The microRNA-regulated SBP-Box transcription factor SPL3 is a direct upstream activator of LEAFY, FRUITFULL, and APETALA1

Abstract

When to form flowers is a developmental decision that profoundly impacts the fitness of flowering plants. In Arabidopsis this decision is ultimately controlled by the induction and subsequent activity of the transcription factors LEAFY (LFY), FRUITFULL (FUL), and APETALA1 (AP1). Despite their central importance, our current understanding of the regulation of LFY, FUL, and AP1 expression is still incomplete. We show here that all three genes are directly activated by the microRNA-targeted transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3). Our findings suggest that SPL3 acts together with other microRNA-regulated SPL transcription factors to control the timing of flower formation. Moreover, the identified SPL activity defines a distinct pathway in control of this vital developmental decision.

Fig. 1. SPL3 activates meristem identity gene expression

(A) Schematic representation of the known interactions involved in the meristem identity (MI) switch (see text for details). Flowering time regulators are indicated in orange, MI regulators in pink. Solid arrows denote direct interactions, dashed arrows denote direct or indirect interactions. (B-D) qRT-PCR analysis of flowering time gene (B) or MI gene expression (C, D) in 35S:SPL3Δ (SPL3OX) compared to wild-type plants. Plants were grown in short-day (SD) for 4 and 10 days (B, C) or in long-day (LD) for 7 and 9 days (D). (E) Temporal expression of SPL3, LFY, FUL and AP1 in wild-type plants grown in LD. The ratio of the expression at each time-point over the final expression level is graphed. (B-E) Values are mean +/- SEM. Black solid horizontal line: two fold increase, black dotted line: two fold decrease. (F) Confocal image of pSPL3:GFP-SPL3 (top view of inflorescence meristem) and in situ hybridization of LFY, FUL and AP1 (longitudinal section through inflorescence meristem). Arrowheads point to the incipient flower primordium, numbers denote stages of flower development. Bar: 50μm.

Fig. 2. The precocious meristem identity phenotype of 35S:SPL3Δ requires a functional copy of LFY, FUL and AP1

Phenotype of 35S:SPL3Δ double mutants with lfy-1 (A), ful-2 (B), and ap1-10 (C). Top: side view of same age plants. Note the increased number of secondary inflorescences in the double mutants (left) compared to 35S:SPL3Δ (right). Bottom: top view of shoot apex close-ups from 5 cm tall primary inflorescences. Note the leafy appearance of 35S:SPL3Δ lfy-1 and 35S:SPL3 ap1-10 due to the presence of additional cauline leaves. Plants were grown in LD. Upper panel bar: 1 cm; lower panel bar: 1mm.

Fig. 3. SPL3 binds regulatory regions of meristem identity genes in vivo

(A) Early flowering phenotype of LD-grown SPL3 overexpressing plants compared to wildtype. The asterisk marks the primary inflorescence bolt. Bar; 1 cm. Fluorescence image of 35S:SPL3Δ-GFP in leaf one (B) and pSPL3:GFP-SPL3 in leaf four (C). Inset in (B); close-up confocal microscopy image. Bar; 1 mm. (D) Schematic of the LFY, FUL and AP1 loci. Pale blue and green boxes represent untranslated regions and exons, respectively. Asterisks indicate computationally identified SPL consensus motifs. Pink horizontal lines: fragments amplified in by qPCR after ChIP. Blue boxes: regulatory regions in the LFY promoter previously identified (Blazquez and Weigel, 2000). (E, F) qPCR of anti-GFP ChIP in 35S:SPL3-GFPΔ (E), pSPL3:GFP-SPL3 and wildtype (F). Plants were grown in SD for 10 days (E) or LD for 7 days (F). Immunoprecipitated DNA enrichment is presented as percent input DNA. Shown is the mean +/- SEM.

Fig. 4. SPL4 and SPL5 upregulate meristem identity genes

qRT-PCR analysis of meristem identity gene expression in 35S:SPL4Δ and 35S:SPL5Δ compared to wild-type plants (A, B) and in 35S:miR156a compared to wild-type plants (C). Plants were grown in SD for 10 days (A) and in LD for 9 days (B) or 10 days (C). Shown is the mean +/- SEM relative to the wildtype. Black solid horizontal line: two fold increase, black dotted line: two fold decrease.

Fig. 5. Regulatory interactions between flowering time and meristem identity regulators

(A) qRT-PCR analysis of the known FT targets LFY, FUL, AP1 and SOC1 as well as SPL3 in 35S:FT-GFP plants grown in LD. Plants were harvested at day 4 and day 6. Shown is the expression mean +/- SEM relative to the wild type. Black solid horizontal line: two fold increase. (B) Phenotypes of F1 progeny of 35S:FT-GFP x 35S:miR156a (top) and 35S:SPL3 × 35S:FT-GFP (bottom). Star: secondary inflorescences, arrow: flower subtended by cauline leaf, asterisk: primary inflorescence. Bar; 1cm. (C) Role of SPL3 in meristem identity transition. Solid arrows denote direct interactions, dashed arrows denote direct or indirect interactions. SPL3 directly regulates LFY, FUL and AP1 transcription (red arrows) and acts in pathway that is partly in parallel with the FT pathway (red dashed arrow).