Cross-species complementation reveals conserved functions for EARLY FLOWERING 3 between monocots and dicots

Abstract

Plant responses to the environment are shaped by external stimuli and internal signaling pathways. In both the model plant Arabidopsis thaliana (Arabidopsis) and crop species, circadian clock factors are critical for growth, flowering, and circadian rhythms. Outside of Arabidopsis, however, little is known about the molecular function of clock gene products. Therefore, we sought to compare the function of Brachypodium distachyon (Brachypodium) and Setaria viridis (Setaria) orthologs of EARLY FLOWERING 3, a key clock gene in Arabidopsis. To identify both cycling genes and putative ELF3 functional orthologs in Setaria, a circadian RNA-seq dataset and online query tool (Diel Explorer) were generated to explore expression profiles of Setaria genes under circadian conditions. The function of ELF3 orthologs from Arabidopsis, Brachypodium, and Setaria was tested for complementation of an elf3 mutation in Arabidopsis. We find that both monocot orthologs were capable of rescuing hypocotyl elongation, flowering time, and arrhythmic clock phenotypes. Using affinity purification and mass spectrometry, our data indicate that BdELF3 and SvELF3 could be integrated into similar complexes in vivo as AtELF3. Thus, we find that, despite 180 million years of separation, BdELF3 and SvELF3 can functionally complement loss of ELF3 at the molecular and physiological level.

Keywords: ELF3; Setaria; circadian RNA‐seq; circadian clock; flowering; growth.

Figure 1

Circadian expression profiles of putative Setaria clock components from Diel Explorer using time‐course RNA‐seq data. Setaria plants were entrained by either photocycle (LDHH) or thermocycle (LLHC), followed by being sampled every 2 hr for 48 hr under constant temperature and light conditions (Free‐Running; F) to generate time‐course RNA‐seq data. Mean values of transcripts per kilobase million (TPM) from two experimental replicates for each timepoints per gene were plotted

Figure 2

ELF3 orthologs suppress hypocotyl elongation defects in elf3‐2. The hypocotyls of 20 seedlings of wild type, elf3‐2 mutant, AtELF3 elf3‐2, BdELF3 elf3‐2, and SvELF3 elf3‐2 (two independent transgenic lines for each ELF3 ortholog) were measured at 4 days after germination under 12‐hr light:12‐hr dark growth conditions at 22°C. Upper panel shows representative seedlings of each genotype, with scale bar equal to 5 mm. Mean and 95% confidence intervals are plotted as crosshairs. This experiment was repeated three times with similar results. ANOVA with Bonferroni correction was used to generate adjusted p values, * <.05, ** <.01, **** <.0001

Figure 3

ELF3 orthologs suppress time to flowering of elf3‐2. 12 wild‐type, elf3‐2 mutant, AtELF3 elf3‐2, BdELF3 elf3‐2, and SvELF3 elf3‐2 seedlings from two independent transformations were measured for days (a) and number of rosette leaves (b) at flowering (1 cm inflorescence). Mean and 95% confidence intervals are plotted as crosshairs. This experiment was repeated twice with similar results. ANOVA with Bonferroni correction was used to generate adjusted p values, **<.01, ***<.001, ****<.0001, of measurements when compared to the elf3‐2 mutant line

Figure 4

ELF3 orthologs can recover CCA1::LUC rhythms and amplitude in elf3‐2 mutants. Eight seedlings of wild type, elf3‐2 mutant, AtELF3 elf3‐2 (a), BdELF3 elf3‐2 (b), and SvELF3 elf3‐2 (c) from two independent transformations were imaged for bioluminescence under constant light after entrainment in 12‐hr light:12‐hr dark growth conditions at 22°C. Each plot shows average bioluminescence of all seedlings along with 95% confidence interval (error bars). This experiment was repeated four times with similar results. Note that wild‐type and elf3‐2 mutant data were plotted on all graphs for comparison. (d) Periods of seedlings. Only periods with a relative amplitude error below 0.5 (see also Fig. S7) were plotted. Mean and 95% confidence intervals are plotted as crosshairs. ANOVA with Bonferroni correction was used to generate adjusted p values, * <.05, ** <.01, *** <.001, **** <.0001, of measurements when compared to the wild type

Figure 5

Both BdELF3 and SvELF3 can directly bind to AtELF4 and AtLUX. Yeast two‐hybrid analysis of testing whether either BdELF3 (a) or SvELF3 (b) can directly interact with either AtELF4, the N‐terminal half of AtLUX (AtLUX‐N, a.a. 1‐143) or the C‐terminal half of AtLUX (AtLUX‐C, a.a. 144‐324). –LW tests for the presence of both bait (DBD) and pray (AD) vectors, while the −LWH + 3AT tests for interaction. Vector alone serves as interaction control. This experiment was repeated twice with similar results