PIF4 and ELF3 Act Independently in Arabidopsis thaliana Thermoresponsive Flowering

Abstract

Plants have evolved elaborate mechanisms controlling developmental responses to environmental stimuli. A particularly important stimulus is temperature. Previous work has identified the interplay of PIF4 and ELF3 as a central circuit underlying thermal responses in Arabidopsis thaliana. However, thermal responses vary widely among strains, possibly offering mechanistic insights into the wiring of this circuit. ELF3 contains a polyglutamine (polyQ) tract that is crucial for ELF3 function and varies in length across strains. Here, we use transgenic analysis to test the hypothesis that natural polyQ variation in ELF3 is associated with the observed natural variation in thermomorphogenesis. We found little evidence that the polyQ tract plays a specific role in thermal responses beyond modulating general ELF3 function. Instead, we made the serendipitous discovery that ELF3 plays a crucial, PIF4-independent role in thermoresponsive flowering under conditions more likely to reflect field conditions. We present evidence that ELF3 acts through the photoperiodic pathway, pointing to a previously unknown symmetry between low and high ambient temperature responses. Moreover, in analyzing two strain backgrounds with different thermal responses, we demonstrate that responses may be shifted rather than fundamentally rewired across strains. Our findings tie together disparate observations into a coherent framework in which multiple pathways converge in accelerating flowering in response to temperature, with some such pathways modulated by photoperiod.

Fig 1. Response to elevated temperature (27°, relative to 22°) among transgenic lines expressing ELF3-polyQ variants.

Mean response and error were estimated by regression, based on two independently-generated transgenic lines for each genotype, with n > = 30 seedlings of each genotype in each condition (S1 Table). WT = Ws, elf3 = elf3 mutant+vector control, 0Q = elf3 mutant+ELF3 transgene lacking polyQ, etc. Error bars indicate standard error of the mean. (A): Ws (Wassilewskija) strain background. Lines are generated in an elf3-4 background. (B): Response in the Col (Columbia) strain background, lines were generated in an elf3-200 background. In both (A) and (B), response is defined as the change in hypocotyl length in mm; **: Bonferroni-corrected p < 0.01, *: Bonferroni-corrected p < 0.05,.: Bonferroni-corrected p < 0.1 in testing the interaction term (different response from WT, Ws or Col). (C): Temperature response is a function of ELF3 functionality (repression of hypocotyl elongation at 22°). Simple means of 22° hypocotyl length, regression estimates of temperature response. PCC = Pearson correlation coefficient; p-value is from a Pearson correlation test.

Fig 2. Adult plant responses to elevated temperature (27°, relative to 22°) in long days among transgenic lines expressing different ELF3-polyQ variants.

(A) and (C): Response in the Ws (Wassilewskija) strain background. Lines are in an elf3-4 background. (B) and (D): Response in the Col (Columbia) strain background, lines are in an elf3-200 background. (A) and (B) display PL:LL temperature response as differences in the PL:LL ratio between temperatures, (C) and (D) display RLN temperature response as differences in the number of leaves between temperatures. Average responses and errors were estimated in a regression model accounting for variation between experiments (S2 Table), based on two to three independently-generated transgenic lines for each genotype. n > = 24 plants overall for each genotype in each condition. PL:LL = petiole to leaf length ratio at 25 days post germination, RLN = rosette leaf number at flowering, WT = wild type, elf3 = elf3 mutant+vector control, 0Q = elf3 mutant+ELF3 transgene with entire polyglutamine removed, etc. Error bars indicate standard error. In each case, **: Bonferroni-corrected p < 0.01, *: Bonferroni-corrected p < 0.05,.: Bonferroni-corrected p < 0.1 in testing the interaction term (different response from WT, Ws or Col).

Fig 3. elf3 and pif4 null mutant phenotypes are independent under LD treatments and robust to conditions.

(A), (B), and (C): 22°: constant 22° LD growth; 27° 14d: transfer from 22° to 27° at 14 days post-germination; 27° 1d: transfer from 22° to 27° at 1 day post-germination. (A): Col (WT), elf3-200, and pif4-2 plants grown under long days with three different temperature regimes were photographed at 20 days post germination. Experiment was repeated with similar results. (B and C): Petiole elongation responses of the indicated genotypes, measured by ratio of petiole to whole leaf length at 25 days post germination. Regression analysis of data in S3 Table. In each case, **: Bonferroni-corrected p < 0.01, *: Bonferroni-corrected p < 0.05, in testing whether the genotype x environment interaction term (difference of 22°-27 response from the Col 22°-27° response) differs from zero. Outliers (defined as >1.5 interquartile ranges away from the median) of each distribution are indicated as points.

Fig 4. Double mutant analysis confirms PIF4 and ELF3 independence in adult temperature responses and non-redundancy of PIF4 with PIF5.

(A): Col, elf3-200, pif4-2, and elf3-200 pif4-2 plants grown under long days with two different temperature regimes were photographed at 25 days post germination. (B): Petiole elongation responses of the indicated genotypes, measured by ratio of petiole to whole leaf length at 25 days post germination. (C) and (D): Flowering temperature response of indicated genotypes, measured by rosette leaf number (RLN) at flowering. (B) and (C): n > 8 plants for each genotype in each treatment. All “27°” plants were seeded and incubated one day at 22° before transfer to 27°. Experiments were repeated with similar results. Regression analysis of data reported in S6 and S7 Tables. In each case, **: Bonferroni-corrected p < 0.01, *: Bonferroni-corrected p < 0.05, in testing whether the genotype x environment interaction term (difference of 22°-27° response from the Col 22°-27° response) differs from zero. Outliers (defined as >1.5 interquartile ranges away from the median) of each distribution are indicated as points.

Fig 5. ELF3 and GI regulate thermoresponsive flowering.

(A): Temperature-responsive expression of photoperiodic pathway components at ZT0. Expression of each gene is quantified relative to levels in Col at 22° (Col 22 = 1.0). Error bars represent SEM across three biological replicates. elf3-4: elf3 null in Ws background; elf3-200: elf3 null in Col background. (B): Thermoresponsive flowering in various flowering mutants. LD RLN = rosette leaf number at flowering under long days. *: Bonferroni-corrected p < 0.05 in testing whether the genotype x environment interaction term (difference of 22°-27° response from the Col 22°-27° response) differs from zero; details of regression model in S9 Table. (C) Thermoresponsive petiole elongation in various flowering mutants. For (B) and (C), n > = 8 plants of each genotype in each condition; white boxes indicate measurements at 22°, red boxes indicate measurements at 27°. gi: gi-2, co: co-101, spy: spy-3, soc1: soc1 T-DNA insertion, elf3: elf3-200. Outliers (defined as >1.5 interquartile ranges away from the median) of each distribution are indicated as points. This experiment was repeated with similar results. (D): Models of thermoresponsive flowering under long and short photoperiods. Dashed edges indicate speculated temperature sensing mechanisms. Edges with increased weight indicate relative increases of influence between conditions. Pathways are indicated, along with other important actors reported elsewhere.