The bZIP3-AS1 complex promotes CONSTANS-induced FLOWERING LOCUS T activation in a daylength-dependent manner

Abstract

Plants monitor daylength to synchronize their flowering time with their surroundings and thus maximize reproductive fitness. In Arabidopsis (Arabidopsis thaliana), CONSTANS (CO) activates the expression of FLOWERING LOCUS T (FT); this activation is a crucial aspect of the daylength-dependent regulation of flowering time. Here, we demonstrate that the basic leucine zipper 3 (bZIP3) transcription factor is important for CO-induced FT expression under long photoperiod conditions in Arabidopsis. We isolated bZIP3 as a CO-interacting protein by yeast two-hybrid screening and verified bZIP3-CO complex formation in Arabidopsis through co-immunoprecipitation assays. The temporal and spatial expression patterns of bZIP3 are very similar to those of CO, and bZIP3 protein levels fluctuate throughout the day, with high abundance in the late afternoon. The bzip3 mutant displayed delayed flowering under long photoperiods, whereas bZIP3 overexpression accelerated flowering regardless of daylength. bZIP3 directly binds to the FT promoter region containing CO-responsive elements in vivo. FT messenger RNA (mRNA) levels in the bzip3 mutant and bZIP3 overexpression lines correlated with their flowering times and changed only during the daytime. bZIP3 overexpression resulted in significantly lower FT transcript levels in the co mutant background than in the wild type. Furthermore, bZIP3 forms a complex with ASYMMETRIC LEAVES1 (AS1), a CO partner that helps CO induce FT expression. The bzip3 as1 double mutant flowered later than the two single mutants under longer daylengths, and FT mRNA levels were much lower in the double mutant than in the bzip3 single mutant. Collectively, our findings uncover a new layer of photoperiod-dependent FT regulation in which bZIP3 facilitates CO to activate FT transcription by forming a complex with AS1.