Supplemental Data. Kang et al. (2009). Cryptochromes, phytochromes and COP1 regulate light-controlled stomatal development in Arabidopsis.

Supplemental Figure 1. Light Spectra.

(A) to (C) The monochromatic light spectra of blue light (30 μmol.m-2.s-1) (A), red

light (50 μmol.m-2.s-1) (B) and far red light (6 μmol.m-2.s-1) (C).

(D) The spectrum of blue light (30μ mol.m-2.s-1) plus red light (50 μmol.m-2.s-1) plus

far red light (6 μmol.m-2.s-1).

(E) The spectrum of blue light (30 μmol.m-2.s-1) plus red light (50 μmol.m-2.s-1).

1

Supplemental Figure 2. The cry1 cry2 Mutant and Transgenic Seedlings

Overexpressing CRY1 Exhibit Dramatic Opposing Stomatal Phenotypes in

Response to Blue Light.

(A) Abaxial cotyledon epidermis of ten-day-old WT, cry1 cry2 and 35Spro–CRY1

seedlings grown under the fluence rates of 0.5, 5 and 50 μmol.m-2.s-1 blue light for 10

days, respectively. Meristemoids are indicated by arrowheads. Scale bars, 20 μm.

(B) Changes in the stomatal index of WT, cry1 cry2 and 35Spro–CRY1 in response to

the increasing blue light intensity.

2

Supplemental Figure 3. The phyB Mutant and Transgenic Seedlings

Overexpressing PHYB Display Opposite Stomatal Phenotypes in Response to

Red Light.

(A) Abaxial cotyledon epidermis of ten-day-old WT, phyB and 35Spro–PHYB

seedlings grown under the fluence rates of 0.5, 5, 50 and 140 μmol.m-2.s-1 red light

3

for 10 days, respectively. Meristemoids are indicated by arrowheads. Scale bars, 20

μm.

(B) Changes in the stomatal index of WT, phyB and 35Spro–PHYB in response to the

increasing red light intensity.

Supplemental Figure 4. The phyA Mutant Shows Little Sign of Stomatal

Development in Response to Far-Red Light.

(A) Abaxial cotyledon epidermis of ten-day-old WT and phyA seedlings grown under

the fluence rates of 0.4, 2, 12 and 40 μmol.m-2.s-1 far-red light for 10 days,

respectively. Meristemoids are indicated by arrowheads. Scale bars, 20 μm.

(B) Changes in the stomatal index of WT and phyA in response to the increasing

far-red light intensity.

4

Supplemental Figure 5. Cryptochromes and Phytochromes Function Additively

to Regulate Stomatal Development and Patterning.

(A) DIC images of the cotyledon epidermis of WT, cry1 cry2, phyA phyB and cry1

phyA phyB seedlings grown under blue light (30 μmol.m-2.s-1) plus red light (50

μmol.m-2.s-1) plus far-red light (6 μmol.m-2.s-1) for 10 days. Scale bars, 20 μm.

(B) DIC images of the true leaf epidermis of WT, cry1 cry2, phyA phyB and cry1

phyA phyB adult plants grown in the same light condition as in (A) for 4 weeks. Scale

bars, 20 μm.

(C) The SI obtained from (A). The SI of cry1 cry2 and phyA phyB is significantly less

great than that of WT, and the SI of cry1 phyA phyB is significantly less great than

that of phyA phyB (**, P<0.01, t-test, n=10).

(D) The SI obtained from (B). The SI of cry1 cry2 and phyA phyB is significantly

less great than that of WT, and the SI of cry1 phyA phyB is significantly less great

than that of phyA phyB (**, P<0.01, t-test, n=5). 5

Supplemental Figure 6. Promoter Activities of CRY1, CRY2, PHYA, PHYB and

COP1 Genes in Cotyledon Epidermal Cells.

GUS staining of the cotyledon epidermis of the seedlings expressing CRY1pro–GUS

(A), CRY2pro–GUS (B), PHYApro–GUS (C), PHYBpro–GUS (D) and

COP1pro–GUS (E) grown under white light (150 μmol.m-2.s-1) for 6 days. M,

meristemoid; GC, mature guard cell; PC, pavement cell. The images were taken under

equal magnification. Scale bar, 20 μm.

6

Supplemental Figure 7. Cryptochromes and Phytochromes Act Additively to

Antagonize TMM in the Regulation of Stomatal Patterning.

(A) DIC images of leaf epidermis of WT, cry1 cry2, phyB, cry1 cry2 phyB, tmm, tmm

cry1 cry2, tmm phyB and tmm cry1 cry2 phyB seedlings. All genotypes were grown

under blue light (30 μmol.m-2.s-1) plus red light (50 μmol.m-2.s-1) for about 4 weeks.

Scale bars: 20 μm.

(B) The SI obtained from (A). The SI of tmm phyB is significantly less great than that

of tmm, the SI of tmm cry1 cry2 phyB is significantly less great than that of tmm phyB,

and the SI of tmm cry1 cry2 phyB is significantly less great than that of cry1 cry2

phyB, respectively (**, P<0.01, t-test, n=5).

7

Supplemental Figure 8. Effects of Light Signaling Components on Relative

Expression of SPCH, MUTE and FAMA Genes.

The cotyledons of six-day-old seedlings were collected and UBQ10 was used as a

control.

(A) Relative expression of SPCH, MUTE and FAMA in WT and cop1-4 seedlings

grown in the dark or under blue light (30 μmol.m-2.s-1) plus red light (50 μmol.m-2.s-1)

plus far-red light (6 μmol.m-2.s-1).

(B) Relative expression of SPCH, MUTE and FAMA in WT, cry1 cry2 and

35Spro–CRY1 seedlings grown under blue light (50 μmol.m-2.s-1).

(C) Relative expression of SPCH, MUTE and FAMA in WT and phyA seedlings

grown under far red light (4 μmol.m-2.s-1).

(D) Relative expression of SPCH, MUTE and FAMA in WT, phyB and 35Spro–PHYB

seedlings grown under red light (50 μmol.m-2.s-1). Average values from three

biological replicates are plotted with SD.

8

Supplemental Figure 9. Promoter Activities of SPCH, MUTE and FAMA Genes

in Different Light Conditions.

(A) and (B) GUS staining of the cotyledon epidermis of the seedlings expressing

SPCHpro–GUS, MUTEpro–GUS and FAMApro–GUS grown under blue light (30

μmol.m-2.s-1) plus red light (50 μmol.m-2.s-1) plus far-red light (6 μmol.m-2.s-1) (A) or

in the dark (B) for 6 days.

(C) GUS staining of the cotyledon epidermis of the seedlings expressing

SPCHpro–GUS, MUTEpro–GUS and FAMApro–GUS grown in the same light

conditions as in (A) for 5 days.

(D) GUS staining of the cotyledon epidermis of the seedlings prepared from (C)

adapted in darkness for 1 day. Scale bar, 20 µm.

9

Supplemental Table 1. Double, Triple and Quadruple Mutants Constructed and

Their Parents

Mutant Constructed Female Parent Male Parent

phyA phyB phyA-211 phyB-9

cry1 phyA phyB cry1-104 cry2-1 phyA-211 phyB-9

cry1 cry2 phyB cry1-104 cry2-1 phyA-211 phyB-9

cry1 cry2 cop1-5 cry1-104 cry2-1 cop1-5/+

phyA cop1-5 phyA-211 cop1-5/+

phyB cop1-5 phyB-9 cop1-5/+

tmm cry1 cry2 cry1-104 cry2-1 tmm-1

tmm phyA phyA-211 tmm-1

tmm phyB phyB-9 tmm-1

tmm cry1 cry2 phyB cry1-104 cry2-1 phyB-9 tmm-1

tmm cop1-4 cop1-4 tmm-1

cop1-5 spch cop1-5/+ spch/+

cop1-5 fama cop1-5/+ fama/+

det1 spch det1-1s/+ spch/+

det1 fama det1-1s/+ fama/+

cop1-5 E1728 cop1-5/+ E1728

det1 E1728 det1-1s/+ E1728

yda-2 E1728 yda-2/+ E1728

10

Supplemental Table 2. Primers for Mutant Genotyping, Plasmid Construction,

RT-PCR and Quantitative RT-PCR

Primer Name DNA Sequence

SPA1-LP CTG AAC TTT GAA TCC CAG AGC

SPA1-RP TGG TTT TCA GAG CAT CTG TCC

SPA2-LP CTG AAC TCT TGC AGC ATT TCC

SPA2-RP GGC TGA AAA TAC GAG CAT GAC

SPA3-LP GAA CCA AAA TCC TCT TCA GGG

SPA3-RP GCG GTA AAG AAG CTT CCT TTC

SPCH-LP GAA AAA CCT AGA TCC TCC CCC

SPCH-RP TCC TAT GAT CGA TGC TTG GTC

FAMA-1-LP TGG TCT TGC TCG TTC TAG CTC

FAMA -1-RP CTA TCT TGC ATG TCT TGC GTC

LBa1 TGG TTC ACG TAG TGG GCC ATC G

LB1 GCC TTT TCA GAA ATG GAT AAA TAG CCT TGC TTC C

LB4 CGT GTG CCA GGT GCC CAC GGA ATA GT

YFP-Kpn1-5’ GG GGT ACC ATG GTG AGC AAG GGC GAG GAG CTG TTC

YFP-Nhe1-Sac1-3 CCC GAG CTC GCT AGC TTA CTT GTA CAG CTC GTC CAT GCC GAG

PHYB-Xba-5’ GC TCT AGA ATG GTT TCC GGA GTC GGG GGT AGT GGC GGT

PHYB-Kpn1-3’ GG GGT ACC ATA TGG CAT CAT CAG CAT CAT GTC ACC ACT TCC ACT

CRY1pro-PstI-5’ AAA CTG CAG ATG AAG ACT ATT CGA CAA CTA C

CRY1pro-SpeI-3’ CG ACT AGT CTC TCA CAA ACT AAA AAA AAC T

CRY2pro-PstI-5’ AAA CTG CAG AAA TTT TCA TCT ATA AGG TGT G

CRY2pro-XbaI-3’ GCT TCT AGA AGT TAT TAT GAT CAC AGA TGA A

PHYApro-pstI-5’ AAA CTG CAG ACA CAT GGT AAT GAA CAG TTG TT

11

PHYApro-BamHI-3’ GCC GGA TCC TTT TTT CCT GAC ACA GAG ACA AG

PHYBpro-HindIII-5’ CCCA AAG CTT TTG TGC ACC ACC GTC TAA GCT AA

PHYBpro-XbaI-3’ GCT TCT AGA GCC GTT TGA TTT TGA ATT TGA GA

COP1pro-PstI-5’ AAA CTG CAG CCT TCC AGG GGA AAA TAA TCT G

COP1pro-XbaI-3’ GCT TCT AGA TTT GCT TTC AAA AGA TTT CTT C

TMMpro-HindIII-5’ CCCA AAG CTT TGT TGC TCC ATG GGC ATG TGC T

TMMpro-PstI-3’ AAA CTG CAG TTC TTA GTT GTT GTT GTT GTG T

sGFP-PstI-5’ AAA CTG CAG ATG GTG AGC AAG GGC GAG GAG

sGFP-SacI-BstEII-3’ CGG AGC TCG GTG ACC TTA CTT GTA CAG CTC GTC CA

MUTE-Xho-5' CCC CTC GAG ATG TCT CAC ATC GCT GTT GAA AGG A

MUTE-Ecor-3' CGG AAT TCG CTT AAG TGA CAC TCC AAT CCT ATC T

MUTE-Sac-5' CGG AGC TCA TGT CTC ACA TCG CTG TTG AAA GGA

MUTE-Spe-3' GGA CTA GTG CTT AAG TGA CAC TCC AAT CCT ATC T

YDA-HindIII-5’ CCCA AAG CTT ATG CCT TGG TGG AGT AAA TCA A

YDA -SpeI-3’ GG ACT AGT GGG TCC TCT GTT TGT TGA TCC G

sGFP-SpeI-5’ GG ACT AGT ATG GTG AGC AAG GGC GAG GAG

GYODA-SpeⅠ-5’ GGA CTA GTA TGC CTT GGT GGA GTA AAT CAA AA

GYODA649-NsiI-3’ GTT ACT ATG CAT TGA CTG ATC CCT ATA CAT AAA GAG

GYODA1097-NsiI-5’ CCA ATG CAT CCT CGA GCT GGA GGG TCA ACT ACT

GYODA-SpeI-3’ GGA CTA GTT TAG GGT CCT CTG TTT GTT GAT CC

YDA-2103-R TGC ATC TTC CGA GTC TAA GCA

Actin8-S GAT GCT GAT GAC ATT CAA CCT

Actin8-A GAA GTG AGA AAC CCT CGT AG

UBC10-FP TCC AGA AGG ATC CAC CTA CAT CA

UBC10-RP CGT TGC CTG CCA GTG AAA C

UBC10-MGB-probe TAG CGC AGG TCC TGT TGC TGA AGA CA

MUTE-FP GAC GAT CAC TTC ATC AGA CAC AAA G

12

MUTE-RP CCT CAA TAT TAG TAG CAT GGA GGA GAC T

MUTE-MGB-probe CAC TCC AAT CCT ATC TTA

SPCH-FP TTC TGC ACT TAG TTG GCA CTC AAT

SPCH-RP GCT GCT CTT GAA GAT TTG GCT CT

SPCH-MGB-probe ATC TTG ATG GTG AAA GAA T

FAMA-FP CTG CTT TGG AGG ATC TTC ATC TCT

FAMA-RP CTT CTG CCG TAA ACC TCG TTT C

FAMA-MGB-probe TCC TTT AAT GTC AAG ATA ACA AG

Supplemental Methods

Construction of Double, Triple and Quadruple Mutants

The spa1 spa2 spa3 triple mutant was obtained by crossing spa1 with spa2 and spa3

sequentially and isolating seedlings of subsequent generations that showed a cop1

mutant-like phenotype in the dark. The genotypes were confirmed by PCR. The phyA

phyB mutant was obtained from F2 seedlings of phyA-211 x phyB-9 that had long

hypocotyls under far-red light and had long petioles and yellow leaves during the

vegetative development stage under white light. The genotype was further confirmed

under red and far-red lights in F3; cry1 cry2 phyB mutant was obtained from F2

seedlings of cry1-104 cry2-1 x phyA-211 phyB-9 that had very long hypocotyls under

white light and long petioles and yellow leaves during the vegetative development

stage under white light. The genotype was confirmed in F3 by PCR genotyping of

CRY1 and CRY2 using the primers described previously (Mao et al., 2005) and by

examining photomorphogenic phenotypes under blue, red, and far-red lights,

respectively. The cry1 phyA phyB mutant was also screened from F2 seedlings of

cry1-104 cry2-1 x phyA-211 phyB-9 and was confirmed by analysis of hypocotyl

phenotype under blue, red, and far-red lights, respectively. The cry1 cry2 cop1-5/+

mutant was obtained from F2 seedlings of cry1-104 cry2-1 x cop1-5/+ that were

13

kanamycin-resistant, had long hypocotyls under blue light, flowered late under 16 hr

light/8 hr dark photoperiod, and had purple seeds in the siliques of mature plants. The

phyB cop1-5/+ mutant was obtained from F2 seedlings of phyB-9 x cop1-5/+ that

were kanamycin-resistant and had long hypocotyls under red light, long petioles and

yellow leaves during the vegetative development stage, and purple seeds in the

siliques of mature plants. The phyA cop1-5/+ mutant was obtained from F2 seedlings

of phyA-211 x cop1-5/+ that had long hypocotyls under far-red light and purple seeds

in the siliques of mature plants. The cry1 cry2 cop1-5, phyB cop1-5, and phyA cop1-5

mutants were identified from offspring of cry1 cry2 cop1-5/+, phyB cop1-5/+, and

phyA cop1-5/+, respectively, that were purple in color.

The procedure for identifying mutations in the CRY1, CRY2, PHYB, and PHYA

loci in tmm cry1 cry2, tmm phyB, tmm phyA, and tmm cry1 cry2 phyB mutants was

similar to that described above, except for mutations in the tmm locus, which were

identified by the stomata cluster phenotype in the leaf epidermis of white light-grown

plants. The tmm cop1-4 mutant was obtained from F2 seedlings of tmm-1 x cop1-4

that had a constitutive photomorphogenic phenotype in the dark and a stomatal cluster

phenotype on the true leaf epidermis of plants grown in white light.

To construct the det1 spch and cop1-5 spch mutants, we first crossed spch/+

with det1-1s/+ and cop1-5/+, respectively. F1 seedlings of det1/+ x spch/+ were

sprayed with basta, and the det1/+ spch/+ mutant was obtained from adult-fertile F2

plants that were basta-resistant and produced purple seeds in the siliques. The

genotype was screened by PCR. F1 seeds of cop1-5/+ x spch/+ were germinated on

MS medium supplemented with 100 mg/mL kanamycin, and the germinated F1

seedlings were sprayed with basta. The cop1-5/+ spch/+ mutant was obtained from

adult-fertile F2 plants that were basta- and kanamycin-resistant and produced purple

seeds in the siliques. The genotype was confirmed by PCR. The det1 spch and cop1-5

spch mutants were identified from the offspring of det1/+ spch/+ and cop1-5/+

spch/+ that were purple in color and had a stomatal phenotype distinguishable from

the det1 and cop1-5 single mutants. The det1 fama and cop1-5 fama mutants were

14

identified using similar procedures, except for fama, which was characterized by

kanamycin resistance and PCR.

Construction of Plant Expression Cassettes

YFP was PCR-amplified, digested with KpnI and SacI, and cloned into

pCAMBIA1302 (CAMBIA, Australia), resulting in pCAMBIA1302–YFP. The

full-length fragment of PHYB was PCR-amplified, digested with XbaI and KpnI, and

cloned into pCAMBIA1302–YFP to generate pCAMBIA1302–PHYB–YFP. The

chimeric PHYB–YFP fragment was excised by digestion with XbaI and NheI and

cloned into the XbaI site of the plant expression vector pKYL71 (Schardl et al., 1987)

to generate pKYL71–35Spro–PHYB–YFP.

To make the vectors for the promoter activity analysis, the promoter sequences

of CRY1 (1491 bp), CRY2 (1502 bp), PHYA (2357 bp), PHYB (2292 bp), COP1 (1143

bp), SPCH (2998bp), MUTE (1953bp) and FAMA (3102bp) were PCR-amplified,

digested with PstI/SpeI, PstI/XbaI, PstI/BamHI, HindIII/XbaI , PstI/XbaI, HindIII/

SpeI , HindIII/XbaI and PstI/BglⅡ, respectively, and ligated into the modified plant

expression vector pCambia1300 (Yang et al., 2005) to generate

pCambia1300–CRY1pro–GUS, –CRY2pro–GUS, –PHYApro–GUS, –PHYBpro–GUS,

–COP1pro–GUS, –SPCHpro–GUS, –MUTEpro–GUS and –FAMApro–GUS,

respectively. GFP was PCR-amplified, digested with PstI and SacI, and cloned into

pBluescript SK (abbreviated as pBS) to yield pBS–GFP. A PCR-amplified fragment

of the TMM promoter was digested with HindIII and PstI and ligated into pBS–GFP

to generate pBS–TMMpro–GFP. The chimeric TMMpro–GFP fragment was excised

by HindIII and BstEII digestion and ligated into pCambia1302, resulting in

pCambia1302–TMMpro–GFP.

To make the RNA-interference (RNAi) construct for MUTE, a 1018 bp fragment

of GUS (nucleotides 792-1809) was PCR-amplified and ligated into the SmaI site of

pBS to generate pBS–ΔGUS. A 540 bp fragment of MUTE was PCR-amplified,

digested with EcoRI and XhoI, and inserted into pBS–ΔGUS to generate

pBS–MUTE–ΔGUS. The corresponding reverse sequence of this fragment (MUTE-rev) 15

was PCR-amplified, excised with SpeI and SacI, and inserted into pBS–MUTE–ΔGUS

to generate pBS–MUTE–ΔGUS–MUTE-rev. The fused MUTE–ΔGUS–MUTE-rev

fragment was excised by XhoI and SacI digestion and cloned into the plant expression

vector pHB (Mao et al., 2005) to generate pHB–35Spro–dsMUTE, which generates

double-stranded RNA of MUTE when expressed in plants.

To make the construct for the study of genetic interaction between COP1 and

YDA, genomic sequences of YDA encoding amino acids 1-178 and 333-883 were

PCR-amplified, respectively. These two fragments were digested with NsiI and then

ligated to each other. The ligation products were used as templates to amplify the

genomic sequence of YDA lacking the N-terminal fragment encoding amino acids

179-332 (ΔN–YDA). The resulting products were digested with SpeI and ligated into

pER8 (Zuo et al., 2000) to generate pER8–XVEpro–ΔN–YDA. All constructs were

confirmed by DNA sequencing.

Transformation and Characterization of Transgenic Plants

The E1728 marker line was transformed with pHB–35Spro–CRY1, and homozygous

35Spro–CRY1 lines expressing E1728 (35Spro–CRY1 E1728) were obtained

according to the procedures described previously (Mao et al., 2005). The cop1-5

E1728 and det1 E1728 mutants were identified from F3 seedlings of E1728 x

cop1-5/+ and E1728 x det1-1s/+, respectively, that were purple in color and whose

cotyledon epidermis expressed GFP fluorescence. The yda-2 E1728 mutant was

identified from F3 seedlings of E1728 x yda-2/+ whose cotyledon epidermis

produced stomata in clusters and expressed GFP fluorescence. Homozygous

35Spro–PHYB lines were obtained from T3 lines transformed with

pKYL71–35Spro–PHYB–YFP that had very short hypocotyls under red light and that

expressed PHYB–YFP fusion protein according to a Western Blot with an anti-GFP

antibody (Santa Cruz Biotechnology). The promoter reporter lines of CRY1pro–GUS,

CRY2pro–GUS, PHYApro–GUS, PHYBpro–GUS, and COP1pro–GUS were

identified from the independent T2 lines by histochemical staining for GUS activity.

16

The 35Spro–dsMUTE, and the cop1-5 35Spro–dsMUTE and det1

35Spro–dsMUTE mutants were identified from siblings of the same T3 lines of

cop1-5/+ transformed with pHB–35Spro–dsMUTE whose adult plants were pale in

color and partially sterile, whose leaf epidermis showed a loss-of-function mute

phenotype, and that had a decrease in MUTE expression analyzed by RT-PCR. cop1-5

35Spro–dsMUTE seedlings were purple in color, whereas 35Spro–dsMUTE and

cop1-5/+ 35Spro–dsMUTE seedlings were pale green and served as controls. The

det1 35Spro–dsMUTE mutant was identified from F3 seedlings of a hybrid between

the homozygous 35Spro–dsMUTE line obtained above and det1-1s/+. Thus,

35Spro–dsMUTE, cop1-5 35Spro–dsMUTE, and det1 35Spro–dsMUTE had the same

35Spro–dsMUTE locus, which is convenient for phenotype comparison.

To generate cop1-5 XVEpro–ΔN–YDA, pER8-XVEpro–ΔN–YDA was used to

transform cop1-5/+, and 33 independent T1 lines that were resistant to both

kanamycin and hygromycin were obtained. In the T2 generation, these 33 cop1-5/+

XVEpro–ΔN–YDA lines were induced with 20 µM β-estradiol. Four of the lines were

not responsive and showed a wild type phenotype, whereas the other 29 lines were

hypersensitive, having major difficulty in germination and growth, and failed to

develop cotyledons. Further efforts were made with these 29 lines using β-estradiol at

concentrations ranging from 0.02 to 2 µM, with no improvement in germination and

growth. However, of these 29 lines, 12 lines segregated pale green seedlings with

cotyledons that were folded and not fully expanded and small and short light purple

seedlings with cotyledons that were folded and not fully expanded without β-estradiol

induction in the T2 generation. The leaky expression of ΔN–YDA in these

XVEpro–ΔN–YDA lines was analyzed by RT-PCR. Actin8 was used as a loading

control. See Supplemental Table 2 for a list of primer sequences. Seedlings with

cotyledons that were folded and not fully expanded were subjected to stomatal

phenotype analysis, and no production of stomata was observed on the cotyledon

epidermis of these seedlings. All these 12 lines contained at least two T-DNA inserts,

based on the ratio of hygromycin-resistant seedlings to hygromycin-sensitive

seedlings in the T2 generation. The results of a representative cop1-5/+ 17

XVEpro–ΔN–YDA line (line 16) are shown in Figures 8I to 8N. Of the 120 T2

seedlings of XVEpro–ΔN–YDA#16 with green cotyledons, 14 seedlings that were pale

green and had cotyledons that were folded and not fully expanded were obtained,

which are regarded as wild type expressing XVEpro–ΔN–YDA#16 (WT

XVEpro–ΔN–YDA#16), and of the 108 T2 seedlings of XVEpro–ΔN–YDA#16 with

small and purple cotyledons, 11 seedlings that were light purple and had cotyledons

that were folded and not fully expanded were obtained, which are regarded as cop1-5

XVEpro–ΔN–YDA#16.

RNA Extraction and Quantitative Real-Time PCR

When the seedlings grew up to 6 days, their cotyledons were collected. Total RNA

was extracted from seedlings using RNArose reagent (Watson, China).cDNA was

synthesized using oligo-(dT)18 primer and ReverTra Ace M-MLV RTase (Toyobo,

Osaka, Japan) according to the manufacture’s recommendation. The primers and

TaqMan-MGB probes were designed and synthesized by Shanghai GeneCore

Company (Shanghai, China). See Supplemental Table 2 for a list of primer sequences.

UBQ10 (At5g53300) was used as a control. The probes were labeled with the

fluorescent reporter dye 6-carboxyfluoroscein on the 5′-end, and with the fluorescent

quencher dye 6-carboxytetramethylrhodamine (TAMRA) and MGB on the 3′-end.

Real-time PCR was performed using the Rotor Gene RG 3000 thermocycler (Corbett

Research, San Francisco, CA, USA), and the data were collected and analysed using

the Rotor Gene 6.0 software (Corbett Research). The gene expression data were

calculated using standard curve methods.

Supplemental References

Mao, J., Zhang, Y.C., Sang, Y., Li, Q.H., and Yang, H.Q. (2005). A role for

Arabidopsis cryptochromes and COP1 in the regulation of stomatal opening.

Proc. Natl. Acad. Sci. USA 102, 12270-12275.

18

Schardl, C.L., Byrd, A.D., Benzion, G., Altschuler, M.A., Hildebrand, D.F., and

Hunt, A.G. (1987). Design and construction of a versatile system for the

expression of foreign genes in plants. Gene 61, 1-11.

Yang, X.H., Xu, Z.H., and Xue, H.W. (2005). Arabidopsis membrane steroid

binding protein 1 is involved in inhibition of cell elongation. Plant Cell 17,

116-131.

Zuo, J., Niu, Q.W., and Chua, N.H. (2000). Technical advance: An estrogen

receptor-based transactivator XVE mediates highly inducible gene expression

in transgenic plants. Plant J. 24, 265-273.

19