Plants are not able to move around and so they need to be able to adapt their growth and development to seasonal changes in their environment. For example, prolonged exposure to cold temperatures during winter can prime some plants to flower when temperatures increase in the spring—a process called vernalization. In these plants, extended periods of cold temperatures lead to lower activity of a gene called FLC, which normally inhibits flowering.

In the plant Arabidopsis thaliana, vernalization requires several months of exposure to temperatures between 0–6°C. Recently, A. thaliana plants from southern Europe were found to vary in the temperature requirements for vernalization, responding to temperatures higher than 6°C. This suggested that plants from northern Europe might vernalize preferentially at lower temperatures. Here, Duncan et al. compared vernalization in a collection of A. thaliana plants (or ‘accessions’) sampled from different regions of Sweden and the UK.

The experiments show that all the accessions needed temperatures above 0°C to vernalize and that vernalization still worked relatively well at temperatures as high as 14°C. The optimal temperature range for vernalization differed between the accessions, but plants from more northern areas did not necessarily vernalize at lower temperatures. For example, for one particular accession from northern Sweden, the temperature that is optimum for vernalization was 8°C, a notably higher temperature than expected.

Historical local climate records suggested that this accession would vernalize before the first snowfall of the winter in North Sweden. Duncan et al. confirmed this proposal with field experiments. Plants were grown in natural field sites in September and then moved into a greenhouse. The experiments show that the plants complete vernalization by November, which strongly suggests that FLC is silenced during autumn rather than during winter, as previously thought. This changed temperature response is due, in part, to a small number of tiny genetic differences in regions of the FLC gene that do not code for protein.

These findings have important implications for future studies of vernalization and flowering time, and for understanding how plants will adapt to on going and future climate change. The next step is to understand what causes these changed temperature responses at a molecular level, which should enable selective breeding for flowering and harvest date in a range of crops.

The sessile nature of plants necessitates that they modulate most aspects of their growth and development in response to external conditions. One aspect of this is the alignment of developmental transitions with seasonal cues. A major seasonal cue is temperature and plants have evolved the ability to integrate daily fluctuations in external temperature in order to monitor long-term trends (Aikawa et al., 2010). Exposure to weeks of cold temperature accelerates the transition to flowering in a process called vernalization. In Arabidopsis thaliana vernalization involves the quantitative epigenetic silencing of FLC (Michaels and Amasino, 1999; Sheldon et al., 1999). Cold exposure promotes a cell-autonomous epigenetic switch at FLC in an increasing proportion of cells (Angel et al., 2011, 2015). This epigenetic switching mechanism requires a Polycomb complex associated with PHD proteins (De Lucia et al., 2008), including the cold-induced VIN3 (Sung and Amasino, 2004). This enables activation of FT, is a potent activator of flowering in A. thaliana (Searle et al., 2006). At a standard vernalization temperature of 5°C, the length of cold required to achieve complete epigenetic silencing varies between A. thaliana accessions and this maps to non-coding cis polymorphisms in FLC (Coustham et al., 2012; Li et al., 2014). Accessions collected from northerly latitudes typically require longer vernalization, for example, the accession Lov-1 originates from near the northerly limit of the Arabidopsis range in Lövvik, North Sweden (62.5°N) and requires three months of vernalization to fully accelerate flowering (Shindo et al., 2006; Coustham et al., 2012).

Effective temperature ranges for vernalization have been determined empirically for different plant species, many of which have been incorporated into chilling unit models that are widely used in agriculture (Byrne and Bacon, 1992). A genetically informed photothermal model for flowering in A. thaliana has assumed that vernalization occurs when daytime hourly temperatures are higher than 0°C and lower than 6°C (Wilczek et al., 2009; Chew et al., 2012, 2014). However, accessions from southern Europe have been found to vernalize at constant temperatures significantly higher than 6°C (Wollenberg and Amasino, 2012), suggesting that accessions from northerly latitudes might vernalize most efficiently at relatively low temperatures. Here, we show this is not the case and find that vernalization in a range of A. thaliana accessions is most effective across a relatively high temperature range with the N. Swedish accession Lov-1, showing maximal vernalization at 8°C. We show that vernalization is complete before snowfall in N. Sweden with the plants flowering immediately upon snowmelt. Vernalization responsiveness in the field matched that when the average monthly temperature was given as constant conditions. Our work has important implications for modeling flowering time and predicting the impact of climate change.

In order to investigate natural variation for vernalization temperature requirement in A. thaliana accessions we selected several genotypes that represent most of the major FLC haplotypes (Li et al., 2014): Lov-1 (Lövvik, N. Sweden—latitude 62.5°N), Var2-6 (Vårhallen, S. Sweden—latitude 55.58°N), Ull2-5 (Ullstorp, S. Sweden—latitude 56.06°N), Edi-0 (Edinburgh, UK—latitude 55.95°N) and the reference Columbia line containing FRIGIDA (Col FRI^Sf2, [Michaels and Amasino, 1999]) (Figure 1—figure supplement 1). All genotypes were vernalized for varying periods at different constant temperatures between 0°C and 14°C and the efficiency of vernalization assayed by determining flowering time (Figure 1A-E). All the genotypes showed limited vernalization after 4 and 6 weeks exposure to 0°C and vernalized more efficiently at all other temperatures. Col FRI^Sf2 and Edi-0 were most effectively vernalized after 4, 6 or 12 weeks at 2°C, 5°C and 8°C and still vernalized relatively efficiently at 12°C and 14°C (Figure 1A and C). Even after 2 weeks of cold at 2°C and 8°C the flowering of Col FRI^Sf2 plants was similar, so lack of any difference was not due to vernalization being close to saturation (Figure 1—figure supplement 2). Ull2-5 showed similar temperature sensitivity to Col FRI^Sf2 and Edi-0, but required 12 weeks for vernalization to be fully effective (Figure 1D). In contrast, Lov-1 and Var2-6 plants showed a differential response to temperature with 2 and 12°C less effective than 5 and 8°C after 6-weeks vernalization (Figure 1B and E). For Lov-1 the only temperature that resulted in flowering after 4 weeks exposure was 8°C and although the enhanced effect of this temperature diminished over time, 8°C consistently resulted in the most effective vernalization (Figure 1B). Thus, the different accessions show distinct temperature profiles for vernalization and all require temperatures higher than 0°C.

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The requirement for longer cold for effective vernalization in the Lov-1 accession has previously been shown to involve quantitative variation in accumulation of epigenetic silencing of FLC (Shindo et al., 2006; Coustham et al., 2012). We compared this quantitative variation in the silencing of Col FRI^Sf2 and Lov-1 FLC alleles after 4 weeks cold exposure at 2, 5, 8, 12 and 14°C (Figure 2—figure supplement 1A and B). In contrast to Col FRI^Sf2, the Lov-1 allele re-activated after 30 days in the warm after vernalization at all the tested temperatures. However, the degree of re-activation was lowest after vernalization at 8°C, consistent with vernalization being most effective at this temperature. Similarly, 6 weeks vernalization at 8°C resulted in lower FLC re-activation post-cold and higher levels of FT induction than 5°C, with similar VIN3 expression (Figure 2A–C). Epigenetic silencing of FLC is associated with Polycomb silencing and accumulation of H3K27me3 over the gene body (Angel et al., 2011; Yang et al., 2014). In Lov-1 it takes longer to accumulate the H3K27me3, mainly due to lower starting levels (Coustham et al., 2012). We found similar accumulation of gene body H3K27me3 in the Col FRI^Sf2 FLC allele at 5, 8 or 14°C, but differential H3K27me3 accumulation in the Lov-1 allele (Figure 2D, Figure 2—source data 1). Vernalization at 8°C resulted in higher levels of H3K27me3 compared to 5 or 14°C (Figure 2D), suggesting that the Polycomb silencing is most effective at 8°C for the Lov-1 FLC allele.

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Primers used for qPCR ChIP.

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The relatively high temperature range for vernalization of the N. Swedish accession was surprising given that flower buds appeared within 2 weeks of the snowmelt on native Arabidopsis at the N. Swedish Lövvik site (Figure 3—figure supplement 1). This early flowering may limit herbivory and help in the competition for nutrients (Kawagoe and Kudoh, 2010; Akiyama and Agren, 2012). A long-term (>5 year study) of the natural populations at several N. Swedish sites throughout the High Coast region, showed most populations behaved as winter annuals with germination occurring predominantly in August and September with no spring germination (Figure 3—source data 1). The rapid flowering after snowmelt suggests that vernalization must have occurred before the end of November given the recurrent snow cover and low temperatures at the Lövvik site over the winter months (Figure 3—figure supplement 2, Figure 3—figure supplement 3A). Hourly climate data collected near Lövvik between 1st August (the earliest germination date observed for natural populations) until snow cover between 2008 and 2013 show an average air temperature of ~8°C (Figure 3—figure supplement 3A). Analysis of national data (1st August—30th November) also revealed an overall average autumn daily average temperature of 8.86°C between 1961 and 2008 (SD = 0.63) with over 86% of days falling within the range identified as being effective for Lov-1 vernalization, (0°C, 15°C) (Figure 3—figure supplement 3B). The agreement of average autumn temperatures with the effective vernalization temperatures identified for the Lov-1 reinforced the view that epigenetic silencing of FLC would occur before snowfall.

We tested the hypothesis of a seasonal shift in the timing of vernalization in N. Sweden by setting up field experiments close to the Lövvik site in autumn 2011 and 2012 (locations shown in Figure 3—figure supplement 4). Seedlings were transplanted into the field at the beginning of September and then transferred to a warmed greenhouse at three time points during autumn (Figure 3A, Figure 3—figure supplement 5A). This enabled us to explicitly test whether 12 weeks of growth preceding winter would be sufficient to fully vernalize Lov-1. Flowering time of the different cohorts showed that vernalization was complete by the end of November in both 2011 (Figure 3B) and 2012 (Figure 3—figure supplement 5B). Furthermore, plants left to overwinter in the field flowered rapidly at snowmelt, at the same time as the native A. thaliana population (Figure 3—figure supplement 6).

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Developmental stage of natural Arabidopsis thaliana populations in spring in the High Coast area of N. Sweden (62.5°N).

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In order to link the flowering time changes with the changed epigenetic silencing at FLC we included a near isogenic line carrying the Lov-1 FLC allele (NIL^Lov-1) in the genetic background of Col FRI^Sf2 in the field experiments. This line was generated through six generations of introgression and had been genotyped with markers to define the introgressed region (Figure 3—figure supplement 7). NIL^Lov-1 took longer to flower than Col FRI^Sf2 after the first two transfers in 2012 (Figure 3—figure supplement 5B). This revealed the clear contribution of the Lov-1 FLC allele to differential vernalization response under field conditions, which likely involves the four non-coding polymorphisms in FLC close to the nucleation site of the PHD-PRC2 previously defined as underpinning the molecular variation in FLC epigenetic silencing between Lov-1 and Col FRI^Sf2 (Coustham et al., 2012).

Expression analysis in the perennial species Arabidopsis halleri growing under natural field conditions has shown that plants average temperature over long-term scales (Aikawa et al., 2010). It was therefore interesting that the optimal vernalizing temperature for Lov-1 matched the average temperature over the 3-month season when vernalization occurred (Figure 3B). We therefore compared vernalization response in the different transplant intervals with vernalization in constant temperatures equivalent to the average temperature of the field conditions (Figure 4—source data 1). The different genotypes showed temporal differences in vernalization responsiveness over the three transplant periods in the field. Remarkably, vernalization responsiveness was very similar when the average field temperature was given as a constant temperature, with each genotype showing a different overall profile (Figure 4). Indeed, the match is remarkable given the daily oscillations in temperature especially in the transplant 1 period (Figure 3A, Figure 3—figure supplement 5A). How plants integrate these fluctuating temperatures over such long timescales is an important area for future molecular dissection.

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Cabinet flowering time data were selected where conditions most closely matched mean temperatures recorded during 2011 and 2012 field experiments.

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All the genotypes analysed were found to vernalize effectively during autumn (Figure 3B, Figure 3—figure supplement 5B), however they have been shown to differ in their seed dormancy (Atwell et al., 2010); accessions from N. Sweden generally have much lower seed dormancy requirement than those from further south (Debieu et al., 2013). Thus, the low seed dormancy of Lov-1 would enable germination to occur early enough to exploit the whole of the N. Swedish autumn for vernalization. The increased seed dormancy of S. Swedish accessions (e.g., Ull2-5) is likely to delay germination leading to the necessity for vernalization in some years to extend into winter in S. Sweden. It is interesting to speculate that the reduced effectiveness of temperatures below 5°C for other Swedish accessions Lov-1 and Var2-6 (Figure 1B,E) could prevent premature vernalization occurring during unseasonal cool periods in early autumn. Our data also show that the (0°C, 6°C) temperature range widely used to estimate vernalization in A. thaliana (Wilczek et al., 2009) would only predict partial vernalization of later flowering accessions during our field experiments (Figure 4—figure supplement 1). Our data suggest that raising the upper threshold temperature to 15°C would improve estimates of vernalization progress for later flowering accessions under natural field conditions.

In summary, we have employed a combination of molecular and ecological approaches to connect temperature-induced molecular changes at FLC with ecologically significant effects in the field. We show that growth at the northern limit of the A. thaliana species range has involved a seasonal shift in the timing of vernalization. Perhaps as a response to selection in these extreme conditions one N. Swedish accession, Lov-1, shows a more distinct vernalization temperature optimum that matches the average historical temperature for August-November in that geographical region (Figure 1, Figure 2, Figure 3—figure supplement 3A). Early germination enables vernalization to complete before snowfall and allows flowering to occur directly after snowmelt when the photoperiod and ambient temperatures increase. Rises in global temperature have already reduced vernalization periods to an extent that has impacted the phenology of a range of plant species (Fitter and Fitter, 2002; Cook et al., 2012). Studies such as this are therefore important to understand how rapidly populations might adapt under future climate scenarios.

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Author details

1. Susan Duncan

   John Innes Centre, Norwich, United Kingdom

   Contribution

   SD, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Svante Holm

   Mid Sweden University, Sundsvall, Sweden

   Contribution

   SH, Designed the field experiments, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Julia Questa

   John Innes Centre, Norwich, United Kingdom

   Contribution

   JQ, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Judith Irwin

   John Innes Centre, Norwich, United Kingdom

   Contribution

   JI, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Alastair Grant

   Department of Environmental Sciences, University of East Anglia, Norwich, United Kingdom

   Contribution

   AG, Conception and design, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Caroline Dean

   John Innes Centre, Norwich, United Kingdom

   Contribution

   CD, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   caroline.dean@jic.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

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