The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis

Abstract

The very late-flowering behavior of Arabidopsis winter-annual ecotypes is conferred mainly by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). A MADS-domain gene, AGAMOUS-LIKE 20 (AGL20), was identified as a dominant FRI suppressor in activation tagging mutagenesis. Overexpression of AGL20 suppresses not only the late flowering of plants that have functional FRI and FLC alleles but also the delayed phase transitions during the vegetative stages of plant development. Interestingly, AGL20 expression is positively regulated not only by the redundant vernalization and autonomous pathways of flowering but also by the photoperiod pathway. Our results indicate that AGL20 is an important integrator of three pathways controlling flowering in Arabidopsis.

Figure 1

Flowering phenotype of lines described in this study. (A) Homozygous fsu1-1D FRI FLC; (B) FRI FLC; (C) Columbia; (D) agl20; (E) 35S::LFY/−; (F) 35S::LFY/− FRI/−; (G) fsu1-1D/− 35S::LFY/− FRI/−; (H) fsu1-1D FRI flc-3; (I) fsu1-1D fri-Col FLC. The insets in (H) and (I) show ectopic flowers subtended by cauline leaves. All plants were grown under long days (16 h light/ 8 h dark). Bars, 1 cm.

Figure 2

Insertion of 35S enhancer in the promoter region of AGL20 causes overexpression. (A) DNA gel blot analysis showing the polymorphism between fsu1-1D FRI FLC (fsu1) and FRI FLC (FRI). (B) Diagram of genomic organization showing the insertion of 35S enhancer into the AGL20 region in fsu1-1D FRI FLC. LB and RB are left border and right border of T-DNA, respectively. A bar represents the DNA fragment obtained by plasmid rescue and used for DNA gel blot analyses. (C) RT-PCR result for SAP18, AGL6, and AGL20 in the leaves of 3-week-old Columbia (Col), FRI FLC (FRI), and fsu1-1D FRI FLC (fsu1). The β-tubulin gene (TUB) was amplified as a quantitative control. (D) The amino acid sequence of AGL20. MADS, I, and K domains are indicated. (E) Neighbor-joining phylogenetic tree for Arabidopsis MADS-domain proteins.

Figure 3

Effect of photoperiod and vernalization on the flowering time of agl20-101D FRI FLC. (A) The effect of photoperiod; (B) the effect of vernalization. agl20-101D FRI FLC is designated as agl20-101D. Flowering time was measured as the number of rosette leaves. agl20-101D was originally designated fsu1-1D.

Figure 4

Analysis of LFY promoter activity in agl20-101D FRI FLC and in situ localization of AGL20 RNA in Ler. Expression of a LFY::GUS transgene introduced into agl20-101D FRI FLC (A) and Columbia (B). The numbers on leaf primordia and floral meristems indicate node numbers above rosette leaves. Arrowheads indicate the position of secondary shoot primordia. In situ localization of AGL20 in the shoot apices of 8-day-old (C) and 12-day-old (D) Ler plants. agl20-101D was originally designated fsu1-1D. Bars, 50 μm.

Figure 5

Analysis of AGL20 expression by RT-PCR. (A) Expression of AGL20 in various organs. (B) A time course of expression of AGL20, FLC, and AP1. Lane numbers indicate days after germination. (C) Comparison of expression levels of AGL20 and FLC in Columbia (Col), FRI FLC (FRI), fri (FN235) FLC (fri), and FRI flc-3 (flc-3). (D) Effect of photoperiod and vernalization on the expression of AGL20 and FLC. To detect FLC expression in Columbia grown under short days and long days, the filter was overexposed. (E) Expression of AGL20 and FLC in agl20-101D FRI FLC (agl20D), 35S::AGL20 FRI FLC (S1, S2, S3), and agl20 knockout mutant. For agl20, the filter was overexposed to detect FLC. (F) Expression of AGL20 in late-flowering mutants in Columbia background. (G) Expression of AGL20 in late-flowering mutants in Ler background. The bar graphs below panels B, F, and G represent the relative expression level of AGL20. Standard deviation obtained from three independent experiments is indicated. The β-tubulin gene (TUB) was amplified as a quantitative control.

Figure 6

Model for the integrative role of AGL20 and the interaction of flowering pathways. The horizontal line represents the vegetative (V) to reproductive (R) transition. Arrows indicate promotion, and T-bars indicate repression. In the autonomous pathway, FRI activates FLC, and FLC represses AGL20. AGL20 acts as a floral activator. Other autonomous pathway genes, such as LD, FCA, and FVE, promote flowering by activating AGL20 through the repression of FLC. Vernalization also promotes flowering by activating AGL20 expression through the repression of FLC. Photoperiod pathway genes, CO and GI, promote flowering by activating AGL20 but act also through other factor(s).