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N 6-methyladenosine (m6A) RNA methylation has been shown to play a crucial role in plant development and floral transition. Two recent studies have identified FIONA1 as an m6A methyltransferase that regulates the floral transition in Arabidopsis through influencing the stability of CONSTANS (CO), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and FLOWERING LOCUS C (FLC). In this study, we confirmed that FIONA1 is an m6A methyltransferase that installs m6A marks in a small group of mRNAs. Furthermore, we show that, in addition to its role in influencing the stability of CO, SOC1, and FLC, FIONA1-mediated m6A methylation influences the splicing of FLC, a key floral repressor, and the stability of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 3 (SPL3) and SEPALLATA3 (SEP3), floral activators, which together play a vital role in floral transition in Arabidopsis. Our study confirms the function of FIONA1 as an m6A methyltransferase and suggests a close molecular link between FIONA1-mediated m6A methylation and the splicing of FLC and the destabilization of SPL3 and SEP3 in flowering time control.

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.

Arabidopsis TBP-ASSOCIATED FACTOR15b (TAF15b) is a plant-specific component of the transcription factor IID (TFIID) complex. TAF15b is involved in the autonomous pathway for flowering and represses transcription of FLOWERING LOCUS C (FLC), a strong floral repressor in Arabidopsis. Although the components of the autonomous pathway for flowering have been studied extensively, little is known about the functional partners of TAF15b and the mechanism of how TAF15b works. Here, we show that C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1) is a physical and functional partner of TAF15b for the transcriptional repression of FLC. CPL1 is a protein phosphatase that dephosphorylates C-terminal domain (CTD) of RNA polymerase II (Pol II). Through the immunoprecipitation and mass spectrometry technique, we fished out the CPL1 protein as an interacting partner of TAF15b. Similar to taf15b, the cpl1 mutant showed a late-flowering phenotype caused by the increase of FLC expression. In addition, FLC increase in cpl1 was correlated with the enrichment of phosphorylated Pol II in FLC chromatin as expected. We also found that CPL1 and TAF15b share additional common target genes through transcriptome analysis. Such results suggest that the TAF15b–CPL1 complex directly represses transcription through dephosphorylation of Pol II, especially at the FLC locus.

N 6-methyladenosine (m 6A) RNA methylation has been shown to play a crucial role in plant development and floral transition. Two recent studies have identified FIONA1 as an m 6A methyltransferase that regulates the floral transition via influencing the stability of CONSTANS (CO), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and FLOWERING LOCUS C (FLC) in Arabidopsis. In this study, we confirmed that FIONA1 is an m 6A methyltransferase that installs m 6A marks in a small group of mRNAs. Furthermore, in addition to its role in influencing the stability of CO, SOC1, and FLC, we show that FIONA1-mediated m 6A methylation influences the splicing of FLC, a key floral repressor, and the stability of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 3 (SPL3) and SEPALLATA3 (SEP3), floral activators, which plays a vital role in the floral transition in Arabidopsis. Our study confirms FIONA1 as an m 6A methyltransferase and suggests a close molecular link between FIONA1-mediated m 6A methylation and the splicing of FLC and the destabilization of SPL3 and SEP3 in flowering time control.

To synchronize flowering time with spring, many plants undergo vernalization, a floral-promotion process triggered by exposure to long-term winter cold. InArabidopsis 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 theFLClocus, has been proposed to facilitateFLCsilencing. Here, we show that C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3′-end ofFLCand CRT/DRE-binding factors (CBFs) are required for cold-mediated expression ofCOOLAIR. CBFs bind to CRT/DREs at the 3′-end ofFLC, bothin vitroandin vivo, and CBF levels increase gradually during vernalization. Cold-inducedCOOLAIRexpression is severely impaired incbfsmutants in which allCBFgenes are knocked-out. Conversely,CBF-overexpressing plants show increasedCOOLAIRlevels even at warm temperatures. We show thatCOOLAIRis induced by CBFs during early stages of vernalization but COOLAIR levels decrease in later phases asFLCchromatin transitions to an inactive state to which CBFs can no longer bind. We also demonstrate thatcbfsandFLCΔCOOLAIRmutants exhibit a normal vernalization response despite their inability to activateCOOLAIRexpression during cold, revealing thatCOOLAIRis not required for the vernalization process.