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  1. eLife assessment

    This important paper advances the understanding, in the model plant Arabidopsis thaliana, of the molecular basis of the promotion of flowering in the spring by exposure to winter cold through a process known as vernalization. In Arabidopsis, there are two classes of long non-coding RNAs produced only when plants are in the cold, and this work provides compelling evidence that the cold-induced expression of one of these (COOLAIR) involves C-repeat binding factor proteins that bind to cognate binding elements in the COOLAIR promoter, but also that COOLAIR is not required for the vernalization-mediated promotion of flowering under standard laboratory conditions in which the vernalization response is measured.

  2. Reviewer #1 (Public Review):

    FLOWERING LOCUS C (FLC) is a key repressor of flowering in Arabidopsis thaliana. FLC expression creates a requirement for vernalization which is the acquisition of competence to flower after exposure to the prolonged cold of winter. Vernalization in Arabidopsis and other Brassicas results in the suppression of FLC expression.

    How exposure to winter cold initiates the vernalization process (i.e., the silencing of FLC) is not fully understood. It is known that cold exposure causes several long non-coding RNAs, including COOLAIR and COLDAIR, to be transcribed from FLC. this work shows that COOLAIR induction by cold results requires the binding of CRT/DRE-binding factors (CBFs) to their cognate recognition elements which reside at the 3' end of the FLC locus. The authors demonstrate this regulation in many ways including studying the effect on vernalization of knocking out all CBFs and also by showing that constitutive CBF expression causes COOLAIR levels to be elevated even without cold exposure. Intriguingly, plants with genetic alterations that eliminate COOLAIR expression (loss of CBF activity and FLC deletion mutants that eliminate COOLAIR expression) do not have a significant impairment in becoming vernalized.

    The work appears to be done properly and provides much important information about how this remarkable environmentally-induced epigenetic switch operates.

  3. Reviewer #2 (Public Review):

    Here the authors questioned the regulation and functional roles of anti-sense transcripts at the 3'end of an important flowering-time regulator FLC.

    The authors present compelling genetic, molecular biology, transgene, and biochemical data on the molecular details of how COOLAIR is induced by cold temperatures. They report that cold-induction of COOLAIR is mediated by C-repeat/dehydration-responsive elements (CRT/DREs) at the 3'-end of the FLC and relatively small deletions of the CRT/DREs prevent cold-induction of COOLAIR. They also report that long-term cold results in an increase in the expression of CRT/DRE BINDING FACTORs (CBFs) that bind to the CRT/DREs and result in the activation of genes containing CRT/DREs.

    Interestingly, in lines in which COOLAIR is not induced the vernalization proceeds normally with respect to flowering behavior and cold-mediated FLC chromatin changes, a result that is at odds with some publications but consistent with other reports.

    The major strength of this research is the comprehensive battery of relevant assays used to address their aim. Using ChIP they demonstrate CBF3 directly binds to the 3'end of FLC in vivo, and of less interest, but still very relevant, CBF3 binds to a CRT/DRE motif containing oligo-nucleotides in vitro using an EMSA. Using CRISPR-mediated genetic deletion of these sequences in vivo, they demonstrated that the downstream antisense transcripts are no longer transcribed. Interestingly, in these CRISPR mutants or genetic mutants of higher-order CBF mutants, the vernalisation response (chromatin modifications) is not impaired. They also show that CBF mRNA transcription occurs in at least two waves, an early peak, and over a prolonged cold period.

    While the CRISPR genetic motif mutants are relatively small, a few hundred base pairs, ideally they would have been smaller if only encompassing the CRT/DRE motif.

    The authors clearly achieved their aims and the presented results strongly support their conclusions. The compelling data clearly questions a widely held view in the vernalisation field. The presented methods can be widely transferable to a broader research community.

  4. Reviewer #3 (Public Review):

    The authors start by examining the COOLAIR promoter and identifying a CRT/DRE motif that is bound by the CBF transcription factor family that is involved in the short-term cold. This is confirmed by gel shift assays and chromatin immunoprecipitation. However, it should be noted that the gel shift assays are an in vitro assay and the chromatin immunoprecipitation is carried out with plants over-expressing CBF3-myc from the pSuper promoter and so do not necessarily reflect the native state. The authors then examine COOLAIR expression in lines over-expressing each of the three CBF proteins of Arabidopsis and found COOLAIR expression elevated in the warm in all three, but with small differences in the variants of COOLAIR that are expressed. Examination of the expression of COOLAIR after short-term cold shows that transcript abundance increases after 6 hours, this expression was not observed in the cbfs-1 where all three CBFs are knocked out. Taken together this provides good evidence that COOLAIR transcription is rapidly induced via CBFs on exposure to cold.

    The authors then go on to look at the roles of CBFs in longer-term cold. COOLAIR has previously been shown to increase during long-term cold (multiple weeks duration), so the question was whether this increase is CBF-dependent. The increase in COOLAIR abundance is similar to other CBF targets but does begin to decline with 40-day cold periods, presumably reflecting the shutdown of the FLC locus. The lack of COOLAIR expression in the cbfs-1 mutant is good evidence that increased COOLAIR expression is CBF-dependent. The authors also present evidence that CBFs are required for COOLAIR induction by the first seasonal frost, which is consistent with this being a short-term cold response.

    The authors then examine deletions of the COOLAIR promoter. In agreement with the hypothesis that CBFs regulate COOLAIR transcription via the CRT/DREs in the COOLAIR promoter, deletions that include the two elements do not show cold induction of COOLAIR, while one that contains them does. It should be noted that these deletions are relatively coarse so could include other elements than the CRT/DREs.

    The authors then use the finding that COOLAIR is not induced in the cbfs-1 mutant or in the deltaCOOLAIR1 and 3 lines to ask whether COOLAIR is required for the repression of FLC in the vernalization response. The data in Figures 6 and 7 show that these lines don't show different responses to vernalization treatment at the FLC expression, FLC chromatin modifications, or flowering time/leaf number to flowering. This supports the conclusion that the COOLAIR transcript does not play an essential role in the vernalization response.

    The Discussion is well-balanced and considers previous publications in this area and highlights differences with this study. The conservation of COOLAIR in other brassica species suggests that it does have a biological function, but the data here suggest it isn't an essential component of the vernalization response. Whether there is a function in more natural conditions where the temperature fluctuates in a diurnal manner during the vernalization period is a possibility that is considered. When the data presented here are taken with other publications, the precise biological role of COOLAIR remains enigmatic.