Mediator subunit18 controls flowering time and floral organ identity in Arabidopsis

Abstract

Mediator is a conserved multi-protein complex that plays an important role in regulating transcription by mediating interactions between transcriptional activator proteins and RNA polymerase II. Much evidence exists that Mediator plays a constitutive role in the transcription of all genes transcribed by RNA polymerase II. However, evidence is mounting that specific Mediator subunits may control the developmental regulation of specific subsets of RNA polymerase II-dependent genes. Although the Mediator complex has been extensively studied in yeast and mammals, only a few reports on Mediator function in flowering time control of plants, little is known about Mediator function in floral organ identity. Here we show that in Arabidopsis thaliana, MEDIATOR SUBUNIT 18 (MED18) affects flowering time and floral organ formation through FLOWERING LOCUS C (FLC) and AGAMOUS (AG). A MED18 loss-of-function mutant showed a remarkable syndrome of later flowering and altered floral organ number. We show that FLC and AG mRNA levels and AG expression patterns are altered in the mutant. Our results support parallels between the regulation of FLC and AG and demonstrate a developmental role for Mediator in plants.

Figure 1. Phenotypes of Mediator subunit 18 (med18) mutants.

(A) Schematic diagram of the MED18 gene showing the locations of T-DNA insertions in the med18 mutants. Black rectangles represent exons, lines represent introns, and triangles represent T-DNA insertions; the med18-1 mutation corresponds to insertion line SAIL_889_C08, whereas the med18-2 mutation corresponds to insertion line, SALK_ 027178. (B, C) 35 day old wild type (B), med18-1 (C) plants. (D, E) Inflorescence of wild type (D), and med18-1 (E) plants. (F–H) Arabidopsis wild type (F), med18-1 (G), and dissected med18-1 (H) flower. (I, J) Transverse section of wild type (I), and med18-1 (J) carpels. (K, L) Anthers of wild type (K) and med18-1 (L) (stained with KI/I₂) at time of flowering. Scale bars: 1 mm in D,E,F,G and H; 100 µm in I,J,K and L.

Figure 2. Expression patterns of MED18.

(A–D) Longitudinal sections of wild type show MED18 mRNA detected in (A) developing seed, (B) inflorescence meristem, floral meristem and sepals, (C, D) petal, stamen, and carpel primordia. (E) Transverse section of a wild type flower shows MED18 mRNA expression in all floral organs. (F) MED18 mRNA sense strand control showing no non-specific hybridization. Numbers in B–D and F indicate flower stages; IM in B and F indicates inflorescence meristem. Scale bars: 100 µm in B–F and 25 µm in A.

Figure 3. MED18 controls flowering time.

(A) MED18 controls flowering time under both long day and short day conditions. Arrow indicates that flowering was not achieved when the experiment was terminated. (B) Transcripts levels of FT and FLC are altered in med18 mutants. (C, D) Flowering time in vernalization (C) and gibberellin (D) treatment. Vm: vernalization, GA: gibberellin. Scale bars indicate mean ± s.e.; **p≤0.01.

Figure 4. Double mutant of med18-1 and floral homeotic genes.

(A) ag-1 flower. (B) med18-1 ag-1 double mutant flower. (C) dissection of med18-1 ag-1 double mutant flower. (D) pi-1 flower. (E) med18-1 pi-1 double mutant flower. (F) ap2-5 flower. (G) med18-1 ap2-5 double mutant flower. Scale bars: 1 mm.

Figure 5. MED18 regulates floral homeotic gene expression.

(A) Relative transcript levels of six floral homeotic genes determined by real-time RT-PCR in wildtype and med18-1 mutants (**p≤0.01, *p≤0.05). (B, D, F, H) AG expression pattern as determined by in situ hybridization in wild type flowers. (C, E, G, I) The AG expression pattern in med18-1 mutant flowers. Numbers in B–I show flower stages, IM in B and C indicates inflorescence meristem. Arrows in (H) and (I) indicate expression in vascular bundles of stamens and carpels, in (F) and (G) showing developing pedals. Scale bars in B–I: 50 µm.

Figure 6. Model of MED18 control flowering time and floral organ identity.

Growth signals are transmitted to the Mediator complex by direct action or through HEN3 on MED18 to regulate the transcription of target genes. Both flowering time integrator FLC and floral organ identity organizer AG transcripts levels are determined by MED18. In addition, MED18 affects the transcription of other target genes, which are flowering time and floral organ identity regulators, such as FT, AP1 and PI. These regulators work together and with other factors to control flowering and floral organ formation.