development 143: doi:10.1242/dev.130211:...

DRACULA2 is a dynamic nucleoporin with a role in regulating the shade

avoidance syndrome in Arabidopsis.

Marçal Gallemí, Anahit Galstyan, Sandi Paulišić, Christiane Then, Almudena

Ferrández-Ayela, Laura Lorenzo-Orts, Irma Roig-Villanova, Xuewen Wang,

Jose Luis Micol, Maria Rosa Ponce, Paul F. Devlin, Jaime F. Martínez-García

SUPPLEMENTARY MATERIALS AND METHODS

Plant material

The PBL transgenic line (also called “Ws-21a”) was in Ws-2 background and

has been described previously (Kozma-Bognar et al., 1999). In this manuscript,

we rename this line as PBL (see Introduction) as it has been used as a control

in our experiments. The mutant dra2-1, originated in our screening, is in Ws-2

background, whereas T-DNA lines in DRA2 are in Col-0 ecotype: dra2-2

(SALK_017077), dra2-3 (SALK_067219), dra2-4 (SALK_015016) and dra2-5

(SAIL_663_D07). Mutant tcu1-1 (in Ler background), tcu1-2/nup58-2

(SALK_099638), tcu1-4 (SAIL_655_C09), nup54-1 (SALK_106346), nup54-2

(SALK_015252), nup62-1 (SALK_037337), and nup62-2 (SAIL_127_F01) have

been described before (Ferrandez-Ayela et al., 2013). Mutant lines sar1-4

(SALK-126801), sar3-1 and sar3-3 (SALK_109959) are in Col-0 background

and have been described previously (Parry et al., 2006; Parry, 2014).

Development 143: doi:10.1242/dev.130211: Supplementary information

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Genetic analyses and positional cloning of the dra2-1 mutation

For genetic analyses, dra2-1 was crossed to the PBL or Ws-2 line to

reduce the number of second-site mutations. Seedlings with the dra2-1

phenotype (long hypocotyl) were counted in the segregating F2 generation to

determine the nature of the mutation. For the positional cloning of the DRA2

gene, we outcrossed the dra2-1 (in the Ws-2 genetic background) to the Col-0

accession. The F2 seeds were sown on GM- plates, stratified as usual and

grown for 7 days under W. Seedlings showing the elongated hypocotyl

phenotype caused by dra2-1 (Figure 1B) were transferred into individual pots

and grown up in the greenhouse. Around 100 mg of rosette leaves from each

individual F2 plant, the corresponding F1 plant and the two parental lines (PBL,

in a Ws-2 background, and Col-0) was harvested for linkage analyses. These

analyses were performed using fluorescently labelled oligonucleotides, as

described (Ponce et al., 1999; Ponce et al., 2006). In brief, for low-resolution

mapping, DNA of 50 F2 phenotypically mutant plants was individually extracted

and used as a template to multiplex PCR co-amplify 26 SSLP and In/Del

molecular markers that were found polymorphic between Ws-2 and Col-0. For

fine mapping, 121 additional F2 plants were used to iteratively assess linkage

between dra2-1 and SSLP, SNP and In/Del molecular markers developed

according to the polymorphisms between Ler and Col-0 described at the

Monsanto Arabidopsis Polymorphism Collection database

(http://www.arabidopsis.org), not all of which were found polymorphic between

Ws-2 and Col-0. Synthetic oligonucleotides for fine mapping are described

below.


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For sequencing of dra2-1 mutation, genomic DNA from Ws-2 and dra2-1

plants was extracted and PCR amplified with oligonucleotides shown below.

PCR products spanning the At1g10390 transcription unit were sequenced, as

described (Barrero et al., 2007).

High Resolution Mapping

Marker Name (Position)

Oligonucleotides

position sequences (5→3)

cer458005

(At1g10560-At1g10570)

Forward CTA-GTT-GAA-GTC-GCA-AAA-TGT-TG

Reverse GAC-TAA-TAG-CAT-GCT-TCC-AAT-TC

cer465532

(At1g10640-At1g10650)

Forward CAT-TAC-ACT-AGA-GAC-TAG-AC

Reverse TAA-TAT-ACA-TGT-GAG-CAT-CCT-

GAC

F16J7-TRB

(At1g11370)

Forward GTG-TCT-TGA-TAC-GCG-TCG-ATC

Reverse TGA-TGT-TGA-GAT-CTG-TGT-GCA-G

JV28/29

(At1g11730-At1g11735)

Forward GAT-ACT-CCT-GTT-TCA-CAT-ATA-TG

Reverse GAG-AGT-CCT-TAT-TGT-TGT-GCC

JV26/27

(At1g11905)

Forward CAT-TCA-AGA-GAT-TGC-AAC-ATC-C

Reverse GGG-TAA-GCT-CCT-TGG-ATC-CG

Candidate gene (At1g10390) sequencing

Name Oligonucleotide sequences (5→3)

At1g10390-F1 CCG-TGA-AGA-TGC-CCT-AAA-TTC

At1g10390-R1 GGA-GCA-AAG-GGA-TTA-TTA-CTA C

At1g10390-F2.1 CTT-GTT-GGT-TTC-CGA-AGC-CAA

At1g10390-R2 CGC-ACC-AAA-AGA-AGG-AGT-ACT-AGA

At1g10390-F2.2 GGT-GCC-ACT-AAC-ACG-CCT-G

At1g10390-F3 GTA-GCA-CTG-GCA-CCA-CGT-TT

At1g10390-R3 GGT-GAT-GGT-GTC-GTT-GTT-CC

At1g10390-F4 CGA-GCT-CAA-CAT-CTA-CCA-ACC

At1g10390-R4 GTA-TGG-GAG-TTG-CAG-AAG-GAA-G


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At1g10390-F5 GTT-GTG-TTA-TTT-TAG-CTT-CAT-CG

At1g10390-R5 GTC-CGA-GAA-TTT-CCA-CAC-ATA-G

At1g10390-F6 CTT-GAT-TAC-TCT-GCG-TGT-GAG

At1g10390-R6 TGG-CAA-CTG-TTA-CTC-TAC-TCG

Oligonucleotides used for genotyping mutant lines

For genotyping the different mutant plants used in this study, specific

oligonucleotide combinations were used to genotype them by PCR analyses:

DRA2 (JO402 + JO403, GO76 + MGO6, MGO7 + GO75, GO74 + GO75, and

GO96 + GO75), dra2-1 (GO97 + GO75), dra2-2 (JO402 + LBb1), dra2-3 (LBb1

+ GO75), dra2-4 (GO76 + LBb1), dra2-5 (MGO7 + LB3), SAR1 (MGO24 +

MGO25), sar1-4 (MGO24 + LBb1), SAR3 (GO104 + GO106 and GO107 +

GO108), sar3-1 (GO105 + GO106), sar3-3 (LBb1 + GO108), TCU1 (GO78 +

GO79), tcu1-2 (LBb1 + GO79), tcu1-4 (GO78 + LB3), NUP54 (GO80 + GO81,

GO82 + GO83), nup54-1 (LBb1 + GO81), nup54-2 (GO82 + LBb1), NUP62

(GO84 + GO85, and MGO10 + MGO5), nup62-1 (GO84 + LBb1) and nup62-2

(MGO10 + LB3).

Name Oligonucleotide sequences (5→3)

JO402 GGT-CGA-AGA-ACG-TGT-GTC-C

JO403 GGT-ACC-AGA-TGA-CTG-TCC

GO74 CAC-TGA-TGA-CGA-AGA-GAG

GO75 CCA-TAA-CCG-TGT-CGT-CCC

GO76 GAT-CTT-CTG-GTT-TTG-GGC-AG

GO78 CCA-AAT-TTG-TTA-AAA-TGT-G

GO79 ACG-ATA-TAC-TCC-ACA-AAC

GO80 CCA-ATG-TTC-GGC-ACT-CCG

GO81 CAT-CTG-ATA-CAG-CTG-CAG-GC


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GO82 CTT-CAG-AGA-CAT-TTG-CAA-GC

GO83 CTA-TGA-GTC-TAG-TGC-CAT-TTC

GO84 GAT-TAT-CAA-GGA-GTG-GAA-TAC

GO85 CAT-TGC-ATC-TCT-AGT-TGA-TAC

GO96 ATA-CGC-CCA-GTT-CAA-CAG-TGG

GO97 ATA-CGC-CCA-GTT-CAA-CAG-TGA

GO104 CAA-TGT-TGT-TGA-TGC-AGC-ATT

GO105 CAA-TGT-TGT-TGA-TGC-AGC-ATA

GO106 TTC-ACA-TCC-TGC-ATC-ACG-TC

GO107 GTA-GAA-CTG-GTA-TGT-CTA-CGT

GO108 CTG-TTT-TAC-TAA-GCT-GAG-ATT-TGG

MGO5 CTT-ATC-AAG-ACA-TCC-AGT-GC

MGO6 CCA-AAA-GCT-GGA-GAC-GAG-CC

MGO7 CCT-GCT-CCG-CTG-AAC-TCT-GTT-G

MGO10 AGC-GCA-CAG-GGA-GAT-TCC-GG

MGO24 CCA-AGT-ATT-TTA-GAT-GGT-TCT-ACG

MGO25 GGT-AGA-TGT-CCA-TCA-CTG-AGG

LBb1 GCG-TGG-ACC-GCT-TGC-TGC-AAC-T

LB3 TAG-CAT-CTG-AAT-TTC-ATA-ACC-AAT-CTC-GAT-ACA-C

Cosegregation analyses of dra2-1 and the mutant-like phenotype.

Seedlings from the F2 generation derived from the dra2-1 x Ws-2 cross

were grown under continuous W for 7 days. Six pools of 5-6 seedlings each

were selected, three displaying wild-type (w1, w2 and w3) and three mutant

(m1, m2, and m3) phenotypes. Genomic DNA was extracted from the pools and

PCR, using specific oligonucleotides, was employed to genotype the DRA2

(GO96 + GO75) and dra2-1 (GO97 + GO75) alleles, that were separated in a

1% (w/v) agarose gel electrophoresis (Figure S1F).


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Complementation of the dra2-1 phenotype with DRA2-GFP

Transgenic lines overexpressing a translational fusion of DRA2 to the

GREEN FLUORESCENT PROTEIN (GFP) marker gene (35S:DRA2-GFP lines)

were generated in the Ws-2 background (Figure S2B). Although no GFP activity

was detected in any independent transgenic line, some displayed a subtle

phenotype (mildly curly and wavy leaves, Figure S2D), suggesting that low

levels of transgenic protein were produced. One of these 35S:DRA2-GFP

transgenic plants (line #01) was crossed to dra2-1, and F2 plants homozygous

for dra2-1 and hemizygous for the transgene were isolated (Figure S2C). In

their selfed F3 progeny, only a fourth of the seedlings were phenotypically

mutant, indicating that DRA2-GFP complements in a dose-dependent,

recessive manner the loss of function caused by dra2-1 (Figure 1D).

Analysis of hypocotyl length of nup62-1 seedlings

We noticed that only homozygous nup62-1 (i.e., not homozygous nup62-

2) plants were sterile and produced no seeds in our growing conditions. To

analyze the hypocotyl elongation of homozygous nup62-1, about 50 seeds

produced by heterozygous nup62-1/+ adult plants (confirmed by PCR

genotyping) were sown in parallel with wild-type (Col-0) seeds, grown as a

control. Simulated shade experiments were performed as indicated, and 10-20

mutant-like (i.e., displaying long hypocotyls and cotyledons curled downwards

under both light treatment) and wild-type seedlings were selected from both W

and W+FR treated plates (day 7) (Figure S4E). This experiment was repeated 3

times giving similar results. Previously, we demonstrated that the selected

mutant-like seedlings were genotyped as homozygous nup62-1.


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Generation of plants that overexpress full length DRA2 in plants

To generate a construct to overexpress DRA2 fused to GFP under the

control of the 35S promoter (35S:DRA2-GFP) the following overall cloning

strategy was implemented: (1) DRA2 full-length coding sequence (3125 bp) was

split up into two fragment named as DRA2n (covering from 1 to 1360 bp) and

DRA2c (covering from 1361 to 3126 bp). (2) Both fragments were subcloned

into a commercial cloning vector (pCRII-TOPO, www.invitrogen.com). They

were sequenced to confirm their identity. (3) DRA2c was fused to GFP by

subcloning it into a binary vector (pCAMBIA1302). (4) DRA2n was fused in front

of DRA2c using the plasmid generated 35S:DRA2c-GFP in the previous step.

As a result, the construct re-established the original full-length cDNA of DRA2

fused to GFP was under the 35S promoter in the pCAMBIA1302-based binary

vector.

To subclone the DRA2n fragment, the respective coding sequence was

PCR-amplified with CTO4 (5´-ggc-cat-ggT-TGG-CTC-ATC-TAA-TCC-TTT-TG-

3’, which introduced an NcoI site and an additional Ala after the Met, that is not

present in the original protein) and CTO5 (5´-ggg-cta-gcA-ATT-GTT-GGG-GTT-

TGA-G-3´, which introduced an NheI site) oligonucleotide combination using

cDNA from Col-0 Arabidopsis plants as a template [sequences corresponding to

the original coding regions are indicated in uppercase, added sequences are

indicated in lowercase and introduced restriction sites used for cloning are

underlined]. The corresponding PCR product, flanked by NcoI and NheI

restriction sites, was directionally subcloned into pCRII-TOPO to give pCT6.

The pCT6 insert was sequenced to confirm its identity. To subclone the DRA2c


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http://www.invitrogen.com/

fragment and to facilitate its fusion to GFP, the original stop codon was

removed and the respective coding sequence was PCR-amplified with CTO6

(5´-gga-cta-gtT-CGA-GTT-TTG-GAA-CGG-3´, which introduced an SpeI site)

and CTO8 (5’-ggt-cta-gAA-ACT-CCA-TCT-TCT-TCA-TCT-TCG-TCG-C-3´,

which introduced an XbaI site) oligonucleotide combination using cDNA from

Col-0 Arabidopsis plants as a template. The corresponding PCR product,

flanked by SpeI and XbaI restriction sites, was directionally subcloned into

pCRII-TOPO to give pCT7. The pCT7 insert was sequenced to confirm its

identity. Non-silent point mutations were found in the DRA2c fragment of pCT7

insert in all the generated clones; these mutations were always located after an

internal BamHI site. To replace the erroneous final part of DRA2c, a smaller C-

terminal fragment DRA2 was PCR-amplified with GO96 and CTO8

oligonucleotide combination using cDNA from Col-0 Arabidopsis plants as a

template. The corresponding PCR product was directionally subcloned into

pCRII-TOPO to give pCT11. The pCT11 insert was sequenced to confirm its

identity and no mutations were found in between the internal BamHI and the C-

terminal XbaI sites. Next, to generate the recomposed DRA2c fragment, pCT11

and pCT7 plasmids were digested with BamHI. Plasmid pCT11, containing the

correct coding sequence of the final part of the DRA2c between BamHI-XbaI

sites, was used as a vector. Fragment from pCT7, containing the correct coding

sequence of the first part of the DRA2c between its SpeI-BamHI sites, was used

as an insert. The resulting plasmid, pCT29, was equivalent to pCT7 containing

the recomposed SpeI-XbaI insert of the DRA2c fragment. Next, the SpeI-XbaI

fragment of pCT29 was subcloned into the binary vector pCAMBIA1302

digested with SpeI to give the pCT8 (35S:DRA2c-GFP). Finally, the NcoI-NheI


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fragment of pCT6 was subloned into pCT8 digested with SpeI, which gave

pCT9 (35S:DRA2-GFP), that allows to overexpress full-length DRA2 fused to

GFP (35S:DRA2-GFP).

The binary plasmid pCT9 was used to transform Arabidopsis Ws-2 plants

via Agrobacterium tumefaciens by the floral dip method (Clough and Bent,

1998). The resulting transgenic plants (named as pCT34) were selected as

hygromycin resistant. Only lines with a single T-DNA insertion (as estimated

from the segregation of the marker gene in T2 populations) were eventually

selected.

Generation of plants that overexpress NtDRA2 in plants

To generate the construct to overexpress the NtDRA2 (M1-W781, 2.343

bp) fused to the GFP under the control of the 35S promoter (35S:NtDRA2-GFP)

the following strategy was used: (1) we used the DRA2n fragment from pCT6,

which covered from M1 to S456; (2) we clone the C-terminal part of NtDRA2,

covering from S457 to Q779, into the binary vector pCAMBIA1302; (3) the full

NtDRA2 was reconstituted by fusing the fragment from pCT6 into the binary

vector generated in step 2.

To generate the C-terminal part of NtDRA2 (977 bp), CTO6 and MGO36

(5’-ggt-cta-gaC-CAC-TGT-TGA-ACT-GGG-CGT-ATA-AC-TAG-AGC-3’, with

introduced XbaI site) oligonucleotides were used using pCT9 as a template.

The PCR fragment cloned on the pCRII-TOPO vector (named as pMG52) was

sequenced to confirm its identity. The SpeI-XbaI fragment from pMG52 was

cloned into pCAMBIA1302 digested with SpeI (construct pMG54). Finally, the

NcoI-NheI fragment from pCT6 was ligated into pMG54 digested with NcoI and


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SpeI, to give pMG56 (35S:NtDRA2-GFP). The binary plasmid pMG56 was used

to transform Arabidopsis plants (Col-0 ecotype) as previously described. The

resulting transgenic plants were selected as hygromycin resistant. Only lines

with a single T-DNA insertion (as estimated from the segregation of the marker

gene in T2 populations) were eventually selected.

Generation of RNAi-DRA2 plants

To generate an RNAi construct to silence the endogenous DRA2, a

fragment of 318 bp was PCR-amplified using the primers GO96 and SPO1 (5’-

AAG-AGC-CTC-GAT-ATC-TGC-AC-3’) and the vector pCT9 as a template. This

DRA2 region was selected because it showed less similarity with DRAL (64.43

% of nucleotide identity) compared to the whole DRA2 and DRAL nucleotide

coding sequences (77.23 % identity); sequence comparison in this region

indicated that identical sequences had a maximum of 12 nucleotides in length,

which likely prevented cross-silencing (Figure S3A). PCR product was

directionally subcloned into pCRII-TOPO to generate pSP30. The pSP30

fragment was sequenced to confirm its identity. A XhoI-BamHI fragment of

pSP30 was subcloned into the same sites of pENTR3C vector (Invitrogen),

flanked by the attL1 and attL2 sites, to give pSP31. Using the Gateway LR

Clonase II (Invitrogen), in vitro recombination with pHELLSGATE12 destination

vector (Wesley et al., 2001), containing attR1 and attR2 sites, generated pSP32

(35S:RNAi-DRA2).

The binary plasmid pSP32 was used to transform Arabidopsis Ws-2

plants via Agrobacterium using the floral dip method (Clough and Bent, 1998).

The resulting transgenic plants were selected as kanamycin resistant.


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Generation of constructs to visualize DRA2 in plants

Because no GFP activity was detected in any independent transgenic

line overexpressing DRA2-GFP, a construct to overexpress a triple fusion GFP-

DRA2-GFP under the control of the 35S promoter (35S:GFP-DRA2-GFP) was

generated. The GFP ORF was PCR-amplified using the primers SPO40 (5’-

GGC-CAT-GGT-AGA-TCT-GAC-TAG-TAA-3’, which introduced an NcoI site)

and SPO41 (5’-GGC-CAT-GGA-CAC-GTG-GTG-GTG-GTG-G-3’, which

introduced an NcoI site) and the vector pCAMBIA1302 as a template. The PCR

product was subcloned into pCRII-TOPO to generate pSP76, whose insert was

sequenced to confirm its identity. A NcoI fragment of pSP76 was directionally

cloned into the same site of pCT9 to give pSP77 (35S:GFP-DRA2-GFP). This

construct was used to transiently express the protein in leaves of Nicotiana

benthamiana.

RNA blot analyses

Total RNA was isolated from seedlings, separated by electrophoresis (10

µg) and blotted as indicated elsewhere (Roig-Villanova et al., 2006). Probe for

At1g10390 (DRA2) was made by amplifying Col-0 genomic DNA with specific

oligonucleotides GO76 (sequence shown above) and GO77 (5’-CAT-TGT-TTG-

TCC-AAA-GGG-AG-3’). PCR product was subcloned into pCRII-TOPO to give

pMG30. Insert was sequenced for identity confirmation. DNA inserts, isolated by

PCR using specific oligonucleotides, were radioactively labeled and purified as

indicated elsewhere (Sorin et al., 2009). Hybridization, washes and exposure

were carried out as described (Roig-Villanova et al., 2006). Images were


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visualized by using a Molecular Imager FX (Bio-Rad, www.bio-rad.com).

Expression levels were normalized with the 25S rRNA signal.

Oligonucleotides used for qPCR analyses

Gene name

and code Name, oligonucleotide sequences (5→3)

HFR1,

At1g02340

BO89, GAT-GCG-TAA-GCT-ACA-GCA-ACT-CGT

BO90, AGA-ACC-GAA-ACC-TTG-TCC-GTC-TTG

PIL1,

At2g46970

BO87, GGA-AGC-AAA-ACC-CTT-AGC-ATC-AT

BO88, TCC-ATA-TAA-TCT-TCA-TCT-TTT-AAT-TTT-GGT-

TTA

PHYB,

At2g18790

MGO16, GCG-ACC-ATT-GTC-AAC-TGC-TAG-T

MGO17, GAG-CTG-AGC-TGA-ACG-CAA-AT

LUC,

no code

MGO18, GCT-GGA-AGA-TGG-AAC-CGC-T

MGO19, CCA-CCT-CGA-TAT-GTG-CAT-CTG-T

DRA2,

At1g10390

SPO17, CAC-CAA-CTG-TTG-AGG-CAG-ACA

SPO18, GGC-AGA-AAT-AGA-TTC-CAA-CTT-TCC

DRAL,

At1g59660

MGO46, ACG-GTG-CAA-TTC-GTG-AAG-CT

MGO47, TTT-TGT-CGC-CTC-CGT-GAT-TT

UBQ10,

At4g05320

BO40, AAA-TCT-CGT-CTC-TGT-TAT-GCT-TAA-GAA-G

BO41, TTT-TAC-ATG-AAA-CGA-AAC-ATT-GAA-CTT

Whole-mount in situ hybridization of polyA RNA

Plant material was mounted in water on glass slides. Poly(A) RNA in situ

hybridization was conducted essentially as described (Gong et al., 2005) with

minor modifications. Briefly, four 7-day-old seedlings at a similar developmental

stage were fixed and dehydrated as described (Gong et al., 2005). Two mL of

Hyb Plus hybridization buffer (Sigma-Aldrich; H-7033, www.sigmaaldrich.com)

was used for the hybridization with 15 pmol of 45-mer oligo(dT) labeled with one


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http://www.sigmaaldrich.com/

molecule of fluoresceine at the 5'-end (synthesized by Sigma-Aldrich Company)

at 50ºC in darkness for more than 8 h. Washes were performed at 50ºC in

darkness, first with 2x SSC, 0.1 % (w/v) SDS (30 min) and then with 0.5x SSC,

0.1 % (w/v) SDS (5 min). After washing, samples were immediately observed

with confocal microscope. Fourteen to sixteen optical sections in 1.5 µm steps

were collected and projected with the LAS AF Lite software (Leica microscope).

Experiments were repeated at least twice with similar results.

Agroinfiltration in tobacco leaves

Nicotiana benthamiana plants were transiently transfected by

agroinfiltration with constructs to express DRA2-GFP, GFP-DRA2-GFP and/or

mCherry-ER proteins. mCherry-ER localizes in the endoplasmic reticulum

(construct C307). For the co-agroinfiltration (Figures 6, S8), equal volumes of

the Agrobacterium transformed cultures (the GFP derived construct and/or the

mCherry; and the strain expressing the HcPro protein) were mixed (Vilela et al.,

2013). Confocal observations were performed 3 days after infiltration. Ten

optical sections in 1.0 µm steps were collected and projected with the Olympus

Fluoview viewer software (Olympus microscope). Experiments were repeated at

least twice with similar results.

Microbombardments of leek epidermal cells

Transient expression of NtDRA2-GFP and dsRED constructs (Figure 5A)

was performed via co-bombardment with the corresponding DNA plasmids of

leek epidermal cells using a Biolistic PDS1000/He system (Bio-Rad) according

to the manufacturer's protocol. After bombardment, epidermal cells were


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recovered on plates of GM- supplemented with 1% (w/v) sucrose for 12-18 h at

22ºC in the dark before analyzing with confocal microscope.

Accession numbers

Sequence data from this paper can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the following accession numbers:

DRA2/NUP98A (At1g10390), DRAL/NUP98B (At1g59660), HFR1 (At1g02340),

NUP58/TCU1 (At4g37130), NUP54 (At1g24310), NUP62 (At2g45000), PHYB

(At2g18790), PIL1 (At2g46970), SAR1/NUP160 (At1g33410), and

SAR3/NUP96 (At1g80680).

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SUPPLEMENTARY FIGURES

Figure S1. Phenotypes and segregation analyses of dra2-1 plants. (A) Aspect

of representative 6-week-old adult PBL and dra2-1 plants grown under SD for 3

weeks and then transferred to LD for 3 additional weeks. (B) Detail of siliques of

the plants shown in B. Bar corresponds to 5 mm. (C) Aspect of representative

6-week-old adult PBL and dra2-1 seedlings grown under SD. (D) Length of

cotyledons and primary leaves (PL) of PBL and dra2-1 in response to W+FR.

Seeds were germinated and grown as indicated in Figure 1C. Different letters

denote significant differences (one-way ANOVA with Tukey test, P<0.05)

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among means, and red asterisks indicate significant differences (two-way

ANOVA, **P<0.01) between the mutant and wild-type genotypes in response to

W+FR. ��%� Mutation found in the DNA sequence of the At1g10390 gene. The

predicted amino acid sequences are shown below. ��&� Cosegregation analyses

of the dra2-1 mutation and the mutant-like phenotype. Seedlings from the F2

generation of the dra2-1 x Ws-2 cross were grown under continuous W for 7

days. PCR products using specific oligonucleotides were subjected to a 1%

(w/v) agarose gel electrophoresis.

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Figure S2. DRA2 corresponds to At1g10390 gene. (A) Map-based cloning

strategy. The molecular markers in chromosome 1 used for linkage analyses

are indicated. Some light-related genes at the top of chromosome 1 are also

indicated. (B) Mutant dra2-1 phenotype is complemented by the overexpression

of At1g10390. Cartoon describing the construct used to complement the dra2-1

mutant phenotype, named as 35S:DRA2-GFP. (C) Diagram shows the cross

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performed between transgenic 35S:DRA2-GFP and mutant dra2-1 plants for

posterior analyses. (D) Aspect of representative 6-week-old adult Ws-2, dra2-1,

35S:DRA2-GFP and dra2-1;35S:DRA2-GFP plants grown under SD for 3

weeks and then transferred to LD for 3 additional weeks. All images are shown

to the same scale. Bar corresponds to 20 mm.

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Figure S3. RNAi-DRA2 plants resemble dra2-1 mutants. (A) Nucleotide

sequence comparison of the region of DRA2 employed to generate the

35S:RNAi-DRA2 and the corresponding region in DRAL. (B) Representative 7-

day-old seedlings (from left to right) of wild type (Ws-2), dra2-1 and two

independent lines of RNAi-DRA2 showing a strong phenotype (lines #05, #15).

(C) Aspect of representative 7-day-old seedlings of Ws-2 and the two

independent RNAi-DRA2 lines (#16 and #27) shown in Figure 1E.

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Figure S4. Structure of DRA2 mutant alleles employed in this work. (A)

Schematic representation of DRA2 (At1g10390) genomic structure, including

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the position of oligonucleotides designed for PCR analyses as arrows. Black

box, covering from GO76 and GO77, indicates the probe employed for the RNA

blot analyses. (B) Expression levels of DRA2 in the corresponding mutant lines

represented in A. Total RNA (10 µg) was extracted from 7-day-old plants. 25S

rRNA levels are shown as a loading control. (C) Hypocotyl length of wild-type

(Col-0) and mutant dra2 seedlings. (D) Backcross of dra2-1 in Col-0 results in

an attenuation of the mutant phenotype. A diagram is shown with the

successive backcrosses of dra2-1 with Col-0. After the first backcross, dra2-

1_BC1 seedlings were selected visually. In the following backcrosses, dra2-1

seedlings were selected after PCR genotyping. In the dra2-1_BC4, the

hypocotyl elongation in response to simulated shade was measured and

compared to that of Col-0 and dra2-4 mutant seedlings. In parts C and D, seeds

were germinated and grown as indicated in Figure 1C. In graphs of sections C

and D, different letters denote significant differences (one-way ANOVA with

Tukey test, P<0.05) among means, and red asterisks indicate significant

differences (two-way ANOVA, *P<0.05, **P<0.01) between the mutant and wild-

type genotypes in response to W+FR.

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Figure S5. Hypocotyl elongation response to simulated shade of NUP-deficient

seedlings. Scheme showing the genomic organization of (A) NUP54 and (B)

NUP62 genes. The location of T-DNA insertions and oligonucleotides used for

genotyping is indicated. Hypocotyl length in response to simulated shade was

measured in Col-0, (C) nup54-1, nup54-2, (D) nup62-1 and nup62-2 mutant

seedlings. Seedlings were grown as described in Figure 1C. Mutant nup62-1

plants were sterile, so we worked with segregating heterozygous plants. (E)

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Representative 7-day-old Col-0 and nup62-1 seedlings grown under W (left) or

W+FR (right), as indicated in part D. (F) Hypocotyl length of wild-type, single

tcu1-2, dra2-3, dra2-5, and double tcu1-2;dra2-3 and tcu1-2;dra2-5 mutants in

response to simulated shade. Seedlings were grown as indicated in Figure 1C.

In sections C, D and F, different letters denote significant differences (one-way

ANOVA with Tukey test, P<0.05) among means, and red asterisks indicate

significant differences (two-way ANOVA, **P<0.01) between the mutant and

wild-type genotypes in response to W+FR.

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Figure S6. Different NUP-deficient mutants display changes in DRAL gene

expression. Expression analysis of DRAL gene in seedlings of wild-type (Col-0

or Ler), tcu1-1, sar3-3, tcu1-2, dra2-4 and the double tcu1-2;dra2-4 mutants.

Seedlings were grown under continuous W for 7 days. Transcript abundance of

DRAL (normalized to UBQ10) is shown. Values are means ± SE of 4-6

independent biological replicates relative to wt values. Asterisks indicate

significant differences (Student’s t test, **P<0.01) relative to the wild-type

seedlings¸and different letters denote significant differences (one-way ANOVA

with Tukey test, P<0.05) among means.

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Figure S7. Shade-induced expression of ATHB2 is not altered on dra2-1.

Expression analysis of ATHB2 in seedlings of wild-type and dra2-1 seedlings

treated for 0, 1, 2 and 4 h with W+FR. Seedlings were grown under continuous

W for 7 days (as in Figure 4). Transcript abundance of ATHB2, normalized to

UBQ10, is shown. Values are means ± SE of three independent quantitative

PCR biological replicates relative to wild-type values at 0 h. Different letters

denote significant differences (one-way ANOVA with Tukey test, P<0.05)

among means, and red line indicate no significant differences (two-way

ANOVA) between the mutant and wild-type genotypes in response to W+FR. D

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Figure S8. DRA2 is localized in the cytoplasm, within the nucleus and in the

nuclear rim. (A) Expression analysis of DRA2 gene in seedlings of wild-type

(Ws-2) and two independent transgenic 35S:DRA2-GFP lines. Seedlings were

grown under continuous W for 7 days. Transcript abundance of DRA2,

normalized to UBQ10, is shown. Values are means ± SE of three independent

quantitative PCR biological replicates relative to wild-type values. Different

letters denote significant differences (one-way ANOVA with Tukey test, P<0.05)

among means. (B) Z stack of confocal images of leaf tobacco cell co-

agroinfiltrated with constructs GFP-DRA2-GFP and mCherry-ER (see Figure

6B, lower images). The 3 shown images (from left to right: green fluorescence,

red and green fluorescence overlay, and bright-field images) are the overlay of

10 optical sections. (C) Series of the optical section images in order from top (1)

to bottom (10) shown as a Z stack in section B. Only the red and green

fluorescence overlay image is shown. Image 5 corresponds to the one shown in

Figure 6B, lower part. All images are shown to the same scale. Scale bar = 20

μm.

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Table S1. Summary of facts of interest of FG-containing genes in Arabidopsis

and human Nup98.

AGI code (name)

Number of FG repeats

Length (in amino acids)

a Location of FG repeats in:

At1g75340 (CG1)

6 435 No (34-360)

At1g55540 (NUP214)

21 1819 C-terminal (553-1801)

At1g10390 (DRA2/NUP98a)

65 1041 N-terminal (2-677)

At1g59660 (DRAL/NUP98b)

52 997 N-terminal (2-661)

At2g45000 (NUP62)

21 739 N-terminal (6-450)

At4g37130 (TCU1/NUP58)

6 513 C-terminal (446-498)

At1g24310 (NUP54)

7 377 N-terminal (2-88)

At3g10650 (NUP136)

26 1309 C-terminal (78-1256)

At1g52380 (NUP50a)

4 440 Central (245-274)

At3g15970 (NUP50b)

3 465 Central (266-298)

Name (Accession)

Number of FG repeats

Length (in amino acids)

Location of FG repeats in the

Human Nup98 (AAH41136)

39 920 N-terminal (6-497)

(a) It refers to whether there is a preferential location of the FG repeats within

the whole amino acid sequence: “No” indicates that FG repeats are spread all

over the amino acid sequence; N-terminal, Central and C-terminal indicate that

most of the FG repeats are located in this region. Brackets indicate the precise

location of the first and last FG repeat in the amino acid sequence.


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Table S2. Summary of Tukey’s Multiple Comparison test for the PHYB, PIL1

and HFR1 expression analyses in Col-0, sar1-4 and sar3-1 seedlings

(experiments shown in Figure 4B). n.s., not significant; *, significant P<0.05.

PHYB:UBQ10 expression. 0 h 1 h 2 h 4 h

sar1-4 sar3-1 Col-0 sar1-4 sar3-1 Col-0 sar1-4 sar3-1 Col-0 sar1-4 sar3-1 0 h Col-0 n.s. n.s. * * * * n.s. n.s. * n.s. n.s.

sar1-4 n.s. * * * * n.s. * * n.s.sar3-1 * * * * * * * *

1 h Col-0 * * * * * * * * sar1-4 n.s. n.s. n.s. n.s. n.s. n.s. n.s.sar3-1 n.s. * n.s. n.s. * *

2 h Col-0 * n.s. n.s. * * sar1-4 n.s. n.s. n.s. n.s.sar3-1 n.s. n.s. n.s.

4 h Col-0 n.s. n.s.sar1-4 n.s.

PIL1:UBQ10 expression. 0 h 1 h 2 h 4 h

sar1-4 sar3-1 Col-0 sar1-4 sar3-1 Col-0 sar1-4 sar3-1 Col-0 sar1-4 sar3-1 0 h Col-0 n.s. n.s. * * * * * * n.s. * *

sar1-4 n.s. * * * * * * n.s. * * sar3-1 * * * * * * n.s. * *

1 h Col-0 n.s. n.s. * n.s. n.s. * n.s. * sar1-4 n.s. * n.s. n.s. * * * sar3-1 * n.s. n.s. * n.s. *

2 h Col-0 * * n.s. n.s. n.s.sar1-4 n.s. * n.s. * sar3-1 * n.s. *

4 h Col-0 n.s.sar1-4 n.s.

HFR1:UBQ10 expression. 0 h 1 h 2 h 4 h

sar1-4 sar3-1 Col-0 sar1-4 sar3-1 Col-0 sar1-4 sar3-1 Col-0 sar1-4 sar3-1 0 h Col-0 n.s. n.s. * * * * * * * * *

sar1-4 n.s. * * * * * * * * * sar3-1 * * * * * * * * *

1 h Col-0 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.sar1-4 n.s. n.s. n.s. n.s. n.s. n.s. n.s.sar3-1 * n.s. * * n.s. n.s.

2 h Col-0 n.s. * n.s. * n.s.sar1-4 n.s. n.s. n.s. n.s.sar3-1 n.s. n.s. n.s.

4 h Col-0 n.s. n.s.sar1-4 n.s.


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development 143: doi:10.1242/dev.130211:...

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