SUPPRESSOR OF FRIGIDA3 encodes a nuclear ACTIN-RELATED PROTEIN6 required for floral repression in Arabidopsis

Abstract

Flowering traits in winter annual Arabidopsis thaliana are conferred mainly by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). FLC acts as a flowering repressor and is regulated by multiple flowering pathways. We isolated an early-flowering mutant, suppressor of FRIGIDA3 (suf3), which also shows leaf serration, weak apical dominance, and infrequent conversion of the inflorescence shoot to a terminal flower. The suf3 mutation caused a decrease in the transcript level of FLC in both a FRI-containing line and autonomous pathway mutants. However, suf3 showed only a partial reduction of FLC transcript level, although it largely suppressed the late-flowering phenotype. In addition, the suf3 mutation caused acceleration of flowering in both 35S-FLC and a flc null mutant, indicating that SUF3 regulates additional factor(s) for the repression of flowering. SUF3 is highly expressed in the shoot apex, but the expression is not regulated by FRI, autonomous pathway genes, or vernalization. SUF3 encodes the nuclear ACTIN-RELATED PROTEIN6 (ARP6), the homolog of which in yeast is a component of an ATP-dependent chromatin-remodeling SWR1 complex. Our analyses showed that SUF3 regulates FLC expression independent of vernalization, FRI, and an autonomous pathway gene, all of which affect the histone modification of FLC chromatin. Subcellular localization using a green fluorescent protein fusion showed that Arabidopsis ARP6 is located at distinct regions of the nuclear periphery.

Figure 1.

Phenotypes of the suf3 Mutant. (A) The wild type (Col:FRI^SF2), Col, and suf3-1 grown under long days. Photographs were taken when flowering initiated. (B) Comparison of leaf shape in the wild type, Col, and suf3-1. The leaves are shown in order of production from the first true leaf at left. (C) Phenotypes of all seven suf3 alleles that show serrated leaves and increased numbers of coflorescence shoots. (D) Conversion of the secondary shoot apex to a terminal flower in suf3. White arrows indicate terminal flowers. (E) suf3-1 flower with five petals.

Figure 2.

Positional Cloning of the SUF3 Gene. (A) The genetic interval, molecular markers, BAC clones, and deletion region are shown. Genomic DNA with marker SH39 could not be amplified by PCR, indicating that the region is deleted in suf3-1. Marker SH39 is located in the first intron of At3g33530. The deletion region in suf3-1 is indicated. (B) SUF3 gene structure showing exons (thick bars) and introns (thin bars). The gray bars represent untranslated regions (UTRs). The underlined region was used to generate AtARP6 RNAi transgenic plants. (C) Phenotype of transgenic plants. (a) Wild type. (b) suf3-1. (c) 35S-ARP6 in suf3-1. (d) ARP6 RNAi in the Col:FRI^SF2 wild type. (e) Flowering time and ARP6 expression level of individual ARP6 RNAi transgenic T1 plants. TUB, TUBULIN.

Figure 3.

Alignment of Deduced Amino Acids of ARP6. The ARP6 homologs of Arabidopsis (At), rice (Oryza sativa; Os), human (Hs), Drosophila (Dm), and yeast (Sc) were aligned with Arabidopsis ACTIN2 (ACT2). ARP6s have a conserved core consisting of two α/β subdomains in the actin family. ARP6s have two peptide insertions (boxed regions) without disrupting the conserved actin fold structure. Dots indicate residues that have structurally equivalent roles in the nucleotide binding site. The amino acid sequences were aligned using ClustalW version 1.7 (Thompson et al., 1994).

Figure 4.

Expression Pattern of SUF3. (A) SUF3 expression in different tissues. Tissues were harvested from 20-d-old Col:FRI^SF2 wild-type plants grown in long days for whole plant (WP), root (RT), shoot apex (SA), and rosette leaves (RL). The tissues for old leaves (OL) were harvested from 60-d-old wild-type plants. The expression level was determined by RNA gel blot analysis. (B) SUF3 expression in the wild type treated with (+) and without (−) vernalization. Total RNAs for RT-PCR analysis were extracted from plants grown at 4°C for 40 d under short days. TUB, TUBULIN. (C) SUF3 expression in FRI or the flowering time mutants ld-1, fca-9, fld-1, gi-2, co-1, ft-1, and soc1-2. All plants were grown for 10 d under long days. (D) In situ hybridization analysis. (a) SUF3 expression in 12-d-old Col plants grown in long days. (b) SUF3 expression in the inflorescence of a Col plant. The asterisk indicates the shoot apical meristem, and the numbers below the flower meristems indicate the floral stages. (c) SUF3 expression in the flower meristem. The numbers inside the flower meristems indicate the floral stages. (d) In situ hybridization with a sense control. No signal was detected.

Figure 5.

FLC Expression in suf3 Mutants and the Effect on Flowering Time. (A) RNA gel blot analysis in all suf3 alleles. Numbers above the lanes are relative transcript levels of FLC compared with the wild type. FLC, SOC1, and SUF3 transcript levels in suf3 alleles were compared with those in the wild type and Col. Plants were grown for 10 d in long days. SUF3 expression was not detected in all suf3 alleles. (B) Flowering time of all suf3 alleles. Error bars indicate sd based on means obtained for 15 plants for each line. (C) FLC expression in different tissues of suf3-1. Numbers above the lanes are relative transcript levels of FLC compared with whole plants from the wild type. WP, whole plant; RT, root; SA, shoot apex; RL, rosette leaves; OL, old leaves. (D) Effect of the suf3 mutation on flowering time of plants with different genetic backgrounds. Flowering times of FRI FLC (wild type), fri FLC (Col), fri flc, and 35S-FLC were compared in the presence or absence of the suf3 mutation. The effect of 5 weeks of vernalization on the flowering time of wild type and suf3 plants was also determined. Black bars represent plants with SUF3, and gray bars represent plants with the suf3 mutation. Error bars are as in (B). (E) FLC and SOC1 transcript levels in double mutants of suf3 and autonomous pathway mutants were determined by RNA gel blot analysis. All plants were grown for 10 d under long days. (F) Flowering time of double mutants of suf3 and autonomous pathway mutants. The suf3 mutation suppressed late flowering of autonomous pathway mutants. Error bars are as in (B).

Figure 6.

Effect of the suf3 Mutation on the Expression of FT, SOC1, and Other Flowering Time Genes. (A) FT and SOC1 expression in wild-type, suf3, Col, and suf3 fri plants grown under long days (LD) for 10 d or under short days (SD) for 20 d were determined by RT-PCR. High transcript levels of FT and SOC1 were detected in suf3 fri under short days. TUB, TUBULIN. (B) Expression of other flowering time genes in suf3-1 and wild-type plants grown for 10 d under long days as determined by RT-PCR.

Figure 7.

Localization of ARP6:GFP and YFP:ARP6 Fusion Proteins in Arabidopsis Protoplast Transient Assay. Chloroplasts appear red or blue (pseudocolor). GFP and YFP fluorescence is green, and RFP fluorescence is red. ARP6:GFP and YFP:ARP6 were localized in distinct regions of the nuclear periphery. (A) Protoplast expressing GFP alone. (B) Protoplast expressing NLS:RFP. (C) Protoplast expressing TFL2:RFP. (D) Protoplast expressing ARP6:GFP. (E) to (H) Protoplasts expressing YFP:ARP6. (E) and (G) are transparent images. (I) to (L) Protoplasts expressing both TFL2:RFP and ARP6:GFP. (I) Section of a protoplast transparent image. (J) ARP6:GFP fluorescence. (K) TFL2:RFP fluorescence. (L) Merged image of TFL2:RFP and ARP6:GFP fluorescence. All images are projections except for (I).