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Transcription

1 AS-NMD modulates FLM-dependent thermosensory flowering response in Arabidopsis NATURE PLANTS 1

2 Supplementary Figure 1. Genomic sequence of FLM along with the splice sites. Sequencing of cdnas reveals FLM undergoes extensive alternative splicing. All the splice sites detected through cloning to be utilized are shown. Donor GT are marked in red and acceptor AG sites in blue. The specific GT donor and AG 2 NATURE PLANTS

3 acceptor sites for which splicing efficiency are represented in Fig 2a are shown in red (GT) or blue (AG) boxes. Exons are shown in black capital letters and introns in green small letters. The 3 UTR as per the TAIR annotation (e.g., for transcripts FLMβ, FLM-δ) is shown in red. Start and stop codons as per TAIR annotation are shown in pink. For all other splice variants the 3 UTR and the stop codons varied as shown in Figure 1b. Only the amplified and cloned region (ATG-3 end) is shown. NATURE PLANTS 3

4 Supplementary Figure 2. A schematic of the predicted proteins likely to be formed due to alternative splicing of FLM. FLM protein belongs to the MIKC type MADS domain proteins, consisting of the DNA binding domain (MADS, red) and intervening region (I, aqua), Keratin like domain (K, pink) and C-terminal domain (C, blue). Predicted protein products from transcripts resulting from temperature-induced splicing changes are shown. For comparison, canonical FLM-β and FLM-δ are also shown. Most of the proteins resulting from alternative splicing will lack the K-domain and C-terminal region, required for dimerisation and specificity. 4 NATURE PLANTS

5 Supplementary Figure 3. Temperature induces enrichment of NMD target transcripts for FLM. (a) Percentage of clones that contain transcripts with or without PTCs in Col-0 and upf1-5 at different temperatures. The effect of temperature is highly significant (chi-square test, p<0.0001) (b) Amplified products using primers described in Pose et al 4 from Col-0 and upf1-5 at 27 ºC short days. The positions of the primers are shown along with the gene structure. FLM-δ primers will not differentiate between FLM-I2RC and FLM-δ. Purple arrow shows the enrichment of the I2RC transcripts in the NMD-defective upf mutants, which are also amplified, NATURE PLANTS 5

6 when the FLM-β primers are used. Multiple lanes represent independent biological replicates. 6 NATURE PLANTS

7 Supplementary Figure 4. Mutations in the NMD pathway abolish thermosensitivity in flowering but not in hypocotyl elongation. (a) Reaction normal of flowering time measured as total leaf number in upf1-5 and upf3-1 mutants compared to Col-0 at 23 ºC and 27 ºC in short days. There is a significant genotype x environment interaction depicting the differential thermo-sensitivity in flowering (p<0.0001, ANOVA model : flowering time ~ genotype + temperature + genotype *temperature) (b) Temperature-induced hypocotyl elongation can still be observed in the upf1-5 mutants, which suggests that the mutations in the NMD pathway does not generally affect thermal response. Neither the genotype nor the genotype x environment had any significant effect. pg X E refers to the p-value for the genotype x environment interaction. NATURE PLANTS 7

8 Supplementary Figure 5. Cycloheximide treatment results in accumulation of FLM transcripts. (a) RT-PCR of full-length FLM transcripts in mock treated or cycloheximide (CHX) treated Col-0 and Sf-2 plants. (b) Quantification of total FLM expression levels through qrt-pcr based on biological triplicates in Col-0 and Sf-2 at 23 ºC or 27 ºC upon cycloheximide treatment. The data are normalized to mock treated Col-0 at 23 ºC (for Col-0) or mock treated Sf-2 at 23 ºC after controlling with tubulin using the 2 -ΔΔcT method. T-test p-values are shown above for each of the comparisons. 8 NATURE PLANTS

9 Supplementary Figure 6. Temperature does not affect levels of FLM-δ. (a) Comparison of reads that map to E1-E2 or E1-E3 junctions from RNA-seq data. No significant differences could be seen. (b) Expression levels of transcripts that contain E1-E3 splice junction including FLM-δ. No significant differences could be detected between temperatures. (c) Flowering time of accessions with deletions that specifically affect FLM-δ at different temperatures. Total leaf numbers after vernalisation is shown. Two different experiments are separated through a break in x-axis. The experiment with Col-0, flm-3, Nd-1 and Ei-6 is shown for reference. A NATURE PLANTS 9

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11 Supplementary Figure 7. Transgenes are expressed in the T1 lines. Expression of specific splice forms in transgenic lines in the flm-3 background at 27 ºC short days. Multiple independent T1 lines per construct are shown as examples. The expression of FLM-I2RC in the Col-0 background is also shown, where the wild-type FLM-β expression can be seen in transgenic lines. NATURE PLANTS 11

12 Supplementary Figure 8. NMD-target transcripts confer early flowering upon overexpression. Confirmation of the overexpression phenotype in T2 lines in the Col-0 background at 27 ºC short days. Flowering time measured as total leaf number in different independent T2 transgenic lines are shown. For each family at least 20 plants were analysed. T-test p-values : *<0.05, **<0.01, ***<0.001, ****< NATURE PLANTS

13 Supplementary Figure 9. Lo-2 harbours a loss of function allele at the FLM locus. (a) Lo-2 fails to complement the flm-3 mutant. Flowering time measured as average total leaf number of Col-0, Lo-2, flm-3 and the F1 between Lo-2 crossed with Col-0 and flm-3 in short days at 23 ºC. (b) Comparison of the effects of FLM-β from Col-0 and Lo-2 in the flm-3 or Lo-2 backgrounds. Average flowering time measured as total leaf number is shown for plants grown in short days at 23 ºC. At least 25 plants for each genotype were phenotyped. Error bars represent standard errors of mean. T-test p-values : *<0.05, **<0.01, ***<0.001, ns-not significant. NATURE PLANTS 13

14 Supplementary Figure 10. flm-3 is epistatic to pif Flowering time measured as total leaf number in different genotypes across multiple conditions. At least 20 plants were phenotyped for each data point. Error bars represent standard errors of mean. T-test p-values : * <0.05, **<0.01, ***<0.001, ****< NATURE PLANTS

15 Supplementary Figure 11. Thermo-sensitivity in the expression levels of NMD target genes. Expression levels of 52 NMD target genes calculated from the RNA- Seq analysis of Col-0 and upf1-5 mutants. The genes are separated as low expression (a, b), medium expression (c, d) and high expression (e, f). The reaction norms for average RPM values from three or two biological replicates for Col-0 and upf1-5 respectively are shown for the two different temperatures. The genes are given in Supplementary Table 6. No general trend can be observed that could suggest a differential regulation of NMD at 23 or 27 ºC. NATURE PLANTS 15

16 Supplementary Tables Supplementary Table 1. Different splice forms identified through Sanger sequencing. ds-splicing refers to downstream splicing events observed after the premature termination codon. Supplementary Table 2. Effect of temperature on splicing at the FLM locus in Col-0. Table shows the number of normalized read counts that spans over exon-exon junctions and the exon-intron junctions in 3 biological replicates at two different temperatures. Supplementary Table 3. Actual counts and normalized counts from RNA-Seq. Reads that map to exons, introns, exon-exon junctions, exon-intron junctions as well as reads that utilize alternative splice sites are given. 23SD and 27SD represents two temperature conditions. Exons are numbered as E1 and E2 and the introns as I1 and I2. Supplementary Table 4. Flowering time measured as total leaf number in accessions that are potential knockouts for FLM-δ. These accessions harbor deletions as per the genome sequence reported in the 1001 genome project. We confirmed the deletion through Sanger sequencing in a subset of strains, which are shown in red. Some accessions did not flower without vernalisation and thus only flowering time after vernalisation is reported. Supplementary Table 5. Primers used in this study. Supplementary Table 6. NMD target genes, whose expression is analysed in Col-0 and upf 1-5 mutants. The average RPM values derived for three (Col-0) or two (upf1-5) biological replicates are given. 16 NATURE PLANTS