Post on 31-Dec-2019
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