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. 2017 Nov 2;68(18):5117-5127.
doi: 10.1093/jxb/erx328.

Contribution of Major FLM Isoforms to Temperature-Dependent Flowering in Arabidopsis Thaliana

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Free PMC article

Contribution of Major FLM Isoforms to Temperature-Dependent Flowering in Arabidopsis Thaliana

Giovanna Capovilla et al. J Exp Bot. .
Free PMC article

Abstract

FLOWERING LOCUS M (FLM), a component of the thermosensory flowering time pathway in Arabidopsis thaliana, is regulated by temperature-dependent alternative splicing (AS). The main splicing variant, FLM-β, is a well-documented floral repressor that is down-regulated in response to increasing ambient growth temperature. Two hypotheses have been formulated to explain how flowering time is modulated by AS of FLM. In the first model a second splice variant, FLM-δ, acts as a dominant negative isoform that competes with FLM-β at elevated ambient temperatures, thereby indirectly promoting flowering. Alternatively, it has been suggested that the induction of flowering at elevated temperatures is caused only by reduced FLM-β expression. To better understand the role of the two FLM splice forms, we employed CRISPR/Cas9 technology to specifically delete the exons that characterize each splice variant. Lines that produced repressive FLM-β but were incapable of producing FLM-δ were late flowering. In contrast, FLM-β knockout lines that still produced FLM-δ flowered early, but not earlier than the flm-3 loss of function mutant, as would be expected if FLM-δ had a dominant-negative effect on flowering. Our data support the role of FLM-β as a flower repressor and provide evidence that a contribution of FLM-δ to the regulation of flowering time in wild-type A. thaliana seems unlikely.

Keywords: Arabidopsis thaliana; CRISPR/Cas9; FLOWERING LOCUS M (FLM); flowering time; splice isoforms; temperature-dependent alternative splicing.

Figures

Fig. 1.
Fig. 1.
Schematic representation of the FLM locus in the two FLM CRISPR lines. The annotated FLM gene structure is shown in black, exons are marked as squares and introns as straight lines. The edited FLM-ΔE2 and FLM-ΔE3 lines are represented in blue and red, respectively. Close-ups provide detailed information on the position of the deletions as determined by Sanger sequencing of the CRISPR lines aligned with wild-type FLM genomic sequences. Position of exons 2 and 3 are marked by thick blue and red lines, respectively. sgRNAs are represented by thin blue and red lines, PAM sites are marked by grey boxes on the wild-type sequences and the position of the expected Cas9 cuts are indicated with black triangles.
Fig. 2.
Fig. 2.
Alternative FLM splice variants detected by Sanger sequencing. The sequences present in the isoforms are aligned to the annotated FLM gene and grey arrows show the positions of the primers used to amplify the cDNA. Stop codons are represented as red stars. Isoforms identified in both Col-0 and at least in one of the CRISPR lines are listed as alternative splice forms (ASF) 1 to 31. The identifiers of isoforms previously described by Sureshkumar and colleagues (Sureshkumar et al., 2016) are given in brackets. 19 new splice variants detected only in the CRISPR lines are listed as cASF. The heat map shows the frequency of each isoform in Col-0, FLM-ΔE2, and FLM-ΔE3 at 16°C, 23°C, and 27°C. The heat map legend shows a gradient of white to green where 0% of the sequences analysed is white and 100% is dark green.
Fig. 3.
Fig. 3.
Relative expression of FLM main isoforms. A) Relative expression of transcripts containing the second (e.g. FLM-β) or third (e.g. FLM-δ) exon measured using the TaqMan assay in Col-0, the CRISPR lines, and the flm-3 mutant at 16°C, 23°C, and 27°C. *P<0.05; **P<0.01;***P<0.001; ****P<0.0001; ns, not significant, using Anova and TukeyHSD test. B) Quantification of splice variants with the second exon and third exon of FLM by the TaqMan assay in a genomic 35S:gFLM overexpression line at 16°C, 23°C, and 27 °C. For each sample three biological replicates of 10 seedlings each were used and each measurement has been replicated three times. Error bars designate the standard deviation between biological replicates. C) Schematic representation of the TaqMan assay designed to detect splice variants with the second exon and third exon of FLM (e.g. FLM-β and FLM-δ) and the normalization control UBC21. Primers are shown as black arrows, probes as black segments. Fluorophores are marked as blue (6-FAM™), green (HEX™2), and orange (CY5®).
Fig. 4.
Fig. 4.
Relative expression of potential protein coding FLM isoforms. A) Representation of the annotated portion of FLM gene, from exon 1 to exon 6, and of the introns or exons included in the isoforms FLM-β, FLM-δ, ASF7, and ASF10. The primers used for the quantification of each isoform are represented as black arrows. B) Relative expression of each isoform at 16°C, 23°C, and 27°C. Error bars designate the standard deviation between three biological replicates. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant, using Anova and TukeyHSD test.
Fig. 5.
Fig. 5.
Flowering time of FLM CRISPR/Cas9 lines. A) Representative pictures of flm-3, FLM-ΔE2, Col-0, FLM-ΔE3, and 35S::gFLM lines grown at 23°C in LD. Scale bar, 1 cm. B) Flowering time given as number of leaves (dark grey, rosette leaves; light grey, cauline leaves) and days to flowering (black) of plants grown at 16°C, 23°C, and 27°C. Error bars indicate standard deviation. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant, using Anova and TukeyHSD test.
Fig. 6.
Fig. 6.
Flowering time of FLM-δ overexpression lines at 16°C. A) Flowering time given as number of leaves (dark grey, rosette leaves; light grey, cauline leaves). B) Days to flowering (black) of plants grown at 16°C. Error bars indicate standard deviation. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant, using Anova and TukeyHSD test.
Fig. 7.
Fig. 7.
Expression of transcripts containing the second exon in natural accessions and flowering time. A) Relative expression of transcripts containing the second exon, e.g. FLM-β, measured using the TaqMan assay in 33 natural A. thaliana accessions. Error bars indicate standard deviation. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001, using Anova and TukeyHSD test. B) Mean flowering time in days and leaf numbers of 33 accessions showing Col-0-like temperature-dependent FLM splicing.

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