Limits...
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.

Show MeSH
Transcriptional activity of PEP1b in A. thaliana FRISFflc-3 PEP1b transgenic plants.(a) Schematic representation of PEP1 locus represented as described in Fig. 2a. The rectangle indicates the extent of 13.4 kb PEP1b transgene transferred into A. thaliana FRISFflc-3 plants. Scale bar, 1 kb. (b) Flowering times of A. thaliana FRISFflc-3 PEP1b independent transformants, FRISFflc-3 parental line and FRISFFLC control line as measured by rosette leaf number (RLN) at bolting±s.d. (n=9–23 individuals). Asterisks indicate flowering time statistically different from the one of FRISFflc-3 plants (Student’s t-test, P value <0.005). (c) PEP1b mRNA levels in A. thaliana FRISFflc-3 PEP1b independent transformants and FLC mRNA levels in the parental FRISFflc-3 line and the control FRISFFLC line. Time points are before vernalization (V0), after 40 days of vernalization (V40), as well as after 40 days of vernalization followed by 10 or 20 days of growth at normal growth temperatures (V40+10 and V40+20). Transcript levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates). Panels b and c share colour-code legends.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109010&req=5

f3: Transcriptional activity of PEP1b in A. thaliana FRISFflc-3 PEP1b transgenic plants.(a) Schematic representation of PEP1 locus represented as described in Fig. 2a. The rectangle indicates the extent of 13.4 kb PEP1b transgene transferred into A. thaliana FRISFflc-3 plants. Scale bar, 1 kb. (b) Flowering times of A. thaliana FRISFflc-3 PEP1b independent transformants, FRISFflc-3 parental line and FRISFFLC control line as measured by rosette leaf number (RLN) at bolting±s.d. (n=9–23 individuals). Asterisks indicate flowering time statistically different from the one of FRISFflc-3 plants (Student’s t-test, P value <0.005). (c) PEP1b mRNA levels in A. thaliana FRISFflc-3 PEP1b independent transformants and FLC mRNA levels in the parental FRISFflc-3 line and the control FRISFFLC line. Time points are before vernalization (V0), after 40 days of vernalization (V40), as well as after 40 days of vernalization followed by 10 or 20 days of growth at normal growth temperatures (V40+10 and V40+20). Transcript levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates). Panels b and c share colour-code legends.

Mentions: To investigate whether the molecular mechanisms regulating expression of FLC orthologues are evolutionarily conserved, A. alpina PEP1b (Fig. 3a), the most highly expressed A. alpina PEP1 gene37 and the one containing the 3′ sequences encoding AaCOOLAIR, was transferred into A. thaliana Columbia FRISFflc-3 (ref. 3). Transgenic FRISFflc-3 plants containing the PEP1b genomic fragment displayed a small but statistically significant (P<0.005) delay in flowering compared with the FRISFflc-3 progenitor line (Fig. 3b, Supplementary Table 2). By contrast, the control line FRISFFLC that carries an active FLC gene flowered much later than FRISFflc-3 (Fig. 3b). PEP1 mRNA was also expressed in FRISFflc-3 PEP1b transgenic lines at lower levels than FLC mRNA in the FRISFFLC control plants (Fig. 3c, V0). These data suggest that the diverged PEP1b promoter might not be efficiently recognized in A. thaliana and cannot promote PEP1 mRNA at the threshold level necessary to strongly delay flowering time. To test the effect of cold on PEP1 mRNA level transgenic plants were vernalized for 40 days and subsequently transferred to warm temperatures for 20 days (Fig. 3c). PEP1 mRNA levels were reduced during vernalization as observed for FLC mRNA in FRISFFLC control plants (V40, Fig. 3c). Interestingly, in this heterologous species PEP1b mRNA level remained low when the transgenic plants were returned to normal growth temperatures after vernalization, similar to what is observed for FLC stable repression (V40+10, V40+20; Fig. 3c and Supplementary Fig. 9a) and in contrast to the unstable repression of PEP1 found in A. alpina. However, the low level of expression of PEP1b in A. thaliana might also reduce the sensitivity of detection of reactivation after vernalization. Despite this reservation, the species-specific variation in trans-acting regulation is likely responsible for the differences in the stability of PEP1 repression observed 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)

Transcriptional activity of PEP1b in A. thaliana FRISFflc-3 PEP1b transgenic plants.(a) Schematic representation of PEP1 locus represented as described in Fig. 2a. The rectangle indicates the extent of 13.4 kb PEP1b transgene transferred into A. thaliana FRISFflc-3 plants. Scale bar, 1 kb. (b) Flowering times of A. thaliana FRISFflc-3 PEP1b independent transformants, FRISFflc-3 parental line and FRISFFLC control line as measured by rosette leaf number (RLN) at bolting±s.d. (n=9–23 individuals). Asterisks indicate flowering time statistically different from the one of FRISFflc-3 plants (Student’s t-test, P value <0.005). (c) PEP1b mRNA levels in A. thaliana FRISFflc-3 PEP1b independent transformants and FLC mRNA levels in the parental FRISFflc-3 line and the control FRISFFLC line. Time points are before vernalization (V0), after 40 days of vernalization (V40), as well as after 40 days of vernalization followed by 10 or 20 days of growth at normal growth temperatures (V40+10 and V40+20). Transcript levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates). Panels b and c share colour-code legends.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109010&req=5

f3: Transcriptional activity of PEP1b in A. thaliana FRISFflc-3 PEP1b transgenic plants.(a) Schematic representation of PEP1 locus represented as described in Fig. 2a. The rectangle indicates the extent of 13.4 kb PEP1b transgene transferred into A. thaliana FRISFflc-3 plants. Scale bar, 1 kb. (b) Flowering times of A. thaliana FRISFflc-3 PEP1b independent transformants, FRISFflc-3 parental line and FRISFFLC control line as measured by rosette leaf number (RLN) at bolting±s.d. (n=9–23 individuals). Asterisks indicate flowering time statistically different from the one of FRISFflc-3 plants (Student’s t-test, P value <0.005). (c) PEP1b mRNA levels in A. thaliana FRISFflc-3 PEP1b independent transformants and FLC mRNA levels in the parental FRISFflc-3 line and the control FRISFFLC line. Time points are before vernalization (V0), after 40 days of vernalization (V40), as well as after 40 days of vernalization followed by 10 or 20 days of growth at normal growth temperatures (V40+10 and V40+20). Transcript levels were measured by RT–qPCR relative to those of the reference gene ACTIN±s.d. (n=3 technical replicates). Panels b and c share colour-code legends.
Mentions: To investigate whether the molecular mechanisms regulating expression of FLC orthologues are evolutionarily conserved, A. alpina PEP1b (Fig. 3a), the most highly expressed A. alpina PEP1 gene37 and the one containing the 3′ sequences encoding AaCOOLAIR, was transferred into A. thaliana Columbia FRISFflc-3 (ref. 3). Transgenic FRISFflc-3 plants containing the PEP1b genomic fragment displayed a small but statistically significant (P<0.005) delay in flowering compared with the FRISFflc-3 progenitor line (Fig. 3b, Supplementary Table 2). By contrast, the control line FRISFFLC that carries an active FLC gene flowered much later than FRISFflc-3 (Fig. 3b). PEP1 mRNA was also expressed in FRISFflc-3 PEP1b transgenic lines at lower levels than FLC mRNA in the FRISFFLC control plants (Fig. 3c, V0). These data suggest that the diverged PEP1b promoter might not be efficiently recognized in A. thaliana and cannot promote PEP1 mRNA at the threshold level necessary to strongly delay flowering time. To test the effect of cold on PEP1 mRNA level transgenic plants were vernalized for 40 days and subsequently transferred to warm temperatures for 20 days (Fig. 3c). PEP1 mRNA levels were reduced during vernalization as observed for FLC mRNA in FRISFFLC control plants (V40, Fig. 3c). Interestingly, in this heterologous species PEP1b mRNA level remained low when the transgenic plants were returned to normal growth temperatures after vernalization, similar to what is observed for FLC stable repression (V40+10, V40+20; Fig. 3c and Supplementary Fig. 9a) and in contrast to the unstable repression of PEP1 found in A. alpina. However, the low level of expression of PEP1b in A. thaliana might also reduce the sensitivity of detection of reactivation after vernalization. Despite this reservation, the species-specific variation in trans-acting regulation is likely responsible for the differences in the stability of PEP1 repression observed 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.

Show MeSH