Regulation ofAPETALA3floral homeotic gene expression by meristem identity genes

Abstract

The Arabidopsis APETALA3 (AP3) floral homeotic gene is required for specifying petal and stamen identities, and is expressed in a spatially limited domain of cells in the floral meristem that will give rise to these organs. Here we show that the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) are required for the activation of AP3. The LFY transcription factor binds to a sequence, with dyad symmetry, that lies within a region of the AP3 promoter required for early expression of AP3. Mutation of this region abolishes LFY binding in vitro and in yeast one hybrid assays, but has no obvious effect on AP3 expression in planta. Experiments using a steroid-inducible form of LFY show that, in contrast to its direct transcriptional activation of other floral homeotic genes, LFY acts in both a direct and an indirect manner to regulate AP3 expression. This LFY-induced expression of AP3 depends in part on the function of the APETALA1 (AP1) floral homeotic gene, since mutations in AP1 reduce LFY-dependent induction of AP3 expression. LFY therefore appears to act through several pathways, one of which is dependent on AP1 activity, to regulate AP3 expression.

The F-box cofactor UFO redirects the LEAFY floral regulator to novel cis-elements

“…To decipher the molecular mechanism underlying the synergistic action of LFY and UFO, we used a Dual Luciferase Reporter Assay (DLRA) to analyze their ability to activate promoters when transiently expressed in Arabidopsis protoplasts. We found that pAP3, the best characterized LFY-UFO target (Hill et al, 1998; Lamb et al, 2002), is strongly activated when LFY (or LFY-VP16, a fusion of LFY with the VP16 activation domain; Parcy et al, 1998) is co-expressed with UFO (or UFO-VP16) but not by either effector alone (Figure 1A and 1E). We also tested the promoter of RABBIT EARS ( RBE ), a gene expressed in petal primordia in a UFO-dependent manner (Krizek et al, 2006).…”

“…Several regulatory regions driving AP3 expression in early floral meristem have been identified, including the Distal Early Element (DEE) and the Proximal Early Element (PEE; Figure 2A; Hill et al, 1998). The DEE contains a predicted canonical LFYBS (Lamb et al, 2002; Figure S2A). Replacing this site by the high-affinity LFYBS from pAP1 did not enhance activation (Figure S2B) and mutating it only slightly reduced pAP3 activation (Figure S2C).…”

“…Replacing this site by the high-affinity LFYBS from pAP1 did not enhance activation (Figure S2B) and mutating it only slightly reduced pAP3 activation (Figure S2C). Hence, in protoplasts like in plants (Lamb et al, 2002), pAP3 LFYBS at DEE is not sufficient to explain pAP3 activation. Previous studies suggested that LFY-UFO activation might involve the PEE and the region directly upstream (Hill et al, 1998; Lamb et al, 2002; Tilly et al, 1998).…”

“…Hence, in protoplasts like in plants (Lamb et al, 2002), pAP3 LFYBS at DEE is not sufficient to explain pAP3 activation. Previous studies suggested that LFY-UFO activation might involve the PEE and the region directly upstream (Hill et al, 1998; Lamb et al, 2002; Tilly et al, 1998). Indeed, in the protoplasts assay, we found that deleting the 107-bp region upstream of the PEE reduced LFY-UFO-dependent pAP3 activation (Figure 2B).…”

“…Other rows represent the different pAP3 versions used in (B-E). LFYBS mutation corresponds to the previously described site1m-site2m mutation (Lamb et al, 2002). The LUBS0 mutation is described in (H).…”