SUPPLEMENTARY INFORMATION

Transcription

1 NaKR1 regulates long-distance movement of FLOWERING LOCUS T in Arabidopsis Supplementary Figure 1. NaKR1 is responsible for the late-flowering phenotype of nakr1-1. a, Schematic diagram shows the site of a single deletion of 7 bp in nakr1-1 and the target site of the artificial microrna (AmiR) in AmiR-nakr1. Exons and introns in the coding region are indicated by black and white boxes, respectively. The start codon (ATG) and stop codon (TGA) are labeled. b, Distribution of flowering time in T1 transgenic plants harboring the NaKR1 genomic fragment (gnakr1) in nakr1-1 background. gnakr1 rescues the late-flowering phenotype of nakr1-1 under LDs. NATURE PLANTS 1

2 Supplementary Figure 2. Effect of NaKR1 on the flowering response to vernalization, GA and changes in ambient temperature. a,b, Flowering time of nakr1-1 and wildtype plants in response to vernalization treatment under SDs (a) and LDs (b). c, Flowering time of nakr1-1 and wild-type plants in response to GA treatment under SDs. Exogenous GA (100 μm) or 0.1% ethanol (mock) was applied weekly from the first week after germination. d, Flowering time of nakr1-1 and wild-type plants in response to changes in ambient temperature under LDs. The ratios of flowering time between 16 C and 23 C (16 C/23 C) and between 23 C and 27 C (23 C/27 C) for wild-type and nakr1-1 are shown in the table. Values were scored from at least 20 plants of each genotype. Error bars in a-d denote SD. 2 NATURE PLANTS

3 Supplementary Figure 3. NaKR1 promotes flowering in Arabidopsis. a, Distribution of flowering time in AmiR-nakr1 T1 transgenic plants grown under LDs. b, Distribution of flowering time in 35S:NaKR1 T1 transgenic plants grown under LDs. c, Downregulation of NaKR1 in independent AmiR-nakr1 T3 transgenic plants under LDs. d, Upregulation of NaKR1 in independent 35S:NaKR1 T3 transgenic plants under LDs. Expression of NaKR1 in c and d was determined by quantitative real-time PCR in independent transgenic seedlings at 9 DAG. Results were normalized against the expression of TUB2 and shown as relative values to the maximal level set at 100%. Error bars in c and d denote SD. NATURE PLANTS 3

4 Supplementary Figure 4. NaKR1 mrna expression is regulated by photoperiod and autonomous pathways. a, NaKR1 expression in photoperiod-pathway mutants at 9 DAG. b, Effect of vernalization treatment on NaKR1 expression. For vernalization treatment, seeds were grown on Murashige and Skoog medium and vernalized at 4 C under low light conditions for 8 weeks. Seedlings at 9 DAG grown under LDs were harvested for expression analysis. c, Effect of GA treatment on NaKR1 expression. For GA treatment, 2-week-old wild-type plants grown under SDs were applied twice weekly with exogenous GA (100 µm) or 0.1% ethanol (Mock). Seedlings treated for 1 week (1W) or 3 weeks (3W) were harvested for expression analysis. d, NaKR1 expression in loss-offunction mutants of several well-known flowering regulators at 9 DAG grown under LDs. e, NaKR1 expression in autonomous-pathway mutants at 9 DAG grown under LDs. Gene expression levels in all panels determined by quantitative real-time PCR were normalized to TUB2 expression and shown as relative values to the maximal level set at 100%. Error bars denote SD. 4 NATURE PLANTS

5 Supplementary Figure 5. NaKR1-4HA is localized in the phloem under shoot apices. a, Analysis of NaKR1-4HA localization by immunogold electron microscopy using anti- HA antibody in companion cells (CCs), sieve elements (SEs) and xylem vessels (XVs) in the vasculature below shoot apices of nakr1-1 gnakr1-4ha seedlings at 11 DAG. Arrows indicate the location of gold particles. Scale bars, 1 μm. b, Quantification of NaKR1-4HA immunogold signals or background signals in sections (as shown in a) probed with anti-ha antibody or mouse IgG control, respectively, in various cells of nakr1-1 gnakr1-4ha. The data are presented as the mean number of gold particles per µm 2 with standard deviation. Statistical analysis was performed using a two-tailed paired Student s t test. The results are considered statistically significant at P < c, Frequency histograms of appearance of NaKR1-4HA immunogold signals or background signals in sections (as shown in a) probed with anti-ha antibody or mouse IgG control, respectively, in various cells of nakr1-1 gnakr1-4ha. NATURE PLANTS 5

6 Supplementary Figure 6. Control experiments for examining NaKR1-4HA localization by immunogold transmission electron microscopy. a, Western blot analysis of NaKR1-4HA expression in nakr1-1 gnakr1-4ha. NaKR1-4HA is specifically detectable by anti-ha antibody in total proteins extracted from nakr1-1 gnakr1-4ha seedlings at 11 DAG. b, Analysis of NaKR1-4HA localization by immunogold electron microscopy using anti-ha antibody in companion cells (CCs), sieve elements (SEs) and xylem vessels (XVs) in the vasculature of the first rosette leaves of wild-type seedlings at 11 DAG. Scale bars, 1 μm. c, Quantification of NaKR1-4HA immunogold signals in sections (as shown in b) or background signals probed with anti- HA antibody or mouse IgG control, respectively, in various cells of wild-type plants. The data are presented as the mean number of gold particles per µm 2 with standard deviation. Statistical analysis was performed using a two-tailed unpaired Student s t test. There are no statistically significant differences in samples probed with anti-ha antibody and mouse IgG control. d, Frequency histograms of appearance of NaKR1-4HA immunogold signals in sections (as shown in b) or background signals probed with anti-ha antibody or mouse IgG control, respectively, in various cells of wild-type plants. 6 NATURE PLANTS

7 Supplementary Figure 7. Subcellular localization of FT-GFP in N. benthamiana leaf epidermal cells. GFP, GFP fluorescence; BF, bright field image; ER-RFP, RFP fluorescence of an ER marker; Merge, merge of GFP, bright field and RFP images. Scale bar, 10 μm. NATURE PLANTS 7

8 Supplementary Figure 8. NaKR1 does not affect TSF expression or interact with TSF. a, TSF expression in nakr1-1 and wild-type plants at 9 DAG grown under LDs. TSF expression levels determined by quantitative real-time PCR were normalized to TUB2 expression and shown as relative values to the maximal level set at 100%. Error bars denote SD. b, Yeast two-hybrid assay shows no interaction between NaKR1 and TSF. Transformed yeast cells containing AD-NaKR1 and BD-TSF constructs did not survive on selective SD/-His/-Leu/-Trp (middle panel) and SD/-His/-Leu/-Trp/-Ade (right panel) medium. Empty refers to AD- or BD- containing vector only. 8 NATURE PLANTS

9 Supplementary Figure 9. Effect of NaKR1 on FT mrna or protein expression. a, Western blot analysis using anti-gfp antibody shows the comparable abundance of FT- GFP protein in SUC2:FT-GFP and nakr1-1 SUC2:FT-GFP plants at 11 DAG (upper panel). Coomassie blue staining of the large Rubisco subunit (rbcl) was used as a loading control (lower panel). The numbers below each lane indicate relative intensity of FT-GFP protein levels in different genetic backgrounds, calculated by first normalizing each signal for FT-GFP against the signal for rbcl and then against the value of SUC2:FT-GFP (second lane). b, Schematic diagrams of native FT and transgenic SUC2:FT-GFP transcripts. Fragments P1, P2 and P3 indicate amplicons in quantitative real-time PCR analyses shown in c. c, Examination of steady-state levels of the native FT or transgenic FT-GFP mrna in various genetic backgrounds. Amplification of P1 fragment, which only detects the amplicon in native FT transcripts, shows that native FT expression is downregulated in nakr1-1, SUC2:FT-GFP or SUC2:FT-GFP nakr1-1. Amplification of P2 fragment, which detects the amplicon in both native FT and transgenic FT-GFP transcripts, shows that although native FT expression is downregulated in nakr1-1 compared with WT, total FT expression (including minor NATURE PLANTS 9

10 native FT and major transgenic FT-GFP expression) is not changed in SUC2:FT-GFP and SUC2:FT-GFP nakr1-1. Amplification of P3 fragment, which only detects the amplicon in transgenic FT-GFP transcripts, shows that transgenic FT-GFP expression is not changed in SUC2:FT-GFP nakr1-1 versus SUC2:FT-GFP. Asterisks indicate undetectable expression of transgenic FT-GFP transcripts in non-transgenic wild-type and nakr1-1 seedlings. Seedlings at 9 DAG grown under LDs were harvested for expression analysis by quantitative RT-PCR. Results were normalized against the expression of TUB2. Error bars denote SD. d, Flowering time of wild-type, nakr1-1 and nakr1-1 SUC2:NaKR1 plants grown under LDs. Values were scored from 22 plants of each genotype. Asterisk indicates a significant difference in flowering time between nakr1-1 and nakr1-1 SUC2:NaKR1 (two-tailed paired Student s t-test, P < 0.001). Error bars denote SD. e,f, NaKR1 (e) and FT (f) expression in wild-type, nakr1-1 and nakr1-1 SUC2:NaKR1 plants at 9 DAG grown under LDs. Gene expression was determined by quantitative real-time PCR and normalized to TUB2 expression. NaKR1 expression levels are shown as fold change numbers compared to the expression level in wild-type plants set as 1 (e), while FT expression levels are shown as relative values to the level in wildtype plants set at 100% (f). Asterisks indicate a significant difference in gene expression between nakr1-1 and nakr1-1 SUC2:NaKR1 (two-tailed paired Student s t-test, P < 0.001). Error bars denote SD. 10 NATURE PLANTS

11 Supplementary Figure 10. NaKR1 does not affect FTIP1 mrna expression or interact with FTIP1. a, Temporal expression of FTIP1 in developing wild-type and nakr1-1 seedlings grown under LDs. Seedlings were harvested at ZT10 at 5, 7, 9, 11 and 13 DAG for expression analysis. FTIP1 expression levels determined by quantitative real-time PCR were normalized to TUB2 expression and shown as relative values to the maximal level set at 100%. Error bars indicate SD. b, Yeast two-hybrid assay shows no interaction between NaKR1 and FTIP1. Transformed yeast cells containing AD-NaKR1 and BD-FTIP1 constructs do not survive on selective SD/-His/-Leu/-Trp (middle panel) and SD/-His/-Leu/-Trp/-Ade (right panel) medium. Empty refers to AD- or BDcontaining vector only. c, Flowering time of ftip1-1, nakr1-1 and ftip1-1 nakr1-1 grown under LDs. Values were scored from at least 20 plants of each genotype. Asterisk indicates a significant difference in flowering time of ftip1-1 nakr1-1 compared with that of ftip1-1 or nakr1-1 (two-tailed paired Student s t-test, P < 0.01). Error bars denote SD. NATURE PLANTS 11

12 Supplementary Figure 11. Measurement of endogenous sucrose contents in shoot apices of wild-type and nakr1-1 developing plants. Shoot apices of the plants grown under LDs were harvested for sugar extraction at ZT16 at 8, 10 and 12 days after germination (DAG). Error bars indicate SD. 12 NATURE PLANTS

13 Supplementary Figure 12. Phylogenetic analysis of NaKR1 orthologs in different plant species. The phylogenetic tree was constructed with the neighbor-joining algorithm using the program MEGA 5.2 based on the alignment of amino acid sequences of NaKR1 orthologs in dicotyledonous and monocotyledonous plants. Each terminal node of the tree is labeled by the two-letter abbreviation of the corresponding species name and the unique GI number. Bootstrap values (>50%) in 500 replicates are indicated next to the nodes. Bd, Brachypodium distachyon; Br, Brassica rapa; Cs, Cucumis sativus; Gm, Glycine max; Jc, Jatropha curcas; Mt, Medicago truncatula; Os, Oryza sativa; Pt, Populus trichocarpa; Sb, Sorghum bicolor; Sl, Solanum lycopersicum; St, Solanum tuberosum; Zm, Zea mays. The scale bar represents 0.05 amino acid substitution. NATURE PLANTS 13

14 Supplementary Table 1. List of primers used in this study Primers for plasmid construction Construct Prime Name Sequence (5-3 ) AmiR-nakr1 35S/SUC2/KN AT1:NaKR1 gnakr1 gnakr1-gus gnakr1-gfp gnakr1-4ha 35S:NaKR1- GFP 35S:cEYFP- NaKR1 1-AmiR-nakr1-s 2-AmiR-nakr1-a 3-AmiR-nakr1-*s 4-AmiR-nakr1-*a NaKR1-F NaKR1-R gnakr1-f gnakr1-r gnakr1-gus-f gnakr1-gus-r gnakr1-gfp-f gnakr1-gfp-r gnakr1-4ha-f gnakr1-4ha-r NaKR1-GFP-F NaKR1-GFP-R NaKR1-BiFC-F NaKR1-BiFC-R GATTGTTATACGACGTCACTCCTTCTCTCTTTTGT ATTCC GAAGGAGTGACGTCGTATAACAATCAAAGAGAA TCAATGA GAAGAAGTGACGTCGAATAACATTCACAGGTCGT GATATG GAATGTTATTCGACGTCACTTCTTCTACATATATA TTCCT CCATCGATATGTTGTGTGCTTCTCAAGC CCCCCGGGTCATTTCTGAATAATCTCAGGCC CCCCCCGGGTCGTCATCGTCTTCTTGATCTC GGACTAGTAAATTTGTGTTTTTGTTTGAATATGCT TAG TGTGTCATTGGAACCACTCAAGATGTTACGTCCT GTAGAAACCCCAAC AGAGCAAACGGCTGGTCTCATTGTTTGCCTCCCT GCTGC TGTGTCATTGGAACCACTCAAGATGAGTAAAGGA GAAGAACTTTTC AGAGCAAACGGCTGGTCTCATTTGTATAGTTCAT CCATGCCAT TGTGTCATTGGAACCACTCAAGTATCCATATGAC GTTCCAGATTACGC AGAGCAAACGGCTGGTCTCAAGATAAACTAGTTC TAGATAATTCGCTGAAATC CCATCGATATGTTGTGTGCTTCTCAAGC CCCCCGGGTTTCTGAATAATCTCAGGCC CCCCCGGGA ATGTTGTGTGCTTCTCAAGCATC CCCCCGGG TCATTTCTGAATAATCTCAGGCC 14 NATURE PLANTS

15 35S:FT-nEYFP AD-NaKR1 AD-N-NaKR1 AD-HMA- NaKR1 BD-TSF NaKR1-GST FT-BiFC-F FT-BiFC-R AD-NaKR1-F AD-NaKR1-R AD-N-NaKR1-F AD-N-NaKR1-R AD-HMA-NaKR1- F AD-HMA-NaKR1- R BD-TSF-F BD-TSF-R NaKR1-GST-F NaKR1-GST-R CCCCCGGGAATGTCTATAAATATAAGAGACCCTC TTATAG CCCCCGGGAAGTCTTCTTCCTCCGCAG CCCCCGGGTATGTTGTGTGCTTCTCAAGCATC CCATCGATTCATTTCTGAATAATCTCAGGCC CCCCCGGTATGTTGTGTGCTTCTCAAGCATC CCATCGATTCATTGATCGGAAGATGAGTTTTTCTC CCCCCGGGATGGTGGTTGTTTTGAGAGTGTCACT CCATCGATTCATTTCTGAATAATCTCAGGCC GGAATTCCATATGTCTTTAAGTCGTAGAGATCCT CTTG CCCCCCGGGCTACGTTCTTCTTCCCCCACAG ATAAGAATGCGGCCGCAATGTTGTGTGCTTCTCA AGCATC ATAAGAATGCGGCCGCTCATTTCTGAATAATCTC AGGCC Primers for quantitative real-time PCR Primer Name Sequence NaKR1-F 5 -ACTCATCTTCCGATCAAGTGGTTG-3 NaKR1-R 5 -CCGTCACGGTAACCTTCTTTGC-3 FTIP1-F 5 -CTAGTGACAGGAGCCAACCA-3 FTIP1-R 5 -TGTTCTTCAAATGGCTCTGC-3 FT-F 5 -TAGTAAGCAGAGTTGTTGGAGACG-3 FT-R 5 -GGGAAGGCCGAGATTGTAGAT-3 P1(FT)-F 5 -CGAGTAACGAACGGTGATGA-3 P1(FT)-R 5 -CGCATCACACACTATATAAGTAAAACA-3 P2(FT)-F 5 -TAGTAAGCAGAGTTGTTGGAGACG-3 P2(FT)-R 5 -GGGAAGGCCGAGATTGTAGAT-3 P3(GFP)-F 5 -GGCATCAAAGCCAACTTCAA-3 P3(GFP)-R 5 -GGCAGATTGTGTGGACAGGT-3 TSF-F 5 -ATCCTTTCACGAGGTTGGTC-3 TSF-R 5 -CGTCTCCTCCAATCTCCACT-3 NATURE PLANTS 15

16 TUB2-F TUB2-R 5'-ATCCGTGAAGAGTACCCAGAT-3' 5'-AAGAACCATGCACTCATCAGC-3' Primer pairs for ChIP assays on the NaKR1 sequence Primer Name Sequence NaKR1-P1 5 -AGCCTGTTACCACAAACACCA-3' 5 -TCTTGCCTCTTGATCTCCGT-3' NaKR1-P2 5 -AATCCACATCCATTCTACTGATGATATC-3' 5 -CAACAAGCTCTCCAAAATGTATGTCT-3' NaKR1-P3 5 -ATCCAATCAGAATCACGCAA-3' 5 -AATAGGCGGCACATCAGTTC-3' NaKR1-P4 5 -TTTCTGGCTCCAAACCCTAA-3' 5 -GAAATAACTATTGGGCTATTACACGG-3' NaKR1-P5 5 -AAAGGTGTGAATAGTGAAAGCTAATACGA-3' 5 -TAGAAAGGCCAATTCTTTTTGCC-3' NaKR1-P6 5 -CAATCTCTCACACATTGGTTTGATAAG-3' 5 -GTCTTTAGTGTTTGCAATGAAAATGTG-3' NaKR1-P7 5 -TTGCTCAACCATGGATCAAA-3' 5 -ACCGTCACGTATGATTGGGT-3' NaKR1-P8 5 -CACCTCAGGGTTCTTCAAGG-3' 5 -TGGTTATCTCCGGTTCATCA-3' NaKR1-P9 5 -GAAACATCTATCGAAATTGAAAGGTAACTAA-3' 5 -TTATACGACGTCACTCCTGCGTT-3' NaKR1-P10 5 -GTGCGTTTATTTGTCTCGGTTATGA-3' 5 -GCTCCACGGTCTTAACAAGCTAATAC-3' NaKR1-P11 5 -AAATTCCCCTGCATTGTGGTTA-3' 5 -CAATTCCAACAAAACGTTCCAAT-3' NaKR1-P12 5 -ATGAGACCAGCCGTTTGCTC-3' 5 -GGTAACATTCTGAAAATCAGGCG-3' TUB2 5 -ATCCGTGAAGAGTACCCAGAT-3 5 -AAGAACCATGCACTCATCAGC-3 16 NATURE PLANTS

17 Extended Material and Methods Yeast two-hybrid assay. To construct the vectors for yeast two-hybrid assay, the coding regions of NaKR1, its truncated versions, FT and TSF were amplified and cloned into pgadt7 or pgbkt7 (Clontech). The yeast two-hybrid assay was performed using the Yeastmaker Yeast Transformation System 2 (Clontech) according to the manufacturer's instructions. In vitro GST pull-down assay. To construct GST-NaKR1, the NaKR1 coding sequence was cloned into pgex-6p-2 vector (Pharmacia). The GST-NaKR1 fusion protein was produced by inducing E.coli BL21 cells harboring the corresponding construct with IPTG at 16 C overnight. The soluble GST fusion proteins were extracted and immobilized on glutathione sepharose beads (Amersham Biosciences) for subsequent pull-down assays. To produce myc-tagged FT protein, the pgbkt7-ft plasmid was added to the TNT T7 Quick Coupled Transcription/Translation Systems (Promega) to synthesize myc-ft. These epitope-tagged proteins were incubated with the immobilized GST and GST- NaKR1 fusion proteins. Proteins retained on the beads were resolved by SDS-PAGE and detected with anti-myc antibody (Santa Cruz Biotechnology). Coimmunoprecipitation experiments. Aerial parts of young seedlings were harvested and ground with mortar and pestle in liquid nitrogen, and total proteins were extracted with the CelLytic P cell lysis reagent (Sigma-Aldrich). Protein extracts were then incubated with anti-myc antibody bound to Protein G PLUS-Agarose (Santa Cruz Biotechnology) at 4 C for 2 h. Immunoprecipitated proteins and total protein extracts as inputs were resolved by SDS-PAGE and detected by anti-ha antibody (Santa Cruz Biotechnology). Sugar measurement. For assays of sugar concentration, 50 shoot apices of plants grown under LDs were harvested to measure the contents of soluble sugars using the D- Glucose/D-Fructose/Sucrose Assay kit according to the manufacturer s instructions (R- Biopharm). After grinding plant materials in liquid nitrogen, soluble sugars were extracted twice with 80% ethanol at 70 C, and the supernatant was collected after centrifugation. Ethanol was evaporated from the supernatant using a vacuum evaporator, and the solution volume was adjusted to 200 μl by adding water for further measurement of sugar contents. NATURE PLANTS 17