to FT and that this module regulates flowering by modulating LFY expression.