Supplemental Data
Supplemental Figure S1.Comparison of VQ genes in Arabidopsis, rice, and moss.
The proportional distribution of the putative number of amino acids (A) and introns (B) in members of the VQ family in Arabidopsis, rice, and moss. The percentages of each group are relative to the total number of VQ genes in the corresponding species.
Supplemental Figure S2. VQ29 protein level in wild type and various point mutants.
To test the protein stability, the wild-type and two point mutations (V70D, Q71L) of VQ29 were fused with GFP, and the fusion proteins or GFP alone were expressed in Arabidopsis protoplasts. Total protein extracts were immunoblotted with anti-GFP antibody. kD, kilodalton. Multiple bands in the blot might be due to degradation of the fusion proteins after extraction.
Supplemental Figure S3.Fluence-rate response under different light conditions.
Seedlings were grown in the indicated light conditions for 5 d.Data are mean ± SD of at least 20 seedlings. Asterisks indicate significant difference from the wild type at P 0.05 (single asterisk) or 0.01 (double asterisks) using Student’s t-test.
Supplemental Figure S4.Hypocotyl length of VQ29 mutant and overexpression plants grown in the media without sucrose.
Seedlings were grown in MS medium without sucrosein darkness, or under low white light (10 µmol m-2 s-1) or far-red (12 µmol m-2 s-1) conditions(A), or different intensities of far-red light (B),for 5 d.Data are mean ± SD of at least 20 seedlings. Asterisks indicate significant difference from the wild type at P 0.05 (single asterisk) or 0.01 (double asterisks) using Student’s t-test.
Supplemental Figure S5.VQ29 protein level during dark-to-light transition.
Five-day-old dark-grown Pro35S:Myc-VQ29 (VQ29-OE line #10) transgenic seedlings were exposed tofar-red light (12 µmol m-2 s-1) for the indicated time period. Dark-grown Col wild-type seedlings were used as control. VQ29 protein level was detected using anti-MYC antibody.Immunoblotting against the tubulin antibody serves as equal loading control. The intensities of the bands were quantified by the ImageJ software.
Supplemental Figure S6.Phenotype in seedling greening and seed germination.
(A)Light-dependent seed germination phenotype of vq29, pif1 and vq29 pif1 mutants and VQ29-OE transgenic plants. Seeds were surface-sterilized and plated on MS medium under white light. One hour after the beginning of seed sterilization, seeds were exposed to 5 min of far-red light (12 µmol m-2 s-1,phyB off) or followed by 5 min of red light (20 µmol m-2 s-1, phyB on), and incubated in the dark for 5 d, and then germination (protrusion of the radicle) rate was scored. Data are mean ± SD of triplicate experiments.
(B)Greening ability of vq29, pif1 and vq29 pif1 mutants and VQ29-OE transgenic plants. Seedlings were grown in the dark for the indicated period and were then exposed to white light (60 µmol m-2 s-1)for 2 d. Greening rate was determined by counting the percentage of dark-green cotyledons from 50 to 80 seedlings of each genotype. Data are mean ± SD of triplicate experiments.
Supplemental Figure S7.Downstream gene expression in vq29 and/or pif1 mutants.
qRT-PCR showing expression of EXT1,EXT3,EXP8 and EXP10 invq29, pif1, vq29 pif1, and Col wild-type seedlings that were grown in darkness for 4 d. Error bars indicate± SD of triplicates.Asterisks indicate significant difference at P 0.05 using Student’s t-test.
Supplemental Figure S8.The effect of VQ29 on PIF1 protein stability.
Pro35S:TAP-PIF1(PIF1-OE)and Pro35S:Myc-VQ29 / Pro35S:TAP-PIF1(VQ29-OEPIF1-OE) transgenic plants were grown in darkness for 5 d, and were then exposed to white light (60 µmol m-2 s-1) for the indicated period. Total proteins were immunoblotted with anti-MYC (TAP-PIF1 contains both Myc and HA tags)or anti-tubulin (control) antibodies. It should be noted that Myc-VQ29 fusion protein was run out of the gel due to its small molecular weight.
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Supplemental Table S1. List of primers used in this study.
Gene / AGI code / Oligo name / Sequence (5’-3’) / PurposeVQ1 / At1g17147 / VQ1-F
VQ1-R / GAAGGATCCATGTCTGCAGGAGTGAGATC
GACCTCGAGATGGTCTGACCAAAGATTATAC / ORF cloning
VQ2 / At1g21320 / VQ2-F
VQ2-R / GAGGAATTCATGGATAATAGATCGCCAAG
GAACTCGAGCCTTCTTCCTCCACGCTGT
VQ3 / At1g21326 / VQ3-F
VQ3-R / GAAGAATTCATGGATAATAGATCGCCAAG
GAACTCGAGAGAATCAAAGAAATTGTTGAAAAG
VQ4 / At1g28280 / VQ4-F
VQ4-R / CAATTGATGGAGAATTCACCGAGATAC
CTCGAGAGAAGTAGAAGCTGATGAAG
VQ5 / At1g32585 / VQ5-F
VQ5-R / CAATTGATGTATCAGCGACCACAAAAT
CTCGAGATTTATTTCGTATGAGAATTCTA
VQ6 / At1g32610 / VQ6-F
VQ6-R / CAATTGATGGATAGGACTTGTTGGTA
CTCGAGGTAACCTCTCCATCTTTGAC
VQ7 / At1g35830 / VQ7-F
VQ7-R / CAATTGATGGATTCTTGTAACAGTGG
CTCGAGATTATTGGAATCTGTAGGGC
VQ8 / At1g68450 / VQ8-F
VQ8-R / GGAATTCATGATTCCAACAAGATGCAATG
GCTCGAGTTATTCGGACTTAAAAACCAAGG
VQ9 / At1g78310 / VQ9-F
VQ9-R / GAGGAATTCATGGATAAGAGCTGTAACTC
GAACTCGAGATGACCTTTGTATTTAGGGC
VQ10 / At1g78410 / VQ10-F
VQ10-R / GAAGAATTCATGTCTGGAAGAGGGAAAGTG
GACCTCGAGATATTCTGACCATAGTTTATAC
VQ11 / At1g80450 / VQ11-F
VQ11-R / GAAGAATTCATGAGTCACCAGCAGCCGC
GTCCTCGAGAGAGTCTCGATGATTATCTTC
VQ12 / At2g22880 / VQ12-F
VQ12-R / GGAATTCATGGAAGCTACTTCACAACCAT
GCTCGAGCTACCATCTTGATAGATTAGCAG
VQ13 / At2g33780 / VQ13-F
VQ13-R / GAGGAATTCATGGAGAAATCACCAAGATAG
GAACTCGAGATGATCATGAGGTGAAGGC
VQ14 / At2g35230 / VQ14-F
VQ14-R / GAGGAATTCATGGATAGGCCTAGACAAAATG
GAACTCGAGGTAATCATTCCATCTTGGACT
VQ15 / At2g41010 / VQ15-F
VQ15-R / GAGGAATTCATGGTGACTTCGGAGGGATT
GAACTCGAGCATAACCTTCCACGATTCAAG
VQ16 / At2g41180 / VQ16-F
VQ16-R / CTCGAGACCATGGAATTCATGGATCAGTCATCATCAACG
CTCGAGACTAGTCCAGAGAGAACCAATGCTTCC
VQ17 / At2g42140 / VQ17-F
VQ17-R / GGAATTCATGGAAATTGAAGCTACTACTGTT
GCTCGAGTTAGGCGTAATGGTGAGGTTC
VQ18 / At2g44340 / VQ18-F
VQ18-R / GCAATTGATGGAGATTACTCAATATCAAAGT
GCTCGAGTTAAGACGAATTATGAGCCTTTGT
VQ19 / At3g15300 / VQ19-F
VQ19-R / GAGGAATTCATGGAGATTTCAACAAACCC
GAACTCGAGCATCTCCGGCGATAATCTCG
VQ20 / At3g18360 / VQ20-F
VQ20-R / GAGGAATTCATGAGCTCAACGTACAAGG
GAACTCGAGAAAATCGCGAAACTCCGTC
VQ21 / At3g18690 / VQ21-F
VQ21-R / GAAGAATTCATGGATCCGTCGGAGTATTT
GAACTCGAGATCTTGATCCCAAATATGACTA
VQ22 / At3g22160 / VQ22-F
VQ22-R / GAAGAATTCATGGCTAACCCCAACGAGTG
GAACTCGAGTTGCAACCTCGAAGAAGAAG
VQ23 / At3g56710 / VQ23-F
VQ23-R / CTCGAGACCATGGAATTCATGGAGTCATCATCGTCGAC
CTCGAGACTAGTGCACATAGAATCGATGCTTCC
VQ24 / At3g56880 / VQ24-F
VQ24-R / GAATTCATGGCGTCGTCGGAGGGATT
GTCGACCATAACTTTCCATGATTCGAG
VQ25 / At3g58000 / VQ25-F
VQ25-R / GGAATTCATGGAAGCCACGATCTTCGA
GCTCGAGTTAAGCGAATTGATCTGAGAAAAC
VQ26 / At3g60090 / VQ26-F
VQ26-R / GGAATTCATGGTGAGAAATTCTATGAAGGCT
GCTCGAGTTATTGTTGTTGAAGATCAAACCATCG
VQ27 / At4g15120 / VQ27-F
VQ27-R / GAAGAATTCATGGCCAACTCTAACAACGA
GACCTCGAGAGAATAATATACCGTTTTTTCC
VQ28 / At4g20000 / VQ28-F
VQ28-R / GAAGAATTCATGAACAACTCTAGAGAAGAC
GAACTCGAGTAAATCGAGATCTCTCATCATA
VQ29 / At4g37710 / VQ29-F
VQ29-R / GGAATTCATGGAAGCAACATCACAACAATT
GCTCGAGCCATCTGGAAATATTTGCAGG
VQ30 / At4g39720 / VQ30-F
VQ30-R / GAATTCATGGAGTCCGGTAATAGTAG
GTCGACTGTTCTCTGGTCGGAAGAC
VQ31 / At5g08480 / VQ31-F
VQ31-R / GAGGAATTCATGAATAGCAAAGGGAGTCA
GAACTCGAGTGGTTTGCCACTCGAATTG
VQ32 / At5g46780 / VQ32-F
VQ32-R / GAAGAATTCATGGATGATCAGAGTAATCG
GAACTCGAGTTGATTTTTCCATAATGGACTA
VQ33 / At5g53830 / VQ33-F
VQ33-R / GAAGAATTCGGATCCATGGAAGTTTCAACATCATCC
GACCTCGAGAGAGTTCCTCGCCGGAGAAGC
VQ34 / At5g65170 / VQ34-F
VQ34-R / CAATTGATGGAATCCGGCAATAGTAG
GTCGACATCAGAAGAAGAGATCCAAG
VQ17 / At2g42140 / VQ17-Fm
VQ17-Rm / GGAAATCGACCAGAATCTCAC
CGGAAGTTGGCGACATCGGTC / Site-directed mutagenesis
VQ18 / At2g44340 / VQ18-Fm
VQ18-Rm / CGTCCTGAGTCTAACCGGG
AGTGAACGAAAGTTCTTGACG
VQ29 / At4g37710 / VQ29-mutant
LBb1 / GTAGATTCTTTCAAGACTGCC
GCGTGGACCGCTTGCTGCAACT / PCR genotyping for vq29-1
VQ29-VQmF
VQ29-VQmR / GTGTTGGATCTGAGGCTAAC
GTTAGCCTCAGATCCAACAC / Site-directed mutagenesis
VQ29-VmD-F
VQ29-VmD-R / GTGTTGGATCAGAGGCTAAC
GTTAGCCTCTGATCCAACAC
VQ29-QmL-F
VQ29-QmL-R / GTGTTGGTTCTGAGGCTAAC
GTTAGCCTCAGAACCAACAC
VQ29-VmA-F
VQ29-VmA-R / GTGTTGGCACAGAGGCTAAC
GTTAGCCTCTGTGCCAACAC
VQ29-Fq
VQ29-Rq / AATTACCTAACTTCGCTCCA
GACTCGGTAGACATGAGGAT / qRT-PCR
VQ29-Fp
VQ29-Rp
VQ29-FNcoI
VQ29-MRSpeI / GTCGACGAATTCAGCTTGAAGCTTACTTGAAG
GGATCCCTCGAGTGAGACTTGAGATGTCGCTT
CCATGGTTATGGAAGCAACATCACAACAA
ACTAGTCCATCTGGAAATATTTGCAGG / ProVQ29:GUS construction
Pro35S:VQ29-GFP construction
PIL1 / At2g46970 / PIL1-Fq
PIL1-Rq
PIL1-Fp
PIL1-Rp / AATTGCTCTCAGCCATTCGT
CATTAGTTTGGCGAGCGATA
ACAAGAAAGAAGGGAGGGAGACA
TTCTCTTTAAATGGGACCCACAAT / qRT-PCR, ChIP
proPIL1-F
proPIL1-R / AAGCTTCCAGAATATTTTGCAGACGTTG
GGATCCTGAAGTAAACTGAACAAAGCTT / ProPIL1:LUCconstruction
XTR7 / At4g14130 / XTR7-Fq
XTR7-Rq
XTR7-Fp
XTR7-Rp / AAGGAGCAACACATGACGAG
GGGTCTCCAGACAATGGAGT
CGCATGCCGGCTGGAATAGATAG
CGACGTGTCACTTCCCTCGTACC / qRT-PCR, ChIP
proXTR7-F
proXTR7-R / AAGCTTTTGAGGTACATATATGCGGAC
GGATCC GGGTTTGGTTGATAGAAATGA / ProXTR7:LUC construction
EXT1 / At1g76930 / EXT1-F
EXT1-R / AATCACCACCTCCTCCTCAC
CCGTCAACGATCTTGTGTCT / qRT-PCR
EXT3 / At1g21310 / EXT3-F
EXT3-R / GGGTCTCCAATGGCCTCTTTA
CGTAGTGCTTCTTAGGTGGTGGT / qRT-PCR
EXP8 / At2g40610 / EXP8-F
EXP8-R / CGCGTGCTATGAGATGAAGT
AAGAGGAGGATTGCACCAAC / qRT-PCR
EXP10 / At1g26770 / EXP10-F
EXP10-R / GTTCCTTGCAGGAGAAGAGG
AGCTTGCCACACTGTTCTTG / qRT-PCR
PIF4 / At2g43010 / PIF4cds-EcoR1-F
PIF4cds-Sal1-R / GAATTCATGGAACACCAAGGTTGGAG
GTCGACGTGGTCCAAACGAGAACCG / pAD-PIF4 construction
PIF5 / At3g59060 / PIF5cds-EcoR1-F
PIF5cds-Sal1-F / GAATTCATGGAACAAGTGTTTGCTG
GTCGACGCCTATTTTACCCATATG / pAD-PIF5 construction
ACTIN / At3g18780 / ACTIN-F
ACTIN-R / GCCATCCAAGCTGTTCTCTC
GCTCGTAGTCAACAGCAACAA / RT-PCR
UBQ1 / At3g52590 / UBQ-F
UBQ-R / TTCCTTGATGATGCTTGCTC
TTGACAGCTCTTGGGTGAAG / qRT-PCR
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