Floral Induction in Arabidopsis by FLOWERING LOCUS T Requires Direct Repression of BLADE-ON-PETIOLE Genes by the Homeodomain Protein PENNYWISE

Abstract

Flowers form on the flanks of the shoot apical meristem (SAM) in response to environmental and endogenous cues. In Arabidopsis (Arabidopsis thaliana), the photoperiodic pathway acts through FLOWERING LOCUS T (FT) to promote floral induction in response to day length. A complex between FT and the basic leucine-zipper transcription factor FD is proposed to form in the SAM, leading to activation of APETALA1 and LEAFY and thereby promoting floral meristem identity. We identified mutations that suppress FT function and recovered a new allele of the homeodomain transcription factor PENNYWISE (PNY). Genetic and molecular analyses showed that ectopic expression of BLADE-ON-PETIOLE1 (BOP1) and BOP2, which encode transcriptional coactivators, in the SAM during vegetative development, confers the late flowering of pny mutants. In wild-type plants, BOP1 and BOP2 are expressed in lateral organs close to boundaries of the SAM, whereas in pny mutants, their expression occurs in the SAM. This ectopic expression lowers FD mRNA levels, reducing responsiveness to FT and impairing activation of APETALA1 and LEAFY. We show that PNY binds to the promoters of BOP1 and BOP2, repressing their transcription. These results demonstrate a direct role for PNY in defining the spatial expression patterns of boundary genes and the significance of this process for floral induction by FT.

Figure 1.

Identification and cloning of a functional suppressor of FT. Phenotypic comparison between lgf58, pGAS1::FT ft-10 tsf-1 and Columbia-0 (Col-0) wild-type plants (A) and their flowering time under LD (n = 10; B). CL, Cauline leaves; RL, rosette leaves. Letters shared in common between the genotypes indicate no significant difference (Student’s t test, P < 0.05). C, Graphic showing the allelic frequency estimations at EMS-induced mutations (AF; y axis) across chromosome 5 (Mb; x axis) of lgf58. AFs were calculated by dividing the number of reads supporting the mutant allele by the number of all reads aligning to a given marker. The color code indicates the resequencing consensus (SHORE) score. EMS mutations showing a SHORE score higher than 25 were selected. AFs in chromosome 5 were higher compared with other regions in the genome (see also Supplemental Fig. S2). D, Scheme of the PNY locus showing the position of the mutation and the sequence change found in lgf58.

Figure 2.

Pattern of expression of PNY during photoperiod flowering. A, PNY mRNA (i–iii) and protein accumulation (iv–vi). Plants were grown for 14 d under SD (i and iv; vegetative stage) and then shifted to LD for 3 (ii and v; floral transition) and 5 additional days (iii and vi; flower development). Scale bars = 50 µm. B, ChIP-quantitative PCR (qPCR) showing binding of SOC1:GFP on PNY promoter. The y axis represents the fold change (FC) of enrichment (percentage of input) between the qPCR results using positive primers (flanking a CArG-box motif) and the negative ones (flanking the coding sequence of PNY). Letters shared in common between the genotypes indicate no significant difference (Student’s t test, P < 0.05). C, Localization of the primers used for ChIP-qPCR of SOC1:GFP on the PNY promoter. Region (+) contains a CArG-box cis-motif, which was enriched after immunoprecipitation of SOC1:GFP. Region (−) was used as a negative control. D, Study of the PNY expression in plants shifted from SDs to LDs. Plants were grown for 2 weeks under SDs and then shifted to LDs for 3, 5, and 7 additional days. RNA was extracted from dissected shoot apices. Error bars indicate sd.

Figure 3.

BOP1/2 genes are important for flowering regulation mediated by PNY. Expression of BOP1 (A) and BOP2 (B) of shoot apices dissected from plants grown under LDs for 7 d. Letters shared in common between the genotypes indicate no significant difference (Student’s t test, P < 0.05). C, In situ hybridizations with BOP2 probe in shoot meristems of Col-0 (i) and pny-58 (ii). Plants were grown for 7 LDs and stayed at the vegetative stage. Expression of BOP2 in Col-0 plants was observed at the boundaries between leaves and the SAM (black arrow). Scale bars = 50 µm. D, Flowering time of the BOP1/2 and PNY mutant combinations grown under LDs. CL, Cauline leaves; RL, rosette leaves. Error bars in A, B, and D indicate sd.

Figure 4.

BOP1/2 genes interfere with FT function and are directly bound by PNY. A, Flowering time of plants misexpressing BOP genes in the presence of high levels of FT. Letters shared in common between the genotypes indicate no significant difference (Student’s t test, P < 0.05). B, Expression of LFY of dissected shoot apices of plants grown for 7 LDs. Asterisks indicate statistical differences between Col-0 and other genotypes (Student’s t test; **, P = 0.0004; *, P = 0.006). C, Expression of AP1 of dissected shoot apices of plants grown for 7 LDs. Asterisks indicate statistical differences between Col-0 and other genotypes (Student’s t test; ***, P = 0.001; **, P = 0.006; *, P = 0.03). ChIP-qPCR of PNY on the promoters of BOP1 (D) and BOP2 (E). x axis indicates the primers used for its qPCR. Asterisks indicate statistical differences between pPNY::Venus:PNY and pny-40126 (Student’s t test; **, P = 0.0001; *, P = 0.02). F, Localization of the primers used for the ChIP-qPCR experiment on the BOP1 (top scheme) and BOP2 (bottom scheme) loci. *, AP1 binding genomic regions according to Kaufmann et al. (2010). Scale bars = 0.5 kb. Error bars in A to E indicate sd.

Figure 5.

BOP1/2 genes regulate the pattern of expression of FD. A, FD expression levels in different plants misexpressing BOP1/2 and PNY. RNA was extracted from shoot apices of plants grown during 7 LDs (vegetative stage), 10 to 13 LDs (floral transition), and 17 LDs (reproductive stage). Asterisks indicate statistical differences between Col-0 and other genotypes (Student’s t test; ***, P = 0.05; **, P = 0.01; *, P = 0.001). B, In situ hybridization of plants grown for 10 LDs showing the expression pattern of FD in Col-0 (i), pny-58 (ii), bop1-3 bop2-1 (iii), and bop1-6D (iv). Bar = 50 µm. Error bars in A to C indicate sd.

Figure 6.

Model explaining the spatial regulation of flowering-related genes by PNY and BOP1/2. It has been suggested that FT is delivered from the phloem to the proximity of the SAM (dashed line; Yoo et al., 2013). Once FT is in the shoot meristem, it is supposed to interact with FD to activate the transcription of AP1 (yellow shade). After the floral induction, BOP1/2 activate the transcription of AP1 mRNA in the FM. We showed that PNY directly represses BOP1/2 expression (blue shade) in the shoot meristem, so that, in the absence of PNY (pny-58, bottom), BOP1/2 pattern of expression becomes broader. We also found that BOP1/2 repress FD expression before floral transition. Thus, the ectopic expression of BOP1/2 in pny mutants leads to the reduction of the expression domain of FD mRNA (green shade) in the shoot meristem. Consequently, FT-FD complex formation might be impaired (gray arrow) and the transcriptional activation of AP1 mRNA reduced.