The two clock proteins CCA1 and LHY activate VIN3 transcription during vernalization through the vernalization-responsive cis-element.

Vernalization, a long-term cold-mediated acquisition of flowering competence, is critically regulated by VERNALIZATION INSENSITIVE 3 (VIN3), a gene induced by vernalization in Arabidopsis. Although the function of VIN3 has been extensively studied, how VIN3 expression itself is upregulated by long-term cold is not well understood. In this study, we identified a vernalization-responsive cis-element in the VIN3 promoter, VREVIN3, composed of a G-box and an evening element (EE). Mutations in either the G-box or the EE prevented VIN3 expression from being fully induced upon vernalization, leading to defects in the vernalization response. We determined that the core clock proteins CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE-ELONGATED HYPOCOTYL (LHY) associate with the EE of VREVIN3, both in vitro and in vivo. In a cca1 lhy double mutant background harboring a functional FRIGIDA allele, long-term cold-mediated VIN3 induction and acceleration of flowering were impaired, especially under mild cold conditions such as at 12°C. During prolonged cold exposure, oscillations of CCA1/LHY transcripts were altered, while CCA1 abundance increased at dusk, coinciding with the diurnal peak of VIN3 transcripts. We propose that modulation of the clock proteins CCA1 and LHY participates in the systems involved in sensing long-term cold for the activation of VIN3 transcription.

MUN (MERISTEM UNSTRUCTURED), encoding a SPC24 homolog of NDC80 kinetochore complex, affects development through cell division in Arabidopsis thaliana.

Kinetochore, a protein super-complex on the centromere of chromosomes, mediates chromosome segregation during cell division by providing attachment sites for spindle microtubules. The NDC80 complex, composed of four proteins, NDC80, NUF2, SPC24 and SPC25, is localized at the outer kinetochore and connects spindle fibers to the kinetochore. Although it is conserved across species, functional studies of this complex are rare in Arabidopsis. Here, we characterize a recessive mutant, meristem unstructured-1 (mun-1), exhibiting an abnormal phenotype with unstructured shoot apical meristem caused by ectopic expression of the WUSCHEL gene in unexpected tissues. mun-1 is a weak allele because of the insertion of T-DNA in the promoter region of the SPC24 homolog. The mutant exhibits stunted growth, embryo arrest, DNA aneuploidy, and defects in chromosome segregation with a low cell division rate. Null mutants of MUN from TALEN and CRISPR/Cas9-mediated mutagenesis showed zygotic embryonic lethality similar to nuf2-1; however, the null mutations were fully transmissible via pollen and ovules. Interactions among the components of the NDC80 complex were confirmed in a yeast two-hybrid assay and in planta co-immunoprecipitation. MUN is co-localized at the centromere with HTR12/CENH3, which is a centromere-specific histone variant, but MUN is not required to recruit HTR12/CENH3 to the kinetochore. Our results support that MUN is a functional homolog of SPC24 in Arabidopsis, which is required for proper cell division. In addition, we report the ectopic generations of stem cell niches by the malfunction of kinetochore components.

Vernalization-triggered expression of the antisense transcript COOLAIR is mediated by CBF genes.

To synchronize flowering time with spring, many plants undergo vernalization, a floral-promotion process triggered by exposure to long-term winter cold. In Arabidopsis thaliana, this is achieved through cold-mediated epigenetic silencing of the floral repressor, FLOWERING LOCUS C (FLC). COOLAIR, a cold-induced antisense RNA transcribed from the FLC locus, has been proposed to facilitate FLC silencing. Here, we show that C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3'-end of FLC and CRT/DRE-binding factors (CBFs) are required for cold-mediated expression of COOLAIR. CBFs bind to CRT/DREs at the 3'-end of FLC, both in vitro and in vivo, and CBF levels increase gradually during vernalization. Cold-induced COOLAIR expression is severely impaired in cbfs mutants in which all CBF genes are knocked-out. Conversely, CBF-overexpressing plants show increased COOLAIR levels even at warm temperatures. We show that COOLAIR is induced by CBFs during early stages of vernalization but COOLAIR levels decrease in later phases as FLC chromatin transitions to an inactive state to which CBFs can no longer bind. We also demonstrate that cbfs and FLCΔCOOLAIR mutants exhibit a normal vernalization response despite their inability to activate COOLAIR expression during cold, revealing that COOLAIR is not required for the vernalization process.

Long spells of cold winter weather may feel miserable, but they are often necessary for spring to blossom. Indeed, many plants need to face a prolonged period of low temperatures to be able to flower; this process is known as vernalization. While the molecular mechanisms which underpin vernalization are well-known, it is still unclear exactly how plants can 'sense' the difference between short and long periods of cold. Jeon, Jeong et al. set out to explore this question by focusing on COOLAIR, one of the rare genetic sequences identified as potentially being able to trigger vernalization. COOLAIR is a long noncoding RNA, a partial transcript of a gene that will not be 'read' by the cell to produce a protein but which instead regulates how and when certain genes are being switched on. COOLAIR emerges from the locus of the FLC gene, which is one of the main repressors of flowering, and it gradually accumulates in the plant when temperatures remain low for a long period. While some evidence suggests that COOLAIR may help to switch off FLC, other studies have raised some doubts about its involvement in vernalization. In response, Jeon, Jeong et al. examined the FLC gene in a range of plants closely related to A. thaliana, and in which COOLAIR also accumulates upon cold exposure. This helped them identify a class of proteins, known as CBFs, which could bind to sequences near the FLC gene to activate the production of COOLAIR when the plants were kept in cold conditions for a while. CBFs were already known to help plants adapt to short cold snaps, but these experiments confirmed that they could act as both short- and long-term cold sensors. This work allowed Jeon, Jeong et al. to propose a model in which CBF and therefore COOLAIR levels increase as the cold persists, until changes in the structure of the FLC gene prevent CBF from binding to it and COOLAIR production drops. Unexpectedly, examining the fate of mutants which could not produce COOLAIR revealed that these plants could still undergo vernalization, suggesting that the long noncoding RNA is in fact not necessary for this process. These results should prompt other scientists to further investigate the role of COOLAIR in vernalization; they also give insight into how coding and noncoding sequences may have evolved together in various members of the A. thaliana family to adapt to the environment.

HEAT SHOCK TRANSCRIPTION FACTOR B2b acts as a transcriptional repressor of VIN3, a gene induced by long-term cold for flowering.

Vernalization, an acceleration of flowering after long-term winter cold, is an intensively studied flowering mechanism in winter annual plants. In Arabidopsis, Polycomb Repressive Complex 2 (PRC2)-mediated suppression of the strong floral repressor, FLOWERING LOCUS C (FLC), is critical for vernalization and a PHD finger domain protein, VERNALIZATION INSENSITIVE 3 (VIN3), recruits PRC2 on FLC chromatin. The level of VIN3 was found to gradually increase in proportion to the length of cold period during vernalization. However, how plants finely regulate VIN3 expression according to the cold environment has not been completely elucidated. As a result, we performed EMS mutagenesis using a transgenic line with a minimal promoter of VIN3 fused to the GUS reporter gene, and isolated a mutant, hyperactivation of VIN3 1 (hov1), which showed increased GUS signal and endogenous VIN3 transcript levels. Using positional cloning combined with whole-genome resequencing, we found that hov1 carries a nonsense mutation, leading to a premature stop codon on the HEAT SHOCK TRANSCRIPTION FACTOR B2b (HsfB2b), which encodes a repressive heat shock transcription factor. HsfB2b directly binds to the VIN3 promoter, and HsfB2b overexpression leads to reduced acceleration of flowering after vernalization. Collectively, our findings reveal a novel fine-tuning mechanism to regulate VIN3 for proper vernalization response.