PEP1 of Arabis alpina is encoded by two overlapping genes that contribute to natural genetic variation in perennial flowering

Abstract

Higher plants exhibit a variety of different life histories. Annual plants live for less than a year and after flowering produce seeds and senesce. By contrast perennials live for many years, dividing their life cycle into episodes of vegetative growth and flowering. Environmental cues control key check points in both life histories. Genes controlling responses to these cues exhibit natural genetic variation that has been studied most in short-lived annuals. We characterize natural genetic variation conferring differences in the perennial life cycle of Arabis alpina. Previously the accession Pajares was shown to flower after prolonged exposure to cold (vernalization) and only for a limited period before returning to vegetative growth. We describe five accessions of A. alpina that do not require vernalization to flower and flower continuously. Genetic complementation showed that these accessions carry mutant alleles at PERPETUAL FLOWERING 1 (PEP1), which encodes a MADS box transcription factor orthologous to FLOWERING LOCUS C in the annual Arabidopsis thaliana. Each accession carries a different mutation at PEP1, suggesting that such variation has arisen independently many times. Characterization of these alleles demonstrated that in most accessions, including Pajares, the PEP1 locus contains a tandem arrangement of a full length and a partial PEP1 copy, which give rise to two full-length transcripts that are differentially expressed. This complexity contrasts with the single gene present in A. thaliana and might contribute to the more complex expression pattern of PEP1 that is associated with the perennial life-cycle. Our work demonstrates that natural accessions of A. alpina exhibit distinct life histories conferred by differences in PEP1 activity, and that continuous flowering forms have arisen multiple times by inactivation of the floral repressor PEP1. Similar phenotypic variation is found in other herbaceous perennial species, and our results provide a paradigm for how characteristic perennial phenotypes might arise.

Figure 1. Flowering behavior of A. alpina accessions.

(A) Accession Paj grown vegetatively for four years in long day glasshouse. Paj has an obligate requirement for vernalization to flower. (B)–(I) Non-vernalization requiring A. alpina accessions at flowering under long days. Accession Dor (B), Tot (C), Wca (D), Cza (E) and Mug (F). (G) Flowering times of non-vernalization requiring A. alpina accessions under long days (16 hours light) compared to pep1-1 mutant and the accession Paj. Flowering time is measured as days to flower (DTF). pep1-1 mutant (H) and the accession Dor (I) flower perpetually after 6 months in long days. (J) Duration of flowering in non-vernalization requiring A. alpina accessions.

Figure 2. Non-vernalization requiring accessions do not rescue the early flowering phenotype of the pep1-1 mutant.

(A) PEP1 mRNA levels in leaves of non-vernalization requiring accessions compared to Paj. (B) Flowering time of F1 hybrids resulted from crosses of non-vernalization requiring accession with pep1-1 mutant and Paj in long days without vernalization. The pep1-1 mutant and Paj were used as controls. Flowering time is measured as days to flower (DTF). (C) PEP1 accumulation in different accessions compared to the accession Paj before vernalization. pep1-1 and Paj after 16 weeks in vernalization were used as negative controls. A cross reacting protein acts as a loading control.

Figure 3. Analysis of sequence variation in PEP1 cDNA and at the genomic locus of Dor accession demonstrates a complex structure for the PEP1 gene.

(A) PEP1 cDNAs in Dor is a mixture of transcripts that contain a G to A substitution in exon 1 compared to Paj or have a similar sequence to accession Paj but have an insertion of 248 bp in the 5′ UTR. (B) Sequence of the PEP1 genomic locus in the accession Paj shows that the locus is highly duplicated. Exons are indicated with black boxes, UTRs with white boxes and solid lines the inter- and intra-genic regions. Upstream and downstream genes of PEP1 are 35 kb apart. Colored boxes indicate relative positions of the duplicated regions. Overlapping boxes indicate overlapping homologous sequences. Numbers besides duplicated boxes show the length of the duplicated fragment and percentage of homology. Duplicated exon 1 copies are indicated as 1a and 1b. Dotted box shows the PEP1 locus region sequenced in the accession Dor. (C) Sequence of the PEP1 genomic locus in the Dor accession reveals that G to A base substitution is in exon 1a. Grey arrows indicate insertions, black arrows indicate deletions and vertical dotted lines indicate SNPs relative to Paj PEP1 locus. The 248 bp insertion upstream in the 5′ UTR is upstream of exon 1b. Colored boxes indicate relative positions of duplicated regions. (D) Structure of the PEP1 locus and predicted splicing events (E) PEP1 transcripts in the accession Dor detected with two different primers in the 5′ UTR using the same reverse primer in the 3′ UTR. Black and grey arrows indicate the position of two different primers in the 5′ UTR relative to the 248 bp insertion. When primer PEP1_5UTRF1 (black) was used most clones contained the G to A substitution in the exon 1. A few clones that did not contain the G to A base substitution also contained a 248 bp insertion in the 5′ UTR. When primer PEP1_5UTRF2 was used most clones did not contain G to A base substitution.

Figure 4. PEP1 locus is tandemly duplicated in several A. alpina accessions.

(A) Structure of the PEP1 locus and the position of exon 1a and exon 1b specific primers. (B) Sequence comparisons of FLC and tandem duplicated copies 1a and 1b in accession Paj (grey to blue boxes in Figure 3A). Vista plot using Calc. window 25, Min cons width 25 and Cons identity 70%. (C) Alignment using part of the 416 bp sequence (yellow box) specific for exon 1a from different FLC homologues. Intr1aR primer was designed in a consensus sequence. (D) PCR test using PEP1a specific primers (Ex1F and Intr1aR) in different accessions. Template used is Dor (1), Tot (2), Wca (3), Cza (4), Mug (5) Paj (6) and water control (7). (E) Alignment using part of the intron sequence downstream of PEP1b. Intr1bR primer was designed in a duplicated region (pink box in Figure 3C, 3B, Figure 4A) conserved in other FLC homologues. (F) PCR test using PEP1b specific primers (Ex1F and Intr1bR) in different accessions. Template used is Dor (1), Tot (2), Wca (3), Cza (4), Mug (5) Paj (6) and water control (7). (G) PEP1 structure of the accessions Paj, Dor, Cza and Tot obtained by sequencing the PEP1 locus.

Figure 5. PEP1a and PEP1b genes in the accession Dor are independently transcribed and have different transcriptional start sites.

(A,B) Number of clones containing G to A polymorphism on exon 1 (A) or not (B) after 5′ RACE using apices from Dor plants growing for 3 weeks in long days. Schematic representation of exon1 and 5′ UTR regions (top), exon1a and exon 1b (black boxes), sequence present in 5′ UTR upstream of both exon 1a and 1b (white box), sequence specific to 5′ UTR upstream of exon 1a (red box), sequence specific to 5′ UTR upstream exon 1b on the 248 bp insertion (grey box). Horizontal lines represent individual clones. Numbers on the top represent bp upstream of ATGs. (B,C) Percentage of clones with the A or G polymorphism after 5′ RACE in apices and leaves before (3 week long days) and after vernalization (5 weeks in long days after 12 weeks vernalization). (D)–(F) PEP1 mRNA levels on 3 week old Dor plants, vernalized for 12 weeks and subsequently grown for several week in long days. (D) PEP1 (a+b) expression, primers used similar as in to detect both transcripts (E) PEP1a expression, primers used to detect only PEP1a transcripts. (F) PEP1b expression, primers used to detect only PEP1b transcripts.