Divergence of annual and perennial species in the Brassicaceae and the contribution of cis-acting variation at FLC orthologues

Abstract

Variation in life history contributes to reproductive success in different environments. Divergence of annual and perennial angiosperm species is an extreme example that has occurred frequently. Perennials survive for several years and restrict the duration of reproduction by cycling between vegetative growth and flowering, whereas annuals live for 1 year and flower once. We used the tribe Arabideae (Brassicaceae) to study the divergence of seasonal flowering behaviour among annual and perennial species. In perennial Brassicaceae, orthologues of FLOWERING LOCUS C (FLC), a floral inhibitor in Arabidopsis thaliana, are repressed by winter cold and reactivated in spring conferring seasonal flowering patterns, whereas in annuals, they are stably repressed by cold. We isolated FLC orthologues from three annual and two perennial Arabis species and found that the duplicated structure of the A. alpina locus is not required for perenniality. The expression patterns of the genes differed between annuals and perennials, as observed among Arabidopsis species, suggesting a broad relevance of these patterns within the Brassicaceae. Also analysis of plants derived from an interspecies cross of A. alpina and annual A. montbretiana demonstrated that cis-regulatory changes in FLC orthologues contribute to their different transcriptional patterns. Sequence comparisons of FLC orthologues from annuals and perennials in the tribes Arabideae and Camelineae identified two regulatory regions in the first intron whose sequence variation correlates with divergence of the annual and perennial expression patterns. Thus, we propose that related cis-acting changes in FLC orthologues occur independently in different tribes of the Brassicaceae during life history evolution.

Keywords: Arabis; FLC; PEP1; annual; flowering time; perennial.

Figure 1

Left: Bayesian phylogenetic analysis of 17 representative annual and perennial species of the Arabideae based on a concatenated alignment of ITS, trnL, trnLF,CHS,ADH and At2g13360. Names of groups and/or larger genera of which only representatives were used in the phylogenetic reconstruction are given in boxes. Annuals are indicated by an encircled 1, posterior probability values are given at all nodes. Arabis alpina and Arabis montbretiana are confirmed to be sister species and together are sisters to the perennial A. nordmanniana. The reconstructed ancestral state for life history (Karl & Koch 2013) has been overlaid on the tree. Expression was studied in species indicated by *. Right: Structural evolution of FLC orthologues in the Arabideae. Throughout the branch leading to A. alpina, the structure of the locus becomes increasingly more complex. However, there is no relation of locus complexity and life cycle as A. nordmanniana loci orthologous to FLC have similar structures to that found in annual A. montbretiana. Colours are explained in the legend below the locus schemes. The sequence labelled as COLDAIR RNA encoding was defined by homology to A. thaliana (Heo & Sung 2011).

Figure 2

Relative expression of FLC orthologues in the perennials A. alpina (PEP1) and tetraploid A. nordmanniana (AnFLC‐A and AnFLC‐B) as well as in the annuals A. montbretiana (AmFLC) and A. auriculata (AauFLC). All examined orthologues are expressed before vernalization (0w = 0 weeks vernalization after growth at warm temperature for 3 or 6 weeks after germination) and repressed by vernalization (12 wv = 12 weeks vernalization at 4 °C). However, only in the perennial species are the FLC orthologues derepressed after vernalization because in the annual species, they stay stably repressed (12 wv + 2/3w = 12 weeks vernalization followed by 2 or 3 weeks at warm temperature). Expression values are relative to the respective orthologues of PP2A or RAN3 (A. nordmanniana) and relative to 0 weeks (0w) of vernalization. For each sample, leaves of four to six plants were pooled, error bars represent standard deviation of three or four technical qPCR replicates (n = 3–4). A biological replicate is given in the supplement (Fig. S4, Supporting information).

Figure 3

Arabis montbretiana (A), A. alpina (B) and A. montbretiana  ×  A. alpina (C) at 50 days after germination. D–F A. montbretiana  ×  A. alpina at (D) the onset of flowering at 99 days after germination, (E) fully flowering but not setting viable seeds after self‐pollination 129 days after germination, (F) perpetually growing and flowering 427 days after germination. (G) Relative expression of AmFLC and PEP1 through a vernalization cycle in two cuttings obtained from the hybrid in D–F. Both FLC orthologues are expressed before vernalization (0w = 0 weeks) and repressed by vernalization (12w = 12 weeks vernalization). After vernalization and return to warm temperatures (12d = 12 days and 19d = 19 days in warm), AmFLC stays stably repressed while PEP1 rises again in expression. Thus, in the interspecies hybrid, both FLC orthologues follow the expression pattern observed in the parents indicating that the differential expression is cis‐mediated. Expression is expressed relative to PP2A and relative to 0 weeks of vernalization as percentage. Error bars represent the propagated error of the standard deviation calculated for two biological replicates with four technical replicates (n = 3–4) each. H‐I Relative expression of PEP1 and/or AmFLC through a vernalization cycle in three introgression lines obtained from back crossing the hybrid in D–F to A. alpina. Expression is relative to PP2A and then expressed as % relative to expression at 0‐w vernalization. Error bars represent the standard deviation derived from three or four technical (n = 3–4) replicates. Different introgression lines represent biological replicates. H Analysis of three independent heterozygous plants (5034, 5002, 5078). The orthologues show different behaviours after vernalization and maintain the same or similar expression pattern that they show in the parental species (Fig. 2). I Analysis of three different AmFLC homozygous plants (5012, 5057, 5006). AmFLC displays the same stable expression pattern after vernalization as in the A. montbretiana control.

Figure 4

Distribution of SNPs (A) and indels (B) among PEP1 and AmFLC analysed by a sliding window approach (window size 100 bp, shift 20 bp). For the analysis of SNP distribution, all indels were excluded, while for the distribution analysis of indels, all indels were coded as 1 irrespective of their length; exons as well as sequence features homologous to regulatory elements known from FLC are annotated on top of the graph as a colour‐coded scheme. Nucleation region is the sequence homologous to the region showing elevated H3K27me3 before vernalization in Arabidopsis (Yang et al. 2014); COLDAIR represents the regions homologous to the COLDAIR RNA‐encoding region (Heo & Sung 2011); vernalization response element (VRE) indicates the region homologous to the VRE of A. thaliana identified in Sung et al. (2006); peak regions with windows containing significantly more SNPs than expected in a random distribution are marked by *.

Figure 5

Comparison of SNP distribution in (A) PEP1 vs. AmFLC and AlFLC1 vs. FLC and (B) AmFLC vs. AauFLC and PEP1 vs. AnFLC‐A by a sliding window analysis (window size 100 bp, shift 1 bp). For the analysis of SNP distribution, all indels were excluded; exons as well as sequence features homologous to regulatory elements known from FLC are annotated on top of the graph as a colour‐coded scheme. Exclusion of gap columns leads to deletion of elements which are duplicated only in PEP1, AmFLC and AnFLC‐A, and therefore, the schematic overview of the locus is shorter than the locus schemes in Fig. 4. Nucleation region is the sequence homologous to the region showing elevated H3K27me3 before vernalization in Arabidopsis (Yang et al. 2014); COLDAIR represents the regions homologous to the COLDAIR RNA‐encoding region (Heo & Sung 2011); vernalization response element (VRE) indicates the region homologous to the VRE of A. thaliana identified in (Sung et al. 2006); dashed lines represent thresholds above or below which more or fewer SNPs than expected by chance occur: (A) grey dashed line at 14 SNPs threshold for more and at two SNPs for fewer SNPs than expected by chance in AlFLC1 vs. FLC; line at six SNPs threshold for more SNPs than expected by chance for PEP1 vs. AmFLC; no window with significantly fewer SNPs, (B) blue dashed line significantly more SNPs than expected by chance for AmFLC vs. AauFLC, orange dashed line significantly more SNPs than expected by chance for PEP1 vs. AnFLC‐A, black dashed line fewer SNPs than expected for both curves. Peak regions with windows containing significantly more SNPs than expected in a random distribution are shaded in yellow; regions containing significantly fewer SNPs than expected in a random distribution are shaded in blue.

Figure 6

Summary of the regulatory regions of FLC and their variation among annual and perennial taxa. Top: Schematic representation of the FLC locus of A. thaliana including the minimal promoter, exon 1, intron 1 and exon 2 indicating the positions of SNPs, binding sites and regulatory elements that were identified in other studies as well as regions containing putative regulatory elements identified in this study. Lower diagrams depict segments of three reporter gene deletions reported by Sheldon et al. (2002) that showed unstable or stable repression after vernalization. The lowest diagram shows a deletion mutant in which FLC was not stably repressed after vernalization and defines the VRE (Sung et al. 2006).