Abstract
Plants monitor many aspects of their fluctuating environments to help align their development with seasons. Molecular understanding of how noisy temperature cues are registered has emerged from dissection of vernalization in Arabidopsis, which involves a multiphase cold-dependent silencing of the floral repressor locus FLOWERING LOCUS C (FLC). Cold-induced transcriptional silencing precedes a low probability PRC2 epigenetic switching mechanism. The epigenetic switch requires the absence of warm temperatures as well as long-term cold exposure. However, the natural temperature inputs into the earlier transcriptional silencing phase are less well understood. Here, through investigation of Arabidopsis accessions in natural and climatically distinct field sites, we show that the first seasonal frost strongly induces expression of COOLAIR, the antisense transcripts at FLC Chamber experiments delivering a constant mean temperature with different fluctuations showed the freezing induction of COOLAIR correlates with stronger repression of FLC mRNA. Identification of a mutant that ectopically activates COOLAIR revealed how COOLAIR up-regulation can directly reduce FLC expression. Consistent with this, transgenes designed to knockout COOLAIR perturbed the early phase of FLC silencing. However, all transgenes designed to remove COOLAIR resulted in increased production of novel convergent FLC antisense transcripts. Our study reveals how natural temperature fluctuations promote COOLAIR regulation of FLC, with the first autumn frost acting as a key indicator of autumn/winter arrival.
Highlights
As sessile organisms, plants have to extract specific temperature cues from fluctuating environments to time their developmental transitions
In some cases, studies of transgenes aimed at attenuating antisense expression have concluded that COOLAIR expression is not required for vernalization (Helliwell et al 2011; Li et al 2018; Luo et al 2019; Luo and He 2020)
Through the analysis of >1000 worldwide Arabidopsis natural accessions, we previously identified five predominant FLOWERING LOCUS C (FLC) haplotypes defined by noncoding single-nucleotide polymorphism (SNP) (Li et al 2014)
Summary
Through the analysis of >1000 worldwide Arabidopsis natural accessions, we previously identified five predominant FLC haplotypes defined by noncoding SNPs (Li et al 2014). Given that the freezing-enhanced COOLAIR induction led to significantly lower FLC mRNA levels (Fig. 1E), these two TEX lines were tested in the temperature regimes of Figure 1B. COOLAIR was more induced in wild-type plants in the freezing regime compared with the other two temperature regimes (Fig. 3H), and FLC mRNA levels in the freezing regimes were lower (Fig. 3I). Type plants, grown without (NV) or with 2 wk of cold treatment (2WV), two major spliced distal COOLAIR forms with a range of transcriptional start sites were identified (Fig. 4A,B), consistent with the previous study (Swiezewski et al 2009). Similar to TEX1.0, antisense transcript start sites were detected in the MAF2-T terminator in the FLC + MAF2-T line (Supplemental Fig. S5D). The generation of novel COOLAIR-CAS transcripts from intragenic regions in FLC at each attempt to remove COOLAIR, including the CRISPR deletion of the endogenous COOLAIR promoter (FLCΔCOOLAIR), suggests a tight interconnection between antisense transcription and chromatin state at the locus
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