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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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Epigenetic control of PEP1b in Arabidopsis FRISFflc-3 PEP1b transgenic plants.(a) PEP1b, AtVIN3 and AaCOOLAIR I and III expression in FRISFflc-3 PEP1b 18-3 transformant before vernalization (V0) after 10, 20, 30 or 40 days of vernalization (V10, V20, V30 or V40) and after 40 days of vernalization followed by 10 or 20 days of growth in normal temperature (V40+10, V40+20). (b) FLC, AtVIN3, AtCOOLAIR I and II expression in FRISFFLC control plants during the vernalization time course described in a. In a and b mRNA levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates) and presented relative to the maximum level of expression of each gene, which is set as 100%. (c) PEP1b mRNA level before vernalization (V0) and after 40 days vernalization followed by 20 days of growth at normal temperature (V40+20) in FRISFflc-3 PEP1b 18-3 parental line and FRISFflc-3 PEP1b 18-3 vrn2-1 fca-1 plants. PEP1b mRNA level is compared with FLC mRNA in FRISFFLC, FRISFflc-3 and vrn2-1 fca-1 plants. mRNA levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates).
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f4: Epigenetic control of PEP1b in Arabidopsis FRISFflc-3 PEP1b transgenic plants.(a) PEP1b, AtVIN3 and AaCOOLAIR I and III expression in FRISFflc-3 PEP1b 18-3 transformant before vernalization (V0) after 10, 20, 30 or 40 days of vernalization (V10, V20, V30 or V40) and after 40 days of vernalization followed by 10 or 20 days of growth in normal temperature (V40+10, V40+20). (b) FLC, AtVIN3, AtCOOLAIR I and II expression in FRISFFLC control plants during the vernalization time course described in a. In a and b mRNA levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates) and presented relative to the maximum level of expression of each gene, which is set as 100%. (c) PEP1b mRNA level before vernalization (V0) and after 40 days vernalization followed by 20 days of growth at normal temperature (V40+20) in FRISFflc-3 PEP1b 18-3 parental line and FRISFflc-3 PEP1b 18-3 vrn2-1 fca-1 plants. PEP1b mRNA level is compared with FLC mRNA in FRISFFLC, FRISFflc-3 and vrn2-1 fca-1 plants. mRNA levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates).

Mentions: Whether A. alpina AaCOOLAIR was generated by mechanisms conserved in A. thaliana was examined. AaCOOLAIR was detected in the FRISFflc-3 PEP1b transformants during vernalization (Fig. 4a), suggesting that trans-acting factors present in A. thaliana can promote AaCOOLAIR expression, including AaCOOLAIR III that is not found at FLC. In the control line FRISFFLC, AtCOOLAIR I and AtCOOLAIR II were induced after 20 days in cold followed by accumulation of AtVIN3 mRNA and stable repression of FLC transcripts (Fig. 4b). Similarly, in transgenic FRISFflc-3 PEP1b plants AaCOOLAIR I and AaCOOLAIR III were also induced by cold and their patterns of expression resembled those of A. thaliana COOLAIR I and II (Fig. 4a,b). Therefore, AaCOOLAIR transcripts are probably controlled by cis-regulatory elements present in PEP1 that are recognized by trans-acting factors conserved between A. thaliana and A. alpina.


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)

Epigenetic control of PEP1b in Arabidopsis FRISFflc-3 PEP1b transgenic plants.(a) PEP1b, AtVIN3 and AaCOOLAIR I and III expression in FRISFflc-3 PEP1b 18-3 transformant before vernalization (V0) after 10, 20, 30 or 40 days of vernalization (V10, V20, V30 or V40) and after 40 days of vernalization followed by 10 or 20 days of growth in normal temperature (V40+10, V40+20). (b) FLC, AtVIN3, AtCOOLAIR I and II expression in FRISFFLC control plants during the vernalization time course described in a. In a and b mRNA levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates) and presented relative to the maximum level of expression of each gene, which is set as 100%. (c) PEP1b mRNA level before vernalization (V0) and after 40 days vernalization followed by 20 days of growth at normal temperature (V40+20) in FRISFflc-3 PEP1b 18-3 parental line and FRISFflc-3 PEP1b 18-3 vrn2-1 fca-1 plants. PEP1b mRNA level is compared with FLC mRNA in FRISFFLC, FRISFflc-3 and vrn2-1 fca-1 plants. mRNA levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates).
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f4: Epigenetic control of PEP1b in Arabidopsis FRISFflc-3 PEP1b transgenic plants.(a) PEP1b, AtVIN3 and AaCOOLAIR I and III expression in FRISFflc-3 PEP1b 18-3 transformant before vernalization (V0) after 10, 20, 30 or 40 days of vernalization (V10, V20, V30 or V40) and after 40 days of vernalization followed by 10 or 20 days of growth in normal temperature (V40+10, V40+20). (b) FLC, AtVIN3, AtCOOLAIR I and II expression in FRISFFLC control plants during the vernalization time course described in a. In a and b mRNA levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates) and presented relative to the maximum level of expression of each gene, which is set as 100%. (c) PEP1b mRNA level before vernalization (V0) and after 40 days vernalization followed by 20 days of growth at normal temperature (V40+20) in FRISFflc-3 PEP1b 18-3 parental line and FRISFflc-3 PEP1b 18-3 vrn2-1 fca-1 plants. PEP1b mRNA level is compared with FLC mRNA in FRISFFLC, FRISFflc-3 and vrn2-1 fca-1 plants. mRNA levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates).
Mentions: Whether A. alpina AaCOOLAIR was generated by mechanisms conserved in A. thaliana was examined. AaCOOLAIR was detected in the FRISFflc-3 PEP1b transformants during vernalization (Fig. 4a), suggesting that trans-acting factors present in A. thaliana can promote AaCOOLAIR expression, including AaCOOLAIR III that is not found at FLC. In the control line FRISFFLC, AtCOOLAIR I and AtCOOLAIR II were induced after 20 days in cold followed by accumulation of AtVIN3 mRNA and stable repression of FLC transcripts (Fig. 4b). Similarly, in transgenic FRISFflc-3 PEP1b plants AaCOOLAIR I and AaCOOLAIR III were also induced by cold and their patterns of expression resembled those of A. thaliana COOLAIR I and II (Fig. 4a,b). Therefore, AaCOOLAIR transcripts are probably controlled by cis-regulatory elements present in PEP1 that are recognized by trans-acting factors conserved between A. thaliana and A. alpina.

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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