DRACULA2 is a dynamic nucleoporin with a role in regulating the shade. Marçal Gallemí, Anahit Galstyan, Sandi Paulišić, Christiane Then, Almudena

Transcription

1 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 ), sar3-1 and sar3-3 (SALK_109959) are in Col-0 background and have been described previously (Parry et al., 2006; Parry, 2014).

2 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 F 2 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 F 2 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 F 2 plant, the corresponding F 1 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 F 2 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 F 2 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 ( not all of which were found polymorphic between Ws-2 and Col-0. Synthetic oligonucleotides for fine mapping are described below.

3 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) cer (At1g10560-At1g10570) cer (At1g10640-At1g10650) F16J7-TRB (At1g11370) JV28/29 (At1g11730-At1g11735) JV26/27 (At1g11905) Oligonucleotides position sequences (5 3) Forward CTA-GTT-GAA-GTC-GCA-AAA-TGT-TG Reverse GAC-TAA-TAG-CAT-GCT-TCC-AAT-TC Forward CAT-TAC-ACT-AGA-GAC-TAG-AC Reverse TAA-TAT-ACA-TGT-GAG-CAT-CCT- GAC Forward GTG-TCT-TGA-TAC-GCG-TCG-ATC Reverse TGA-TGT-TGA-GAT-CTG-TGT-GCA-G Forward GAT-ACT-CCT-GTT-TCA-CAT-ATA-TG Reverse GAG-AGT-CCT-TAT-TGT-TGT-GCC 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

4 At1g10390-F5 At1g10390-R5 At1g10390-F6 At1g10390-R6 GTT-GTG-TTA-TTT-TAG-CTT-CAT-CG GTC-CGA-GAA-TTT-CCA-CAC-ATA-G CTT-GAT-TAC-TCT-GCG-TGT-GAG 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 JO402 JO403 GO74 GO75 GO76 GO78 GO79 GO80 GO81 Oligonucleotide sequences (5 3) GGT-CGA-AGA-ACG-TGT-GTC-C GGT-ACC-AGA-TGA-CTG-TCC CAC-TGA-TGA-CGA-AGA-GAG CCA-TAA-CCG-TGT-CGT-CCC GAT-CTT-CTG-GTT-TTG-GGC-AG CCA-AAT-TTG-TTA-AAA-TGT-G ACG-ATA-TAC-TCC-ACA-AAC CCA-ATG-TTC-GGC-ACT-CCG CAT-CTG-ATA-CAG-CTG-CAG-GC

5 GO82 GO83 GO84 GO85 GO96 GO97 GO104 GO105 GO106 GO107 GO108 MGO5 MGO6 MGO7 MGO10 MGO24 MGO25 LBb1 LB3 CTT-CAG-AGA-CAT-TTG-CAA-GC CTA-TGA-GTC-TAG-TGC-CAT-TTC GAT-TAT-CAA-GGA-GTG-GAA-TAC CAT-TGC-ATC-TCT-AGT-TGA-TAC ATA-CGC-CCA-GTT-CAA-CAG-TGG ATA-CGC-CCA-GTT-CAA-CAG-TGA CAA-TGT-TGT-TGA-TGC-AGC-ATT CAA-TGT-TGT-TGA-TGC-AGC-ATA TTC-ACA-TCC-TGC-ATC-ACG-TC GTA-GAA-CTG-GTA-TGT-CTA-CGT CTG-TTT-TAC-TAA-GCT-GAG-ATT-TGG CTT-ATC-AAG-ACA-TCC-AGT-GC CCA-AAA-GCT-GGA-GAC-GAG-CC CCT-GCT-CCG-CTG-AAC-TCT-GTT-G AGC-GCA-CAG-GGA-GAT-TCC-GG CCA-AGT-ATT-TTA-GAT-GGT-TCT-ACG GGT-AGA-TGT-CCA-TCA-CTG-AGG GCG-TGG-ACC-GCT-TGC-TGC-AAC-T 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 F 2 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).

6 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 F 2 plants homozygous for dra2-1 and hemizygous for the transgene were isolated (Figure S2C). In their selfed F 3 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 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.

7 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, 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

8 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

9 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 T 2 populations) were eventually selected. Generation of plants that overexpress NtDRA2 in plants To generate the construct to overexpress the NtDRA2 (M 1 -W 781, 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 M 1 to S 456 ; (2) we clone the C-terminal part of NtDRA2, covering from S 457 to Q 779, 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

10 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 T 2 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.

11 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

12 visualized by using a Molecular Imager FX (Bio-Rad, Expression levels were normalized with the 25S rrna signal. Oligonucleotides used for qpcr analyses Gene name and code HFR1, At1g02340 PIL1, At2g46970 PHYB, At2g18790 LUC, no code DRA2, At1g10390 DRAL, At1g59660 UBQ10, At4g05320 Name, oligonucleotide sequences (5 3) BO89, GAT-GCG-TAA-GCT-ACA-GCA-ACT-CGT BO90, AGA-ACC-GAA-ACC-TTG-TCC-GTC-TTG BO87, GGA-AGC-AAA-ACC-CTT-AGC-ATC-AT BO88, TCC-ATA-TAA-TCT-TCA-TCT-TTT-AAT-TTT-GGT- TTA MGO16, GCG-ACC-ATT-GTC-AAC-TGC-TAG-T MGO17, GAG-CTG-AGC-TGA-ACG-CAA-AT MGO18, GCT-GGA-AGA-TGG-AAC-CGC-T MGO19, CCA-CCT-CGA-TAT-GTG-CAT-CTG-T SPO17, CAC-CAA-CTG-TTG-AGG-CAG-ACA SPO18, GGC-AGA-AAT-AGA-TTC-CAA-CTT-TCC MGO46, ACG-GTG-CAA-TTC-GTG-AAG-CT MGO47, TTT-TGT-CGC-CTC-CGT-GAT-TT 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, was used for the hybridization with 15 pmol of 45-mer oligo(dt) labeled with one

13 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

14 recovered on plates of GM- supplemented with 1% (w/v) sucrose for 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). SUPPLEMENTARY REFERENCES Barrero, J. M., Gonzalez-Bayon, R., del Pozo, J. C., Ponce, M. R. and Micol, J. L. (2007) 'INCURVATA2 encodes the catalytic subunit of DNA Polymerase alpha and interacts with genes involved in chromatin-mediated cellular memory in Arabidopsis thaliana', The Plant Cell 19(9): Clough, S. J. and Bent, A. F. (1998) 'Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana', The Plant Journal 16(6): Ferrandez-Ayela, A., Alonso-Peral, M. M., Sanchez-Garcia, A. B., Micol-Ponce, R., Perez-Perez, J. M., Micol, J. L. and Ponce, M. R. (2013) 'Arabidopsis TRANSCURVATA1 Encodes NUP58, a Component of the Nucleopore Central Channel', PLoS One 8(6): e67661.

15 Gong, Z., Dong, C. H., Lee, H., Zhu, J., Xiong, L., Gong, D., Stevenson, B. and Zhu, J. K. (2005) 'A DEAD box RNA helicase is essential for mrna export and important for development and stress responses in Arabidopsis', The Plant Cell 17(1): Kozma-Bognar, L., Hall, A., Adam, E., Thain, S. C., Nagy, F. and Millar, A. J. (1999) 'The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B', Proceedings of the National Academy of Sciences, USA 96(25): Parry, G. (2014) 'Components of the Arabidopsis nuclear pore complex play multiple diverse roles in control of plant growth', J Exp Bot 65(20): Parry, G., Ward, S., Cernac, A., Dharmasiri, S. and Estelle, M. (2006) 'The Arabidopsis SUPPRESSOR OF AUXIN RESISTANCE proteins are nucleoporins with an important role in hormone signaling and development', The Plant Cell 18(7): Ponce, M. R., Robles, P., Lozano, F. M., Brotons, M. A. and Micol, J. L. (2006) 'Low-resolution mapping of untagged mutations', Methods Mol Biol 323: Ponce, M. R., Robles, P. and Micol, J. L. (1999) 'High-throughput genetic mapping in Arabidopsis thaliana', Mol Gen Genet 261(2): Roig-Villanova, I., Bou, J., Sorin, C., Devlin, P. F. and Martinez-Garcia, J. F. (2006) 'Identification of primary target genes of phytochrome signaling. Early transcriptional control during shade avoidance responses in Arabidopsis', Plant Physiology 141(1): Sorin, C., Salla-Martret, M., Bou-Torrent, J., Roig-Villanova, I. and Martinez- Garcia, J. F. (2009) 'ATHB4, a regulator of shade avoidance, modulates hormone response in Arabidopsis seedlings', The Plant Journal 59(2):

16 Vilela, B., Moreno-Cortes, A., Rabissi, A., Leung, J., Pages, M. and Lumbreras, V. (2013) 'The maize OST1 kinase homolog phosphorylates and regulates the maize SNAC1-type transcription factor', PLoS One 8(2): e Wesley, S. V., Helliwell, C. A., Smith, N. A., Wang, M. B., Rouse, D. T., Liu, Q., Gooding, P. S., Singh, S. P., Abbott, D., Stoutjesdijk, P. A. et al. (2001) 'Construct design for efficient, effective and high-throughput gene silencing in plants', The Plant Journal 27(6):

17 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)

18 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.

19 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 At1g Cartoon describing the construct used to complement the dra2-1 mutant phenotype, named as 35S:DRA2-GFP. (C) Diagram shows the cross

20 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.

21 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.

22 Figure S4. Structure of DRA2 mutant alleles employed in this work. (A) Schematic representation of DRA2 (At1g10390) genomic structure, including

23 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 wildtype genotypes in response to W+FR.

24 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)

25 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.

26 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.

27 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.

28 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 coagroinfiltrated 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.

29 Table S1. Summary of facts of interest of FG-containing genes in Arabidopsis and human Nup98. AGI code (name) At1g75340 (CG1) At1g55540 (NUP214) At1g10390 (DRA2/NUP98a) At1g59660 (DRAL/NUP98b) At2g45000 (NUP62) At4g37130 (TCU1/NUP58) At1g24310 (NUP54) At3g10650 (NUP136) At1g52380 (NUP50a) At3g15970 (NUP50b) Number of FG repeats Length (in amino acids) a Location of FG repeats in: No (34-360) C-terminal ( ) N-terminal (2-677) N-terminal (2-661) N-terminal (6-450) C-terminal ( ) N-terminal (2-88) C-terminal ( ) Central ( ) Central ( ) Name (Accession) Human Nup98 (AAH41136) Number of FG Length (in Location of FG repeats amino acids) repeats in the 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.

30 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.