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Evolutionary conservation of cold-induced antisense RNAs of FLOWERING LOCUS C in Arabidopsis thaliana perennial relatives.

Castaings L, Bergonzi S, Albani MC, Kemi U, Savolainen O, Coupland G - Nat Commun (2014)

Bottom Line: Study of the A. alpina orthologue, PERPETUAL FLOWERING 1 (PEP1), demonstrates that AaCOOLAIR is induced each winter of the perennial life cycle.Introduction of PEP1 into A. thaliana reveals that AaCOOLAIR cis-elements confer cold-inducibility in this heterologous species while the difference between PEP1 and FLC mRNA patterns depends on both cis-elements and species-specific trans-acting factors.Thus, expression of COOLAIR is highly conserved, supporting its importance in FLC regulation.

View Article: PubMed Central - PubMed

Affiliation: 1] Max Planck Institute for Plant Breeding Research, Carl von Linné Weg 10, D-50829 Cologne, Germany [2].

ABSTRACT
Antisense RNA (asRNA) COOLAIR is expressed at A. thaliana FLOWERING LOCUS C (FLC) in response to winter temperatures. Its contribution to cold-induced silencing of FLC was proposed but its functional and evolutionary significance remain unclear. Here we identify a highly conserved block containing the COOLAIR first exon and core promoter at the 3' end of several FLC orthologues. Furthermore, asRNAs related to COOLAIR are expressed at FLC loci in the perennials A. alpina and A. lyrata, although some splicing variants differ from A. thaliana. Study of the A. alpina orthologue, PERPETUAL FLOWERING 1 (PEP1), demonstrates that AaCOOLAIR is induced each winter of the perennial life cycle. Introduction of PEP1 into A. thaliana reveals that AaCOOLAIR cis-elements confer cold-inducibility in this heterologous species while the difference between PEP1 and FLC mRNA patterns depends on both cis-elements and species-specific trans-acting factors. Thus, expression of COOLAIR is highly conserved, supporting its importance in FLC regulation.

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Cold-induced antisense non-coding RNAs are transcribed at the PEP1 locus in A. alpina and follow a seasonal pattern.(a) Class I (AaCOOLAIR I), class II (AaCOOLAIR II) and class III (AaCOOLAIR III) antisense RNAs transcribed at PEP1 locus. Black boxes are exons; lines are introns and other non-coding regions. Grey boxes indicate the region that is duplicated in tandem. Arrows indicate transcriptional start sites at PEP1. Dots show PEP1 antisense transcript polyadenylation sites; blue, Class I; light orange, Class III; dark orange, Class II. Asterisks indicate the most abundant form among splicing variants obtained by 3′ RACE (Classes I and III) or RT–qPCR product sequencing (Class II). Scale bar, 1 kb. (b) AaCOOLAIR I, II and III cold-inducibility. RT–qPCR on leaf RNA of A. alpina plants exposed to 0, 7, 21, 35 and 84 days of vernalization (V0, V7, V21, V35 and V84) followed by 7, 21 and 35 days of growth in normal growth temperatures (V+7, V+21 and V+35). Transcript levels were expressed relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). (c) AaCOOLAIR I, AaCOOLAIR III, PEP1 and AaVIN3 expression patterns in A. alpina leaves during the vernalization time course shown in b. Transcripts levels were measured by RT–qPCR relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). The maximum level of expression of each gene is set at 100%. (d) Seasonal expression patterns of PEP1, AaCOOLAIR I, AaCOOLAIR III and AaVIN3 over two successive vernalization treatments. A. alpina plants were vernalized for 0, 7, 21, 35 and 84 days (V0, V7, V21, V35 and V84) followed by 7, 21 and 35 days of growth in normal temperature (V+7, V+21 and V+35), then vernalized again for 7, 21, 35, 56 and 84 days (VV7, VV21, VV35, VV56 and VV84) and grown for 7, 21 and 35 more days in normal growth temperatures (VV+7, VV+21 and VV+35). Grey areas indicate the cold treatments. Transcript levels were measured by RT–qPCR relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). The maximum level of expression of each gene is set at 100%. A biological replicate is shown in Supplementary Fig. 8.
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f2: Cold-induced antisense non-coding RNAs are transcribed at the PEP1 locus in A. alpina and follow a seasonal pattern.(a) Class I (AaCOOLAIR I), class II (AaCOOLAIR II) and class III (AaCOOLAIR III) antisense RNAs transcribed at PEP1 locus. Black boxes are exons; lines are introns and other non-coding regions. Grey boxes indicate the region that is duplicated in tandem. Arrows indicate transcriptional start sites at PEP1. Dots show PEP1 antisense transcript polyadenylation sites; blue, Class I; light orange, Class III; dark orange, Class II. Asterisks indicate the most abundant form among splicing variants obtained by 3′ RACE (Classes I and III) or RT–qPCR product sequencing (Class II). Scale bar, 1 kb. (b) AaCOOLAIR I, II and III cold-inducibility. RT–qPCR on leaf RNA of A. alpina plants exposed to 0, 7, 21, 35 and 84 days of vernalization (V0, V7, V21, V35 and V84) followed by 7, 21 and 35 days of growth in normal growth temperatures (V+7, V+21 and V+35). Transcript levels were expressed relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). (c) AaCOOLAIR I, AaCOOLAIR III, PEP1 and AaVIN3 expression patterns in A. alpina leaves during the vernalization time course shown in b. Transcripts levels were measured by RT–qPCR relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). The maximum level of expression of each gene is set at 100%. (d) Seasonal expression patterns of PEP1, AaCOOLAIR I, AaCOOLAIR III and AaVIN3 over two successive vernalization treatments. A. alpina plants were vernalized for 0, 7, 21, 35 and 84 days (V0, V7, V21, V35 and V84) followed by 7, 21 and 35 days of growth in normal temperature (V+7, V+21 and V+35), then vernalized again for 7, 21, 35, 56 and 84 days (VV7, VV21, VV35, VV56 and VV84) and grown for 7, 21 and 35 more days in normal growth temperatures (VV+7, VV+21 and VV+35). Grey areas indicate the cold treatments. Transcript levels were measured by RT–qPCR relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). The maximum level of expression of each gene is set at 100%. A biological replicate is shown in Supplementary Fig. 8.

Mentions: In A. thaliana cold-induced repression of FLC mRNA requires cis-regulatory sequences in the promoter and first intron1314. Whether these cis-regulatory elements are evolutionarily conserved was tested by comparing genomic sequences of FLC orthologues from a range of Brassicaceae species (Methods; Fig. 1; Supplementary Figs 1–3).With the exception of PEP1 in A. alpina, FLC orthologues from A. lyrata, Capsella rubella and Thellungiella halophila exhibit the same gene structure as A. thaliana FLC. A. alpina PEP1 harbours a tandem duplication (PEP1a and PEP1b) containing part of the promoter, the first exon and part of intron 1 (Fig. 2a; ref. 21). The two copies of exon 1 have individual transcriptional start sites and each is spliced to the unique exon 2 giving rise to two overlapping transcripts37. Two tandemly arranged full-length copies of FLC, called AlFLC1 and AlFLC2, are present in A. lyrata, and both are transcriptionally active2638. Promoter sequences between the translational start site (ATG) of FLC and the end of the next upstream gene were compared revealing two main blocks of homology (Fig. 1a and Supplementary Fig. 1). The first block was ~400 bp long and included the proximal promoter as well as the 5′ untranslated leader. In A. alpina, homology in this region was reduced and mainly restricted to the untranslated leaders of PEP1a and PEP1b (Supplementary Fig. 1g). The second region was ~2 kb further upstream, and in A. alpina was found only in PEP1a. Apart from these two conserved blocks, the FLC promoter has diverged rapidly during evolution, even between the closely related species A. thaliana and A. lyrata, as observed previously38.


Evolutionary conservation of cold-induced antisense RNAs of FLOWERING LOCUS C in Arabidopsis thaliana perennial relatives.

Castaings L, Bergonzi S, Albani MC, Kemi U, Savolainen O, Coupland G - Nat Commun (2014)

Cold-induced antisense non-coding RNAs are transcribed at the PEP1 locus in A. alpina and follow a seasonal pattern.(a) Class I (AaCOOLAIR I), class II (AaCOOLAIR II) and class III (AaCOOLAIR III) antisense RNAs transcribed at PEP1 locus. Black boxes are exons; lines are introns and other non-coding regions. Grey boxes indicate the region that is duplicated in tandem. Arrows indicate transcriptional start sites at PEP1. Dots show PEP1 antisense transcript polyadenylation sites; blue, Class I; light orange, Class III; dark orange, Class II. Asterisks indicate the most abundant form among splicing variants obtained by 3′ RACE (Classes I and III) or RT–qPCR product sequencing (Class II). Scale bar, 1 kb. (b) AaCOOLAIR I, II and III cold-inducibility. RT–qPCR on leaf RNA of A. alpina plants exposed to 0, 7, 21, 35 and 84 days of vernalization (V0, V7, V21, V35 and V84) followed by 7, 21 and 35 days of growth in normal growth temperatures (V+7, V+21 and V+35). Transcript levels were expressed relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). (c) AaCOOLAIR I, AaCOOLAIR III, PEP1 and AaVIN3 expression patterns in A. alpina leaves during the vernalization time course shown in b. Transcripts levels were measured by RT–qPCR relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). The maximum level of expression of each gene is set at 100%. (d) Seasonal expression patterns of PEP1, AaCOOLAIR I, AaCOOLAIR III and AaVIN3 over two successive vernalization treatments. A. alpina plants were vernalized for 0, 7, 21, 35 and 84 days (V0, V7, V21, V35 and V84) followed by 7, 21 and 35 days of growth in normal temperature (V+7, V+21 and V+35), then vernalized again for 7, 21, 35, 56 and 84 days (VV7, VV21, VV35, VV56 and VV84) and grown for 7, 21 and 35 more days in normal growth temperatures (VV+7, VV+21 and VV+35). Grey areas indicate the cold treatments. Transcript levels were measured by RT–qPCR relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). The maximum level of expression of each gene is set at 100%. A biological replicate is shown in Supplementary Fig. 8.
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f2: Cold-induced antisense non-coding RNAs are transcribed at the PEP1 locus in A. alpina and follow a seasonal pattern.(a) Class I (AaCOOLAIR I), class II (AaCOOLAIR II) and class III (AaCOOLAIR III) antisense RNAs transcribed at PEP1 locus. Black boxes are exons; lines are introns and other non-coding regions. Grey boxes indicate the region that is duplicated in tandem. Arrows indicate transcriptional start sites at PEP1. Dots show PEP1 antisense transcript polyadenylation sites; blue, Class I; light orange, Class III; dark orange, Class II. Asterisks indicate the most abundant form among splicing variants obtained by 3′ RACE (Classes I and III) or RT–qPCR product sequencing (Class II). Scale bar, 1 kb. (b) AaCOOLAIR I, II and III cold-inducibility. RT–qPCR on leaf RNA of A. alpina plants exposed to 0, 7, 21, 35 and 84 days of vernalization (V0, V7, V21, V35 and V84) followed by 7, 21 and 35 days of growth in normal growth temperatures (V+7, V+21 and V+35). Transcript levels were expressed relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). (c) AaCOOLAIR I, AaCOOLAIR III, PEP1 and AaVIN3 expression patterns in A. alpina leaves during the vernalization time course shown in b. Transcripts levels were measured by RT–qPCR relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). The maximum level of expression of each gene is set at 100%. (d) Seasonal expression patterns of PEP1, AaCOOLAIR I, AaCOOLAIR III and AaVIN3 over two successive vernalization treatments. A. alpina plants were vernalized for 0, 7, 21, 35 and 84 days (V0, V7, V21, V35 and V84) followed by 7, 21 and 35 days of growth in normal temperature (V+7, V+21 and V+35), then vernalized again for 7, 21, 35, 56 and 84 days (VV7, VV21, VV35, VV56 and VV84) and grown for 7, 21 and 35 more days in normal growth temperatures (VV+7, VV+21 and VV+35). Grey areas indicate the cold treatments. Transcript levels were measured by RT–qPCR relative to those of the reference gene RAN3±s.d. (n=3 technical replicates). The maximum level of expression of each gene is set at 100%. A biological replicate is shown in Supplementary Fig. 8.
Mentions: In A. thaliana cold-induced repression of FLC mRNA requires cis-regulatory sequences in the promoter and first intron1314. Whether these cis-regulatory elements are evolutionarily conserved was tested by comparing genomic sequences of FLC orthologues from a range of Brassicaceae species (Methods; Fig. 1; Supplementary Figs 1–3).With the exception of PEP1 in A. alpina, FLC orthologues from A. lyrata, Capsella rubella and Thellungiella halophila exhibit the same gene structure as A. thaliana FLC. A. alpina PEP1 harbours a tandem duplication (PEP1a and PEP1b) containing part of the promoter, the first exon and part of intron 1 (Fig. 2a; ref. 21). The two copies of exon 1 have individual transcriptional start sites and each is spliced to the unique exon 2 giving rise to two overlapping transcripts37. Two tandemly arranged full-length copies of FLC, called AlFLC1 and AlFLC2, are present in A. lyrata, and both are transcriptionally active2638. Promoter sequences between the translational start site (ATG) of FLC and the end of the next upstream gene were compared revealing two main blocks of homology (Fig. 1a and Supplementary Fig. 1). The first block was ~400 bp long and included the proximal promoter as well as the 5′ untranslated leader. In A. alpina, homology in this region was reduced and mainly restricted to the untranslated leaders of PEP1a and PEP1b (Supplementary Fig. 1g). The second region was ~2 kb further upstream, and in A. alpina was found only in PEP1a. Apart from these two conserved blocks, the FLC promoter has diverged rapidly during evolution, even between the closely related species A. thaliana and A. lyrata, as observed previously38.

Bottom Line: Study of the A. alpina orthologue, PERPETUAL FLOWERING 1 (PEP1), demonstrates that AaCOOLAIR is induced each winter of the perennial life cycle.Introduction of PEP1 into A. thaliana reveals that AaCOOLAIR cis-elements confer cold-inducibility in this heterologous species while the difference between PEP1 and FLC mRNA patterns depends on both cis-elements and species-specific trans-acting factors.Thus, expression of COOLAIR is highly conserved, supporting its importance in FLC regulation.

View Article: PubMed Central - PubMed

Affiliation: 1] Max Planck Institute for Plant Breeding Research, Carl von Linné Weg 10, D-50829 Cologne, Germany [2].

ABSTRACT
Antisense RNA (asRNA) COOLAIR is expressed at A. thaliana FLOWERING LOCUS C (FLC) in response to winter temperatures. Its contribution to cold-induced silencing of FLC was proposed but its functional and evolutionary significance remain unclear. Here we identify a highly conserved block containing the COOLAIR first exon and core promoter at the 3' end of several FLC orthologues. Furthermore, asRNAs related to COOLAIR are expressed at FLC loci in the perennials A. alpina and A. lyrata, although some splicing variants differ from A. thaliana. Study of the A. alpina orthologue, PERPETUAL FLOWERING 1 (PEP1), demonstrates that AaCOOLAIR is induced each winter of the perennial life cycle. Introduction of PEP1 into A. thaliana reveals that AaCOOLAIR cis-elements confer cold-inducibility in this heterologous species while the difference between PEP1 and FLC mRNA patterns depends on both cis-elements and species-specific trans-acting factors. Thus, expression of COOLAIR is highly conserved, supporting its importance in FLC regulation.

Show MeSH
Related in: MedlinePlus