Molecular basis of flowering under natural long-day conditions in Arabidopsis

Abstract

Plants sense light and temperature changes to regulate flowering time. Here, we show that expression of the Arabidopsis florigen gene, FLOWERING LOCUS T (FT), peaks in the morning during spring, a different pattern than we observe in the laboratory. Providing our laboratory growth conditions with a red/far-red light ratio similar to open-field conditions and daily temperature oscillation is sufficient to mimic the FT expression and flowering time in natural long days. Under the adjusted growth conditions, key light signalling components, such as phytochrome A and EARLY FLOWERING 3, play important roles in morning FT expression. These conditions stabilize CONSTANS protein, a major FT activator, in the morning, which is probably a critical mechanism for photoperiodic flowering in nature. Refining the parameters of our standard growth conditions to more precisely mimic plant responses in nature can provide a powerful method for improving our understanding of seasonal response.

Fig. 1:. The florigen FT gene is induced in the morning in natural LD.

a, Changes in light intensity and temperature on the days near the summer solstice in 2013 when the samples were harvested. For outside conditions, Zeitgeber time 0 (ZT0) was set as the sunrise time (i.e. 5 AM in Seattle from 6/23/13 to 6/25/13). Light intensity results are means ± SEM from different growth areas (n=3). Temperature data were obtained from a nearby weather station. b, c, Expression profiles of CO (b) and FT (c) under the conditions shown in (a). All gene expression results (means ± SEM) in this manuscript were normalized against IPP2 and PP2A (n=3 biologically independent samples). d, Flowering time results of plants grown outside in June and in lab LD. Each box is located between the upper and the lower quartiles, and the whiskers indicate 1.5-times interquartile ranges. The thick horizontal lines in the boxes represent the median, and open diamonds represent the mean. Outliers are indicated by circles. (12≤n≤100, *** p<0.001, ns: non-significant, linear models or generalized linear models were used throughout the manuscript. See detail statistical information in Supplementary Table 3). e, FT expression profiles in wild-type (WT: Col-0) plants, fkf1, and gi-2 mutants grown outside around the summer solstice. f, FT expression profiles in WT grown at different times in spring. g, FT expression profiles in WT grown around the summer solstice in Seattle and Zürich. (for e-g, n=3 biologically independent samples). h, Flowering phenotypes of WT plants grown in different months and locations in spring. The details of the box plots are the same as those in Fig. 1d (n≥11, ** p<0.01, *** p<0.001, ns: non-significant, statistical information in Supplementary Table 3).

Fig. 2:. Adjusting the R/FR ratio to 1 and changing the daily temperature of the lab growth conditions are sufficient to recreate the FT profiles and flowering of plants grown in natural LD.

a, b, FT expression profiles in LD and LD+FR (a), and in LD, LD+FR+temp, and outside in 2014 (b). The results represent means ± SEM (n=3 biologically independent samples). c, Flowering phenotypes of WT accessions and photoperiodic mutants in LD+FR+temp. Each box is located between the upper and the lower quartiles, and the whiskers indicate 1.5-times interquartile ranges. The thick horizontal lines in the boxes represent the median, and open diamonds represent the mean. Outliers are indicated by circles. (n≥11, * p<0.05, ** p<0.01, *** p<0.001, ns: non-significant, statistical information in Supplementary Table 3). d, Spatial expression patterns of FT in LD+FR+temp. FT:GUS plants were grown in LD+FR+temp for two weeks and harvested at ZT4 (n=4–5 independent plants, repeated twice biologically). As a comparison, the FT:GUS plants were grown in LD and harvested at ZT4 (n=5 independent plants). The staining patterns of GUS activity in the LD-grown samples harvested in ZT4 resembled those in the ones harvested at the end of the day (ZT16) (Supplementary Fig. 13), most likely due to the very stable nature of the GUS protein. Scale bar=1 mm.

Fig. 3:. Morning induction of florigen expression occurs under both natural LD and LD+FR+temp conditions, and is a common response in wild-type accessions.

a, The upregulated genes of RNA-seq results in two-week-old samples harvested at ZT4 in 2013, 2014, and LD+FR+temp conditions compared with the ZT4 samples in LD (n=3 biologically independent samples). The GO term categories enriched in the 57 genes are shown. The p-values represent one-tail Fisher Exact Probability Values. See Supplementary Table 1 for the actual values. b, Flowering-related genes in FLOR-ID were extracted from the dataset shown in (E). c, FT expression levels in the morning (ZT4) and at dusk (ZT16) in 20 Arabidopsis WT accessions (Supplementary Fig. 15) in LD and LD+FR+temp.

Fig. 4:. phyA and ELF3 are involved in the regulation of morning FT expression in LD+FR+temp.

a-c, FT expression profiles in WT plants, co-101, and phyA-211 mutants in LD+FR+temp (a), LD (b), and LD+FR (c). d-f, FT expression profiles in WT plants, phyB-9, and elf3–1 mutants in LD+FR+temp (d), LD (e), and LD+FR (f). g, h, FT levels in WT plants, phyA-211, and elf3–1 mutants in LD with different R/FR ratios. The levels of FT in these plants in the morning, ZT4 (g), and at dusk, ZT16 (h). For a-h, the results represent means ± SEM (n=3 biologically independent samples). i, Daily accumulation patterns of phyA protein in LD and LD+FR+temp. j, Daily accumulation patterns of ELF3 protein in ELF3:ELF3–6H3F plants in LD and LD+FR+temp. For both (i) and (j), the representative blot images are shown. Actin was used as a loading control. The protein quantification results (relative values against the loading control) represent means ± SEM (n=6 biologically independent samples).

Fig. 5:. CO protein stability was increased in LD+FR+temp during the morning.

a, b, CO protein accumulation patterns in CO:HA-CO plants in LD and LD+FR+temp. Histone H3 was used as a loading control. The quantification results represent means ± SEM [n=5 (a) and n=3 (b) biologically independent samples]. c, Coimmunoprecipitation analysis of ELF3 and CO proteins. 35S:ELF3–6H3F, 35S:3HA-CO, and 35S:3HA-CO/35S:ELF3–6H3F plants were grown in LD, LD+FR (labeled as FR), or LD+FR+temp (FR+temp) and harvested in the morning (ZT4). The experiments were repeated three times independently, and similar results were obtained. d, CO protein accumulation patterns in CO:HA-CO and CO:HA-CO/elf3–1 plants grown in LD+FR+temp. The quantification results represent means ± SEM (n=5 biologically independent samples). e, f, A model for CO-dependent FT regulation under natural LD conditions. This model shows temporal expression patterns of CO protein (top) and FT transcripts (bottom) under lab LD (e) and natural LD (f) conditions. e, Under artificial lab LD conditions in which the R/FR ratio is equal to or greater than 2 and the temperature is constant, CO protein appears to immediately accumulate after light onset and then rapidly degrade, resulting in low levels of CO protein in the morning and early afternoon. During this period, ELF3 protein inhibits FT expression through an unknown mechanism. CO protein peaks again at the end of the day, which directly activates FT transcription under these conditions. f, Under natural LD conditions, the R/FR ratio is 1 and the ambient temperature oscillates throughout the day. The amount of phyA protein increases in the morning, whereas the amount of ELF3 protein decreases (Fig. 3i, j). CO protein accumulates rapidly at high levels after sunrise, and CO protein degrades more slowly under natural LD conditions than under lab LD conditions. This CO accumulation might be important for morning induction of FT. In addition to the CO protein stability changes, there might be other factors (depicted as “X”) involved in the induction of morning FT under natural LD conditions. The phyA signal is positively involved in FT induction under these conditions. ELF3 negatively acts on FT regulation under these conditions. In addition, the temperature oscillations strongly repress FT transcription in the evening. Therefore, although CO protein abundance is high even at dusk, the levels of FT expression remain relatively low around dusk compared to morning. We showed that we can recreate these FT expression profiles in the lab by simply adjusting the R/FR ratio of light source and temperature conditions.