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

Abstract

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 the present 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 function 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.

Heat Shock Transcription Factor B2b Acts as a Transcriptional Repressor of Vin3, a Gene Induced by Long-term Cold for Flowering

“…The circadian clock was previously demonstrated to be involved in the regulation of VIN3, and components of the circadian clock, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), directly regulate the diurnal rhythm of VIN3 during vernalization 23,26 . One of the circadian clock regulators, PRR7, has also been reported to be a transcription factor repressed by HsfB2b 22 , which is required for proper abiotic stress responses.…”

“…To identify upstream regulators of VIN3, the GUS reporter line (-0.2kb pVIN3_U_I::GUS) 23 was mutagenised with ethyl methanesulfonate (EMS). A total of 3,412 M1 lines were generated and plants showing enhanced GUS signals were isolated as hypersensitive mutants.…”

“…Yeast one-hybrid assay was performed following the previously described method with some modifications 23 . For the reporter constructs used in the Y1H analysis, four tandem repeats containing HSEVIN3 (5'-TTAGAAACATCTAGAAAAAACAAA-3') were cloned into the pHisi vector.…”

“…VIN3 expression is reported to show a circadian rhythm and HsfB2b acts as a negative regulator of the circadian clock regulator, PSEUDO RESPONSE REGULATOR7 (PRR7) 22,23,26 . Thus, we verified whether the VIN3 rhythm was affected by hsfb2b during cold treatment.…”

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

“…For example, NAC WITH TRANSMEMBRANE MOTIF 1-LIKE 8 (NTL8) has been identified as a direct regulator of VIN3 expression through its accumulation during long-term cold exposure 14 . Moreover, two circadian clock regulators, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) , were identified as direct regulators of VIN3, which presumably render diurnal rhythms of VIN3 expression 15 . Such findings provide supportive explanations for VIN3 regulation under multiple thermosensory pathways which was previously constructed by mathematical modeling of VIN3 dynamics 16 , 17 .…”