Nonadditive regulation of FRI and FLC loci mediates flowering-time variation in Arabidopsis allopolyploids

Abstract

Allopolyploidy is formed by combining two or more divergent genomes and occurs throughout the evolutionary history of many plants and some animals. Transcriptome analysis indicates that many genes in various biological pathways, including flowering time, are expressed nonadditively (different from the midparent value). However, the mechanisms for nonadditive gene regulation in a biological pathway are unknown. Natural variation of flowering time is largely controlled by two epistatically acting loci, namely FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FRI upregulates FLC expression that represses flowering in Arabidopsis. Synthetic Arabidopsis allotetraploids contain two sets of FLC and FRI genes originating from Arabidopsis thaliana and A. arenosa, respectively, and flower late. Inhibition of early flowering is caused by upregulation of A. thaliana FLC (AtFLC) that is trans-activated by A. arenosa FRI (AaFRI). Two duplicate FLCs (AaFLC1 and AaFLC2) originating from A. arenosa are expressed in some allotetraploids but silenced in other lines. The expression variation in the allotetraploids is associated with deletions in the promoter regions and first introns of A. arenosa FLCs. The strong AtFLC and AaFLC loci are maintained in natural Arabidopsis allotetraploids, leading to extremely late flowering. Furthermore, FLC expression correlates positively with histone H3-Lys4 methylation and H3-Lys9 acetylation and negatively with H3-Lys9 methylation, epigenetic marks for gene activation and silencing. We provide evidence for interactive roles of regulatory sequence changes, chromatin modification, and trans-acting effects in natural selection of orthologous FLC loci, which determines the fate of duplicate genes and adaptation of allopolyploids during evolution.

Figure 1.

Flowering-time variation correlated with FLC expression in Arabidopsis allotetraploids and their progenitors. (A) A total of 25 nascent allotetraploids (F₁) were produced by direct hybridization between A. thaliana (Ler) autotetraploid (At4) and A. arenosa autotetraploid (Aa). Allo733, -738, and -745 were produced by self-pollination, and Allo745 was outcrossed to A. suecica in S3. (B) Natural A. suecica (As) was formed by combining A. thaliana and A. arenosa ancestral genomes. Three strains are shown. All photos were taken at 100 days after seed germination. (C) Flowering-time variation in A. thaliana (Ler) autotetraploid (At4), A. arenosa (Aa), Allo733, -738, -745, and natural A. suecica strains (As9502, AsLC1, and As13).

Figure 2.

Phylogeny of FLC/MADS box gene family in Arabidopsis and Brassica. (A) Phylogenetic tree of FLC loci constructed using the maximum parsimony method (see materials and methods). The box with dashed lines indicates the phylogenetic location of three new FLC alleles detected in A. thaliana-related species. The GenBank accession numbers are provided in the supplemental data (http://www.genetics.org/supplemental/). (B) Partial sequence alignment of AtFLC, AaFLC1, AaFLC2, and AsFLC1. Letters and – indicate changes and no changes, respectively, in AaFLC1, AaFLC2, and AsFLC1 compared to AtFLC. The forward and reverse primers used in RT–PCR analysis and the ClaI site (ATCGAT) in AtFLC are underlined. Allele-specific TaqMan probes (boldface type) contain the dinucleotide polymorphism (boxed) in quantitative RT–PCR analysis. The GenBank accession numbers of FLC loci are as follows: AtFLC (At5G10140), AaFLC1 (DQ167446), AsFLC1 (DQ167447), AaFLC2 (DQ167444), BrFLC1-1 (AY364013), BrFLC1-2 (AY273165), BrFLC1-3 (AY273164), BnFLC3 (AY036890), BoFLC1 (AY273161), BoFLC3 (AY306123), RsFLC (AY273160), BnFLC4 (AY036891), BoFLC4 (AY306122), BnFLC2 (AY036889), BnFLC1 (AY036888), BnFLC5 (AY036892), MAF2 (At5g65050), AGL31 (AF312667), MAF3 (At5g65060), MAF1 (AF342808), MAF4 (At5g65070), and MAF5 (AY231455).

Figure 3.

Sequence changes in the promoter regions and first introns of FLC loci. (A) Diagrams showing sequence variation in the promoters and first introns of FLC in A. thaliana, A. arenosa, and A. suecica. Open boxes and solid lines indicate exons and genomic fragments, respectively. ATG and TAG codons are shown. AaFLC/AsFLC had a 253-bp promoter fragment, whereas AtFLC had a 1.5-kbp fragment. Transposon insertion in the first intron (1.3 kbp) was detected in AtFLC (Ler) but not in A. thaliana (Col), A. arenosa, or A. suecica. Locations of the PCR primers (arrows and expected size of the fragments) in the first intron are indicted below each diagram. A 557-bp deletion in the first intron of AaFLC1, -2, and AsFLC2 is shown. Dashed line in AsFLC1 indicates sequence variation compared to other FLC loci. (B) PCR amplification of the intron fragments using the primers indicated above. The corresponding FLC loci are indicated on the right. The same strains as in A were used for genotyping. M, molecular size markers.

Figure 4.

Expression of AaFLC and AtFLC in A. thaliana tetraploid, A. arenosa, synthetic allotetraploids, and A. suecica. (A) RT–PCR and cleaved amplified polymorphic sequence analysis of allele-specific expression of FLC. FLC was amplified using the primers indicated in Figure 2B (top), and the amplified fragments were digested with ClaI (middle). ACT2 was amplified as a control for mRNA and PCR amplification (bottom). (B) Quantitative RT–PCR (qRT–PCR) analysis of AtFLC and AaFLC/AsFLC expression in A. suecica strains, allotetraploids, and their progenitors using allele-specific probes (Figure 2B). At2SF2, A. thaliana SF2, a late-flowering ecotype. Solid and open bars indicate relative expression levels of AtFLC and AaFLC/AsFLC, respectively.

Figure 5.

(A) AaFRI confers late-flowering phenotype in rapid cycling A. thaliana Col. The construct for transgenic plants is shown at the top. LB, left border; p35S, 35S CaMV promoter; AaFRI, full-length AaFRI cDNA; OCS, octopine gene; pNOS, NOS promoter; NPTII, selective marker; NOS, terminator; RB, right border. Restriction sites for cloning are shown. The transgenic plants (Col-AaFR1#1 and -#2, T1) grew slowly. A late-flowering SF2 and an early-flowering Col are shown. (B) Activation of AtFLC in the transgenic plants. Relative expression levels of FLC and FRI were determined by qRT–PCR analysis in SF2, Col, and transgenic plants.

Figure 6.

(A) Expression variation detected by semiquantitative RT–PCR and SSCP analyses using primers shown in Figure 2B. Products in lanes (AaFLC1 and AaFLC2) were amplified from inserts in plasmid DNA as molecular size markers. The corresponding loci indicated on the right were determined by cloning and sequencing individual cDNA fragments (data not shown). Note that two different-size bands in AtFLC or AsFLC1 represented the same allele. (B) ChIP analysis of promoter regions of AtFLC (left) and AaFLC/AsFLC (right). FLC expression is associated positively with histone H3-Lys4 dimethylation and H3-Lys9 acetylation and negatively with H3-Lys9 dimethylation. The immunoprecipitates were heated to reverse the crosslinks and amplified by PCR using the A. thaliana and A. arenosa promoter-specific primers (supplemental Figure 1, http://www.genetics.org/supplemental/) so that only the AtFLC promoter was amplified in the left and the AaFLC in the right. The controls were the amplified DNA, respectively, from the chromatin fractions prior to antibody incubation (Input) and from those that were precipitated without antibodies (mock).

Figure 7.

Vernalization reduces FLC expression (RNA blot analysis, middle) and induces early flowering in A. arenosa (Aa) and A. suecica 9502 (As9502) (top). Agarose gel showing total RNA as a loading control for RNA blot analysis (bottom).