BACKGROUND
in analogy to photomorphogenesis, the term thermomorphogenesis describes the effect of temperature on morphogenesis  <cit> .
hypocotyl elongation  <cit>  and leaf hyponasty  <cit>  belong to the most sensitive thermomorphogenic changes in plant development.
physiologically, these coordinated responses likely enhance evaporative leaf cooling  <cit>  and thus enable plants to adapt to warmth.
within the context of globally increasing ambient temperatures, it is imperative to improve our understanding of the basic processes plants employ to react to such environmental perturbations.
a major hub in the ambient temperature signaling network is the basic helix-loop-helix  transcription factor phytochrome-interacting factor <dig> .
pif <dig> protein binds to the promoters of auxin biosynthesis and response genes .
it thereby transcriptionally activates auxin responses, resulting in elongation growth.
pif <dig> itself seems to be transcriptionally regulated in a temperature-dependent manner by the bzip transcription factor elongated hypocotyl <dig>   <cit> .
accumulating data on pif <dig> regulation from light signaling, photomorphogenesis and the circadian clock  indicate a more complex regulation of pif <dig> activity on several levels.
the objective of this study was to exploit natural variation within the gene pool of arabidopsis thaliana to identify additional components of the complex signaling network that plants use to adapt growth to changes in ambient temperature.
based on a quantitative genetic approach, we here show that two naturally occurring alleles of early flowering <dig>  cause a differential response in thermomorphogenesis.
RESULTS
we previously observed extensive natural variation for the thermomorphogenic signature phenotype we termed temperature-induced hypocotyl elongation .
to identify the underlying genetic variants, we performed quantitative trait locus  analyses based on two natural accessions from geographically distant locations.
when comparing growth at  <dig> and 28 °c, the selected accessions bay- <dig>  and sha  differed significantly in several thermomorphogenic responses .
identification of the underlying genetic variants would help to improve our understanding of how ambient temperature changes are translated into growth responses.fig.
<dig> quantitative trait locus analysis of temperature-induced growth responses in arabidopsis thaliana.
a relative hypocotyl length  of 10 days-old a. thaliana accessions bay- <dig> and sha.
b relative petiole length and leaf expansion of 12 days-old seedlings.
a,b box plots show medians , interquartile ranges , and data ranges .
black dots mark outliers .
asterisks mark significant differences in temperature responses  as assessed by two-way anova  of the absolute data presented in additional file  <dig>  c frequency plot of phenotypic classes observed in a bay- <dig> x sha-derived recombinant inbred line population for hypocotyl length of 10 days-old seedlings grown at 20 °c  or 28 °c , and for the ratio of  <dig> vs. 20 °c  means.
parental phenotype classes are indicated to illustrate the transgression effects within the population.
d lod scores  from composite interval mapping  and multiple qtl mapping  are plotted against all chromosomes .
tick marks on the x axis correspond to molecular markers in the genetic map.
colored dots on the x axis show co-variates set for cim.
thresholds are based on  <dig> permutations and an alpha of  <dig> .
the 28 °c vs. 20 °c gir <dig>  qtl, which is the subject of this study, is highlighted
qtl analysis of temperature-induced hypocotyl elongation
we phenotyped a bay- <dig> x sha recombinant inbred line population  <cit>  for the tihe response.
we grew seedlings in different ambient temperatures  under a long-day diurnal cycle.
qtl analysis based on composite interval and multiple qtl mapping using r/qtl was performed for hypocotyl length at either temperature alone or the ratio between hypocotyl length at  <dig> and 20 °c .
in total, we identified  <dig> different qtls .
focusing on growth differences between high and low temperature  identified five qtls, which were named giraffe1/ <dig> / <dig> / <dig> / <dig>  , according to their respective chromosomal location.
together the five qtls explained ~43 % of the phenotypic variation within the mapping population.
the strongest qtl, gir <dig>  , explained ~18 % of this variation , suggesting that a sizeable part of the natural variation between bay- <dig> and sha can be attributed to this locus.
to facilitate map-based cloning of gir <dig> , we first validated it using heterogeneous inbred families .
phenotypic differences between two hif lines, carrying either parental allele in the target region, while being otherwise genetically identical, can be attributed to genetic variation in the target qtl interval.
in addition to gir <dig> , we also included gir <dig> and gir <dig>  in this analysis.
we were unable to validate gir <dig> , but observed significant differences in tihe for the hif lines separating the two parental alleles for gir <dig>  and gir <dig>   under long-day photoperiod .
due to the high impact on the phenotypic variation further analyses focused on gir <dig> .
here, the sha allele conferred long hypocotyls and the bay- <dig> allele conferred short hypocotyls .
we found that the differences in tihe between the gir <dig>  hif lines did not persist under continuous light, darkness or short-day conditions .
hence, diurnal cycling with an extensive light phase seems to be necessary for natural variation in tihe caused by gir <dig> .
furthermore, parental differences under monochromatic lights seem to be independent of gir <dig>  .fig.
<dig>
gir <dig>  validation and photoperiod specificity.
a haplotype overview of the heterogeneous inbred family   <dig> that segregates for bay- <dig> and sha alleles within the gir <dig>  interval.
b box plots show relative hypocotyl length  of seedlings grown for 8 days under long-day , continuous light , in darkness  or short-day  photoperiods.
horizontal bars, boxes, and whiskers show medians, interquartile ranges , and data ranges, respectively.
black dots mark outliers .
different letters denote statistical differences in temperature responses  as assessed by two-way anova  of the absolute data presented in additional file 5
gir <dig>  possibly constitutes a ghost qtl
an f <dig> derived by crossing 84-b with 84-s showed that the long-hypocotyl phenotype inherited by the sha allele is dominant over the bay- <dig> short-hypocotyl phenotype .
in the process of fine-mapping the gir <dig>  interval, genotyping of f <dig> and f <dig> recombinants with long hypocotyls  at 28 °c revealed several plants for which the long-hypocotyl phenotype could be attributed to different, non-overlapping sha intervals within the gir <dig>  region.
this indicated that the exact localization of the gir <dig>  lod score peak was possibly caused by two or more contributing loci.
this phenomenon is frequently observed in qtl analysis and has been called ghost qtl  <cit> .
recomputing the qtl analysis with additional co-variates separated this peak into two neighbors, supporting this scenario .
interestingly, jiménez-gómez et al.
<cit>  reported a similar phenomenon for this region in the same bay- <dig> × sha population for the regulation of shade avoidance responses, which phenocopies the high temperature response.
however, as we only reproducibly observed long hypocotyls for one of the two sha intervals , we focused subsequent analyses on this robust interval.
a single nucleotide polymorphism in early flowering <dig> is underlying the gir <dig>  qtl
knowing that a diurnal photoperiod was a prerequisite for tihe differences between bay- <dig> and sha , we identified early flowering <dig>  as a candidate gene located in the gir <dig>  target interval.
elf <dig> is a component of the circadian clock  <cit>  that functions in the evening complex to repress growth  <cit>  and had previously been shown to regulate hypocotyl elongation in response to shade avoidance  <cit> .
using a transgenic approach, anwer et al.
<cit>  recently showed that the elf3bay- <dig> and elf3sha alleles differentially regulate period length of the circadian clock.
consistent with a role of elf <dig> in thermomorphogenesis, we found that elf3- <dig> null mutants conferred long hypocotyls in comparison to their ws- <dig> wild-type .
to investigate whether tihe differences between the bay- <dig> and sha hif lines can indeed be attributed to elf <dig>  we followed a transgenic complementation approach.
we took advantage of the same transgenic lines generated by anwer et al.
<cit>  that contain either the elf3bay- <dig> or the elf3sha allele in the elf3- <dig> mutant genome , which enabled us to study allele-specific tihe effects in an independent elf <dig> loss-of-function background.
figure 3b shows that transgenic lines carrying either parental allele complemented the elf3- <dig> phenotype at 20 °c.
at 28 °c, however, transgenics carrying elf3bay- <dig>  repressed hypocotyl elongation significantly more than those carrying the elf3sha allele .
this demonstrated that allelic variation in elf <dig> affects tihe in the bay × sha population.fig.
<dig> transgenic complementation of elf3-mediated tihe variation.
a overview of transgenic constructs used for complementation of the elf3- <dig> null mutation .
b box plot shows absolute hypocotyl length of 8 days-old seedlings grown in ld at  <dig> or 28 °c, respectively.
horizontal bars, boxes, and whiskers show medians, interquartile ranges , and data ranges, respectively.
black dots mark outliers .
different letters denote statistical differences as assessed by one-way anova and tukey hsd
bay- <dig> and sha elf <dig> variants display several promoter polymorphisms  <cit>  and differ  in a nonsynonymous snp causing an amino acid change at position  <dig>  encoding an alanine-to-valine transition , and  in the length of a c-terminal glutamine stretch  <cit> .
although this might depend on the genetic background  <cit> , tajima et al.
<cit>  suggested that there was no apparent correlation between the length of the polyglutamine region and hypocotyl elongation.
we therefore focused on the a362v polymorphism and investigated its potential role in conferring allelic differences in thermomorphogenesis.
again, we made use of transgenic lines in the elf3- <dig> mutant background generated by anwer et al.
<cit> .
we inspected tihe in transgenic elf3- <dig> lines carrying either the probay-0:elf3bay- <dig> allele or the probay-0:elf3bay allele, differing only in the a362v snp  <cit> .
we found that temperature-induced hypocotyls in transgenic lines carrying a valine at position  <dig>  were similar to the elf3sha allele and, importantly, significantly longer than those with its alanine counterpart at the same position .
theoretically, differences in thermomorphogenesis among elf3- <dig>  and elf3- <dig>  might also be affected by circadian or other differences in elf <dig> expression.
the significant differences observed between elf3- <dig> plants expressing the probay:elf3bay and probay-0:elf3bay, however, demonstrate that the snp underlying the a362v change in elf <dig> causes phenotypic variation in tihe and establishes elf <dig> as a negative regulator of thermomorphogenesis.
differential transcriptional responses caused by natural elf <dig> variants
it has recently been shown that the evening complex of the circadian clock consisting of elf <dig>  elf <dig>  and lux arrhythmo  underlies the molecular basis for circadian gating of hypocotyl growth by directly down-regulating the expression of pif <dig> in the early evening  <cit> .
as a result, hypocotyl elongation peaks at dawn under diurnal cycles.
we, therefore, tested the hypothesis that adopts this model for ambient temperature signaling and investigated whether pif <dig> expression and possibly also the transcript levels of pif4-regulated genes that mediate cell elongation are affected in response to elevated temperature.
to assess temperature responsiveness, we grew seedlings for 7 days at 20 °c under long-day photoperiods, kept control plates at 20 °c, and shifted the remaining seedlings to 28 °c at lights off .
control and 28 °c seedlings were subsequently harvested 4 h after the shift.
we found that loss of elf <dig> in elf3- <dig> results in up-regulation of pif <dig> transcript levels at both temperatures .
at 20 °c, we observe that complementation of elf3- <dig> with either transgenic allele ) restores wild-type pif <dig> levels , demonstrating functionality of the constructs, but suggesting that allelic differences are absent at 20 °c.
this observation is consistent with similar hypocotyl length of the three lines at 20 °c .
at 28 °c, however, pif <dig> expression levels in elf3- <dig>  and elf3- <dig>  are significantly higher than in elf3- <dig>  , again reflecting the hypocotyl phenotype .
importantly, this expression behavior at  <dig> vs. 28 °c explains the detection of the gir <dig>  qtl , at 28 °c and the absence of a qtl at  <dig> °c .
together, this suggests that the natural variation observed for tihe is attributable to temperature-dependent differences in pif <dig> expression levels caused by the a362v snp in elf3sha.fig.
<dig> effects of elf <dig> allelic variation on the expression of pif <dig> and auxin-responsive genes.
qrt-pcr analysis of  pif <dig>   saur <dig>   saur <dig>  and  athb <dig> expression in wt  and transgenic complementation lines .
seedlings were grown for 7 days at 20 °c and transferred to 28 °c or kept at 20 °c .
seedlings were harvested after 4 h in the middle of the 8 h dark period.
relative expression levels of three biological replicates per treatment were assessed using at1g <dig> as control gene.
bar plots show means and sem.
different letters denote statistical differences among samples as analyzed by one-way anova and tukey hsd test
we then connected this scenario to the level of pif <dig> target genes responsible for cell elongation.
genes involved in cell elongation such as the small auxin upregulated rna  family or arabidopsis thaliana homeobox protein <dig>  have previously been shown to be activated by pif <dig> in a temperature-dependent manner  <cit> .
as fig.
4b–d shows, we found that temperature-induced expression levels of the tested genes in elf3- <dig>  were significantly higher than those in elf3- <dig> .
this strongly suggested that the a362v snp causes natural variation of temperature-induced pif <dig> expression levels directly resulting in alterations of auxin-mediated cell elongation.
discussion
our findings shown here illustrate the power of natural variation approaches and support elf <dig> as a negative regulator of ambient temperature signaling.
physiological and gene expression data indicate that elf <dig> protein might be involved in down-regulating transcript levels of the major ambient temperature signaling hub pif <dig>  and thereby affect thermo-responsive growth.
transgenic complementation assays furthermore demonstrated that a nonsynonymous snp between the natural accessions bay- <dig> and sha significantly affects the ability of elf <dig> to regulate temperature-induced pif <dig> transcript levels, its target genes, and hypocotyl elongation.
in general, different types of polymorphisms, such as nonsynonymous snps or expression level polymorphisms, can contribute to the expression of a particular trait  <cit> .
in line with this phenomenon, distinct types of naturally occurring elf <dig> polymorphisms seem to contribute to hypocotyl elongation in response to different temperatures.
box et al.
<cit>  recently used a different quantitative genetic approach based on the magic lines  <cit> , and elegantly showed that both protein-coding and expression level polymorphisms in elf <dig> are likely responsible for tihe differences in natural accessions.
the authors presented convincing evidence that warmth relieves the gating of growth by elf <dig> at night.
specifically, elf <dig> gating of transcriptional targets responds rapidly to changes in temperature by temperature-dependent binding of elf <dig> to target promoters including pif <dig>  together with box et al.’s  <cit>  non-transgenic quantitative complementation assays, our transgenic complementations unequivocally establish the role of elf <dig> in thermomorphogenesis signaling.
intriguingly, the elf <dig> qtls in both studies were identified in different photoperiods.
elf <dig> polymorphisms causal for variation within the magic population were identified under short-day conditions.
in contrast, our study identified the elf <dig> polymorphism under a long-day photoperiod and subsequent analysis of hif lines showed long-day specificity .
furthermore, a direct comparison of bay- <dig> and sha with two of the parental lines used in the study of box et al.
<cit>  revealed the short-day-specificity of the sf- <dig> and zu- <dig> alleles in promoting hypocotyl elongation .
this photoperiod specificity of the different natural alleles represents an interesting observation in itself requesting further investigations.
another unexpected difference between the two studies relates to the growth temperature at which the elf <dig> qtl was detected.
whereas we identified the elf <dig> qtl peak for hypocotyl growth at 28 °c, but not at 20 °c , box et al.
<cit>  did not detect elf <dig> at high temperature but rather at 22 °c standard growth conditions.
this difference could be attributed to the differential integration of temperature and photoperiod by natural elf <dig> alleles.
alternatively, the genetic backgrounds and interactions with other contributing loci might be involved.
in support of this, it is known that the capacity of elf <dig> to mediate growth depends on the context of the genome  <cit> .
hence, box et al.
<cit>  and this study confer complementary evidence for a central role of elf <dig> as a major signaling hub acting upstream of pif <dig> in the ambient temperature signaling network and add yet another layer to its complex regulation .fig.
<dig> simplified model of ambient temperature signaling.
elf <dig> functions as a transcriptional repressor of pif <dig> and integrates temperature and photoperiod information in the regulation of thermomorphogenesis.
in addition, pif <dig> regulation in response to temperature involves the regulatory components of the photomorphogenesis pathway det <dig>  cop <dig>  and hy <dig>  <cit> .
pif4-mediated transcriptional regulation of target genes can be terminated by the rna binding protein fca, causing dissociation of pif <dig> from target gene promoters  <cit> .
additional mechanisms previously shown to contribute to pif <dig> regulation e.g., transcriptional regulation of target genes via the bap/hlh/bhlh module of transcription factors  <cit> .
might contribute also to the regulation of temperature responses.
other putatively involved modulators of pif <dig> activity known from other biological contexts are depicted by gray dashed lines.
these involve direct binding of det <dig> to pif <dig>  <cit> , competition of pif <dig> and other transcription factors for similar binding sites  <cit> , pif <dig> protein sequestration by hfr <dig>  <cit>  and phyb-mediated phosphorylation and degradation of pif <dig>  <cit>
anwer et al.
<cit>  recently identified elf <dig> as a qtl for the regulation of the circadian clock in the same bay- <dig> × sha population.
in fact, they showed that the elf3sha protein variant failed to properly localize to the nucleus and its ability to accumulate in the dark was compromised.
if the same scenario holds for ambient temperature responses, then elf3sha might fail to accumulate in the nucleus during nighttime when hypocotyl growth peaks.
as a consequence of a weaker potential of elf3sha to repress pif <dig> expression, a temperature increase could result in a much earlier activation of pif4-mediated signaling processes during the night.
indeed, box et al.
<cit>  demonstrated that hypocotyl growth dynamics at elevated temperatures are considerably different from those at standard conditions and show a prolonged growth throughout the first night and an additional growth peak in the beginning of the dark period in subsequent nights.
however, it is currently unknown in which manner temperature might affect elf <dig> protein localization.
CONCLUSIONS
in summary, remarkable progress has been made in understanding the functions of elf <dig>  in combination with recent studies on the role of elf <dig> in the shade avoidance response  <cit>  and the circadian clock  <cit> , this work contributes to understanding its role in the fine-tuned integration of a variety of environmental stimuli that in concert regulate plant growth and development .
natural variation in thermomorphogenesis caused by elf <dig> variants could be mediated at several levels.
first, transcriptional regulation of elf <dig> itself caused by expression level polymorphisms can result in varying amounts of pif4-repressing elf <dig> protein  <cit> .
in addition, coding sequence polymorphisms might affect the ability of elf <dig> protein to interact with pif <dig> protein and thereby inhibit its transcriptional activity, as shown by nieto et al.
<cit> .
however, it is unknown whether this protein-protein interaction is temperature-dependent and the existence of natural variation for this mechanism has yet to be reported.
lastly, nonsynonymous snps may affect nuclear accumulation of elf <dig>  which - like expression level polymorphisms described above - would result in variation of the amount of nuclear elf <dig>  <cit>  capable of transcriptionally repressing pif <dig>  and other targets.
possibly, the latter mechanism is responsible for natural variation between bay- <dig> and sha thermomorphogenesis reported here.
our work in combination with the study of box et al.
<cit>  adds further insight into the essential role of elf <dig> in integrating multiple signals to promote architectural changes.
the photoperiod-specific function of natural elf <dig> alleles could provide new avenues to elucidate the clock-mediated growth regulation in general and the elf <dig> mode of action specifically.

 in analogy to photomorphogenesis, the term thermomorphogenesis describes the effect of temperature on morphogenesis. hypocotyl elongation and leaf hyponasty belong to the most sensitive thermomorphogenic changes in plant development. physiologically, these coordinated responses likely enhance evaporative leaf cooling and thus enable plants to adapt to warmth. within the context of globally increasing ambient temperatures, it is imperative to improve our understanding of the basic processes plants employ to react to such environmental perturbations. a major hub in the ambient temperature signaling network is the basic helix-loop-helix transcription factor phytochrome-interacting factor. pif protein binds to the promoters of auxin biosynthesis and response genes. it thereby transcriptionally activates auxin responses, resulting in elongation growth. pif itself seems to be transcriptionally regulated in a temperature-dependent manner by the bzip transcription factor elongated hypocotyl. accumulating data on pif regulation from light signaling, photomorphogenesis and the circadian clock indicate a more complex regulation of pif activity on several levels. the objective of this study was to exploit natural variation within the gene pool of arabidopsis thaliana to identify additional components of the complex signaling network that plants use to adapt growth to changes in ambient temperature. based on a quantitative genetic approach, we here show that two naturally occurring alleles of early flowering cause a differential response in thermomorphogenesis.  we previously observed extensive natural variation for the thermomorphogenic signature phenotype we termed temperature-induced hypocotyl elongation. to identify the underlying genetic variants, we performed quantitative trait locus analyses based on two natural accessions from geographically distant locations. when comparing growth at and 28 °c, the selected accessions bay- and sha differed significantly in several thermomorphogenic responses. identification of the underlying genetic variants would help to improve our understanding of how ambient temperature changes are translated into growth responses.fig. quantitative trait locus analysis of temperature-induced growth responses in arabidopsis thaliana. a relative hypocotyl length of 10 days-old a. thaliana accessions bay- and sha. b relative petiole length and leaf expansion of 12 days-old seedlings. a,b box plots show medians, interquartile ranges, and data ranges. black dots mark outliers. asterisks mark significant differences in temperature responses as assessed by two-way anova of the absolute data presented in additional file c frequency plot of phenotypic classes observed in a bay- x sha-derived recombinant inbred line population for hypocotyl length of 10 days-old seedlings grown at 20 °c or 28 °c, and for the ratio of vs. 20 °c means. parental phenotype classes are indicated to illustrate the transgression effects within the population. d lod scores from composite interval mapping and multiple qtl mapping are plotted against all chromosomes. tick marks on the x axis correspond to molecular markers in the genetic map. colored dots on the x axis show co-variates set for cim. thresholds are based on permutations and an alpha of. the 28 °c vs. 20 °c gir qtl, which is the subject of this study, is highlighted qtl analysis of temperature-induced hypocotyl elongation we phenotyped a bay- x sha recombinant inbred line population for the tihe response. we grew seedlings in different ambient temperatures under a long-day diurnal cycle. qtl analysis based on composite interval and multiple qtl mapping using r/qtl was performed for hypocotyl length at either temperature alone or the ratio between hypocotyl length at and 20 °c. in total, we identified different qtls. focusing on growth differences between high and low temperature identified five qtls, which were named giraffe1/ / / /, according to their respective chromosomal location. together the five qtls explained ~43 % of the phenotypic variation within the mapping population. the strongest qtl, gir, explained ~18 % of this variation, suggesting that a sizeable part of the natural variation between bay- and sha can be attributed to this locus. to facilitate map-based cloning of gir, we first validated it using heterogeneous inbred families. phenotypic differences between two hif lines, carrying either parental allele in the target region, while being otherwise genetically identical, can be attributed to genetic variation in the target qtl interval. in addition to gir, we also included gir and gir in this analysis. we were unable to validate gir, but observed significant differences in tihe for the hif lines separating the two parental alleles for gir and gir under long-day photoperiod. due to the high impact on the phenotypic variation further analyses focused on gir. here, the sha allele conferred long hypocotyls and the bay- allele conferred short hypocotyls. we found that the differences in tihe between the gir hif lines did not persist under continuous light, darkness or short-day conditions. hence, diurnal cycling with an extensive light phase seems to be necessary for natural variation in tihe caused by gir. furthermore, parental differences under monochromatic lights seem to be independent of gir.fig. gir validation and photoperiod specificity. a haplotype overview of the heterogeneous inbred family that segregates for bay- and sha alleles within the gir interval. b box plots show relative hypocotyl length of seedlings grown for 8 days under long-day, continuous light, in darkness or short-day photoperiods. horizontal bars, boxes, and whiskers show medians, interquartile ranges, and data ranges, respectively. black dots mark outliers. different letters denote statistical differences in temperature responses as assessed by two-way anova of the absolute data presented in additional file 5 gir possibly constitutes a ghost qtl an f derived by crossing 84-b with 84-s showed that the long-hypocotyl phenotype inherited by the sha allele is dominant over the bay- short-hypocotyl phenotype. in the process of fine-mapping the gir interval, genotyping of f and f recombinants with long hypocotyls at 28 °c revealed several plants for which the long-hypocotyl phenotype could be attributed to different, non-overlapping sha intervals within the gir region. this indicated that the exact localization of the gir lod score peak was possibly caused by two or more contributing loci. this phenomenon is frequently observed in qtl analysis and has been called ghost qtl. recomputing the qtl analysis with additional co-variates separated this peak into two neighbors, supporting this scenario. interestingly, jiménez-gómez et al. reported a similar phenomenon for this region in the same bay- × sha population for the regulation of shade avoidance responses, which phenocopies the high temperature response. however, as we only reproducibly observed long hypocotyls for one of the two sha intervals, we focused subsequent analyses on this robust interval. a single nucleotide polymorphism in early flowering is underlying the gir qtl knowing that a diurnal photoperiod was a prerequisite for tihe differences between bay- and sha, we identified early flowering as a candidate gene located in the gir target interval. elf is a component of the circadian clock that functions in the evening complex to repress growth and had previously been shown to regulate hypocotyl elongation in response to shade avoidance. using a transgenic approach, anwer et al. recently showed that the elf3bay- and elf3sha alleles differentially regulate period length of the circadian clock. consistent with a role of elf in thermomorphogenesis, we found that elf3- null mutants conferred long hypocotyls in comparison to their ws- wild-type. to investigate whether tihe differences between the bay- and sha hif lines can indeed be attributed to elf we followed a transgenic complementation approach. we took advantage of the same transgenic lines generated by anwer et al. that contain either the elf3bay- or the elf3sha allele in the elf3- mutant genome, which enabled us to study allele-specific tihe effects in an independent elf loss-of-function background. figure 3b shows that transgenic lines carrying either parental allele complemented the elf3- phenotype at 20 °c. at 28 °c, however, transgenics carrying elf3bay- repressed hypocotyl elongation significantly more than those carrying the elf3sha allele. this demonstrated that allelic variation in elf affects tihe in the bay × sha population.fig. transgenic complementation of elf3-mediated tihe variation. a overview of transgenic constructs used for complementation of the elf3- null mutation. b box plot shows absolute hypocotyl length of 8 days-old seedlings grown in ld at or 28 °c, respectively. horizontal bars, boxes, and whiskers show medians, interquartile ranges, and data ranges, respectively. black dots mark outliers. different letters denote statistical differences as assessed by one-way anova and tukey hsd bay- and sha elf variants display several promoter polymorphisms and differ in a nonsynonymous snp causing an amino acid change at position encoding an alanine-to-valine transition, and in the length of a c-terminal glutamine stretch. although this might depend on the genetic background, tajima et al. suggested that there was no apparent correlation between the length of the polyglutamine region and hypocotyl elongation. we therefore focused on the a362v polymorphism and investigated its potential role in conferring allelic differences in thermomorphogenesis. again, we made use of transgenic lines in the elf3- mutant background generated by anwer et al.. we inspected tihe in transgenic elf3- lines carrying either the probay-0:elf3bay- allele or the probay-0:elf3bay allele, differing only in the a362v snp. we found that temperature-induced hypocotyls in transgenic lines carrying a valine at position were similar to the elf3sha allele and, importantly, significantly longer than those with its alanine counterpart at the same position. theoretically, differences in thermomorphogenesis among elf3- and elf3- might also be affected by circadian or other differences in elf expression. the significant differences observed between elf3- plants expressing the probay:elf3bay and probay-0:elf3bay, however, demonstrate that the snp underlying the a362v change in elf causes phenotypic variation in tihe and establishes elf as a negative regulator of thermomorphogenesis. differential transcriptional responses caused by natural elf variants it has recently been shown that the evening complex of the circadian clock consisting of elf elf and lux arrhythmo underlies the molecular basis for circadian gating of hypocotyl growth by directly down-regulating the expression of pif in the early evening. as a result, hypocotyl elongation peaks at dawn under diurnal cycles. we, therefore, tested the hypothesis that adopts this model for ambient temperature signaling and investigated whether pif expression and possibly also the transcript levels of pif4-regulated genes that mediate cell elongation are affected in response to elevated temperature. to assess temperature responsiveness, we grew seedlings for 7 days at 20 °c under long-day photoperiods, kept control plates at 20 °c, and shifted the remaining seedlings to 28 °c at lights off. control and 28 °c seedlings were subsequently harvested 4 h after the shift. we found that loss of elf in elf3- results in up-regulation of pif transcript levels at both temperatures. at 20 °c, we observe that complementation of elf3- with either transgenic allele ) restores wild-type pif levels, demonstrating functionality of the constructs, but suggesting that allelic differences are absent at 20 °c. this observation is consistent with similar hypocotyl length of the three lines at 20 °c. at 28 °c, however, pif expression levels in elf3- and elf3- are significantly higher than in elf3-, again reflecting the hypocotyl phenotype. importantly, this expression behavior at vs. 28 °c explains the detection of the gir qtl, at 28 °c and the absence of a qtl at °c. together, this suggests that the natural variation observed for tihe is attributable to temperature-dependent differences in pif expression levels caused by the a362v snp in elf3sha.fig. effects of elf allelic variation on the expression of pif and auxin-responsive genes. qrt-pcr analysis of pif saur saur and athb expression in wt and transgenic complementation lines. seedlings were grown for 7 days at 20 °c and transferred to 28 °c or kept at 20 °c. seedlings were harvested after 4 h in the middle of the 8 h dark period. relative expression levels of three biological replicates per treatment were assessed using at1g as control gene. bar plots show means and sem. different letters denote statistical differences among samples as analyzed by one-way anova and tukey hsd test we then connected this scenario to the level of pif target genes responsible for cell elongation. genes involved in cell elongation such as the small auxin upregulated rna family or arabidopsis thaliana homeobox protein have previously been shown to be activated by pif in a temperature-dependent manner. as fig. 4b–d shows, we found that temperature-induced expression levels of the tested genes in elf3- were significantly higher than those in elf3-. this strongly suggested that the a362v snp causes natural variation of temperature-induced pif expression levels directly resulting in alterations of auxin-mediated cell elongation. discussion our findings shown here illustrate the power of natural variation approaches and support elf as a negative regulator of ambient temperature signaling. physiological and gene expression data indicate that elf protein might be involved in down-regulating transcript levels of the major ambient temperature signaling hub pif and thereby affect thermo-responsive growth. transgenic complementation assays furthermore demonstrated that a nonsynonymous snp between the natural accessions bay- and sha significantly affects the ability of elf to regulate temperature-induced pif transcript levels, its target genes, and hypocotyl elongation. in general, different types of polymorphisms, such as nonsynonymous snps or expression level polymorphisms, can contribute to the expression of a particular trait. in line with this phenomenon, distinct types of naturally occurring elf polymorphisms seem to contribute to hypocotyl elongation in response to different temperatures. box et al. recently used a different quantitative genetic approach based on the magic lines, and elegantly showed that both protein-coding and expression level polymorphisms in elf are likely responsible for tihe differences in natural accessions. the authors presented convincing evidence that warmth relieves the gating of growth by elf at night. specifically, elf gating of transcriptional targets responds rapidly to changes in temperature by temperature-dependent binding of elf to target promoters including pif together with box et al.’s non-transgenic quantitative complementation assays, our transgenic complementations unequivocally establish the role of elf in thermomorphogenesis signaling. intriguingly, the elf qtls in both studies were identified in different photoperiods. elf polymorphisms causal for variation within the magic population were identified under short-day conditions. in contrast, our study identified the elf polymorphism under a long-day photoperiod and subsequent analysis of hif lines showed long-day specificity. furthermore, a direct comparison of bay- and sha with two of the parental lines used in the study of box et al. revealed the short-day-specificity of the sf- and zu- alleles in promoting hypocotyl elongation. this photoperiod specificity of the different natural alleles represents an interesting observation in itself requesting further investigations. another unexpected difference between the two studies relates to the growth temperature at which the elf qtl was detected. whereas we identified the elf qtl peak for hypocotyl growth at 28 °c, but not at 20 °c, box et al. did not detect elf at high temperature but rather at 22 °c standard growth conditions. this difference could be attributed to the differential integration of temperature and photoperiod by natural elf alleles. alternatively, the genetic backgrounds and interactions with other contributing loci might be involved. in support of this, it is known that the capacity of elf to mediate growth depends on the context of the genome. hence, box et al. and this study confer complementary evidence for a central role of elf as a major signaling hub acting upstream of pif in the ambient temperature signaling network and add yet another layer to its complex regulation.fig. simplified model of ambient temperature signaling. elf functions as a transcriptional repressor of pif and integrates temperature and photoperiod information in the regulation of thermomorphogenesis. in addition, pif regulation in response to temperature involves the regulatory components of the photomorphogenesis pathway det cop and hy. pif4-mediated transcriptional regulation of target genes can be terminated by the rna binding protein fca, causing dissociation of pif from target gene promoters. additional mechanisms previously shown to contribute to pif regulation e.g., transcriptional regulation of target genes via the bap/hlh/bhlh module of transcription factors. might contribute also to the regulation of temperature responses. other putatively involved modulators of pif activity known from other biological contexts are depicted by gray dashed lines. these involve direct binding of det to pif, competition of pif and other transcription factors for similar binding sites, pif protein sequestration by hfr and phyb-mediated phosphorylation and degradation of pif anwer et al. recently identified elf as a qtl for the regulation of the circadian clock in the same bay- × sha population. in fact, they showed that the elf3sha protein variant failed to properly localize to the nucleus and its ability to accumulate in the dark was compromised. if the same scenario holds for ambient temperature responses, then elf3sha might fail to accumulate in the nucleus during nighttime when hypocotyl growth peaks. as a consequence of a weaker potential of elf3sha to repress pif expression, a temperature increase could result in a much earlier activation of pif4-mediated signaling processes during the night. indeed, box et al. demonstrated that hypocotyl growth dynamics at elevated temperatures are considerably different from those at standard conditions and show a prolonged growth throughout the first night and an additional growth peak in the beginning of the dark period in subsequent nights. however, it is currently unknown in which manner temperature might affect elf protein localization.  in summary, remarkable progress has been made in understanding the functions of elf in combination with recent studies on the role of elf in the shade avoidance response and the circadian clock, this work contributes to understanding its role in the fine-tuned integration of a variety of environmental stimuli that in concert regulate plant growth and development. natural variation in thermomorphogenesis caused by elf variants could be mediated at several levels. first, transcriptional regulation of elf itself caused by expression level polymorphisms can result in varying amounts of pif4-repressing elf protein. in addition, coding sequence polymorphisms might affect the ability of elf protein to interact with pif protein and thereby inhibit its transcriptional activity, as shown by nieto et al.. however, it is unknown whether this protein-protein interaction is temperature-dependent and the existence of natural variation for this mechanism has yet to be reported. lastly, nonsynonymous snps may affect nuclear accumulation of elf which - like expression level polymorphisms described above - would result in variation of the amount of nuclear elf capable of transcriptionally repressing pif and other targets. possibly, the latter mechanism is responsible for natural variation between bay- and sha thermomorphogenesis reported here. our work in combination with the study of box et al. adds further insight into the essential role of elf in integrating multiple signals to promote architectural changes. the photoperiod-specific function of natural elf alleles could provide new avenues to elucidate the clock-mediated growth regulation in general and the elf mode of action specifically. 
in addition, natural elf alleles seem to differentially integrate temperature and photoperiod information to induce architectural changes. we identified giraffe, a major qtl explaining ~18 % of the phenotypic variation for temperature-induced hypocotyl elongation in the bay- x sha recombinant inbred line population. the source of variation could be allocated to a single nucleotide polymorphism in the elf coding region, resulting in differential expression of pif and its target genes, likely causing the observed natural variation in thermo-responsive growth. perception and transduction of temperature changes result in altered growth enabling plants to adapt to increased ambient temperature. while phytochrome-interacting factor has been identified as a major ambient temperature signaling hub, its upstream regulation seems complex and is poorly understood. in combination with other recent studies, this work establishes the role of elf in the ambient temperature signaling network. ambient temperature signalingauxinelf3pif4thermomorphogenesisissue-copyright-statement© the author 2015 here, we exploited natural variation for thermo-responsive growth in arabidopsis thaliana using quantitative trait locus analysis. natural variation of elf3-mediated gating of pif expression during nightly growing periods seems to be affected by a coding sequence quantitative trait nucleotide that confers a selective advantage in certain environments. transgenic complementation demonstrated that allelic variation in the circadian clock regulator early flowering is underlying this qtl.
 perception and transduction of temperature changes result in altered growth enabling plants to adapt to increased ambient temperature. while phytochrome-interacting factor has been identified as a major ambient temperature signaling hub, its upstream regulation seems complex and is poorly understood. here, we exploited natural variation for thermo-responsive growth in arabidopsis thaliana using quantitative trait locus analysis.  we identified giraffe, a major qtl explaining ~18 % of the phenotypic variation for temperature-induced hypocotyl elongation in the bay- x sha recombinant inbred line population. transgenic complementation demonstrated that allelic variation in the circadian clock regulator early flowering is underlying this qtl. the source of variation could be allocated to a single nucleotide polymorphism in the elf coding region, resulting in differential expression of pif and its target genes, likely causing the observed natural variation in thermo-responsive growth.  in combination with other recent studies, this work establishes the role of elf in the ambient temperature signaling network. natural variation of elf3-mediated gating of pif expression during nightly growing periods seems to be affected by a coding sequence quantitative trait nucleotide that confers a selective advantage in certain environments. in addition, natural elf alleles seem to differentially integrate temperature and photoperiod information to induce architectural changes. thus, elf emerges as an essential coordinator of growth and development in response to diverse environmental cues and implicates elf as an important target of adaptation. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords ambient temperature signalingauxinelf3pif4thermomorphogenesisissue-copyright-statement© the author 2015 