Natural variation reveals that intracellular distribution of ELF3 protein is associated with function in the circadian clock

  1. Muhammad Usman Anwer
  2. Eleni Boikoglou
  3. Eva Herrero
  4. Marc Hallstein
  5. Amanda Melaragno Davis
  6. Geo Velikkakam James
  7. Ferenc Nagy
  8. Seth Jon Davis  Is a corresponding author
  1. Max Planck Institute for Plant Breeding Research, Germany
  2. University of York, United Kingdom
  3. Biological Research Centre of the Hungarian Academy of Sciences, Hungary
14 figures, 4 tables and 3 additional files

Figures

Illustrative and statistical features of CCR2 periodicity post-photic vs post-thermal entrainment in BxS populations.

(A) Normal frequency distribution of CCR2 periodicity in BxS individuals. Blue-colored bars represent periodicity after photic entrainment, and magenta-colored bars represent periodicity after …

https://doi.org/10.7554/eLife.02206.003
Map-based cloning identifies ELF3 as a candidate for chr2 QTL.

Period estimates of CCR2::LUC expression in (A) three independent HIFs (57, 92, and 343) harboring either the Bay-0 or the Sha allele at QTL confidence interval (B) parental accessions and NIL-S …

https://doi.org/10.7554/eLife.02206.007
Transgenic complementation confirms ELF3 as QTG underlying Chr2 QTL.

Free-running profile of CCR2::LUC expression in Bay-0, Sha, and NIL in darkness (A), Left y-axis shows the luminescence measures of Bay and NIL, and the right y-axis shows the luminescence measures …

https://doi.org/10.7554/eLife.02206.008
Flowering time and hypocotyl length measurements for ELF3-Sha.

(A) Flowering time of HIF 89-S, HIF 539-B, NIL, and Bay-0 under long day (16L:8D) and short day (8L:16D). The flowering time was counted as the number of days at the appearance of 1 cm bolt. (B) …

https://doi.org/10.7554/eLife.02206.009
Molecular basis of ELF3-Sha phenotypes.

(A) Schematic diagram explaining the different promoter-coding combinations used in (B). The Sha promoter of ELF3 fused with different coding regions is shown (for details see 'Materials and …

https://doi.org/10.7554/eLife.02206.010
Alterations in the ELF3-Sha oscillator and clock resetting.

(A) Free-running profile of LHY::LUC expression in ELF3-Bay and ELF3-A362V in a 15-day continuous experiment under consecutive light and in dark conditions. The plants were entrained for 7 days …

https://doi.org/10.7554/eLife.02206.011
Alanine sustains robust oscillator in darkness.

(AC) Scatter plot for R.A.E. against period showing estimates of individual ELF3-Bay and ELF3-A362V lines shown in (Figure 5A). Only lines with R.A.E. < 1.0 were plotted.

https://doi.org/10.7554/eLife.02206.012
Circadian oscillator does not abolish in ELF3-Sha in darkness.

(A) Phase shifts in dark-adapted seedlings after resumption to LL. ELF3-Bay and ELF3-A362V plants entrained for 7 days under light/dark cycles (LD) were transferred in DD for 1 day and then …

https://doi.org/10.7554/eLife.02206.013
ELF3-Sha is short-period under a range of light intensities.

Free-running period of Bay and NIL-S under different intensities of (A) red (B) blue and (C) red+blue lights (∼3:1). Plants were entrained for 7 days under 12 hr LD cycles (white light) before …

https://doi.org/10.7554/eLife.02206.014
Compromised clock network in ELF3-Sha. Luciferase expression profile of different clock genes in NIL, elf3-1, elf3-4 and Bay-0 under LL (left panel, AF) and in DD (right panel, GL).

(A and G) CCA1::LUC, (B and H) LHY::LUC, (C and I) TOC1::LUC, (D and J) GI::LUC, (E and K) PRR7::LUC, and (F and L) PRR9::LUC. Error bars represent SEM and are shown on every third reading. Note …

https://doi.org/10.7554/eLife.02206.015
Transcript accumulation pattern of different clock genes under LL.

Transcript accumulation of different clock genes in Bay-0, Sha, NIL-S and elf3-4 under LL. (A) CCA1::LUC, (B) LHY::LUC, (C) PRR7::LUC, and (D) PRR9::LUC, (E) GI::LUC, and (F) TOC1::LUC. Error bars …

https://doi.org/10.7554/eLife.02206.016
Sub-cellular localization defects of ELF3-Sha.

(A) and (B) show maximum intensity projection of ELF3-YFP localization in root cells of ELF3-Bay-YFP (A) and ELF3-Sha-YFP (B). Arrows indicate the nuclei that are magnified four times and shown in …

https://doi.org/10.7554/eLife.02206.018
ELF3 cyclic accumulation is altered in ELF3-Sha.

Accumulation pattern of ELF3 transcript and encoded protein in ELF3-Bay-YFP and ELF3-Sha-YFP lines under LD (A and B), LL (C and D) and in DD (E and F). For LD, plants were grown under 12L:12D …

https://doi.org/10.7554/eLife.02206.017
Distribution of the ELF3 locus.

(A) Neighbor-joining tree showing the phylogenetic relationship of ELF3 coding sequence of 251 accessions. All accessions harboring ELF3-Sha allele were grouped in a single clade indicated by …

https://doi.org/10.7554/eLife.02206.019

Tables

Table 1

Circadian periodicity analysis of the BxS RILs after photic or thermal entrainment

https://doi.org/10.7554/eLife.02206.004
ZeitgeberMean*S. E95% confidence interval
Lower boundUpper bound
BxSLD26.7020.03526.63326.771
TMP26.2690.03426.20226.335
LD-TMPBxS
Pairwise comparisonsMean difference (hr)0.433
S. E0.047
Significance<0.001
CorrelationsCorrelation coefficient0.86
Significance0.001
  1. LD stands for photic-zeitgeber and TMP for thermal-zeitgeber.

  2. *

    denotes the modified population marginal mean for the 95% Confidence Interval.

  3. S.E. denotes standard error.

  4. LD-TMP denotes the pairwise comparisons such that TMP period is subtracted from LD period.

Table 2

Statistical analysis of CCR2 circadian periodicity after the two zeitgeber protocols for BxS population

https://doi.org/10.7554/eLife.02206.005
Overall modelUnivariate BxS
FactorFP
Genotype7.753<0.001
Environment36.413<0.001
Environment*Genotype2.183<0.001
Univariate LDCV LDUnivariate TMPCV TMP
FPFP
BxSRIL10.387<0.00118.0437.382<0.00116.498
TRANS (RIL)1.513<0.0011.915<0.001
  1. F = mean sum of squares\error sum of squares. P = significance value of the F-ratio.

  2. Genotype denotes RIL, ENVIRONMENT denotes the different entrainments, TRANS denotes independent transformants of each genotype.

  3. *

    denotes the testing of an interaction between two factors, whereas B(A) denotes the testing main factor A in which a factor B is nested.

  4. CV is the coefficient of genetic variation, LD stands for photic and TMP stands for thermal entrainment.

  5. NS denotes nonsignificance.

Table 3

Localization of the main QTLs for the BxS population after photic vs thermal zeitgeber protocols

https://doi.org/10.7554/eLife.02206.006
ZeitgeberH2ChromosomePositionLOD score (*)% expl varianceFp value2a
(cM)(h)
LD0.76I63.73.2114.97.7040.007−0.614
II34.55.7227.320.784<0.0011.003
IV69.93.8917.114.003<0.0010.833
TMP0.73II34.54.5625.525.033<0.0010.988
IV69.93.2616.26.5010.0140.508
  1. H2 denotes broad sense heritability.

  2. (cM) denotes centiMorgan.

  3. (*) LOD- score threshold was determined at 2.4.

  4. % expl variance is the percent of explained variance.

  5. F = mean sum of squares\error sum of squares.

  6. P denotes the significance value of the F-ratio.

  7. 2a denotes the additive effect of Bay allele when the effect of the Sha allele on period is subtracted.

  8. (h) denotes the effect in hours.

  9. − denotes that the Sha allele displayed longer period than that of Bay allele.

Table 4

Geographical location of the 15 accessions with ELF3-Sha allele

https://doi.org/10.7554/eLife.02206.020
AbbreviationNameStock IDCountryLocationLatitudeLongitudeAltitude
ICE130Kolyvan/Kly-4CS76384RussiaKolyvan51.3282.55505
ICE138Lebjashje/Leb-3CS76426RussiaAltaijskij Kraj51.6580.82301
ICE70Borskoje/Borsk-2CS76421RussiaSamarskaja Oblast53.0451.7575
ICE71Shiguljovsk/Shigu-1CS76375RussiaSamarskaja Oblast53.3349.48181
ICE72Shiguljovsk/Shigu-2CS76374RussiaSamarskaja Oblast53.3349.48181
Kar1KarakolCS76522KyrgyzstanSusamyr village/West Karakol river42.374.362503
KondaraKondaraCS76532TadjikistanKhurmatov38.568.5815
Kz9KazakhstanCS76537KazakhstanKaragandy49.6773.33535
NemrutNemrut-1CS76398TurkeyNemrut Dag38.6442.232249
Neo6Neo6CS76560TajikistanJawshangoz village37.3572.463467
RubenzhoeRubenzhoe-1CS76594UkraineRubezhnoe49.0138.3656
ShaShakdaraCS76382TadjikistanPamiro-Alay38.9069.053439
SorboSorboCS22653Tadjikistan38.8269.481751
Westkar4West KarakolCS76629KyrgyzstanKarakol valley42.3742187
Zal1Zal1CS76634KyrgyzstanTchong-Kemin valley/Djachyl-Kul lake42.876.352230

Additional files

Supplementary file 1

RIL periodicity of CCR2::LUC in BxS after photic entrainment.

https://doi.org/10.7554/eLife.02206.021
Supplementary file 2

RIL periodicity of CCR2::LUC in BxS after thermal entrainment.

https://doi.org/10.7554/eLife.02206.022
Supplementary file 3

Detail of primers used.

https://doi.org/10.7554/eLife.02206.023

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