Vernalization-repression of Arabidopsis FLC requires promoter sequences but not antisense transcripts

Abstract

The repression of Arabidopsis FLC expression by vernalization (extended cold) has become a model for understanding polycomb-associated epigenetic regulation in plants. Antisense and sense non-coding RNAs have been respectively implicated in initiation and maintenance of FLC repression by vernalization. We show that the promoter and first exon of the FLC gene are sufficient to initiate repression during vernalization; this initial repression of FLC does not require antisense transcription. Long-term maintenance of FLC repression requires additional regions of the gene body, including those encoding sense non-coding transcripts.

Figure 1. FLC is transiently repressed by extended cold in the flc-20 mutant.

A, B and C) qPCR quantification of proximal antisense (A), distal antisense (B) , and FLC 5′ unspliced transcript (C) is shown for C24 and flc-20 for plants grown for 12 long days in warm conditions (12LD), 12 days in warm and transferred to cold conditions for 4 weeks (12LD+4V) or cold treated then returned to warm conditions for 2 days (12LD+4V+2LD). D, E and F) H3K27me3 ChIP-qPCR for amplicon 2 (D), amplicon 5b (E) and amplicon 6 (F) in C24 and flc-20, on same plant samples used in A–C. G) Diagram of the FLC gene and associated lncRNAs. Exons of the sense transcript are shown boxed, the exons of mature antisense transcripts are shown in bold, introns in antisense transcripts are shown as dotted lines. Transcription starts indicated by arrows, triangle marks Ds insertion in flc-20. The Ds inserted in intron 1 of FLC in flc-20 contains a GUS reporter gene and nptII resistance gene . It is likely that the transcript detected with the distal primers in flc-20 originates from within the Ds insertion as no PCR product is obtained using a primer set that spans the large intron in the distal antisense transcripts .

Figure 2. COOLAIR lncRNAs are not required for cold-repression of FLC.

A, B and C) qPCR quantification of antisense (A, B), and FLC 5′ unspliced transcript (C) is shown for ColFRI, SALK_092716, SALK_140021 and SALK_131491 for plants grown for 12 long days in warm conditions (12LD), 12 days in warm and transferred to cold conditions for 4 weeks (12LD+4V) or cold treated then returned to warm conditions for 2 or 5 days (12LD+4V+2LD, 12LD+4V+5LD). D, E, F and G) H3K27me3 ChIP-qPCR for amplicon 2 (D), amplicon 5b (E), amplicon 6 (F) and amplicon 11 (G) in ColFRI and T-DNA insertion lines. H) Diagram of FLC gene, triangles mark T-DNA insertion sites. n.d.; not determined.

Figure 3. Initial repression of FLC transcription is not dependent on VIN3 or PRC2.

A and B) qPCR of FLC 5′ unspliced (A) and mature FLC mRNA (B)in ColFRI, vin3-4 and swn7clf28 plants grown for 12 long days in warm conditions (12LD), 12 days in warm and transferred to cold conditions for 1–4 weeks (12LD+1V, 2V, 3V, 4V) or cold treated then returned to warm conditions for 2 days (12LD+4V+2LD).