Supporting Online Material

Materials and Methods

Seedling growth and measurements

Seeds were surface sterilized and plated on growth medium (GM)

without sucrose as described by Huq and Quail (2002) (S3). The

red and far-red light sources have been described by Wagner et

al. (1991) (S4). Fluence rates of various lights were measured

using a spectroradiometer (model LI-1800, LiCor, NE). The T-DNA

tagged seeds were obtained from the Arabidopsis Biological

Resource Center (Columbus, OH) and Torrey Mesa Research

Institute, (San Diego, CA) (S5-S6). Hypocotyl length

measurements are performed as described by Huq and Quail (2002)

(S3).

Transformation of Arabidopsis and transgenic plant analysis

The PIF1 open reading frame from the full-length cDNA was

amplified using PFU Turbo polymerase (Stratagene) with forward

and reverse primers, which included restriction sites for

cloning. The resulting fragments were cloned into the pKF111

vector (S7) in both orientations and sequenced. These constructs

were introduced into GV3101 (MP90) Agrobacterium and used for

transformation of wild type Col-O by the floral dip method (S8).

Transgenic seeds were plated on GM-Suc plates containing 5 µg/ml

of glufosinate-ammonium (Riededel-de Haen, Germany), and the

2

resistant seedlings were transplanted to soil and grown in the

greenhouse.

In vitro co-immunoprecipitation assay

For in vitro co-immunoprecipitation experiments, all proteins

were expressed from T7 promoters in the TnT in vitro

transcription/translation system (Promega) in the presence of

35S-methionine. The DNA constructs for expressing phyA, phyB,

GADPIF4, and GADPIF3, are described by Huq and Quail (2002)

(S3). The GAD:PIF1 vector was constructed by PCR amplification

of GAD and PIF1 separately, followed by three fragment ligation

into a pET17b vector (Invitrogen), and confirmed by sequencing.

In vitro co-immunoprecipitation experiments, sample preparation

and quantification were performed according to Huq and Quail

(2002) (S3).

RNA isolation and Northern blotting

Total RNA was isolated from 4-day-old seedlings of wild type

and pif1 mutants using Qiagen RNeasy Plant Mini Kit (Qiagen,

Valencia, CA). Full-length PIF1 ORF was used as a probe to

detect message levels in the wild type, pif1-1 and pif1-2 mutant

backgrounds.

Subcellular Localization of PIF1

For subcellular localization, full-length PIF1 open reading

frame was amplified using PCR primers containing restriction

3

sites (Cla I - Sal I), and fused to the C-terminus of GUS in the

TEX3 vector digested with Cla I - Sal I (S3). The resulting GUS-

PIF1 fusion construct was transiently transfected into onion

epidermal cells and stained for GUS activity according to Huq

and Quail (2002) (S3).

Construction of plasmids and transactivation assays

For Gal4 DNA binding domain (GBD) fusions, the PIF1 open

reading frame was cloned as a SmaI – KpnI fragment into pMN6

(S9) in frame to GBD in front of a full-length cauliflower

mosaic virus (CaMV) 35S promoter. pMN6 alone was used as a

negative control. For detection, the Dual – Luciferase system

was used (Promega). The LUC+ gene in pGLL was driven by a

chimeric promoter containing two copies of the lac operator and

four copies of the Gal4 binding site fused upstream of a minimal

35S promoter (-98) fragment. Renilla lucerifase driven by a full

length CaMV 35S promoter was used as internal control (pRNL).

Three-day-old etiolated Arabidopsis Col wild type or phyB-9 or

phyA-211 mutants were grown on 0.8% GM-S plates on filter paper.

Before bombardment the hypocotyls were laid flat to create a

continuous square of seedlings. 10 µg of effector plasmids, 10 µg

of pGLL reporter and 0.5 µg of pRNL internal control were co-

precipitated in 10 mM spermidine and 1.25 M CaCl2 onto 1 µm gold

particles, washed, dried in ethanol, split in two and delivered

4

to two tissue plates by particle bombardment at 1100 psi using a

Dupont Biolistic particle delivery system. The tissue was then

treated with 15 min far-red light (730 nm, 20 µmolm-2s-1) and then

transferred either to darkness, continuous red (660 nm, 13 µmolm-

2s-1) or continuous far-red (730 nm, 20 µmolm-2s-1) light for 16

hrs. Alternatively, the seedlings were exposed to a pulse regime

of either 9x 5 min red pulses (55 µmolm-2s-1) or 9x 5 min red

pulse followed by a 5 min far-red pulse (40 µmolm-2s-1) every 2

hours.

The tissue was extracted in LUC extraction buffer (200 mM

NaPO4 pH 7.8, 4 mM EDTA, 2 mM DTT, 5% glycerol, 1 mg/ml BSA, 2 mM

PMSF and 1xcomplete protease inhibitor cocktail (Roche) at 3x

w/v. Debris was separated by centrifugation at 14,000 rpm and 20

µl of the supernatant were used to measure firefly and Renilla

luciferase activity. In each experiment eight replica plates

were used for each effector and treatment.

Supporting Figures

Supplementary Material Figure 1: PIF1 is not necessary for

phytochrome-mediated hypocotyl suppression. A) Mean hypocotyl

lengths of wild type Col, transgenic overexpression lines of

PIF1, two pif1 T-DNA insertion lines and their wild type

siblings grown under dark (D), red (Rc, 32 µmolm-2s-1) and far-red

light (FRc, 17 µmolm-2s-1) conditions for four days. B) Visible

5

phenotypes of seedlings grown under the same conditions as

described in (A).

Supplementary Material Figure 2: Photobleaching of pif1 mutants

under prolonged incubation in light. A) In vivo low temperature

(77K) fluorescence emission spectra of cotyledons of 4 day old

etiolated seedlings of wild type (WT) and pif1 that were kept in

the dark or exposed for 10 min, 6 h, 15 h and 24 h to white

light (125 µE m-2 sec-1). Single cotyledons were cut from each

seedling after the light treatment and frozen immediately in

liquid nitrogen for the measurement of the emission spectra. The

four major peaks represented by downward arrows shows the

positions of free protochlorophyllide (Peak 1),

protochlorophyllide bound with protochlorophyllide

oxidoreductase enzyme (POR) (Peak 2), chlorophyll/photosystem II

(Peak 3) and photosystem I (Peak 4), respectively. The

excitation wavelength was 433 nm and the emission was recorded

between 600 and 800 nm. B) In vitro fluorescence emission

spectra of acetone extracts of WT and pif1 mutant seedlings

grown in the dark for 4 days and exposed to white light for 15

and 24 h, respectively. Extracts of 10 seedlings were used for

each time point. The excitation wavelength was 433 nm and the

emission was recorded between 600 and 700 nm. The low

temperature fluorescence spectra were recorded as described

6

earlier (S1). Extraction and measurement of tetrapyrroles are

according to op den Camp et al. (2003) (S2).

Supplementary Material Figure 3: Cotyledon areas of wild type,

pif1, phyA and phyB mutant seedlings. A) Wild type and pif1

seedlings have similar cotyledon area. Cotyledon area of two-

day-old dark-grown, or dark-grown seedlings transferred to white

light (50 µmolm-2s-1) for 5 hrs were measured for wild type and

pif1 mutants. Digital pictures were taken from at least 30

cotyledons of each genotype and area was measured using NIH

ImageJ software. B) Wild type, phyA and phyB mutant seedlings

have similar cotyledon areas. Digital pictures were taken for

three-day-old dark-grown, or dark-grown seedlings transferred to

white light for 5 hrs and cotyledon area was measured as

described in A.

Supplementary Material Figure 4: PIF1 is localized to the

nucleus. Top: Schematic representation of GUS only and GUS-PIF1

fusion constructs used in transient assays in onion epidermal

cells. Bottom: Left pair of panels show GUS staining for GUS-

PIF1 and GUS only control. Middle pair of panels shows DAPI

staining for nuclei in the left panels. Right pair of panels

shows superimposition of the GUS and DAPI staining. GUS-PIF1, β-

glucuronidase fused to the amino-terminus of the full-length

PIF1 protein.

7

Supplementary Material Figure 5: PIF1 binds to the G-box DNA

motif present in various light-regulated promoters. A) PIF1

binds to the G-box in a sequence-specific manner. One µl of TnT-

expressed PIF1 was used in each lane indicated (+). Thirty

thousand cpm of labeled probe was used in each lane. The binding

conditions, amount of probe used, and the gel compositions are

described in Huq and Quail, 2002 (S3). FP, free probe. B) G-box-

bound PIF1 does not bind to phyA and phyB. Numbers on the top

are µl of each component added in each lane. The binding

conditions, amount of probe used, and the gel compositions are

described in Huq and Quail, 2002 (S3).

0

2

4

6

8

10

12

14 D Rc FRc

Col

PIF1Ox 1

P IF 1Ox2

pif1- 1

pif1- 2

WTSib ling

WTS ibling

Col

PIF 1Ox1

PIF1Ox2

pif1- 1

p if1-2

WTSibling

WTS ib ling

MMeeaannHHyyppoocc oottyylllleenngg tthh ss

(( mmmm))

AA BB

RRcc

FFRRcc

DD

Figure S1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2600

615

629

644

658

673

687

702

716

731

745

760

774

789

R.F.U

pif DD

wt DD

pif DDL 10'

wt DDL 10'

pif DDL 6h

wt DDL 6h

pif DDL 15h

wt DDL 15h

pif DDL 24h

wt DDL 24h

in vivo

1

2

3

4

0

5

10

15

20

25

600

608

615

623

630

638

645

653

660

668

675

683

690

698

pif light 15h

wt light 15 h

pif light 24h

wt light 24h

in vitro

Figure S2

A

B

Wavelength (nm)

Wavelength (nm)

Rela

tive

Fluo

resc

ence

Uni

tsRe

lativ

e Fl

uore

scen

ce U

nits

Figure S3

PIF1GUS

GUS

GUS DAPI SUPERIMPOSED

GUS-PIF1

GUS

Figure S4

FP

PIF1

PIF1TnT

Probe G-wt G-mut cold

competitorA

+ ++++++

1 2 3 4 5 6 7 8 9

RFR

FP

- 1 1 1 1 1 - - - -- - - - - - 1 1 1 -- - - - 4 4 - 1 1 -- - 4 4 - - - - - -

TnT

phyBPIF3PIF1

Light

PIF3+PfrBPIF3PIF1

- 4 - - - - - - - 4phyA

RFR RFR

1 2 3 4 5 6 7 8 9 10

B

Figure S5

13

Supporting References:

S1. N.N., Lebedev, P., Siffel, A.A. Krasnovskii, Photosynthetica

19, 183 (1985).

S2. R.G.L. op den Camp et al. Plant Cell 15, 2320 (2003).

S3. E. Huq, P. H. Quail, EMBO J. 21, 2441 (2002).

S4. D. Wagner, J. M. Tepperman, P. H. Quail, Plant Cell 3, 1275

(1991).

S5. A. Sessions et al., Plant Cell 14, 2985 (2002).

S6. J.M. Alonso et al., Science 301, 653 (2003).

S7. M. Ni, J. M. Tepperman, P. H. Quail, Cell 95, 657 (1998).

S8. S. J. Clough, A. F. Bent, Plant J. 16, 734 (1998).

S9. M. Ni, K. Dehesh, J. M. Tepperman, P. H. Quail, Plant Cell

8, 1041 (1996).