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Natural haplotypes of FLM non-coding sequences fine-tune flowering time in ambient spring temperatures in Arabidopsis

  1. Ulrich Lutz
  2. Thomas Nussbaumer
  3. Manuel Spannagl
  4. Julia Diener
  5. Klaus FX Mayer
  6. Claus Schwechheimer Is a corresponding author
  1. Technische Universität München, Germany
  2. Helmholtz Zentrum München, German Research Center for Environmental Health, Germany
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Cite as: eLife 2017;6:e22114 doi: 10.7554/eLife.22114

Abstract

Cool ambient temperatures are major cues determining flowering time in spring. The mechanisms promoting or delaying flowering in response to ambient temperature changes are only beginning to be understood. In Arabidopsis thaliana, FLOWERING LOCUS M (FLM) regulates flowering in the ambient temperature range and FLM is transcribed and alternatively spliced in a temperature-dependent manner. We identify polymorphic promoter and intronic sequences required for FLM expression and splicing. In transgenic experiments covering 69% of the available sequence variation in two distinct sites, we show that variation in the abundance of the FLM-ß splice form strictly correlate (R2 = 0.94) with flowering time over an extended vegetative period. The FLM polymorphisms lead to changes in FLM expression (PRO2+) but may also affect FLM intron 1 splicing (INT6+). This information could serve to buffer the anticipated negative effects on agricultural systems and flowering that may occur during climate change.

https://doi.org/10.7554/eLife.22114.001

Introduction

Plants are sessile organisms that have adapted to their habitats to optimize flowering time and thereby guarantee reproductive success and survival. Temperature is one major cue controlling flowering time, particularly before the winter but also during spring when ambient cool temperatures generally delay and warm temperatures promote flowering. Different molecular pathways controlling flowering in different temperature ranges and environments have been genetically dissected (Capovilla et al., 2015; Verhage et al., 2014).

In many plant species and in many accessions of the plant model species Arabidopsis thaliana (Arabidopsis), the well-studied vernalization pathway prevents premature flowering before the long cold periods of the winter (Song et al., 2012). In Arabidopsis, flowering of winter-annual accessions without vernalization is strongly delayed by the MADS-box transcription factor FLOWERING LOCUS C (FLC) (Song et al., 2012; Michaels and Amasino, 1999, 2001; Johanson et al., 2000). FLC forms a repressor complex through interactions with the MADS-box transcription factor SVP (SHORT VEGETATIVE PHASE) to repress the transcription of the flowering promoting genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) (Li et al., 2008; Lee et al., 2007). As a result of the prolonged exposure to cold temperatures during winter, FLC abundance is gradually reduced, predominantly through epigenetic mechanisms, and flowering repression is gradually relieved (Song et al., 2012). Substantial natural variation in the vernalization-dependent expression of FLC has already been described and characterized in many Arabidopsis accessions (Coustham et al., 2012; Li et al., 2014).

During spring, ambient temperature is an important climatic factor. Temperature changes by only a few degree Celsius (°C) during cold or warm spring periods shift flowering time in many plant species. Since temperature changes associated with global warming could lead to similar flowering changes and threaten agricultural production systems, elucidating the ambient temperature pathway has recently received increased attention (Moore and Lobell, 2015; Wheeler and von Braun, 2013; Jagadish et al., 2016).

The complexities of the ambient temperature flowering pathway are just beginning to be understood (Capovilla et al., 2015; Verhage et al., 2014). After vernalization or in vernalization-insensitive accessions, flowering in ambient temperatures is largely under control of the FLC-related FLOWERING LOCUS M (FLM) and residual FLC retains only a minor role (Gu et al., 2013; Balasubramanian et al., 2006; Blázquez et al., 2003; Lee et al., 2013). FLM controls flowering in the range between 5°C and 23°C and FLM can form, just like FLC, a flowering repressive complex with SVP (Lee et al., 2013; Posé et al., 2013). Besides FLM, as the dominant regulator, ambient temperature flowering is also regulated by the FLM-homologs MAF2 - MAF4 (Li et al., 2008; Gu et al., 2013; Lee et al., 2013; Ratcliffe et al., 2003; Airoldi et al., 2015). Conversely, loss-of-function mutations of SVP lead to early and temperature-insensitive flowering in the range between 5°C and 27°C (Lee et al., 2007, 2013).

Within the ambient temperature range, FLM is differentially spliced in a temperature-dependent manner through the alternative use of the exons 2 (FLM-ß) and 3 (FLM-δ). Based on transgenic experiments, a model was proposed according to which FLM-ß and FLM-δ function antagonistically with SVP. Accordingly, FLM-ß engages in flowering repressive interactions with SVP in cooler temperatures. Conversely, FLM-δ would outcompete FLM-ß in warmer temperatures and form heterodimers with SVP unable to bind DNA and repress flowering (Lee et al., 2013; Posé et al., 2013). Recent data suggest that this attractive model, based solely on transgenic experiments, may not be valid in natural contexts (Sureshkumar et al., 2016; Lutz et al., 2015).

As yet, it has remained largely unknown whether genetic variation of the FLM gene locus plays a role in flowering time regulation across Arabidopsis accessions and if so, which variations determine basal and temperature-dependent FLM expression and splicing. Although two FLM deletion alleles conferring temperature-insensitive early flowering were identified in the Arabidopsis accessions Niederzenz-1 (Nd-1) and Eifel-6 (Ei-6), their limited demographic and genetic spread indicated that FLM deletions may be disadvantageous (Werner et al., 2005; Balasubramanian and Weigel, 2006). A first FLM expression variant was determined from the early flowering accession Killian-0 (Kil-0). In Kil-0, a LINE retrotransposon insertion in FLM intron 1 caused premature transcription termination, aberrant FLM splicing, consequently reduced FLM expression and earlier flowering. This phenotype was especially prominent at a temperature of 15°C , which is closer to the average temperature in the native range of the species than the commonly used 21°C (Lutz et al., 2015; Hoffmann, 2002; Weigel, 2012). The subsequent identification of nine further accessions with an identical LINE insertion was suggestive for a recent adaptive selective sweep (Lutz et al., 2015). It was thus concluded that FLM expression-modulating alleles are advantageous for flowering time adaptation in the ambient temperature range, particularly since there are no other pleiotropic growth changes observed in FLM mutant alleles.

Previous work had shown that an intronless FLM cDNA expressed from a FLM promoter fragment was unable to rescue the flm mutant phenotype. Since this suggested that important information for FLM expression may reside in FLM non-coding sequences, we examined non-coding sequence polymorphisms with a potential role in controlling FLM expression and splicing. Using phylogenetic footprinting, we found conserved FLM promoter and intron 1 regions essential for FLM expression. By association analysis using polymorphism data from ≈800 Arabidopsis accessions, we identified a small polymorphic region in the FLM promoter and a highly polymorphic nucleotide triplet in FLM intron 6 controlling basal and temperature-dependent FLM expression. Small changes in the relative abundance of the FLM-ß splice variant dynamically modulated flowering at 15°C over a range of 15 leaves. When tested in a homogenous genetic background, FLM abundance correlated almost perfectly with flowering time (R2 = 0.94) and contributed (R2 = 0.21) to flowering time variation in heterogeneous natural Arabidopsis populations. Our data suggest that FLM-ß is an important determinant of flowering during cool or warm spring periods.

Results

Intronic sequences are required for basal and temperature-sensitive FLM expression

Temperature-dependent changes in FLM abundance and alternative splicing are critical for flowering time control in ambient temperatures in Arabidopsis. How FLM expression is regulated and whether Arabidopsis accessions have employed differential FLM expression and splicing to adapt to different temperature climates remained to be shown. We previously found that Arabidopsis transgenic lines expressing an intronless FLM from a functional FLM promoter fragment cannot express FLM to detectable levels and fail to rescue the flowering time phenotype of a flm-3 loss-of-function mutant (Lutz et al., 2015). We subsequently compared FLM expression in transgenic lines expressing a genomic FLM fragment, obtained from the Columbia-0 (Col-0) wild type, containing all six introns (pFLM::gFLM; FLMCol-0) with lines expressing the FLM-ß (pFLM::FLM-ß) or FLM-δ (pFLM::FLM-δ) splice variants retaining intron 1 but lacking all other introns (Figure 1A). The presence of intron 1 was sufficient to restore the basal expression of FLM at the ambient temperatures 15°C, 21°C, and 27°C, but introns 2–6 were required for the temperature-sensitive regulation of the splice variants FLM-ß and FLM-δ (Figure 1B). We concluded that intron 1 may contain critical information for FLM expression and introns 2–6 may contribute to temperature-dependent FLM expression.

FLM intronic sequences determine basal and temperature-dependent FLM expression.

(A) Schematic representation of the pFLM::gFLM (FLMCol-0), pFLM::FLM-ß, and pFLM::FLM-δ constructs. Scale is set to 1 according to the A of the ATG start codon. Black boxes represent exons, grey lines 5’- or 3’-untranslated regions, dark-grey lines introns. Arrows indicate primer binding sites; the forward primer F1 was used to amplify FLM-ß and FLM-δ with reverse primers R1 and R2, respectively. (B) Mean and SD (three biological replicates) of qRT-PCR analyses of FLM-ß and FLM-δ expression in ten day-old plants harbouring the transgenes shown in (A). For normalization, the respective values measured at 21°C were set to 1.

https://doi.org/10.7554/eLife.22114.002

Phylogenetic footprinting pinpoints essential regions for basal expression in the promoter and the first intron

To identify non-coding regions important for FLM expression, we performed multiple sequence alignments of Arabidopsis FLM with its closest sequence homologue MAF3 and FLM homologues from five other Brassicaceae species (Figure 2—figure supplement 1A,B) (Martinez-Castilla and Alvarez-Buylla, 2003; Van de Velde et al., 2014). Within the non-coding sequences, we detected one promoter region of 250 bp and two intron 1 regions of 373 and 101 bp with increased sequence conservation (>60%) (Figure 2A and Figure 2—figure supplement 1). We then generated PROΔ225bp, INT1Δ373bp, and INT1Δ101bp transgenic lines expressing pFLM::gFLM variants (FLMCol-0) with deletions of the three regions in the FLM deletion accession Nd-1 to measure the effects on FLM-ß and FLM-δ expression at 15°C and 23°C (Figure 2B and Figure 2—figure supplement 2) (Werner et al., 2005). To normalize for variability between the transgenic lines, we examined pools of independent T2 segregating lines (n = 21–34). We validated this pooling strategy by demonstrating that the established behaviour of FLM in the Col-0 and Kil-0 accessions could be faithfully recapitulated when performing equivalent analyses with T2 lines expressing gFLMCol-0 and gFLMCol-0 bearing the Kil-0 LINE insertion (Lutz et al., 2015) (Figure 2—figure supplement 2C,D). While we detected a strong downregulation of FLM at 15°C and 23°C in PROΔ225bp and INT1Δ373bp lines, the deletion in INT1Δ101bp had comparably minor effects on FLM expression (Figure 2C,D). This indicated that the proximal 225 bp promoter and the 337 bp intron 1 regions contained important sequences for basal FLM expression. Subsequent experiments showed that the 254 bp promoter fragment alone was, however, not sufficient to confer FLM expression of a genomic FLM fragment (PRO254bp; Figure 2B–D).

Figure 2 with 2 supplements see all
Phylogenetic footprinting identifies promoter and intron 1 regions required for FLM expression.

(A) Phylogenetic footprinting of promoter and genomic regions of FLM and putative FLM orthologs from six Brassicaceae species. Coverage is shown in blue, identities are shown in ocre (≥30%) and red (<30%). Exons are displayed in green, regions with high non-coding sequence conservation are displayed in orange. (B) Schematic illustration of the FLM genomic region and FLMCol-0 transgenic variants used for expression analysis. (C) and (D) Mean and SD (four replicate pools with five to ten independent T2 transgenic lines) of qRT-PCR analysis of FLM-β (C) and FLM-δ (D) expression at 15°C and 23°C in ten day-old seedlings of 21–40 bulked T2 transgenic lines. Student’s t-tests: *, p≤0.05; **, p≤0.01; ***, p≤0.001; n.s., not significant.

https://doi.org/10.7554/eLife.22114.003

Non-coding sequence variation of major FLM haplotypes influences FLM expression

To find further non-coding determinants for FLM expression, we analysed FLM nucleotide variation in 776 sequenced Arabidopsis accessions (The 1001 Genomes Consortium, 2016). We identified 45 promoter and intronic SNPs with a minor allele frequency (MAF)≥5% and used these to define ten major haplotypes (H1–-H10) representing 379 (49%) accessions (Figure 3—figure supplement 1A,B; Supplementary file 1). We defined an initial set with 41 accessions by selecting five to twelve accessions from six of the ten haplotype groups (Figure 3A). Since introns 2–6 seemed important for temperature-sensitive FLM expression, we added 11 accessions with varying intron 2–6 haplotypes (HI2-6) but identical intron 1 haplotype (HI1; Figure 3A and Figure 3—figure supplement 1D–G). Finally, we added Col-0 (H3) and Kil-0 (H1) due to their well characterized FLM regulation to obtain a representative FLM haplotype set with ultimately 54 accessions (Supplementary file 2). We assured, by analytical PCR, that none of these accessions, except Kil-0, carried the previously described intron 1 LINE insertion (Lutz et al., 2015).

Figure 3 with 7 supplements see all
FLM haplotype analysis of 776 accessions identifies major haplotypes determined by non-coding variation.

(A) Haplotype group affiliation of 54 accessions of the FLM haplotype set based on 45 SNPs for either the 7 kb FLM locus (H7kb), intron 1 (HI1) or introns 2–6 (HI2-6). Group numbering and colouring are according to group size. (B) Summarized expression values of total FLM (exon 1), FLM-ß, and FLM-δ expression of the FLM haplotype set. Outliers were determined based on 1.5 x IQR (interquartile range). The coefficient of variation (CV) is shown in the lower graph.

https://doi.org/10.7554/eLife.22114.006

To find polymorphisms regulating FLM expression and splicing, we performed a genotype-phenotype association analysis. Since the vernalization pathway strongly delayed flowering in 24 of the 54 accessions and consequently suppressed FLM effects, we used FLM expression as a phenotype for the association analysis (Figure 3—figure supplement 2A). To ascertain that FLM expression was not affected by FLC, we examined FLM in non-vernalized wild type Col-0 and flc-3 mutants as well as in Col-0 carrying a functional vernalization module (Michaels and Amasino, 1999). Concurrent with previous reports, we did not detect an influence of FLC on FLM transcript abundance in our conditions (Figure 3—figure supplement 2B) (Scortecci et al., 2001; Ratcliffe et al., 2001). We then obtained FLM expression data from the FLM haplotype set and measured total FLM transcript levels as well as FLM-ß and FLM-δ levels at 15°C or 23°C (Supplementary file 3). In line with the reported behaviour of FLM in Col-0, we observed that FLM-ß expression decreased (on average 0.6 fold) and FLM-δ increased (on average 1.4 fold) with increasing temperature in most accessions (Figure 3B and Supplementary file 3) (Lee et al., 2013; Posé et al., 2013; Lutz et al., 2015). At the same time, we also observed substantial variation in FLM-ß and FLM-δ expression between the accessions of the FLM haplotype set, inviting the conclusion that non-coding sequence variation modulates FLM expression (Figure 3B and Supplementary file 3). Since total FLM transcript levels strongly correlated with FLM-ß but not with FLM-δ abundance at 15°C and at 23°C, it could be suggested that FLM-ß represents the major FLM form among the FLM transcripts (Figure 3—figure supplement 3A,B).

Association analysis identifies polymorphic sites with potential FLM regulatory functions

Using the FLM haplotype set, we next performed association tests between FLM expression at 15°C and 23°C or expression ratios derived from these values and an extended set of 119 polymorphisms along the 7 kb FLM region, which included the 45 SNPs, but also low frequent SNPs (MAF>1%) and indels (≤2 bp; MAF>1%) (Figure 3—figure supplement 4). The association analysis detected polymorphisms with significant associations (p<0.001) (Figure 3—figure supplement 5 and Supplementary file 4). We decided to investigate the potential role of a single base pair deletion (PRO1T/-; bp −215) and a genetically slightly linked SNP (PRO2A/C; bp −93), because they were both located in the proximal part of the FLM promoter. Further, we investigated three genetically unlinked nucleotides because they were positioned as a highly diverse nucleotide triplet in intron 6 (INT6A/C-A/C-A/T/C; bp +3975–+ 3977) in an otherwise conserved sequence context (Figure 4A, Figure 3—figure supplements 57, Supplementary file 5).

Figure 4 with 1 supplement see all
Polymorphisms in PRO2+ and INT6+ sites influence basal and temperature-dependent FLM expression.

(A) Schematic representation of the FLM genomic locus as shown in Figure 1. Green dots indicate the positions of the PRO1, PRO2, and INT6 sites that were chosen for further investigation. Sequence logos display the allele frequencies at these sites among the 54 accessions of the FLM haplotype set. (B) Sequence logos and allele frequency distribution of PRO2+ and INT6+ polymorphisms among 840 Arabidopsis accessions. All polymorphic residues are marked with asterisks and the respective allele frequencies are indicated by the sequence logo. The Col-0 reference haplotype is marked in yellow, haplotypes chosen for further investigation are marked in green. (C) and (D) Alignment of the PRO2+ and INT6+ polymorphisms (C) and schematic representation of the FLMCol-0 reference construct (pFLM::gFLM) as well as the FLMCol-0 variants selected for transgenic analysis in the FLM deletion accession Nd-1. Bases that differ between FLMCol-0 and the variants are coloured. Deletions in the deletion constructs of the PRO2+ and INT6+ sites as well as the PolyA motifs are displayed with red lines (not drawn to scale). (E) and (F) Mean and SD (four replicate pools with four to eleven independent T2 transgenic lines) of qRT-PCR analyses of FLM-β (E) and FLM-δ (F). For easier comparison, the values obtained with FLMCol-0 are displayed as red and blue dotted lines. Student’s t-tests: *p≤0.05; **p≤0.01; ***p≤0.001; n.s., not significant.

https://doi.org/10.7554/eLife.22114.014

Strikingly, the PRO2 and INT6 sites were directly flanked or in close proximity to a total of four PolyA motifs of variable length ([A]7-11), one of which represented a so-called CArG-box, a potential binding site for MADS-box transcription factors (Zhang et al., 2016). Two more PolyA motifs resided in introns 3 and 5 (Figure 4D and Figure 4—figure supplement 1A). Since PolyA motifs had been reported to be important for gene expression, we reasoned that these motifs could be relevant for FLM regulation, in isolation or in combination with the highly significant non-coding variations (O'Malley et al., 2016; Horton et al., 2012).

To understand the sequence variation surrounding the PRO1, PRO2 and INT6 sites, we reanalysed available Arabidopsis genome sequences (The 1001 Genomes Consortium, 2016). Besides the PRO1 1 bp deletion, the region surrounding PRO1 was conserved and we therefore focussed on the PRO1T/- deletion polymorphism (Figure 4A). Few bases up- and downstream of PRO2 (bp −102 to bp −92), we detected four additional highly diverse SNPs and designated this region PRO2+. We also identified additional frequent haplotypes of the INT6 triplet (INT6+). Apart from the two PolyA motifs located at PRO2+ and INT6+, the four remaining PolyA motifs were conserved across all accessions examined (Figure 4B).

FLM non-coding sequence variation regulates FLM abundance in a homogenous transgenic background

To examine the effects of the non-coding sequence variations in a homogenous background, we transformed the deletion accession Nd-1 with FLMCol-0 variants carrying the PRO1, PRO2+ and INT6+ polymorphisms or deletions of PRO2+ (PRO2+Δ16bp) and INT6+ (INT6+Δ17bp) (Figure 4C,D). Additionally, we transformed variants with deletions of between two and six PolyA stretches (PolyA_2xΔa, PolyA_2xΔb, PolyA_3xΔ, PolyA_4xΔ, PolyA_5xΔ, PolyA_6xΔ; Figure 4D and Figure 4—figure supplement 1A). Using pooled T2 segregating lines (n = 18–45) for each of the transgenes, we analysed the effects of the sequence variation on FLM-ß and FLM-δ expression at 15°C and 23°C. The PRO1- deletion, as present in the FLMCol-0 reference, did not reveal differences in FLM transcript levels when compared to PRO1T (FLMCol-0) (Fig. Figure 4—figure supplement 1B,C). However, two PRO2+ (PRO2+GGAAC, PRO2+AAACC) and three INT6+ variants (INT6+ACA, INT6+CAC and INT6+AAA) displayed an upregulation of FLM-ß at 15°C when compared to FLMCol-0 (Figure 4E). Increases and decreases of FLM-δ levels largely followed those of FLM-ß levels except for INT6+AAA, which showed an upregulation exclusively of FLM-ß (Figure 4E,F). The deletion variant PRO2+Δ16bp showed upregulation of FLM-ß but INT6+Δ17bp did not have significantly altered FLM-ß and FLM-δ levels when compared to FLMCol-0 (Figure 4E,F). We concluded that the identity of these regions rather than their presence controlled FLM expression and temperature-sensitive FLM regulation. The PolyA motif deletion variants showed gradually decreasing FLM-ß and FLM-δ levels with an increasing number of deletions while the deletion of all six PolyA motifs (PolyA_6xΔ) had an especially strong effect on FLM-ß expression and its temperature-sensitive expression (Figure 4E,F and Figure 4—figure supplement 1B,C). Thus, the PolyA motifs possess a regulatory role but may function in a redundant manner.

FLM-ß levels and flowering time strongly correlate in a linear manner

The phylogenetic footprinting and association analyses resulted in the identification of polymorphisms and regions that significantly altered FLM expression when present in a homogenous molecular and genomic context. To correlate FLM expression with flowering, we measured flowering time in T2 transgenic lines of eight variants with significantly different FLM abundance (Figure 5A and Figure 5—figure supplement 1A–C). We detected a very strong correlation between FLM-ß transcript levels and flowering time (R2 = 0.94) in plants grown at 15°C. Flowering time responded in a linear manner to FLM-ß levels with 17 to 30 rosette leaves until flowering in the range of relative transcript levels from 0.6 to 2.4 (Figure 5B). Further, the low expression variants PolyA_6xΔ, INT1Δ373bp, and PROΔ225bp flowered as early as the deletion accession Nd-1 suggesting that there must be a critical lower threshold for FLM-ß to be effective (Figure 5A,B). The correlation was much lower for FLM-δ (R2 = 0.70) suggesting that FLM-ß is the major determinant for flowering time in these conditions (Figure 5—figure supplement 1D). Within the range of lines examined, we did not detect a saturating effect at the upper expression level.

Figure 5 with 1 supplement see all
FLM-ß expression shows high correlation with flowering time of transgenic plants.

(A) Box plot of quantitative flowering time analysis (rosette leaf number) of independent T2 transgenic lines at 15°C and long day photoperiod. Ten plants of three replicate pools were analysed for each construct. The data were corrected for the anticipated 25% of non-transgenic Nd-1 segregants (see Figure 5—figure supplement 1D for the uncorrected analysis). Single values are shown as jittered dots, the colour represents the type of variant as introduced in Figure 4D, the median of the FLMCol-0 reference is indicated as dotted line. Wilcoxon rank test: *p≤0.05; **p≤0.01; ***p≤0.001; n.s., not significant. (B) Correlation (simple linear regression) between FLM-ß and qRT-PCR expression data as presented in Figure 5—figure supplement 1A,B and flowering time analysis as shown in (A). Datapoints of INT6+ variants with contrasting FLM expression and FLMCol-0 are designated. The color code corresponds to (A). The variants PolyA_6xΔ, INT1Δ373bp, and PROΔ225bp, which do not respond in a linear manner are shown as dotted circles. The shaded areas indicate the 95% confidence intervals; p<0.0001. Note, that the flowering time data shown in (A) were corrected by removing values of non-transgenic T2 segregants. An analysis using the uncorrected data is shown in Figure 5—figure supplement 1E,F.

https://doi.org/10.7554/eLife.22114.016

The INT6+CAA polymorphism confers temperature-insensitive FLM expression and flowering

Introns 2–6 were required for temperature-sensitive FLM expression (Figure 1). When we statistically tested the interaction between genotype and temperature with a multiple linear model, we found that temperature-sensitive FLM-ß regulation was significantly reduced in INT6+CAA between 15°C and 23°C and when compared to the FLMCol-0 control variant (p=0.012) (Figures 4E and 6A,B). In line with the prediction, flowering of this variant was indeed less sensitive to temperature changes when tested at 15°C and 23°C in homozygous T3 progeny plants and compared to the FLMCol-0 reference (Figure 6C,D). Thus, temperature-independent FLM-ß expression changes correlate with temperature-insensitive flowering in the selected temperature range.

The INT6+CAA polymorphism reduces temperature-sensitivity of FLM expression and flowering.

(A) qRT-PCR analysis and (B) expression ratios of FLM-β of FLMCol-0 (INT6+AAT) and INT6+CAA grown in 15°C and 23°C. Statistical tests of temperature-sensitivity are described in the text. (C) Means and SD of quantitative flowering time analysis (rosette leaf number). n = 5 (15°C) and 8 (23°C) replicates from five independent homozygous T3 transgenic lines grown at 15°C and 23°C in long day photoperiod. (D) Mean and SD of ratios of rosette leaf numbers from the analysis shown in (C). Student’s t-test: *p<0.05; **p≤0.01.

https://doi.org/10.7554/eLife.22114.018

Differential abundance of FLM splice forms can correlate with changes in FLM transcription or alternative splicing

To understand whether the same or different molecular mechanisms are the basis of altered FLM expression in FLM variants, we estimated FLM transcription of variants with strongly altered abundance of processed FLM abundance by measuring levels of unprocessed FLM pre-mRNA from plants grown at 15°C. When compared to the levels of processed FLM mRNA, the PROΔ225bp and INT1Δ373bp variants showed similarly strong reductions of unprocessed pre-mRNA, suggesting that the respective deletion polymorphisms directly affect FLM transcription (Figures 4E,F and and 7A). In turn, the INT6+CAA and PolyA_6xΔ lines had reduced FLM mRNA levels but, when compared to FLMCol-0, did not show substantial changes in unprocessed pre-mRNA levels (Figures 4E,F and and 7B). Since this indicated that post-transcriptional events may be affected in these variants, we tested for the abundance of differential polyadenylated splice variants after semi-quantitative 3’-RACE-PCR and sequencing of the cloned PCR products (Figure 7—figure supplement 1A). There, we detected a relative reduction of FLM-ß transcripts in INT6+CAA and PolyA_6xΔ that was accompanied by increases in the abundance of two polyadenylated transcripts containing exon 1 and intron 1 (E1I1p) that had already been noted in an earlier publication (Figure 7C,D) (Lutz et al., 2015). Importantly, we did not identify a single FLM-δ clone among the 163 sequenced cDNAs. The relative increase in E1I1p transcripts could also be independently confirmed by E1I1p-specific qRT-PCRs and suggested in summary that splicing site choice at the exon 1 - intron 1 junction is changed in the INT6+CAA and PolyA_6xΔ alleles (Figure 7—figure supplement 1B,C). In relation to all exon 1-containing transcripts, the overall abundance of these intron 1-containing transcripts was comparatively low (Figure 7—figure supplement 1C).

Figure 7 with 1 supplement see all
FLM polymorphisms affect FLM transcription or splicing at the expense of FLM-ß.

(A) and (B) Mean and SD (n = 3) of FLM pre-mRNA levels. Student’s t-test: ***p≤0.001; n.s. = not significant. (C) Schematic representation of FLM cDNAs detected more than once (n = 163). FLM-δ transcripts were not detected and are only shown for completeness. Grey areas correspond to FLM exons of the FLM-ß and FLM-δ gene models as specified in Figure 1A. The arrow indicates the position of the 3' RACE primer used in combination with an oligo(dT) reverse primer to detect polyadenylated transcripts. (D) Frequency distribution of the transcripts displayed in (B) with total number of sequences depicted in the graph.

https://doi.org/10.7554/eLife.22114.019

PRO2+ and INT6+ polymorphisms contribute to global variation of FLM levels

The nine PRO2+ and INT6+ haplotypes tested in transgenic experiments were present in 579 (69%) of all 840 accession with available genome sequence information (Figure 8—figure supplement 1A). To examine whether these haplotypes explain natural variation of FLM-ß levels in natural accessions, we randomly selected an experimental population of 94 accessions (2 to 14 accessions per PRO2+/INT6+ group, average 10) with a broad genetic and geographic distribution (Figure 8—figure supplement 1B,C). We next determined FLM-ß expression and, following quality filtering, grouped 85 accessions according to their PRO2+ or INT6+ haplotypes (Supplementary file 6A).

We found that the PRO2+/INT6+ haplotype significantly affected FLM-ß transcript levels (Figure 8—figure supplement 1D). When we integrated the average values from these natural accessions with the respective values from the transgenic analysis, we detected a positive, however not significant correlation, a likely consequence of the small number of datapoints (FLM-ß, R2 = 0.13) (Figure 8—figure supplement 1E).

To examine the correlation between FLM-ß expression and flowering time, we determined flowering time of accessions at 15°C. To avoid strong interference from the vernalization pathway, we selected 27 genetically diverse summer-annual accessions, which initiate flowering without the need of vernalization (Figure 8—figure supplement 2A, Supplementary file 6B). We measured FLM-ß transcript levels and flowering time at 15°C. As residual FLC transcript in these summer-annual accessions may still affect flowering time, we also determined FLC transcript levels (Coustham et al., 2012; Li et al., 2014; Duncan et al., 2015). Using a multiple linear regression approach, we found that FLM-ß and FLC explained 32.9% (R2 = 0.329, p=0.0083) of flowering time variation, with FLM-ß significantly explaining a subfraction of 21.0% (R2 = 0.210, p=0.011) and FLC only 11.9%, however not significantly (R2 = 0.119, p>0.05) (Figure 8B and Figure 8—figure supplement 2B). Further, by integration of expression data and flowering time data into a multiple linear model and comparison of the slopes, we found that flowering time responded stronger to FLM-ß levels in the accessions than in the transgenic variants (p=0.0321) (Figure 5B and Figure 8). Taken together, we concluded that variations in FLM-ß levels account for flowering time in cool ambient spring temperatures in a diverse population of summer-annual Arabidopsis accessions (Figure 9).

Figure 8 with 2 supplements see all
FLM-ß expression shows high correlation with flowering time in Arabidopsis accessions.

(A) Correlation (simple linear regression) of FLM-ß transcript levels detected in transgenic plants and natural accessions with identical PRO2+ and INT6+ haplotypes. When the contribution of individual haplotypes was assessed, PRO2+AAAAC INT6+CAA showed an exceptional response in accessions and was excluded. Figure 8—figure supplement 1E shows the complete analysis. Horizontal and vertical error bars depict SD of four replicate transgenic line pools or SD of the accessions. Data of the transgenic lines was taken from Figure 4E. The grey area indicates the 95% confidence interval. (B) Correlation of FLM-ß transcript levels with flowering time (n = 8–10) as measured in rosette leaf number of summer-annual accessions. p=0.011.

https://doi.org/10.7554/eLife.22114.021
Model of the proposed role of PRO2+ and INT6+ haplotypes and temperature on FLM-ß abundance and flowering.

The abundance of the flowering repressor FLM-ß decreases in response to higher temperature and flowering is consequently accelerated (Figures 1B and 4E) (Lee et al., 2013; Posé et al., 2013; Lutz et al., 2015). Note, that previous studies showed an especially prominent effect of FLM in a range from 9°C to 21°C (long-day photoperiod) (Lee et al., 2013; Posé et al., 2013; Lutz et al., 2015). At the genetic level, FLM-ß abundance is triggered by the PRO2+ (purple) and/or the INT6+ (pink) haplotype and flowering time correlated to FLM-ß abundance (Figure 4E). Among the PRO2+/INT6+ combinations tested, the Col-0 (grey) reference allele (PRO2+AAAAC/INT6+AAT) showed intermediate FLM-ß levels (Figure 4E). We suggest, that changes in flowering time due to changing ambient temperature can be precisely buffered by modifying the PRO2+ and INT6+ regions, as illustrated by similar plant symbols.

https://doi.org/10.7554/eLife.22114.024

Discussion

Ambient temperature during spring is a major cue determining flowering time. Cool temperatures generally delay and warm temperatures promote flowering time of Arabidopsis. The FLM locus explains flowering time variation in different ambient temperatures but the underlying genetic bases of FLM-dependent flowering remained largely unclear (Salomé et al., 2011; el-Lithy et al., 2006; O'Neill et al., 2008).

We found that, besides the FLM promoter, also intron 1 sequences were essential for FLM basal expression and subsequently identified by phylogenomic footprinting a conserved 373 bp intron 1 region essential for FLM basal expression (Figure 2). Further, through association analyses using genomic sequence information, we uncovered FLM regulatory regions (PRO2+ and INT6+) that control temperature-dependent FLM expression in a haplotype-specific manner (Figure 4). While most PRO2+ and INT6+ haplotypes displayed differential basal but temperature-sensitive FLM expression, correlating strongly with changes in flowering time, one INT6+ variant, INT6+CAA, was strongly compromised in temperature-sensitive FLM regulation and flowering pointing at the importance of this intronic region for flowering time adaptation (Figures 4 and 6).

In all our experiments, FLM-ß highly correlated (R2 = 0.94) with flowering time over a broad vegetative range (15–30 rosette leaves) when determined at 15°C and tested in a homozygous background, regardless of the type of variant (Figure 5). Our finding that FLM-ß had a stronger effect in the control of flowering time than FLM-δ indicates that previously phrased functional models proposing that FLM-δ had an antagonistic activity to FLM-ß need to be corrected (Posé et al., 2013; Sureshkumar et al., 2016). This is further supported by the fact that our results estimate that FLM-δ levels are overall very low and that we did not identify a single FLM-δ clone in a directed sequencing approach that identified 53 FLM-ß clones. Our findings thus support more recent studies suggesting that the FLM-δ splice variant may be biologically irrelevant (Sureshkumar et al., 2016; Lutz et al., 2015). One of these studies also identified a large number of biologically irrelevant transcripts that could be identified with the primer combination used here for the detection of FLM-δ (Sureshkumar et al., 2016). Our data thus support the conclusion that none of these amplification products has a biologically important function (Sureshkumar et al., 2016).

Whereas the PRO2+ and INT6+ polymorphisms tested affected FLM transcript abundance, the molecular causes of these transcription changes varied among the different polymorphisms (Figures 4 and 7). Deletion of a 225 bp promoter region including the PRO2+ polymorphic site was associated with tenfold reduced levels of FLM pre-mRNA indicating that this promoter region was essential for FLM expression (Figure 7). A twofold relative difference of FLM-ß levels was found when comparing the PRO2+GATAC and PRO2+AAACC variants with the lowest and highest FLM-ß expression, respectively, and our linear model would predict a flowering time difference of 14.4 leaves at 15°C (Figures 4 and 5). The PRO2+ region harbours a predicted MADS-box transcription factor binding site and several instances of auto- or cross-regulation of MADS-box factors have been described (de Folter et al., 2005; Kaufmann et al., 2009; Smaczniak et al., 2012). It can therefore be envisioned that FLM expression is regulated by MADS-box transcription factors and that PRO2+ polymorphisms modulate the efficiency or specificity of these binding events and thereby modulate basal FLM expression.

Similarly, the deletion of a 373 bp fragment in FLM intron 1 resulted in an elevenfold reduction in FLM expression and earlier flowering by 11.5 leaves as experimentally determined. This effect size is much smaller than predicted by the linear model. However, as proposed earlier, this may likely be due to a critical lower threshold for FLM-ß to become effective (Figures 4 and 5). Intronic cis-regulatory transcription factor binding sites have been identified in other MADS-box transcription factors and interactions of enhancer and silencer elements that reside in the promoter sequence or the 3’-end of the first intron were reported (Hong et al., 2003; Schauer et al., 2009). Thus, similar mechanisms may govern the expression of FLM at intron 1 sites that may act in isolation or together with binding events at the FLM promoter.

Interestingly, natural polymorphisms in intron 6 (INT6+) led to about fourfold differences in the abundance of FLM-ß, which could, as predicted by the linear model, relate to a flowering time delay by 28.8 leaves, suggesting that INT6+ harbours extensive potential to fine-tune flowering (Figures 4 and 5). Importantly, this molecular effect appears to be mediated, in the case of INT6+CAA, by effects on the splicing efficiency and specificity at the distal intron 1. There, INT6+CAA promotes the formation of short intron 1-containing transcripts, at the expense of FLM-ß, that are likely subjected for degradation by nonsense-mediated decay (Figure 7) (Lutz et al., 2015).

The INT6+ site is directly flanked by a short PolyA motif and such sites represent potential recognition sites of hnRNP (heterogeneous nuclear ribonucleoprotein) splicing factors. The predicted hnRNP binding pattern at INT6+ depended indeed on the INT6+ haplotype, when predicted by web-based algorithms, and their binding preference and activity, in concert with other splicing or transcriptional regulators, may ultimately be the basis of the splicing changes observed here (Piva et al., 2012; Carrillo Oesterreich et al., 2011; Reddy et al., 2013).

The cooccurrence of PolyA motifs ([A]7-11) with the PRO2+ and the INT6+ sites had attracted our attention (Figure 4—figure supplement 1). Combinatorial deletions of all six FLM PolyA motifs led to gradual decreases in FLM-ß abundance, which could be explained be altered intron 1 splicing (Figure 7). This ultimately resulted in FLM-ß levels below a lower effective threshold in the PolyA_6xΔ lines and consequently very early flowering (Figure 4—figure supplement 1). This suggested that the PolyA motifs may modulate FLM expression by altering FLM splicing. In support of this conclusion, we found that an extended PolyA motif, as it is present in the INT6+AAA variant when compared to the Col-0 reference variant INT6+AAT, correlated with strong increases in FLM-ß but not in FLM-δ abundance (Figure 4). PolyA motifs are known to prevent nucleosome binding and changes of chromatin architecture may influence splicing (Reddy et al., 2013; Suter et al., 2000; Wijnker et al., 2013). The knowledge about the underlying molecular mechanisms and the identity and specificity of the splicing regulators in plants is still very limited. We noted with interest, however, that several hnRNPs have a reported role in flowering time regulation in Arabidopsis and wheat (Kippes et al., 2015; Fusaro et al., 2007; Streitner et al., 2012; Xiao et al., 2015).

The nine PRO2+/INT6+ haplotype combinations included in our transgenic experiments represent 69% of the world-wide PRO2+/INT6+ variation (Figure 8—figure supplement 1A,B). We found, that FLM-ß explained around 21% of flowering time in a genetically heterogeneous population of summer-annual Arabidopsis accessions (Figure 8). Hence, PRO2+ and INT6+ haplotypes regulate FLM-ß abundance and, in turn, contribute to flowering time regulation in Arabidopsis accessions (Figures 8 and 9). We consider it not surprising that the correlation of flowering time with FLM-ß levels (21% versus 94%) as well as the responsiveness of flowering time to FLM-ß levels differ between a genetically heterogeneous natural population and the homogenous transgenic population. First, we showed that residual FLC transcript may also slightly contribute to variation of flowering time of summer-annual accessions, possibly interfering with the effects of FLM-ß (Balasubramanian et al., 2006; Li et al., 2006). Further, additional sequence variation in FLM in natural accessions may contribute to relevant expression changes, e.g. in the part of intron 1 identified as essential for FLM expression but not analysed in further detail here (Figure 2). Then, it is possible that these accessions harbour variation in the FLM-related MAF2 - 4, and this variation may differentially affect flowering time, in isolation or in combination with variation of their common interaction partner SVP (Ratcliffe et al., 2003; Airoldi et al., 2015; Scortecci et al., 2003). Bearing these possible genetic and environmental interferences in mind, we regard the detected effect of FLM-ß on flowering (21%) in cool (15°C) temperatures as considerable and suggest that it is rather an underestimation.

We found a uniform distribution of nine PRO2+/INT6+ haplotype combinations among the genetic clusters that have recently been established based on the analysis of genomic sequences from 1135 Arabidopsis accessions (The 1001 Genomes Consortium, 2016). Interestingly, the PRO2+GGAAC/INT6+AAT haplotype was overrepresented among the relict accessions and the PRO2+AAAAC/INT6+AAA haplotype was overrepresented among the Asian group (The 1001 Genomes Consortium, 2016) (Figure 8—figure supplement 1). In our experiments, both of these haplotypes were associated with increased FLM-ß transcript levels and consequently late flowering (Figure 4). Since the relict and Asian accessions have been proposed to originate from glacial refugia from where central Europe was recolonized after the last ice age, these late-flowering PRO2+/INT6+ haplotypes may represent original haplotypes (Sharbel et al., 2000; Schmid et al., 2006). The late flowering phenotype associated with these haplotypes would be in line with the hypothesis that late flowering alleles are more ancient and that early flowering alleles were derived only during the more recent Arabidopsis evolution (Toomajian et al., 2006). Further, genetic linkage among the PRO2+ and INT6+ polymorphic sites was overall low (R2 = 0.11–0.55) and the sequences surrounding PRO2+ and INT6+ were comparatively conserved. Taken together, this suggests that mutations in the PRO2+ and INT6+ sites independently arose multiple times and that these sites were preferentially selected during Arabidopsis flowering adaptation. All PRO2+/INT6+ alleles displayed a broad geographic distribution, except PRO2+GGAAC/INT6+AAT, which was overrepresented in Spain (Figure 8—figure supplement 1). Likely, microclimates at specific geographic locations rather than general climate conditions at broad geographic regions may be important to drive adaptation of flowering to changing ambient temperature, as it was previously suggested for broadly distributed non-coding haplotypes of FLC that explain variation in vernalization (Li et al., 2014; Weigel, 2012; Shindo et al., 2006).

Mutations in cis-regulatory regions are important for adaptation and phenotypic evolution and have a low probability to generate negative pleiotropic effects (Swinnen et al., 2016; Meyer and Purugganan, 2013; Wray, 2007). FLM may be an ideal candidate for flowering time adaptation through non-coding sequence variation at PRO2+ and INT6+ sites since changes in FLM-ß abundance precisely modulate flowering while maintaining phenotypic plasticity and without generating negative pleiotropic effects (Figure 9). Changes at the PRO2+ and INT6+ sites should allow adapting flowering time in response to altered geographic distribution and consequently climate conditions as well as during changing global environments (Figure 9). The role of FLM orthologues in other Brassicaceae, including a number of agronomically important species, is as yet not understood. It is thus at present not possible to predict the role of Arabidopsis FLM polymorphisms for flowering time adaptation and plant breeding in this plant family. However, in view of the availability of new genome editing methodologies, the knowledge about important non-coding regions may be useful to fine-tune flowering time (Shan et al., 2013). This may allow buffering the anticipated negative effects on agricultural systems that occur as a consequence of small temperature changes during climate change (Moore and Lobell, 2015; Wheeler and von Braun, 2013).

Materials and methods

Biological material

All Arabidopsis thaliana accessions used in this study as well as flm-3 (Salk_141971; Col-0) and Nd-1 (N1636) were provided by the Nottingham Arabidopsis Stock Centre (NASC; Nottingham, UK). flc-3 and FRISF-2 FLC (Michaels and Amasino, 1999) were a gift from Franziska Turck and George Coupland (Max-Planck Institute of Plant Breeding Research, Cologne, Germany). pFLM::gFLM (pDP34), pFLM::FLM-ß (pDP96), and pFLM::FLM-δ (pDP97) were previously reported (Posé et al., 2013). Primers to screen the accessions for the FLMLINE insertion were used as previously described (Lutz et al., 2015).

Physiological experiments

For flowering time analyses, plants were grown under long day-conditions with 16 hr white light (110–130 μmol m−2 s−1)/8 hr dark in MLR-351 SANYO growth chambers (Ewald, Bad Nenndorf, Germany). Plants were randomly arranged in trays and trays were rearranged every day. Water was supplied by subirrigation. Flowering time was quantified by counting rosette leaf numbers (RLN). Consistent with previous reports, we observed a strong correlation between days to bolting and rosette leaf number of Arabidopsis accessions (Figure 8—figure supplement 2C) (Atwell et al., 2010). Student’s t-tests (normally distributed values), Wilcoxon rank tests (not normally distributed values), and multiple regression models were calculated with R (http://www.r-project.org/).

Cloning procedure

A previously described construct with the Col-0 genomic FLM fragment pFLM::gFLM template (pDP34) (Posé et al., 2013) was recombined into a pDONR201 destination vector using the Gateway system (Life Technologies, Carlsbad, CA). This vector was used as a template (FLMCol-0) to generate mutations using either a single phosphorylated primer or a combination of forward and reverse primers (Sawano and Miyawaki, 2000; Hansson et al., 2008). In case of variants with multiple modifications, individual mutations were introduced one at a time. The mutated inserts were recombined to the pFAST-R07 expression vector using the Gateway system (Life Technologies, Carlsbad, CA) (Shimada et al., 2010). All expression constructs were verified by sequencing and transformed into Agrobacterium tumefaciens strain GV3101. Nd-1 plants were transformed using the floral-dip method (Clough and Bent, 1998) and transgenic plants were identified based on seed fluorescence (Shimada et al., 2010). Segregation of T2 lines was examined and lines with single insertion events were selected for further analysis based on segregation ratios (Shimada et al., 2010). A list of primers and expression constructs is listed in Supplementary file 7.

Quantitative real-time PCR

For qRT-PCR analyses of homozygous lines, total RNA was isolated from three biological replicates using the NucleoSpin RNA kit (Machery-Nagel, Düren, Germany). DNA was removed by an on-column treatment with rDNase (Machery-Nagel, Düren, Germany). 2–3 μg total RNA were reverse transcribed with M-MuLV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA) using an oligo(dT) primer. The cDNA equivalent of 30–50 ng total RNA was used in a 12 μl PCR reaction with SsoAdvancedUniversal SYBR Green Supermix (BioRad, München, Germany) in a CFX96 Real-Time System Cycler (BioRad, München, Germany). The relative quantification was calculated with the ΔΔCt method using ACT8 (AT1G49240) as a standard (Pfaffl, 2001). For the analysis of the pooled independent T2 transgenic lines, a quarter of all available independent lines per construct (18 to 45) was sampled resulting in four replicate pools comprising between four and eleven lines. Around 1000 seeds were used for pooling per line. One RNA sample was extracted per replicate pool and processed as described above.

The large scale expression experiments were performed using a previously described 96-well format (Figure 8 and Supplementary file 3) (Box et al., 2011). DNA was digested with DNaseI (Thermo Fisher Scientific, Waltham, USA) and reverse transcription and qPCR reactions were performed as described above using a CFX384 Real-Time System Cycler (BioRad, München, Germany). ACT8 (AT1G49240) and BETA-TUBULIN-4 (AT5G44340) were used as reference genes. Student’s t-tests were calculated with Excel (Microsoft). qRT-PCR primers are listed in Supplementary file 7.

Data retrieval from the 1001 Arabidopsis thaliana genome project

Sequence information from datasets of 776 and 840 accessions, respectively, have been available that were used in this study. The genomic sequences of a comprehensive set of 1135 Arabidopsis accessions have just recently been released and were used for concluding analyses (The 1001 Genomes Consortium, 2016). Genomic FLM sequences (7 kb; Chr1: 28953637–28960296; including 2 kb upstream and 0.2 kb downstream sequence) were extracted from 776 accessions from the 1001 Genomes portal (http://1001genomes.org/datacenter/) using the Wang dataset (343 accessions), the GMI dataset (180 accessions), the Salk dataset (171 accessions) and the MPI dataset (80 accessions) (The 1001 Genomes Consortium, 2016; Schmitz et al., 2013; Long et al., 2013; Cao et al., 2011). 45 SNPs with a MAF>5% were extracted and used for haplotype analysis of the FLM locus (Figure 3A and Figure 3—figure supplement 1). 31 SNPs were polymorphic between the 54 selected accessions represented in the FLM haplotype set (Supplementary file 1).

To obtain an extended set of sequences that included not only SNPs but also information about insertions and deletions, we extracted FLM genomic sequences (including 2 kb upstream and 0.38 kb downstream sequence) from the GEBrowser 3.0 resource (http://signal.salk.edu/atg1001/3.0/gebrowser.php). A set of 850 sequences was manually curated and aligned using MEGA7.0.14 (Tamura et al., 2011). Some sequences were excluded since they possessed a high number of ambiguous bases. The core sequence set consisted of 840 sequences. This sequence set was used for association analysis (Figure 3—figure supplements 47) and all further analysis on FLM PRO2+ and INT6+ variation (Figure 8 and Figure 8—figure supplement 1). Accession identifiers, geographic data, and ADMIXTURE group membership (k = 9) were obtained from the 1001 genomes tool collection (http://tools.1001genomes.org/) (The 1001 Genomes Consortium, 2016).

Haplotype analysis

Haplotype analyses were performed with DNaSP 5.10 (Rozas and Rozas, 1995) using the SNP dataset of the above-described set of 776 sequences. Invariable sites were removed and networks were generated using FLUXUS network software (Bandelt et al., 1999).

Phylogenetic tree and alignments

Alignments were calculated using ClustalW2 or MUSCLE and Neighbor-Joining trees (Maximum Composite Likelihood method, 1000 bootstrap replicates) were constructed with Geneious vR7.0.5 (Biomatters Limited, Auckland, New Zealand) and MEGA7.0.14 (Tamura et al., 2011).

LD analysis

Linkage (R2) between the polymorphic sites that were used as input for the simple single locus association test was calculated using the LD function in GGT v2.0 (van Berloo, 2008). Only diallelic SNPs (109 of 119) were considered.

Kruskal-Wallis test

To detect variants with significant effects on FLM transcript levels (simple single locus association test), 119 variants of a 7 kb FLM locus were extracted from a set of 840 sequences retrieved from the GEBrowser 3.0, as described above. To examine whether a variant showed a significant effect on FLM expression, the qRT-PCR expression values of the 54 accessions from the FLM haplotype set were used as input data (Supplementary file 4) to run Kruskal-Wallis tests using R (http://www.r-project.org/). p-values from all comparisons were corrected following the Benjamini-Hochberg multiple testing correction and resulting values were -log(10) transformed and plotted along the gene model (Figure 3—figure supplement 5 and Supplementary file 5).

Phylogenetic footprinting

FLM orthologues were identified first by OrthoMCL (V2.0) clustering (PMID: 12952885; standard parameters, inflation value = 1.5, BLAST e-value-cutoff = e-05), incorporating predicted protein sequences from Arabidopsis thaliana (TAIR10; AT1G77080.4, AT5G65060.1), Arabidopsis lyrata (V1.0; fgenesh2_kg.2__2000__AT1G77080, fgenesh2_kg.8__2543__AT5G65050, fgenesh2_kg.233__4__AT5G65050), Boechera stricta (V1.2; Bostr.4104s0001.1), Brassica rapa (V1.3; Brara.B03928.1.p, Brara.F02378.1.p), Brassica oleracea (V2.1; Bo2g166500.1, Bo2g166560.1), Capsella grandiflora (V1.1; Cagra.0450s0030.1, Cagra.0917s0081.1), Capsella rubella (V1.0; Carubv10020979m, Carubv10027327m), Gossypium raimondii (V2.1), Medicago truncatula (V4.0), Oryza sativa (MSU7), Populus trichocarpa (V3.0) and Solanum lycopersicum (ITAG2.3). Data sets were downloaded from Phytozome (Goodstein et al., 2012), PGSB PlantsDB (Spannagl et al., 2016) and Ensembl Plants (Kersey et al., 2016). Cluster(s) containing FLM were extracted and, in case of the presence of multiple group members from individual species, filtered further using the best bi-directional BLAST hit criterion. Genomic sequences and 2 kb upstream region were aligned with ClustalW2 (Larkin et al., 2007). Conserved regions in the alignment were manually annotated.

FLM molecular analyses

qRT-PCRs for FLM pre-mRNA abundance and 3' RACE PCR were performed as previously described (Lutz et al., 2015). Primers sequences are listed in Supplementary file 7.

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Decision letter

  1. Daniel J Kliebenstein
    Reviewing Editor; University of California, Davis, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Natural haplotypes of FLM non-coding sequences fine-tune flowering time in ambient spring temperatures in Arabidopsis" for consideration by eLife. Your article has been favorably evaluated by Ian Baldwin (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal his identity: Dan Runcie (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The manuscript describes an interesting illustration of how to utilize genome wide association in combination with a variety of genomic tools and thoughts to begin to improve the model of how FLM integrates temperature into the flowering time decision.

There are four key concerns that need to be addressed prior to any final decision.

1) The FLM expression to flowering correlation is not sufficient. The exclusion of multiple accessions was considered not justified. A multiple regression approach with FLC expression included in the model is a way that may account for this and would be a more correct statistical test of the hypothesis.

2) There are other significant statistical concerns that may affect the core claims from the manuscript such as temperature sensitivity, etc. These must be corrected and the claims either supported or changed pending the results.

3) The role of vernalization in FLM expression and variation thereof needs to be assessed to allow for the data to be properly integrated into the broader model. Given that the integration into the broader model is the critical interest in this paper.

4) There was concern about including FLMd if it is not found in the measurements? This should be addressed.

Essential revisions:

Please also address the following concerns that will improve the manuscript.

Reviewer #2:

The authors present a detailed search for the genetic basis of variation in ambient temperature sensitivity for flowering time in Arabidopsis. Focusing specifically on the control of the expression of FLM which has been previously linked to temperature sensitivity, the authors draw on a range of modern and innovative tools to pinpoint alleles conferring variation FLM expression within the species. They identify the major alleles controlling both basal expression and temperature sensitivity of FLM segregating at high frequency in the species and confirm effects of these alleles using transgenics. Overall, this study is important in its demonstration of how modern tools in Arabidopsis can be used to rapidly identify the specific base-pairs underlying natural variation in important traits. While I think that the major conclusions seem robust and well-supported, this is a very complicated paper and I do have concerns about specific analyses and interpretations.

1) My main concern is with the central focus on the comparison between two isoforms of FLM (FLMb and FLMd). A recent study showed that the qPCR assays targeting these isoforms actually pick up other isoforms, many of which produce truncated proteins (Sureshkumar et al. 2016 Nature Plants). Here, the authors note that they can't even find a true FLMd transcript (out of 163 tested). It is not clear what if any conclusions can be drawn from the FLMd assay, and so devoting so much space throughout the article and figures to FLMd is not useful and distracting. On the other hand, this and earlier studies the FLMb assay does seem to be useful for assaying FLM activity, so I am comfortable with these results.

2) Secondly, I am concerned with the results presented in Figure 8 – the correlations between FLM expression in accessions and a) expression in constructs and b) flowering times of the accessions. In order to find a correlation between FLM expression in accessions and in the transgenic lines with the corresponding constructs, the authors dropped an "outlier" point (1 each for FLMb and FLMd). No justification for this is given beyond the point not fitting expectations. It's not clear from Figure 8—figure supplement 1 that these points are really more outliers than any others. And, it's not that this is really a single point – on the y-axis this is the average expression of ~10 accessions, so why should they all be dropped?

3) For the flowering time comparison, the lines were vernalized. However, FLM is known to be repressed by vernalization (as the authors note), so how could it be affecting flowering, and might there be variation among lines in how FLM responds to the vernalization? It seems more likely that other loci that are correlated with FLM are controlling flowering time here. The authors should either measure FLM expression in these vernalized plants and correlate that with flowering, or repeat the experiment without vernalization where FLM and FLC expression in these lines has already been measured. A better way to control for FLC variation would be to statistically account for its effects with multiple regression.

4) Finally, on the statistical side, the conclusions about "temperature insensitivity" for INT6+ alleles are not backed by statistics – the authors should explicitly test for a change in the temperature sensitivity between FLM-Col0 and INT6+. From the graphs, it appears temperature sensitivity is reduced, but certainly not eliminated (Figure 6).

Reviewer #3:

In “Natural haplotypes of FLM non-coding sequences fine-tune flowering time in ambient spring temperatures in Arabidopsis" Lutz et al. address the biological relevance of FLM splice variants for variation in flowering time in natural accessions and identify non-coding sequence polymorphisms that contribute to the regulation of basal expression levels and the differential, thermo-responsive generation of the β and δ splice variants. As such, this work provides novel insights into the regulation of flowering in the ambient temperature range and manages to dissect polymorphisms which are relevant for basal FLM expression and temperature-induced effects on the β and δ splice variants.

The manuscript is overall very well written and the authors provide extensive data which has apparently been subjected to comprehensive and sound statistical analyses. The data is presented in an attractive manner. Main manuscript figures are supported by extensive supplementary material including the presentation of extensive control data, which is greatly appreciated. In some cases the figure descriptions or legends should be extended. Due to the vast amount of data condensed in the figures, the descriptions (at least in the text) should be detailed enough for a wide readership to follow the authors through their argumentation.

1) The authors state that “The presence of intron 1 is sufficient to restore the basal expression of FLM” – while it does restore expression levels to detectable levels, the expression level of the β splice variant at 15°C is considerably lower than the WT, whereas the FLMδ levels are similar the WT at this temperature. This would indicate that intron 1 alone cannot fully account for basal levels of FLM β.

2) The use of pools of independent segregating T2 lines for transcriptional analysis seems unusual. While the controls presented in Figure 2—figure supplement 2 suggest that this can recapitulate the “natural” situation (why not subjected to statistical analysis?), it seems nonetheless risky to deliberately tolerate the presence of non-transgenics in the mix. Could the authors state their reasoning for this unusual procedure and provide the missing statistics here? Also, the phrase “replicate pools compromising…” should probably read “comprising”.

3) I had a hard time to comprehensively grasp the information provided in Figure 3, especially when it comes to the color code and its use in Figure 3B. Figure 3—figure supplement 1 provides additional information, but still the color coding of haplotypes which is provided for three different regions (full length, intron 1 or intron 6) is confusing as the composition of the different haplotypes varies depending on the selected gene region (Figure 3—figure supplement.1G). The authors may want to better explain these figures to enable a wide readership to follow their analysis and key points here. Also, in the caption of Figure 3—figure supplement 1G the haplotype “Numbers on the left” probably refer to (D) not (E)?

4) Figure 5B and C: Please specify again in the figure caption that this data corresponds to 40 selected accessions. Why was the FLM β level not determined in the vernalized plants? Even though FLC effects seem to be generally negligible, it would have been as easy to do the analysis on the same material.

5) In Figure 5—figure supplement 1D/E: removing the lower quartile (or more!) of flowering time data for the correlation analysis by simple assumption that these represent the non-transformed Nd-1 individuals of the segregating population is questionable. While this may be an attractive assumption, it negates the fact that each line pool may represent its individual set of variances (also present in ND-1 itself). This would at least require a spot check for the presence of transgenes in this pool or the percentage of “clean” Nd-1 individuals. In principle, a simple test by PCR would have provided a solid answer to that. As is, the authors should include these lines in the correlation analysis as the factual genotype is not known.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Natural haplotypes of FLM non-coding sequences fine-tune flowering time in ambient spring temperatures in Arabidopsis" for further consideration at eLife. Your revised article has been favorably evaluated by Ian Baldwin (Senior Editor), a Reviewing Editor, and two reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

There are still some key issues on interpretation and analysis that need to be addressed. None of these require new wet lab experiments and are relatively easy and quick to clean up. All felt that this is necessary.

Reviewer #2:

I thank the authors for their improvements to the paper – the figures especially are more clear now. I still have the following concerns about the overall conclusions and analysis. These relate to three important conclusions:

1) That FLM variation contributes to flowering time variation across a diverse panel of summer annual accessions.

The new experiment to test this is very helpful, and does provide good evidence that FLMB expression level is important for flowering time variation. My concern is with interpretation: The authors provide several explanations for why the correlation between flowering and FLMB expression in this accession set is lower than in the transgenics. This is trivial because there is much more variation within each haplotype class in the accessions than the transgenics, because they vary at many loci across the genome. I think the real question should be: Is the slope of the relationship between FLMB and flowering time significantly less in the accessions? This would ask if the equivalent change in FLMB expression would cause the same change in flowering. This is a more meaningful metric than change in R2. The correct test would be to include both accessions and transgenics in the same model (flowering_time ~ FLMB * genotype_class), and ask if the interaction is significant.

2) That the 9 promoter haplotypes explain the variation in FLMB expression in the accessions.

This is the aim of the analysis in Figure 8A and Figure 8—figure supplement 1D/E. The main claim is that the expression variation induced by these haplotypes in the transgenics is correlated with the expression variation of accessions carrying the same haplotypes. This conclusion only holds when one of the 9 haplotypes is excluded. The explanation given for excluding this haplotype is that the correlation goes up after removing this. I did a quick simulation study and found that using this algorithm you'd expect that the best correlation found by dropping each of the 9 pairs would be greater than 46% about 21% of the time even if there were actually no correlation at all. So, this is really not very strong evidence that the haplotype effects are similar. Certainly, the statement "We found that the PRO2+/INT6+ effects, as detected in transgenic experiments (R2 = 0.94), partially explain the FLM-ß expression variation in natural Arabidopsis accessions (R2 = 0.46)" is not supported, because this 46% number is only true when you exclude those accessions that don't fit.

As above, I think a more appropriate analysis would be to ask if the sizes of the differences between the haplotypes is the same in the transgenics and the accessions (i.e., is the slope of the graph different from 1?). A more straightforward answer to the question of how much variation in FLMB is explained by these haplotypes is simply to report the R2 for the analysis shown in Figure 8—figure supplement 1D.

3) That the INT6+(CAA) haplotype controls temperature sensitivity.

This conclusion is important because of the model that FLM controls temperature sensitivity of flowering. New data are presented here relative to the previous version, though where they came from is not clear. The correct test for a change in temperature sensitivity is to ask if the interaction between genotype and temperature is significant (expression ~ Genotype + Temp + Genotype:Temp). The n.s. effect of temperature on expression for INT6+ is not evidence of no temperature effect, only a lack of evidence for a temperature effect. The figure caption states the statistics are done based on a t-test. A t-test can't be used to test for an interaction (except in 6C/D if the 5 transgenic lines per genotype were used as the replicates, n = 5). An ANOVA is needed to conclude that this haplotype affects temperature sensitivity.

Reviewer #3:

The authors have generally met my major concerns and provide additional data as well as detailed explanations for the changes in the manuscript.

Remaining concerns:

1) The authors have clarified that the major concerns about the exclusion of accessions from the correlation of FLM expression and flowering time was largely based on a misunderstanding of the method and intention of the analysis. The changes to the text makes this much more clear now. The exclusion of the “exceptional" haplotypes raises the correlation from 0.13 to 0.46 in a linear regression analysis. Please also provide the corresponding p-values here to make sure that the p-values reflect a robust correlation effect (as is done in the further analysis of FLMß/FLC expression via multiple regression analysis). Please also describe how p-values were obtained for both cases then.

2) I appreciate the explanation regarding the use of the pooling strategy and the inclusion of statistics. However, no information on the performed test is given in the figure caption. was this also a t-test as mentioned in the methods? If so, please state whether a two-sided t-test and correction for multiple testing was implicated. Otherwise perform an ANOVA with suitable post-hoc test.

https://doi.org/10.7554/eLife.22114.032

Author response

[…] There are four key concerns that need to be addressed prior to any final decision.

1) The FLM expression to flowering correlation is not sufficient. The exclusion of multiple accessions was considered not justified. A multiple regression approach with FLC expression included in the model is a way that may account for this and would be a more correct statistical test of the hypothesis.

Please see our reply to reviewer 2, comment 2 and Comment 3.

2) There are other significant statistical concerns that may affect the core claims from the manuscript such as temperature sensitivity, etc. These must be corrected and the claims either supported or changed pending the results.

Please see our reply to reviewer 2, comment 3 and reviewer 3 comment 2.

3) The role of vernalization in FLM expression and variation thereof needs to be assessed to allow for the data to be properly integrated into the broader model. Given that the integration into the broader model is the critical interest in this paper.

Please see our reply to reviewer 2 comment 3.

4) There was concern about including FLMd if it is not found in the measurements? This should be addressed.

See our reply to reviewer 2 comment 1.

Essential revisions:

Please also address the following concerns that will improve the manuscript.

Reviewer #2:

The authors present a detailed search for the genetic basis of variation in ambient temperature sensitivity for flowering time in Arabidopsis. Focusing specifically on the control of the expression of FLM which has been previously linked to temperature sensitivity, the authors draw on a range of modern and innovative tools to pinpoint alleles conferring variation FLM expression within the species. They identify the major alleles controlling both basal expression and temperature sensitivity of FLM segregating at high frequency in the species and confirm effects of these alleles using transgenics. Overall, this study is important in its demonstration of how modern tools in Arabidopsis can be used to rapidly identify the specific base-pairs underlying natural variation in important traits. While I think that the major conclusions seem robust and well-supported, this is a very complicated paper and I do have concerns about specific analyses and interpretations.

1) My main concern is with the central focus on the comparison between two isoforms of FLM (FLMb and FLMd). A recent study showed that the qPCR assays targeting these isoforms actually pick up other isoforms, many of which produce truncated proteins (Sureshkumar et al. 2016 Nature Plants). Here, the authors note that they can't even find a true FLMd transcript (out of 163 tested). It is not clear what if any conclusions can be drawn from the FLMd assay, and so devoting so much space throughout the article and figures to FLMd is not useful and distracting. On the other hand, this and earlier studies the FLMb assay does seem to be useful for assaying FLM activity, so I am comfortable with these results.

The major concept of our study had been to elucidate regulatory regions of FLM transcription and this also included analysis of FLMd. Rejecting the importance of FLMd was not a prerequisite but an important result of the study. It is true that the finding that FLMd may not have a function in flowering time control had already been a conclusion from our earlier work (Lutz et al., 2015). On the other side, the two papers that led to the original model, which now has to be considered invalid, were published very prominently (Nature, Science), reached a very large readership and are still frequently discussed and cited, e.g. in a very recent review (Deng and Cao, 2016, Current Opinion in Plant Biology). We therefore also felt that it may be used as an argument against our current data analysis and manuscript if we ignored FLMd.

We also felt that the two recent studies that put the model that attributed a functional role to FLMd into question (Lutz et al., 2015; Sureshkumar et al.,2016) did not yet convincingly proof that any regulatory role for FLMd could be completely rejected. We therefore included FLMd in the present study to test a putative regulatory role of FLMd with our new genetic materials and testing FLM expression in different environmental conditions. We had discussed internally and before submission of the original manuscript whether it was possible to reduce the data load by omitting FLMd data but came to the conclusion that this would weaken the significance of the data set. We have now reexamined this point and made the following changes to reduce the FLMd data load:

We have moved the FLMd data of Figure 5C to Figure 5—figure supplement1D. We removed the FLMd data from Figure 8—figure supplement 1E/F and the respective section in the text since, at this point, the conclusion that FLMd was not important had already been phrased.

That the primers used to detect FLMd may also amplify other minor isoforms was published comparatively late during our analysis (Sureshkumar et al., 2016). We agree that this issue needs to be addressed in the manuscript and discuss this point briefly in the Discussion:

“Our findings thus support more recent studies suggesting that the FLM-δ splice variant may be biologically irrelevant {Lutz, 2015 #118;Sureshkumar, 2016 #96}. One of these studies also identified a large number of biologically irrelevant transcripts that could be identified with the primer combination used here for the detection of FLM-δ {Sureshkumar, 2016 #96}. Our data thus support the conclusion that none of these amplification products has a biologically important function {Sureshkumar, 2016 #96}."

We have also modified Figure 1 and now show the position of the primers and specify in the graphs, which primer combinations had been used for the respective amplification product.

2) Secondly, I am concerned with the results presented in Figure 8 – the correlations between FLM expression in accessions and a) expression in constructs and b) flowering times of the accessions. In order to find a correlation between FLM expression in accessions and in the transgenic lines with the corresponding constructs, the authors dropped an "outlier" point (1 each for FLMb and FLMd). No justification for this is given beyond the point not fitting expectations. It's not clear from Figure 8—figure supplement 1 that these points are really more outliers than any others. And, it's not that this is really a single point – on the y-axis this is the average expression of ~10 accessions, so why should they all be dropped?

We apologize for having used the term “outlier” in an apparently misleading manner. In this analysis, we asked whether one of the nine haplotype groups influenced FLMb levels in a different manner from the others. We thereby examined whether it had a kind of outlying regulatory role. For this purpose, we performed the analysis by excluding, one by one, each of the nine haplotypes and calculated R2. None of these exclusions substantially changed R2 except when PRO2+AAAAC/INT6+CAA was excluded. When this haplotype was excluded R2 increased from 0.13 to 0.46 indicating that this haplotype substantially and negatively influenced our analysis. Herein, our aim had not been to force the linear model to fit our expectations but to identify a PRO2+/INT6+ haplotype, which possesses a differential effect when tested in transgenic lines when compared to accessions. We have now clarified the legend of Figure 8A, removed the term outlier, and improved the description of the findings in the text. We also removed the correlation analysis of FLMd from Figure 8—figure supplement 1.

Legend 8A:

Figure 8. FLM-ß expression shows high correlation with flowering time in Arabidopsis accessions. […] Data of the transgenic lines was taken from Figure 4E. The grey area indicates the 95% confidence interval.”

Results:

PRO2+ and INT6+ polymorphisms contribute to global variation of FLM levels: The nine PRO2+ and INT6+ haplotypes tested in transgenic experiments were present in 579 (69%) of all 840 accession with available genome sequence information (Figure 8—figure supplement 1A). […] This indicated that PRO2+AAAAC INT6+CAA has an exceptional effect on FLM-ß in the accessions than cannot be predicted based on the results from the transgenics (Figures 4E, 8A and Figure 8—figure supplement 1E).”

3) For the flowering time comparison, the lines were vernalized. However, FLM is known to be repressed by vernalization (as the authors note), so how could it be affecting flowering, and might there be variation among lines in how FLM responds to the vernalization? It seems more likely that other loci that are correlated with FLM are controlling flowering time here. The authors should either measure FLM expression in these vernalized plants and correlate that with flowering, or repeat the experiment without vernalization where FLM and FLC expression in these lines has already been measured. A better way to control for FLC variation would be to statistically account for its effects with multiple regression.

We have extracted from the literature that the role of vernalization on FLM expression is rather unclear. Ratcliffe et al. (2001, Plant Phys.) observed decreased FLM levels after six to eight weeks of vernalization, even though FLM seems not to be as strongly repressed by vernalization as FLC. Scortecci et al. (2001, Plant Journal) did not find an effect on FLM transcript abundance after 30 days of vernalization. However, this observation was revisited in Sung et al. (2006, Genes and Development), who showed that FLM is repressed by vernalization through epigenetic mechanisms. Finally, Lee et al. (2013, Science) did not observe any effect of vernalization on FLM abundance whereby no information on the duration of the vernalization treatment was provided. Even though a repressive effect of vernalization on FLM abundance seems to be the consensus we suppose that the effect of vernalization on FLM abundance needs much more detailed investigation. Especially the duration of vernalization may be a critical factor. We had summarized this literature and findings in our original manuscript. Since we now present an improved analysis based on improved experimental design as described below, we have deleted the respective sections since they were no longer relevant.

Regarding the improved experiment as suggested by the reviewer, we considered it not insightful to conduct an experiment with vernalized plants (and including winter annuals) since the FLC-dependent vernalization pathway is epistatic to the effects of FLM.

Instead, we have now performed an experiment with summer annuals not requiring vernalization (all carrying PRO2+/INT6+ haplotypes of the described range). As many summer-annual accessions carry weak FLC alleles with residual low FLC abundance, which still are able to contribute to repression of flowering time {Duncan, 2015 #156;Coustham, 2012 #30;Li, 2014 #111}, we measured FLM and FLC transcript levels and tested their combined and single contribution to flowering time (rosette leaf numbers). Flowering time data was generated in a new experiment. Interestingly, we found a comparable significant contribution of FLMb to flowering time than in the first experiment (R2 = 0.21, before R2= 0.15), with a smaller, however not significant, contribution of the residual FLC levels (R2 = 0.119). We integrated the findings of this new experiment into Figure 8B, changed Figure 8—figure supplement 2, changed the Results, Discussion, and figure legends.

Results:

“To examine the correlation between FLM-ß expression and flowering time, we determined flowering time of accessions at 15°C. […] Taken together, we concluded that PRO2+ and INT6+ alleles contribute to variations in FLM-ß levels and that FLM-ß accounts for flowering time in cool ambient spring temperatures in a diverse population of summer-annual Arabidopsis accessions (Figure 9).”

Discussion:

“The nine PRO2+/INT6+ haplotype combinations included in our transgenic experiments represent 69% of the world-wide PRO2+/INT6+ variation (Figure 8—figure supplement 1A). […] Bearing these possible genetic and environmental interferences in mind, we regard the detected effect of FLM-ß on flowering (21%) in cool (15°C) temperatures as considerable and suggest that it is rather an underestimation.”

Figure legends:

Figure 8. FLM-ß expression shows high correlation with flowering time in Arabidopsis accessions. […] (B) Correlation of FLM-ß transcript levels with flowering time (n = 8 – 10) as measured in rosette leaf number of summer-annual accessions. p = 0.011.”

Figure 8—figure supplement 2: Geographic distribution of summer-annual accessions. (A) Geographic distribution of the 27 summer-annual accessions as described in Figure 8B. (B) Correlation of FLC transcript levels with flowering time data (n = 8 – 10) as measured in rosette leaf number of summer-annual accessions. p > 0.05.”

4) Finally, on the statistical side, the conclusions about "temperature insensitivity" for INT6+ alleles are not backed by statistics – the authors should explicitly test for a change in the temperature sensitivity between FLM-Col0 and INT6+. From the graphs, it appears temperature sensitivity is reduced, but certainly not eliminated (Figure 6).

To address this comment, we have re-evaluated the observation with a new measurement and corrected the calculation and integrated statistical tests. The results and the conclusions were found to be consistent with those presented in the initial manuscript.

Reviewer #3:

In “Natural haplotypes of FLM non-coding sequences fine-tune flowering time in ambient spring temperatures in Arabidopsis" Lutz et al. address the biological relevance of FLM splice variants for variation in flowering time in natural accessions and identify non-coding sequence polymorphisms that contribute to the regulation of basal expression levels and the differential, thermo-responsive generation of the β and δ splice variants. As such, this work provides novel insights into the regulation of flowering in the ambient temperature range and manages to dissect polymorphisms which are relevant for basal FLM expression and temperature-induced effects on the β and δ splice variants.

The manuscript is overall very well written and the authors provide extensive data which has apparently been subjected to comprehensive and sound statistical analyses. The data is presented in an attractive manner. Main manuscript figures are supported by extensive supplementary material including the presentation of extensive control data, which is greatly appreciated. In some cases the figure descriptions or legends should be extended. Due to the vast amount of data condensed in the figures, the descriptions (at least in the text) should be detailed enough for a wide readership to follow the authors through their argumentation.

1) The authors state that “The presence of intron 1 is sufficient to restore the basal expression of FLM” – while it does restore expression levels to detectable levels, the expression level of the β splice variant at 15°C is considerably lower than the WT, whereas the FLMδ levels are similar the WT at this temperature. This would indicate that intron 1 alone cannot fully account for basal levels of FLM β.

The reviewer is correct in stating that Figure 1 may invite the conclusion that intron 1 does not fully account for basal FLMb levels. However, as we used transgenic lines for this analysis we suggest that no conclusions about the basal expression levels of native FLMb nor FLMd can be drawn from this analysis. Nevertheless, the relative change of transcript levels in response to changing temperature can be detected and compared to the WT construct, which is the main aim of this analysis. To avoid any misunderstandings, we normalized the transcript levels measured at 21°C to “1” and provided an improved description in the legend.

Figure legend:

Figure 1. FLM intronic sequences determine basal and temperature-dependent FLM expression. […] For normalization, the respective values measured at 21°C were set to 1.”

2) The use of pools of independent segregating T2 lines for transcriptional analysis seems unusual. While the controls presented in Figure 2—figure supplement 2 suggest that this can recapitulate the “natural” situation (why not subjected to statistical analysis?), it seems nonetheless risky to deliberately tolerate the presence of non-transgenics in the mix. Could the authors state their reasoning for this unusual procedure and provide the missing statistics here? Also, the phrase “replicate pools compromising…” should probably read “comprising”.

By adopting this pooling strategy, we have followed procedures that were described in publications from leading flowering time labs (e.g. Coustham, 2012, Science; Caroline Dean lab). This is an attractive way to reduce the variability of transgene expression that result from positional effects in different transgenic lines. In view of the ample analysis of transgenic lines presented in our manuscript, this presented the most efficient way for reducing cost and time while still producing a meaningful data set. If we had used T3 generation plants, we would have had to propagate and test the segregation of over 3000 individual plants. We have modified (and thereby improved) the above-described strategy by pooling plants into four pools, which we designate replicate pools. We have also verified the trustworthiness of this pooling system by demonstrating that it is possible to precisely recapitulate the effects observed with the Col-0 and Kil-0 FLM alleles (Figure 2—figure supplement 2C and D). The statistical analysis has now been integrated into the figure as requested by the reviewer.

It should also be noted that we verified that the lines are single insertion lines by scoring their segregation, which was possible due to the use of the FAST vector system, which allows the identification of transgenes by seed fluorescence (Shimada, 2010). Further, we compared the transcript levels to a Col-0 allele reference transgenic construct, which was obtained by the exact same procedure of pooling etc.

"Compromising" has been corrected to "comprising".

3) I had a hard time to comprehensively grasp the information provided in Figure 3, especially when it comes to the color code and its use in Figure 3B.

Yes, it is true that the use of the same color in both panels may imply that the two data sets are related to each other, but they are not. We have now changed the colors in 3B to avoid this confusion.

Figure 3—figure supplement 1 provides additional information, but still the color coding of haplotypes which is provided for three different regions (full length, intron 1 or intron 6) is confusing as the composition of the different haplotypes varies depending on the selected gene region (Figure 3—figure supplement 1G). The authors may want to better explain these figures to enable a wide readership to follow their analysis and key points here.

The reviewer is correct in stating that using similar colors for different haplotype regions may be confusing. Here, we used the same color code for each grouping by using the same color for the respective largest group of each the three comparisons. To clarify this, we have now adjusted the shade of these color codes and provided a better description.

Figure legend:

Figure 3—figure supplement 1: Haplotype analysis based on 45 SNPs from 776 accessions. […] The color coding for haplotype group numbers, which illustrates the group number and group size as shown in (D) and (E) is specified below the graph.”

Also, in the caption of Figure 3—figure supplement 1G the haplotype “Numbers on the left” probably refer to (D) not (E)?

Yes, thank you. We have corrected this.

4) Figure 5B and C: Please specify again in the figure caption that this data corresponds to 40 selected accessions. Why was the FLM β level not determined in the vernalized plants? Even though FLC effects seem to be generally negligible, it would have been as easy to do the analysis on the same material.

We have improved the experimental design and have now included an improved data set by using summer annual accessions with minor FLC activity. Such an experiment had been suggested by reviewer #2. Please also see our reply to reviewer 2, comment 3.

5) In Figure 5—figure supplement 1D/E: removing the lower quartile (or more!) of flowering time data for the correlation analysis by simple assumption that these represent the non-transformed Nd-1 individuals of the segregating population is questionable. While this may be an attractive assumption, it negates the fact that each line pool may represent its individual set of variances (also present in ND-1 itself). This would at least require a spot check for the presence of transgenes in this pool or the percentage of “clean” Nd-1 individuals. In principle, a simple test by PCR would have provided a solid answer to that. As is, the authors should include these lines in the correlation analysis as the factual genotype is not known.

We have provided the full data set without corrections (Figure 5—figure supplement 1D/E) as well as the corrected data set (Figure 5A). For the data presented in Figure 5A, we precisely removed the lower quartile and emphasize that we had confirmed that all lines carried single locus inserinots, using a fluorescence marker. Thus, 25% of the plants should be wild type segregants from the transgenic lines and these should correspond to the early flowering individuals since the genetic background for the transgenic analysis had been the FLM deletion accession Nd-1. In this analysis, the homozygous wild type lines Nd-1 and Col-0 are not included since they do not segregate and the correction procedure of removing the lower quartile could not be applied.

Regardless of this correction, very similar R2 values were obtained with the corrected (R2 = 0.94) and uncorrected data (R2 = 0.89). We assume that the high correlation between FLMb levels and flowering time is generally very high, regardless of the only small improvements in the correlation analysis when using the corrected or uncorrected flowering time values.

We have clarified the text and added further detailed information.

Figure legend:

Figure 5. FLM-ß expression shows high correlation with flowering time of transgenic plants. […] An analysis using the uncorrected data is shown in Figure 5—figure supplement 1E,F.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

There are still some key issues on interpretation and analysis that need to be addressed. None of these require new wet lab experiments and are relatively easy and quick to clean up. All felt that this is necessary.

Reviewer #2:

I thank the authors for their improvements to the paper – the figures especially are more clear now. I still have the following concerns about the overall conclusions and analysis. These relate to three important conclusions:

1) That FLM variation contributes to flowering time variation across a diverse panel of summer annual accessions.

The new experiment to test this is very helpful, and does provide good evidence that FLMB expression level is important for flowering time variation. My concern is with interpretation: The authors provide several explanations for why the correlation between flowering and FLMB expression in this accession set is lower than in the transgenics. This is trivial because there is much more variation within each haplotype class in the accessions than the transgenics, because they vary at many loci across the genome. I think the real question should be: Is the slope of the relationship between FLMB and flowering time significantly less in the accessions? This would ask if the equivalent change in FLMB expression would cause the same change in flowering. This is a more meaningful metric than change in R2. The correct test would be to include both accessions and transgenics in the same model (flowering_time ~ FLMB * genotype_class), and ask if the interaction is significant.

We performed the suggested test for interaction FLMB:genotype_class and found that the slopes differ (p = 0.0321). We included this analysis in the results (subsection “PRO2+ and INT6+ polymorphisms contribute to global variation of FLM levels”, last paragraph) and Discussion.

2) That the 9 promoter haplotypes explain the variation in FLMB expression in the accessions.

This is the aim of the analysis in Figure 8A and Figure 8—figure supplement 1D/E. The main claim is that the expression variation induced by these haplotypes in the transgenics is correlated with the expression variation of accessions carrying the same haplotypes. This conclusion only holds when one of the 9 haplotypes is excluded. The explanation given for excluding this haplotype is that the correlation goes up after removing this. I did a quick simulation study and found that using this algorithm you'd expect that the best correlation found by dropping each of the 9 pairs would be greater than 46% about 21% of the time even if there were actually no correlation at all. So, this is really not very strong evidence that the haplotype effects are similar. Certainly, the statement "We found that the PRO2+/INT6+ effects, as detected in transgenic experiments (R2 = 0.94), partially explain the FLM-ß expression variation in natural Arabidopsis accessions (R2 = 0.46)" is not supported, because this 46% number is only true when you exclude those accessions that don't fit.

As above, I think a more appropriate analysis would be to ask if the sizes of the differences between the haplotypes is the same in the transgenics and the accessions (i.e., is the slope of the graph different from 1?). A more straightforward answer to the question of how much variation in FLMB is explained by these haplotypes is simply to report the R2 for the analysis shown in Figure 8—figure supplement 1D.

We understand the reviewer's concern and deleted the analysis excluding the exceptional haplotype from Figure 8 and reported the R2 of the full dataset shown in Figure 8—figure supplement 1E (R2 = 0.13) as suggested. We changed the respective parts of the text, worded the related conclusions less strongly (subsection “PRO2+ and INT6+ polymorphisms contribute to global variation of FLM levels” and Discussion) and changed Figure 8 accordingly.

3) That the INT6+(CAA) haplotype controls temperature sensitivity.

This conclusion is important because of the model that FLM controls temperature sensitivity of flowering. New data are presented here relative to the previous version, though where they came from is not clear. The correct test for a change in temperature sensitivity is to ask if the interaction between genotype and temperature is significant (expression ~ Genotype + Temp + Genotype:Temp). The n.s. effect of temperature on expression for INT6+ is not evidence of no temperature effect, only a lack of evidence for a temperature effect. The figure caption states the statistics are done based on a t-test. A t-test can't be used to test for an interaction (except in 6C/D if the 5 transgenic lines per genotype were used as the replicates, n = 5). An ANOVA is needed to conclude that this haplotype affects temperature sensitivity.

The new data was generated by measuring FLM-β levels with qRT-PCR using the same samples. As suggested by the reviewer, we performed an interaction test between genotype and temperature of the data shown in Figure 6A. We found a significant change in temperature sensitivity (p = 0.012259). We integrated this test in the Results section subsection “The INT6+CAA polymorphism confers temperature-insensitive FLM expression and flowering”) and the figure legend. As the change in temperature sensitivity was now reported by testing the interaction of genotype to temperature, we removed the t-tests from Figure 6A and B.

Reviewer #3:

The authors have generally met my major concerns and provide additional data as well as detailed explanations for the changes in the manuscript.

Remaining concerns:

1) The authors have clarified that the major concerns about the exclusion of accessions from the correlation of FLM expression and flowering time was largely based on a misunderstanding of the method and intention of the analysis. The changes to the text makes this much more clear now. The exclusion of the “exceptional" haplotypes raises the correlation from 0.13 to 0.46 in a linear regression analysis. Please also provide the corresponding p-values here to make sure that the p-values reflect a robust correlation effect (as is done in the further analysis of FLMß/FLC expression via multiple regression analysis). Please also describe how p-values were obtained for both cases then.

Please see our reply to comment #2 of reviewer #2. According to his comment and reasoning, removing the exceptional haplotype may not be appropriate. Therefore, we decided to simply report the R2 shown in Figure 8—figure supplement 1D in the text. For further details please see our answer to comment #2 of reviewer #2.

2) I appreciate the explanation regarding the use of the pooling strategy and the inclusion of statistics. However, no information on the performed test is given in the figure caption. was this also a t-test as mentioned in the methods? If so, please state whether a two-sided t-test and correction for multiple testing was implicated. Otherwise perform an ANOVA with suitable post-hoc test.

A t-test was applied previously. We now performed an ANOVA and Tukey HSD post-hoc test. The interpretation remained unchanged compared to the previously applied t-tests. We changed Figure 2—figure supplement 2C, D and the respective figure legend accordingly.

https://doi.org/10.7554/eLife.22114.033

Article and author information

Author details

  1. Ulrich Lutz

    1. Plant Systems Biology, Technische Universität München, Freising, Germany
    Contribution
    UL, Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0003-3625-3440
  2. Thomas Nussbaumer

    1. Plant Genome and Systems Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany
    Present address
    1. Computational Systems Biology, University of Vienna, Vienna, Austria
    Contribution
    TN, Data curation, Formal analysis
    Competing interests
    The authors declare that no competing interests exist.
  3. Manuel Spannagl

    1. Plant Genome and Systems Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany
    Contribution
    MS, Data curation, Formal analysis
    Competing interests
    The authors declare that no competing interests exist.
  4. Julia Diener

    1. Plant Systems Biology, Technische Universität München, Freising, Germany
    Contribution
    JD, Data curation, Formal analysis
    Competing interests
    The authors declare that no competing interests exist.
  5. Klaus FX Mayer

    1. Plant Genome and Systems Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany
    Contribution
    KFXM, Data curation, Supervision, Funding acquisition, Methodology
    Competing interests
    The authors declare that no competing interests exist.
  6. Claus Schwechheimer

    1. Plant Systems Biology, Technische Universität München, Freising, Germany
    Contribution
    CS, Conceptualization, Supervision, Funding acquisition, Project administration, Writing—review and editing
    For correspondence
    1. claus.schwechheimer@wzw.tum.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0003-0269-2330

Funding

Deutsche Forschungsgemeinschaft (SPP1530)

  • Claus Schwechheimer

Deutsche Forschungsgemeinschaft (SFB924)

  • Klaus FX Mayer
  • Claus Schwechheimer

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors would like to thank Detlef Weigel (MPI Developmental Biology, Tübingen, Germany), Markus Schmid (MPI Developmental Biology, Tübingen, Germany; Umea Plant Science Center, Umea, Sweden) and David Posé (MPI Developmental Biology, Tübingen, Germany; University of Malaga, Spain) for materials and discussions. We thank Thomas Regnault and Alexandre Magalhães for discussion and critical reading of the manuscript. Paula Thompson (Technical University of Munich) is thanked for language editing. This work was supported by grants from the Deutsche Forschungsgemeinschaft as part of the SPP1530 ‘Flowering time control: from natural variation to crop improvement.’ to Claus Schwechheimer and as part of the Sonderforschungsbereich 924 ‘Molecular mechanisms regulating yield and yield stability’ to Claus Schwechheimer and Klaus FX Mayer.

Reviewing Editor

  1. Daniel J Kliebenstein, Reviewing Editor, University of California, Davis, United States

Publication history

  1. Received: October 5, 2016
  2. Accepted: March 9, 2017
  3. Accepted Manuscript published: March 15, 2017 (version 1)
  4. Version of Record published: April 11, 2017 (version 2)

Copyright

© 2017, Lutz et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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