Vernalization-triggered expression of the antisense transcript <i>COOLAIR</i> is mediated by <i>CBF</i> genes.

Abstract

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