A putative CCAAT-binding transcription factor is a regulator of flowering timing in Arabidopsis

Abstract

Flowering at the appropriate time of year is essential for successful reproduction in plants. We found that HAP3b in Arabidopsis (Arabidopsis thaliana), a putative CCAAT-binding transcription factor gene, is involved in controlling flowering time. Overexpression of HAP3b promotes early flowering while hap3b, a null mutant of HAP3b, is delayed in flowering under a long-day photoperiod. Under short-day conditions, however, hap3b did not show a delayed flowering compared to wild type based on the leaf number, suggesting that HAP3b may normally be involved in the photoperiod-regulated flowering pathway. Mutant hap3b plants showed earlier flowering upon gibberellic acid or vernalization treatment, which means that HAP3b is not involved in flowering promoted by gibberellin or vernalization. Further transcript profiling and gene expression analysis suggests that HAP3b can promote flowering by enhancing expression of key flowering time genes such as FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1. Our results provide strong evidence supporting a role of HAP3b in regulating flowering in plants grown under long-day conditions.

Figure 1.

Delayed flowering in hap3b mutants and early flowering in HAP3b-overexpression plants grown under a 16-h/8-h light/dark photoperiod. A, Delayed flowering in hap3b. B, hap3b has a T-DNA insertion at 9 bp after the first ATG. C, No transcript was detected in hap3b using RT-PCR using two independent plants; there is no intron in HAP3b and the PCR products are the same when using genomic DNA as a template (gDNA). D, hap3b developed more leaves before flowering compared with wild-type plants (wt); overexpression plants (P_actin:HAP3b in wild-type background) flowered earlier with fewer leaves compared with control plants (C1: P_actin:GUS in wild-type background). The data are means ± se (n = approximately 30) from three independent experiments. **, Indicates P < 0.001 compared with wild type.

Figure 2.

Localization of HAP3b in nuclei using GFP as a reporter gene: leaf epidermal cells (A) and root tip cells (B).

Figure 3.

Flower timing assays showing that HAP3b function is restricted to modifying a long-day photoperiod flowering timing pathway. A and B show that the hap3b mutants delay in flowering (under long-day conditions) is not reversed by GA (A) or vernalization (B), whereas mutant hap3b and wild-type plants grown under a short-day photoperiod show no differences. *, Indicates P < 0.01; **, indicates P < 0.001 compared with minus GA control (A) or minus vernalization control (B) or wild type (C).

Figure 4.

SOC1 (A) and FT (B) genes were down-regulated in hap3b but up-regulated in HAP3b-overexpression plants. The transcript level of each gene was determined using a qPCR method and was then normalized with the transcript level of ACT2. The fold-change was calculated by dividing the normalized transcript level of hap3b by that of wild-type plants (wt) or the normalized transcript level of overexpression plants (P_actin:HAP3b) by that of C1 (P_actin:GUS) plants. The data are means ± se of three independent experiments. The pattern was reproducible in each experiment.

Figure 5.

A proposed model for how HAP3b expression can promote early flowering. [See online article for color version of this figure.]