Analysis of the molecular basis of flowering time variation in Arabidopsis accessions

Abstract

Allelic variation at the FRI (FRIGIDA) and FLC (FLOWERING LOCUS C) loci are major determinants of flowering time in Arabidopsis accessions. Dominant alleles of FRI confer a vernalization requirement causing plants to overwinter vegetatively. Many early flowering accessions carry loss-of-function fri alleles containing one of two deletions. However, some accessions categorized as early flowering types do not carry these deletion alleles. We have analyzed the molecular basis of earliness in five of these accessions: Cvi, Shakhdara, Wil-2, Kondara, and Kz-9. The Cvi FRI allele carries a number of nucleotide differences, one of which causes an in-frame stop codon in the first exon. The other four accessions contain nucleotide differences that only result in amino acid substitutions. Preliminary genetic analysis was consistent with Cvi carrying a nonfunctional FRI allele; Wil-2 carrying either a defective FRI or a dominant suppressor of FRI function; and Shakhdara, Kondara, and Kz-9 carrying a functional FRI allele with earliness being caused by allelic variation at other loci including FLC. Allelic variation at FLC was also investigated in a range of accessions. A novel nonautonomous Mutator-like transposon was found in the weak FLC allele in Landsberg erecta, positioned in the first intron, a region required for normal FLC regulation. This transposon was not present in FLC alleles of most other accessions including Shakhdara, Kondara, or Kz-9. Thus, variation in Arabidopsis flowering time has arisen through the generation of nonfunctional or weak FRI and FLC alleles.

Figure 1.

Nucleotide and amino acid changes in the FRI gene of the five accessions Cvi, Wil-2, Shakhdara, Kondara, and Kz-9 relative to the active FRI allele from H51 (accession no. AF228499). Sequences containing a stop codon are underlined. a.a., Amino acid; Nucl., nucleotide; nd, not determined. The asterisk indicates an insertion. ^*a, ^*b, and ^*c are the first, second, and third nucleotide insertions after nucleotide 459 from H51. These changes have been used to infer the evolutionary relationship between the different FRI alleles, and this is represented schematically. The Wil-2 allele is represented as arising from a recombination event between a Cvi-like and a Shakhdara/Kondara/Kz-9-like allele.

Figure 2.

A, Flowering time analysis of the five accessions Cvi, Wil-2, Shakhdara, Kondara, and Kz-9 assayed as total leaf number at flowering. B, Northern analysis of FRI and FLC in the five accessions Cvi, Wil-2, Shakhdara, Kondara, and Kz-9.

Figure 3.

Transposon Insertion in FLC. A, Untranslated (gray boxes) and coding regions (white boxes) and start and stop codons (ATG and TAG, respectively) of FLC are indicated. The position of the 30-bp repeat is indicated by black arrows, and the insertion site of the transposon in Ler, DiG, and Di2 is indicated by a triangle. Single-nucleotide substitutions are shown by asterisks. Numbering is based on the A of the ATG start codon as +1. B, Sequence of the 30-bp sequence repeated n times, where n = 1 or 2. C, Sequence flanking the insertion site in Ler is shown in plain text, the 9-bp target site duplication (TSD) is underlined, and the transposon sequence is indicated in bold. The Col sequence shown corresponds to bacterial artificial chromosome T31P16 (accession no. AL356332), positions 40,620 to 40,591. D, Inverted repeat of the TE showing the first and last 50 bp of the transposon in Ler, with complementary residues indicated by vertical lines.