Transcription Factor Interplay between LEAFY and APETALA1/CAULIFLOWER during Floral Initiation

Abstract

The transcription factors LEAFY (LFY) and APETALA1 (AP1), together with the AP1 paralog CAULIFLOWER (CAL), control the onset of flower development in a partially redundant manner. This redundancy is thought to be mediated, at least in part, through the regulation of a shared set of target genes. However, whether these genes are independently or cooperatively regulated by LFY and AP1/CAL is currently unknown. To better understand the regulatory relationship between LFY and AP1/CAL and to obtain deeper insights into the control of floral initiation, we monitored the activity of LFY in the absence of AP1/CAL function. We found that the regulation of several known LFY target genes is unaffected by AP1/CAL perturbation, while others appear to require AP1/CAL activity. Furthermore, we obtained evidence that LFY and AP1/CAL control the expression of some genes in an antagonistic manner. Notably, these include key regulators of floral initiation such as TERMINAL FLOWER1 (TFL1), which had been previously reported to be directly repressed by both LFY and AP1. We show here that TFL1 expression is suppressed by AP1 but promoted by LFY. We further demonstrate that LFY has an inhibitory effect on flower formation in the absence of AP1/CAL activity. We propose that LFY and AP1/CAL act as part of an incoherent feed-forward loop, a network motif where two interconnected pathways or transcription factors act in opposite directions on a target gene, to control the establishment of a stable developmental program for the formation of flowers.

Figure 1.

Genes controlled by LFY in the absence of AP1/CAL function. A, Experimental set-up of the genome-wide analyses using inflorescence-like meristems from p35S:LFY-GR ap1 cal plants. CHX, cycloheximide; Dex, dexamethasone. B, K-means clustering (k = 5) of 669 DEGs identified in the p35S:LFY-GR ap1 cal time course experiment. Log₂-transformed expression ratios (dexamethasone/mock) for the different time points (as indicated) were used for the analysis. C, Gene Ontology terms enriched among the DEGs at different time points (as indicated). Negative decadal logarithms of P values are shown. D, Response of selected LFY targets (as indicated) to an activation of LFY-GR in ap1 cal inflorescences. E and F, Response of AP3 (E) and AG (F) to an activation of LFY-GR and AP1-GR, respectively, in ap1 cal inflorescences. Data for AP1-GR were taken from Kaufmann et al. (2010). In D to F, log₂-transformed fold-change values (dexamethasone/mock) from microarray experiments are shown.

Figure 2.

Comparison of gene expression programs acting downstream of LFY and AP1. A and B, Overlap between genes differentially expressed after AP1-GR and LFY-GR activation in ap1 cal inflorescences. Overlaps for different time points (as indicated) are shown in A for the LFY-GR and in B for the AP1-GR time course experiments. C to F, Antagonistic regulation of selected genes after activation of AP1-GR and LFY-GR in ap1 cal inflorescences. Log₂-transformed expression ratios (dexamethasone/mock) for the different time points (as indicated) are shown. Data for AP1-GR were taken from Kaufmann et al. (2010). C, TFL1. D, FD. E, TEMPRANILLO1. F, AP2.

Figure 3.

Direct activation of TFL1 by LFY. A and B, Relative expression of TFL1 (A) and LMI1 (B) in p35S:LFY-GR ap1 cal inflorescences after mock treatment (mock), treatments with cycloheximide (CHX) or dexamethasone (DEX) alone, or after a combined treatment with both cycloheximide and dexamethasone (CHX/DEX). C, Results of ChIP-qPCR experiments. Binding of LFY to selected known target genes (on the left, as indicated) and negative control regions (on the right; MU through TUB1) was tested after activation of LFY-GR in ap1 cal inflorescences. Error bars indicate ses of four biologically independent measurements. D, Results of ChIP-Seq experiments. Binding of LFY to the TFL1 locus was tested after activation of LFY-GR in ap1 cal inflorescences. A peak was detected ∼2.8 to 3.3 kb downstream of the transcribed region of the gene.

Figure 4.

Ectopic expression of TFL1 in LFY-VP16 lines. A, pTFL1:GUS activity in a wild-type inflorescence. Staining was restricted to the shoot apical meristem. B to D, pTFL1:GUS activity in pLFY:LFY-VP16 inflorescences. B, Staining was observed in the center of the shoot apical meristem as well as in flowers. C and D, Staining at the base of the gynoecium (C) and along pedicels (D) in cleared flowers. Arrows point to ectopic pTFL1:GUS activity in flowers. E and F, Ectopic activation of pTFL1:GUS in plants expressing LFY-VP16 from a heat shock promoter. E, Seedlings grown at ambient temperature with pTFL1:GUS activity in the shoot apical meristem. F, Heat-shocked seedling with additional staining in cotyledons, leaves, and roots. Asterisks in panels A, B, E, and F mark the position of the shoot apical meristem.

Figure 5.

Antagonistic activities of LFY and AP1. A to D, Effects of LFY-GR and AP1-GR activation, or of a simultaneous activation of both fusion proteins in ap1 cal inflorescences on the expression of selected genes (as indicated). Tissue was collected 3 h after treating plants with a dexamethasone-containing solution. Log₂-transformed fold-change values (dexamethasone/mock) from qRT-PCR experiments are shown. Error bars indicate ses from four independent measurements.

Figure 6.

Activation of LFY in ap1 cal mutants delays the onset of flowering. A, Response of p35S:AP1-GR ap1 cal (on the left) and p35S:LFY-GR ap1 cal (on the right) plants to treatments with a dexamethasone-containing (Dex) or a mock solution. Images of inflorescences 4, 6, 10, and 18 d after the treatment are shown. Note the complete lack of floral structures in dexamethasone-treated p35S:LFY-GR ap1 cal plants at the 18-d time point. Scale bars = 1 mm. B, Onset of flowering in dexamethasone- and mock-treated p35S:LFY-GR ap1 cal plants. Percentage of plants in a treatment population with visible floral structures are shown. Error bars indicate ses calculated from three independent measurements. C, Model for the activities of LFY and AP1 (and CAL) during floral initiation. Left: gene interactions in the wild type; right: gene interactions after inactivation of AP1/CAL. Font size changes for gene symbols and small arrows indicate up- and down-regulation, respectively. TFL1, LFY, and AP1/CAL appear to be part of an incoherent feed-forward loop, where LFY promotes expression of TFL1 but is also involved in activating the TFL1 repressors, AP1/CAL. At the same time, LFY and AP1/CAL are also part of a positive feedback loop and reinforce each other’s expression through direct regulation.