elf3_pif4_project_data_code

data and code pertinent to submitted manuscript

<i>elf3</i> and <i>pif4</i> null mutant phenotypes are independent under LD treatments and robust to conditions.

<p>(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), <i>elf3-200</i>, and <i>pif4-2</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161791#pone.0161791.s007" target="_blank">S3 Table</a>. 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.</p

elf3 and pif4 are independent in flowering, data and code

new fileset containing data and code associated with the newest version of manuscript: http://biorxiv.org/content/early/2016/03/22/038257 (this is still the old manuscript version, new one to be posted soon). <div><br></div><div><br></div

PIF4 and ELF3 Act Independently in <i>Arabidopsis thaliana</i> Thermoresponsive Flowering

<div><p>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 <i>Arabidopsis thaliana</i>. 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.</p></div

Current and suggested GWAS approaches.

<p>(A) Current approach. GWAS identify variants that are overrepresented in cases. Rare variants of large effect (red square, blue star) may escape detection, thereby contributing to missing heritability. Common variants that are overrepresented in cases (small yellow bar, 6 versus 2) do not contribute strongly to disease risk. A cryptic disease-related variant does not show significant overrepresentation in cases (open circle). (B) Suggested approach. Individuals are first analyzed for phenotypic robustness (bold box) and then for variants associated with disease. Rare variants of large effect will be enriched in robust cases, although they may also be present in nonrobust cases. Variants that are overrepresented in all cases (robust, nonrobust) will show higher penetrance in nonrobust individuals (large yellow bars). The formerly cryptic, disease-related variant (open circle) is significantly enriched in nonrobust cases versus nonrobust controls (and robust cases) and can therefore be identified. Together, heritability significantly increases. The formerly cryptic genetic variant and higher penetrance variant can be thought of as “disease-specifiers” as they determine the specific disease phenotype of individuals carrying them. Note symbols represent highly simplified frequencies of specific variant in indicated groups and not individuals carrying certain variants.</p

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

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161791#pone.0161791.s005" target="_blank">S1 Table</a>). WT = Ws, <i>elf3</i> = <i>elf3</i> mutant+vector control, 0Q = <i>elf3</i> mutant+<i>ELF3</i> transgene lacking polyQ, etc. Error bars indicate standard error of the mean. (A): Ws (Wassilewskija) strain background. Lines are generated in an <i>elf3-4</i> background. (B): Response in the Col (Columbia) strain background, lines were generated in an <i>elf3-200</i> 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.</p

ELF3 and GI regulate thermoresponsive flowering.

<p>(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. <i>elf3-4</i>: <i>elf3</i> null in Ws background; <i>elf3-200</i>: <i>elf3</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161791#pone.0161791.s013" target="_blank">S9 Table</a>. (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°. <i>gi</i>: <i>gi-2</i>, <i>co</i>: <i>co-101</i>, <i>spy</i>: <i>spy-3</i>, <i>soc1</i>: <i>soc1</i> T-DNA insertion, <i>elf3</i>: <i>elf3-200</i>. 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.</p

Summary of combined statistical and functional support for loci underlying root length.

<p>Four epistatic pairs, involving seven unique loci (connected boxes), were identified at a genome-wide significance-threshold in a two-dimensional genome-wide GWA scan. Green, yellow, and red lines connect pairs of loci with very low (p = 0.003), intermediate (0.28 < p < 0.37), and high (p = 0.95) risk for the interaction being a false-positive when accounting for population size. The risk of the statistical epistatic association resulting from high-order LD to an unobserved functional variant in the genome (i.e. “apparent epistasis”) is illustrated by arrow color, in which yellow indicates an intermediate risk (0.18 < p < 0.42) and green a very low risk (p = 0.0021). Green boxes indicate loci for which the T-DNA insertion line analyses suggest the named genes to be involved in root development. When considering the joint statistical and functional results, two pairs emerge as highly likely true positive two-locus associations: 3_66596/3_9273674 due to very strong statistical support and one identified functional candidate gene, and 3_10891195/5_1027939 where the identification of functional candidate genes at both loci suggest that the two-locus association in the original genome-wide scan is true despite the lower statistical support in after the conservative statistical correction for sample-size. For the other two pairs, the results are inconclusive. There is strong support for one of the two associated loci (3_66596 from its statistical interaction with 3_9273674 and 1_17257526 by the detection of the a functional candidate gene in the T-DNA analysis), but weaker support for the second locus. Further work is thus needed to conclude whether these pairs represent true positive two-locus associations, or whether they are false-positives due to the small population-size or high-order LD (“apparent epistasis”) to unknown functional variants.</p

Function and expression patterns of genes in LD with leading SNP from epistatic GWAS analysis.

<p>¹ Locations of high expression were obtained from the BAR eFP <i>Arabidopsis</i> Browser [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005541#pgen.1005541.ref044" target="_blank">44</a>].</p><p>The gene names or proposed function is listed for all genes harboring polymorphisms in LD with the leading epistatic SNPs in the whole genome interaction analysis.</p

Estimated narrow (h²) and broad sense (H²) heritabilities of root length mean and variance in a population of 93 natural <i>A</i>. <i>thaliana</i> accessions.

<p>¹Estimated using ANOVA on accession,</p><p>²Estimated using the R/hglm package (hglm) by fitting a linear mixed model including both additive and epistatic kinship matrices as random effects,</p><p>³p-values from Wald tests for the heritability being larger than zero, and s.e. are the standard errors estimated via jackknife resampling.</p><p>The broad-sense heritability estimates obtained using the phenotypic variances within and between accessions (H² = V_G/V_P; ANOVA) and the genomic relationships of the accessions (H² = (V_A + V_AA)/V_P; hglm) were similar and intermediate. The narrow-sense heritability (h² = V_A/V_P), estimated based on the genomic relationships between the accessions, was negligible.</p