PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis
- Donald Danforth Plant Science Center, United States
- University of California at Riverside, United States
- Michigan State University, United States
- Mary Institute and Saint Louis Country Day School, United States
Figures
Figure 1 with 5 supplements
PCH1 (At2g16365.2) encodes a conserved evening-phased protein.
(A) Time-course gcRMA (GeneChip Robust Multiarray Averaging) values of PCH1 expression (from Diurnal database, http://diurnal.mocklerlab.org/, Mockler et al., 2007) under short day, 12L:12D and long …
Figure 1—figure supplement 1
At2g16365.2 is the predominant transcript of PCH1.
(A) Schematic structures of four splice variants of At2g16365. Solid boxes represent exons (red) and UTR (blue), while introns are shown as lines. The T-DNA insertion site of SALK_024229 (pch1), two …
Figure 1—figure supplement 2
Peptides identified by ELF3/4 AP-MS only mapped to the protein encoded by At2g16365.2.
Comparison of the amino acid sequences of protein encoded by At2g16365.2 and At2g16365.1 (amino acids encoded by exon 3 and 4 were in gray), with peptides that were identified by ELF3 or ELF4 AP-MS …
Figure 1—figure supplement 3
Available RNAseq data suggest only At2g16365.2 is expressed.
A screen capture from IGB (Integrated Genome Browser) analyzing a publically available RNAseq dataset (Gulledge et al., 2014). RNAseq reads are represented by solid green blocks and only mapped to …
Figure 1—figure supplement 4
PCH1 levels peak at dusk under 12L:12D or long day conditions.
Time-course protein extracts (from ZT 0 to 24, with 3 hr intervals) from 4-day-old PCH1p::PCH1 and PCH1ox3 lines grown under either 12L:12D (A) or long day (B) conditions were immunoblotted with …
Figure 1—figure supplement 5
Multiple sequence alignments of PCH1 orthologs at the C-terminus.
The last 201 amino acids of PCH1 orthologs from Arabidopsis thaliana (AtPCH1, At2g16365.2), Brachypodium distachyon (BdPCH1, Bradi2g46850) (International Brachypodium Initiative, 2010), Oryza sativa …
Figure 2 with 1 supplement
PCH1 regulates the photoperiodic response of hypocotyl elongation in the evening.
(A) Hypocotyl lengths of 4-day-old WT, pch1, PCH1ox3, elf3-2, elf4-2 and phyB-9 seedlings grown under long day, 12L:12D and short day conditions. Mean ± 95% confidence interval (CI) (n=20). (B) pch1 …
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Figure 2—source data 1
Raw measurements of hypocotyl lengths for Figure 2A.
Hypocotyl lengths (mm) of 4-day-old WT, pch1, PCH1ox3, elf3-2, elf4-2 and phyB-9 seedlings grown under long day, 12L:12D and short day conditions, with n = 20.
- https://doi.org/10.7554/eLife.13292.010
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Figure 2—source data 2
ANOVA analyses and Bonferroni's multiple comparison tests for Figure 2A.
2-way ANOVA analyses were carried out using GraphPad Prism (version 6.0, Graphpad.com, La Jolla, California), with Bonferroni's multiple comparisons test results.
- https://doi.org/10.7554/eLife.13292.011
Figure 2—figure supplement 1
PCH1 levels regulate hypocotyl length under short day conditions.
Hypocotyl lengths of short-day-grown, 4-day-old plants of WT, pch1, two independent overexpression lines (PCH1ox3 and 4, in WT) and two complementation lines (PCH1p::PCH1-7 and -8, in pch1) were …
Figure 3 with 1 supplement
Phenotypic characterization of pch1 and PCH1ox3 in circadian and flowering pathways.
(A) Seedlings of WT, pch1, or PCH1ox3 carrying the CCA1:LUC luciferase reporter were grown under 12L:12D conditions for five days before transferring to continuous white light. Bioluminescence were …
Figure 3—figure supplement 1
Modulating PCH1 levels does not affect the circadian period.
Comparison of WT, pch1, and PCH1ox3 periods using data from Figure 3A and B. Mean ± 95% CI (n = 8). ns = not significantly different than WT. Experiments were repeated at least three times.
Figure 4 with 1 supplement
PCH1 directly interacts with phyB in a light-dependent manner.
(A) and (B) yeast two-hybrid between PCH1 (fused to GAL4 DNA binding domain, DBD) and preys (ELF3, ELF4, N-/C- termini (Nt or Ct) and full length (FL) LUX, COP1, TZP and the Ct of phyA, B, C, D, and …
Figure 4—figure supplement 1
Interaction map of PCH1-associated proteins.
An updated interaction map from previous described (Huang et al., 2015), which integrate our AP-MS and protein-protein interaction data to illustrate both directly and indirectly interacting …
Figure 5 with 1 supplement
PCH1 is localized in the nucleus to stabilize phyB-containing photobodies.
(A) PCH1-YPet is nuclear localized when transiently expressed in tobacco and co-localizes with phyB-CFP to photobodies. YPet alone was used as control. Scale bars = 25 µm. (B) Representative …
Figure 5—figure supplement 1
Fewer large photobodies were detected in pch1 with higher intensity of red light treatment.
(A) Representative confocal images showing phyB-containing photobodies in phyB-9, pch1 phyB-9 and PCH1ox3 phyB-9 plants expressing a phyB-GFP (PBG) fusion protein. Plants were entrained by the short …
Figure 6 with 1 supplement
pch1 exhibits defects in red light responsive hypocotyl growth and expression of downstream transcription factors.
(A) Hypocotyl lengths of 4-day-old WT, pch1, PCH1ox3, PCH1p::PCH1 and phyB-9 seedlings grown under either dark or constant red light of various intensities (25, 40 and 100 µmol·m-2·s-1). Mean ± 95% …
Figure 6—figure supplement 1
pch1 does not affect far-red or blue light mediated hypocotyl elongation.
(A) Hypocotyl measurements of 4-day-old WT, pch1, PCH1ox3 and phyA-211 seedlings grown under either dark or constant far-red light conditions (FRc, 25 µmol·m-2·s-1). Mean ± 95% CI (n = 20). (B) …
Figure 7
pch1 affects PIF4 levels and PIFs are required for the hypocotyl phenotype in pch1.
(A) qPCR of PIF4 using time-course cDNA samples of short-day grown, 4-day-old WT and pch1 seedlings. Expression was normalized to IPP2 and APA1. Mean ± SD (n=3 biological reps). Grey shading …
Figure 8 with 1 supplement
A model of PCH1-regulated day-length specific growth.
A proposed model illustrates the role of PCH1 in controlling the photoperiodic hypocotyl elongation response. In short days, PCH1 peaks at dusk (ZT 8), maintains phyB photobody formation to suppress …
Figure 8—figure supplement 1
Expression of PIF3, PIF4 and PIF5 under multiple photoperiods.
Daily expression (represented as gcRMA values) of PIF3, PIF4, and PIF5 under short day, 12L:12D (Col_LDHH) and long day conditions were generated from microarray data (Diurnal database, http://diurna…
Tables
Table 1
Proteins Co-Purified by PCH1 AP-MS in WT and phyB-9. Proteins co-purified with PCH1 were identified from affinity purification coupled with mass spectrometry (AP-MS) analyses using 12L:12D grown, …
| AGI number | Protein name | ELF3 AP-MSb | Exclusive unique peptide count/Percent coveragea | ||||
|---|---|---|---|---|---|---|---|
| PCH1ox3 in WT | PCH1ox3 in phyB-9 | ||||||
| rep1 | rep2 | rep3 | rep1 | rep2 | |||
| At2g16365 | PCH1c | Y | 30/73% | 41/85% | 37/79% | 40/85% | 32/77% |
| At2g18790 | phyB | Y | 46/69% | 47/65% | 41/60% | — | — |
| At5g35840 | phyC | Y | 31/44% | 23/28% | 25/29% | — | — |
| At4g16250 | phyD | Y | 22/47% | 19/34% | 20/38% | 22/38% | 6/11% |
| At4g18130 | phyE | Y | 41/55% | 40/52% | 45/60% | 49/60% | 31/41% |
| At1g09570 | phyA | Y | 31/46% | 36/49% | 35/45% | 36/48% | 29/39% |
| At2g37678 | FHY1 | N | 2/22% | 2/21% | 4/28% | 4/28% | 2/21% |
| At3g42170 | DAYSLEEPER | Y | 5/13% | — | 4/11% | 3/8% | 2/6% |
| At1g09340 | CRB | Y | —d | —d | 4/17% | 6/24% | 3/13% |
| At5g43630 | TZP | Y | 9/15% | 6/12% | 12/23% | — | — |
| At2g32950 | COP1 | Y | 7/15% | 8/16% | 8/18% | — | — |
| At2g46340 | SPA1 | Y | 8/14% | 5/7% | 8/12% | — | — |
| At2g25930 | ELF3 | Y | 6/12% | 11/25% | 12/26% | — | — |
| At2g40080 | ELF4 | Y | —d | 4/60% | 3/42% | — | — |
| At3g46640 | LUX | Y | 2/6% | —d | 4/15% | — | — |
| At3g03940 | MLK2 | Y | —d | 2/6% | 2/6% | — | — |
| At1g15750 | TPL | N | —d | 3/3% | 4/5% | 2/2% | — |
| At1g47128 | RD21a | Y | —d | 3/8% | 2/4% | —d | —d |
| Also see Table 1—source data 1 | |||||||
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a All listed proteins match 99% protein threshold, minimum number peptides of 2 and peptide threshold as 95%. Proteins not matching the criteria were marked with "—".
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b ELF3 AP-MS (Huang et al., 2015) was used for comparison.
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c Percent coverage for PCH1 is calculated using protein encoded by At2g16365.2.
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d Only one exclusive unique peptide was detected.
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Table 1—source data 1
The full list of proteins identified by AP-MS, listing unique peptides and the percent coverage.
The full list is generated and exported by Scaffold (Proteome Software Inc., Portland, Oregon; v.4.4.3) showing all co-purified proteins from all replicates of PCH1ox3 AP-MS and the GFP Control. The file contains reports on exclusive unique peptide counts and percent coverage for each co-purified proteins, with their names, accession numbers and molecular weight.
- https://doi.org/10.7554/eLife.13292.016
Table 2
Proteins Co-Purified by PCH1 AP-MS in elf4-2 and elf3-2, compared to WT. Proteins co-purified with PCH1 were identified from affinity purification coupled with mass spectrometry (AP-MS) analyses …
| AGI number | Protein name | ELF3 AP-MSb | Exclusive unique peptide count/Percent coveragea | ||||||
|---|---|---|---|---|---|---|---|---|---|
| PCH1ox3 in WTc | PCH1ox3 in elf4-2 | PCH1ox3 in elf3-2 | |||||||
| rep1 | rep2 | rep3 | rep1 | rep2 | rep1 | rep2 | |||
| At2g16365 | PCH1d | Y | 30/73% | 41/85% | 37/79% | 29/77% | 34/78% | 42/82% | 36/82% |
| At2g18790 | phyB | Y | 46/69% | 47/65% | 41/60% | 47/70% | 31/46% | 42/63% | 40/56% |
| At5g35840 | phyC | Y | 31/44% | 23/28% | 25/29% | 30/43% | 13/16% | 20/28% | 15/18% |
| At4g16250 | phyD | Y | 22/47% | 19/34% | 20/38% | 20/42% | 12/25% | 20/37% | 16/30% |
| At4g18130 | phyE | Y | 41/55% | 40/52% | 45/60% | 42/57% | 37/50% | 43/55% | 40/53% |
| At1g09570 | phyA | Y | 31/46% | 36/49% | 35/45% | 32/47% | 24/34% | 34/49% | 27/38% |
| At2g37678 | FHY1 | N | 2/22% | 2/21% | 4/28% | —e | 3/21% | —e | 3/21% |
| At3g42170 | DAYSLEEPER | Y | 5/13% | — | 4/11% | 3/7% | 5/11% | 2/5% | —e |
| At1g09340 | CRB | Y | —e | —e | 4/17% | 2/9% | 5/22% | 3/13% | 3/13% |
| At5g43630 | TZP | Y | 9/15% | 6/12% | 12/23% | 4/7% | —e | 12/21% | 2/3% |
| At2g32950 | COP1 | Y | 7/15% | 8/16% | 8/18% | 3/7% | —e | 12/25% | 7/11% |
| At2g46340 | SPA1 | Y | 8/14% | 5/7% | 8/12% | 5/10% | 2/4% | 17/26% | 7/11% |
| At2g25930 | ELF3 | Y | 6/12% | 11/25% | 12/26% | 4/9% | 3/6% | — | — |
| At2g40080 | ELF4 | Y | —e | 4/60% | 3/42% | — | — | — | —e |
| At3g46640 | LUX | Y | 2/6% | —e | 4/15% | —e | — | — | — |
| At3g03940 | MLK2 | Y | —e | 2/6% | 2/6% | — | — | 2/4% | — |
| At1g15750 | TPL | N | —e | 3/3% | 4/5% | 2/3% | 3/3% | 4/5% | 4/5% |
| At1g47128 | RD21a | Y | —e | 3/8% | 2/4% | —e | 2/8% | 3/8% | —e |
| Also see Table 1—source data 1 | |||||||||
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a All listed proteins match 99% protein threshold, minimum number peptides of 2 and peptide threshold as 95%. Proteins not matching the criteria were marked with "—".
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b ELF3 AP-MS (Huang et al., 2015) was used for comparison.
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c PCH1ox3 in WT is as shown in Table 1, for comparison with PCH1ox3 in elf4-2 and elf3-2.
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d percent coverage for PCH1 is calculated using protein encoded by At2g16365.2
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e only one exclusive unique peptide was detected.
Table 3
Primers used in this study.
| Primers used for cloning PCH1 and PCH1 promoter a | ||
|---|---|---|
| Amplified Fragments | Forward primer (5'->3') | Reverse primer (5'->3') |
| PCH1-stop | CACCATGTCTGAACATGTTATGGTTTTGG | CTACCTCAAATCCCTTGCATTCCA |
| PCH1-nonstop | CACCATGTCTGAACATGTTATGGTTTTGG | CCTCAAATCCCTTGCATTCCAAAC |
| PCH1-promoter b | AAGCTTAGTTTCCTCATCATTTGCTATTG | GCGTAAATCCTCACCGGTCTT |
| Primers used to generate yeast two-hybrid constructs, all with a stop codon a | ||
| Amplified fragments | Forward primer (5'->3') | Reverse primer (5'->3') |
| PCH1 | CACCATGTCTGAACATGTTATGGTTTTGG | CTACCTCAAATCCCTTGCATTCCA |
| ELF3 | CACCATGAAGAGAGGGAAAGATGAG | CTAAGGCTTAGAGGAGTCATAGCGTTT |
| ELF4 | CACCATGAAGAGGAACGGCGAGACGA | TTAAGCTCTAGTTCCGGCAGCACC |
| LUX (full length) | CACCATGGGAGAGGAAGTACAAA | TTAATTCTCATTTGCGCTTCCACCT |
| LUX-Nt (amino acids 1-143) | CACCATGGGAGAGGAAGTACAAA | CTATTTAAGTGTTTTCCCAGATAG |
| LUX-Ct (amino acids 144-324) | CACCATGCGACCGCGTTTAGTGTGGACA | TTAATTCTCATTTGCGCTTCCACCT |
| phyA-Ct (amino acids 606-1123) | CACCATGGATCTCAAAATTGATGGTATACAA | CTACTTGTTTGCTGCAGCGAGTTC |
| phyB-Ct (amino acids 640-1173) | CACCATGGCGGGGGAACAGGGGATTGATGAG | CTAATATGGCATCATCAGCATCATGTCA |
| phyC-Ct (amino acids 592-1112) | CACCATGGATAATAGGGTTCAGAAGGTAGAT | TCAAATCAAGGGAAATTCTGTGAGGATCAC |
| phyD-Ct (amino acids 645-1165) | CACCATGGTACAGCAAGGGATGCAG | TCATGAAGAGGGCATCATCATCA |
| phyE-Ct (amino acids 583-1113) | CACCATGAATGGCGTAGCAAGAGATGC | CTACTTTATGCTTGAACTACCCTCTGT |
| COP1 | CACCATGGAAGAGATTTCGACGGA | TCACGCAGCGAGTACCAGAACTTTG |
| TZP | CACCATGGGAGATGGAGATGAGCAA | CTAAAAGCCTAACATTTTTCTCTGCTGA |
| Primers used for qPCR | ||
| Gene | Forward primer (5'->3') | Reverse primer (5'->3') |
| PCH1 set A | CCGGCTCCATTTCTTCGTCA | TCCGGAACAAGAGGTGGTTCT |
| PCH1 set B | GAAGTTATTGTTGTCGCCCT | GGGAAATCCAAAGCGGTATT |
| IPP2 | CTCCCTTGGGACGTATGCTG | TTGAACCTTCACGTCTCGCA |
| APA1 (At1g11910) c | CTCCAGAAGAGTATGTTCTGAAAG | TCCCAAGATCCAGAGAGGTC |
| HFR1 | TAAATTGGCCATTACCACCGTTTA | ACCGTGAAGAGACTGAGGAGAAGA |
| ATHB-2 | GAAGCAGAAGCAAGCATTGG | CGACGGTTCTCTTCCGTTAG |
| PIF4 | GTTGTTGACTTTGCTGTCCCGC | CCAGATCATCTCCGACCGGTTT |
| Primers for genotyping | ||
| Mutant | for wild type PCR (5'->3') | for mutant PCR (5'->3') |
| pch1 (SALK_024229) | TGTCAGGTATTTCGGTCCTTG (LP) and CACTTGCTTGATGCTCATGAG (RP) | AAGAACCGGCAAAGATACCAC (RP) and ATTTTGCCGATTTCGGAAC (LBb 1.3) |
| pif3 (SALK_081927C) | AGTCTGTTGCTTCTGCTACGC (LP) and AAGAACCGGCAAAGATACCAC (RP) | ACATACAGATCTTTACGGTGG (RP)and ATTTTGCCGATTTCGGAAC (LBb 1.3) |
| pif4 (pif4-101) d | CTCGATTTCCGGTTATGG (SL42) and CAGACGGTTGATCATCTG (SL43) | GCATCTGAATTTCATAACCAATC (PD14) and CAGACGGTTGATCATCTG (SL43) |
| pif5 (pif5-1) d | TCGCTCACTCGCTTACTTAC (SL46) and TCTCTACGAGCTTGGCTTTG (SL47) | TCGCTCACTCGCTTACTTAC (SL46) and GGCAATCAGCTGTTGCCCGTCTCACTGGTG (JMLB1) |
| elf3-2 c | TGAGTATTTGTTTCTTCTCGAGC and CATATGGAGGGAAGTAGCCATTAC | TGGTTATTTATTCTCCGCTCTTTC and TTGTTCCATTAGCTGTTCAACCTA |
| elf4-2 c | ATGGGTTTGCTCCCACGGATTA and CAGGTTCCGGGAACCAAATTCT, cut with HpyCH4V. WT has 5 cuts while elf4-2 has 4 cuts to give a unique 689 bp band. | |
| phyB-9 | GTGGAAGAAGCTCGACCAGGCTTTG and GTGTCTGCGTTCTCAAAACG, cut with MnlI, phyB-9 gives 167+18 bp bands, WT gives a 185 bp band. | |
| Primers for making pB7SHHc and pB7YSHHc | ||
| Primer Name | Sequence (5'->3') | |
| pDAN0193 | TGCCCGCCTGATGAATGCTC | |
| pDAN0202 | GCGGGATATCACCACCCTAGGCACCACTTTGTACAAGAAAGCTGA | |
| pDAN201 | TCAGCTTTCTTGTACAAAGTGGTGCCTAGGGTGGTGATATCCCGC | |
| pDAN0223 | ATTCTCATGTATGATAATTCGAGG | |
| pDAN0242 | TACAAAGTGGTGCCTAGGGGTGGAAGCTGGAGCCACCCTC | |
| pDAN0241 | GCGGGATATCACCACCCTAGTGATGGTGATGGTGATGAGCG | |
| pDAN0249 | GCTTTCTTGTACAAAGTGGTGCCTGCTGCTGCTGCC | |
| pDAN0250 | GGTGGCTCCAGCTTCCACCCCCCTTATAGAGCTCGTTC | |
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a CACC (underscored) were added to forward primers for cloning into the pENTR/D-TOPO vector.
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b a Hind III restriction site (in bold) was added to the forward primer.
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c (Nusinow et al., 2011).
