Mechanisms of Age-Dependent Response to Winter Temperature in Perennial Flowering of Arabis alpina

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Science  31 May 2013:
Vol. 340, Issue 6136, pp. 1094-1097
DOI: 10.1126/science.1234116

Multiple Inputs to Flowering

Perennial plants need to cycle through an extended vegetative phase, in a process known as vernalization, before they initiate flowering. Bergonzi et al. (p. 1094) and Zhou et al. (p. 1097) studied how molecular signals translate environmental information—such as exposure to a winter season or changes in daylength and physiological information, such as age of the plant—into signals that promote flowering. In both Arabis alpina and Cardamine flexuosa, age and vernalization pathways are integrated through the regulation of microRNAs miR156 and miR172.


Perennial plants live for more than 1 year and flower only after an extended vegetative phase. We used Arabis alpina, a perennial relative of annual Arabidopsis thaliana, to study how increasing age and exposure to winter cold (vernalization) coordinate to establish competence to flower. We show that the APETALA2 transcription factor, a target of microRNA miR172, prevents flowering before vernalization. Additionally, miR156 levels decline as A. alpina ages, causing increased production of SPL (SQUAMOSA PROMOTER BINDING PROTEIN LIKE) transcription factors and ensuring that flowering occurs in response to cold. The age at which plants respond to vernalization can be altered by manipulating miR156 levels. Although miR156 and miR172 levels are uncoupled in A. alpina, miR156 abundance represents the timer controlling age-dependent flowering responses to cold.

Polycarpic perennial plants live for many years and flower each year. To maintain resources and sustain multiple flowering cycles, they tightly control the extent and duration of flowering. In particular, perennials flower only when they reach a certain age and then are described as competent to flower (1). Environmental signals induce flowering of older plants, whereas younger plants remain vegetative under the same conditions. Other age-dependent developmental processes, such as the transition from juvenile to adult vegetative phase, are regulated by microRNAs miR156 and miR172 (2, 3). As plants age, the abundance of miR156 declines while miR172 levels increase (46), and these opposing effects are mechanistically coupled in Arabidopsis thaliana (6). In addition, miR156 and miR172 respectively repress or promote the transition from vegetative growth to flowering (2). In A. thaliana, the targets of miR156 are the mRNAs of 11 genes encoding SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors (79), whereas miR172 regulates six members of the APETALA2 (AP2) transcription factor family (1012). Involvement of miR156 and miR172 in regulating age-related competence to flower is difficult to test in the model annual species A. thaliana because it responds to environmental cues to induce flowering soon after germination (13, 14). Mutants in SPL genes are less sensitive to inductive photoperiods, but how this is related to age-dependent flowering is unclear (15). We studied these processes in the model perennial Arabis alpina accession Pajares that flowers only if exposed to winter cold (vernalization) when at least 5 weeks old (16).

To identify genes that regulate flowering and perennial traits, we first screened a mutagenized population of A. alpina (17). The perpetual flowering 2-1 (pep2-1) mutant flowered without vernalization and showed perpetual flowering (Fig. 1, A and B). These phenotypes were described previously for pep1-1, which is caused by a mutation in the A. alpina ortholog of the A. thaliana FLOWERING LOCUS C (FLC) gene (17). The pep2-1 mutation is not allelic to pep1-1 (Fig. 1C), and using whole-genome sequencing as well as genetic linkage analysis, we showed that the pep2-1 phenotype is caused by an amino acid substitution in the DNA binding domain of A. alpina APETALA2 (AaAP2) (fig. S1A) (18). Based on conservation of synteny of flanking regions, PEP2 is the ortholog of A. thaliana AP2 (fig. S1B). The AP2 transcription factor represses flowering and confers floral organ identity in A. thaliana (19, 20), and pep2-1 shows similar floral defects (18) (fig. S1C). Additional mutagenesis screens for enhancers of pep1-1 identified six mutants carrying nonsynonymous changes in AaAP2 (Fig. 1D, pep2-2 to pep2-7) (18). These pep2 pep1-1 double mutants flowered earlier than pep1-1 (Fig. 1, E and F, and fig. S1D) and showed floral phenotypes similar to that of pep2-1 (Fig. 1, G and H), supporting the idea that the pep2-1 phenotype is due to the mutation in AaAP2. Although AP2 delays flowering of A. thaliana, it was not previously reported to confer a vernalization requirement (19).

Fig. 1

PEP2 regulates flowering in response to vernalization and is the A. alpina ortholog of APETALA2. (A) The pep2-1 mutant flowers without vernalization. WT, wild-type; bar, 9 cm. (B) The pep2-1 mutant flowers perpetually. (C) The pep2-1 and pep1-1 mutations are not allelic. Flowering time in long days of pep1-1, pep2-1, and F1 plants derived from crossing the mutants. NF, never flowers. (D) Mutations in the A. alpina ortholog of APETALA 2 (AaAP2) in pep2 mutants. The pep2-1 allele (red) is in wild type whereas pep2-2 to pep2-7 (blue) are in pep1-1 mutant. (E to H) Mutations in PEP2 enhance the early flowering of pep1-1 mutants, and the double mutants show floral homeotic conversion. The pep1-1 and pep2-2 pep1-1 mutants 10 weeks after germination (E); flowering time in long days (F). Flower phenotype of pep1-1 (G) and pep2-2 pep1-1 (H). Error bars in (C) and (F) indicate standard deviation.

We examined whether PEP2 acts in the same molecular pathway as PEP1. PEP1 mRNA levels were lower in 2-week-old pep2-1 seedlings compared to wild type, whereas PEP2 mRNA levels were not reduced in pep1-1 mutants (Fig. 2A). PEP1 represses flowering through the A. alpina SOC1 ortholog (AaSOC1) (16), which is a promoter of flowering in A. thaliana (21, 22). AaSOC1 mRNA levels were approximately twice as high in pep2-1 seedlings compared to wild type (Fig. 2A). Therefore, PEP2 likely confers a vernalization requirement in A. alpina in part by increasing transcription of PEP1. However, PEP2 must also delay flowering by other means because pep1-1 and pep2 mutations have additive effects on flowering time (Fig. 1, E and F, and fig. S1, D to F). PEP2 mRNA levels did not change during prolonged exposure to cold (fig. S2A) or as plants aged and became sensitive to vernalization (Fig. 2, B to D).

Fig. 2

PEP2 and miR172 expression patterns and the effect of pep2-1 on PEP1 mRNA. (A) PEP1, PEP2, and AaSOC1 mRNA levels in 2-week-old pep2-1 (black), wild-type (white), and pep1-1 (gray) seedlings. (B) miR172 (blue) and PEP2 mRNA (purple) levels in apices of plants growing in long days (LDs). (C and D) In situ hybridization of PEP2 mRNA in the main shoot apices of 2-week-old (C) and 8-week-old (D) plants. Bar, 100 μm. (E) miR172 abundance in apices of plants exposed to cold. Plants grown for 2 weeks (white) or 8 weeks (black) before exposure to cold. AaLEAFY mRNA level (red line) in 8-week-old plants exposed to cold. (F and G) In situ hybridization of inflorescence meristems. miR172 locked nucleic acid (LNA) probe (F) and PEP2 mRNA probe (G) hybridized to subsequent sections of the same apex. Plants were grown for 8 weeks, exposed to cold for 12 weeks, and returned to long days for 2 weeks. Bar, 100 μm. Error bars in (A), (B), and (E) indicate standard deviation.

AP2 activity is regulated by miR172, and in several species the abundance of this miRNA increases as plants age (4, 5, 10, 23, 24). PEP2 mRNA also contains a binding site for miR172 (Fig. 1D and fig. S2B). During vegetative growth, miR172 levels did not change, either in A. alpina apices (Fig. 2B) or in young plants exposed to cold for several weeks (Fig. 2E). By contrast, the abundance of miR172 increased during cold treatment of older plants that flowered in response to vernalization (Fig. 2E). This increase occurred during flower development, recognized by higher expression of A. alpina LEAFY (Fig. 2E) (16). In the inflorescence meristem, miR172 and PEP2 showed complementary spatial expression patterns, with miR172 accumulating mainly in the center and PEP2 mRNA in the lateral floral primordia (Fig. 2, F and G), as described in A. thaliana (10, 23). Thus, miR172 and PEP2 appear to have conserved roles in flower development of A. alpina but do not contribute to the age-dependent initiation of flowering.

To identify genes that confer age-dependent sensitivity to vernalization, we compared global gene expression patterns between apices of 2-week-old and 8-week-old A. alpina plants (25) (table S1). Expression of several genes encoding SPL transcription factors was increased in apices of older plants (table S2). In A. alpina, 15 genes show high sequence similarity and conserved synteny to the A. thaliana SPL genes (fig. S3 and table S3), and these fell into three major groups on the basis of their temporal expression patterns (Fig. 3A). The first of these groups comprised six AaSPL genes that were more highly expressed in apices of older plants that flower in response to vernalization.

Fig. 3

miR156 abundance is reduced as A. alpina plants age and is regulated by cold. (A) Heat map of microarray hybridization levels for SPL genes of A. alpina in apices of 2-week-old (2wo) and 8-week-old (8wo) plants before and after 4 weeks at 4°C (+ cold) (25). SPL genes are placed in three groups (I, II, III) according to their expression pattern. Asterisks: A. alpina genes containing a miR156 binding site. (B) miR156 abundance in the main shoot apex of plants growing in long days (LDs). (C and D) In situ hybridization of miR156 (LNA probe) in apices of 2-week-old (C) and 8-week-old (D) plants growing in long days. Bar, 100 μm. (E) miR156 abundance in apices of 2-week-old (white) or 8-week-old (black) plants exposed to cold. Time points as in Fig. 2E. Asterisk: miR156 levels in plants able to flower after a second cold treatment. (F) miR156 abundance in apices of 2-week-old plants exposed to cold for up to 60 weeks (white) compared to levels in 8-week-old plants (black). Error bars in (B), (E), and (F) indicate standard deviation.

All of the AaSPL genes up-regulated in apices of older A. alpina plants before or during vernalization contained miR156 binding sites (Fig. 3A). Therefore, we tested the abundance of miR156 at the shoot apex and found that it declined as plants aged, similarly to other species (Fig. 3, B to D). Trough levels of miR156 occurred about 5 weeks after germination, around the time A. alpina plants acquired sensitivity to vernalization (16). These results suggested that at the main shoot apex, reduction in miR156 accumulation to trough levels sets the age at which plants become sensitive to vernalization. Axillary branches attain the ability to respond to vernalization independently of the main shoot apex, and this also correlates with a reduction in miR156 levels in the axillary shoot apices (fig. S4).

Exposure of young plants to cold increases the age at which they become able to flower in response to vernalization. Two-week-old A. alpina seedlings exposed for more than a year to cold continued to grow but did not flower when returned to warm conditions (fig. S5, A and B). The high miR156 abundance observed in 2-week-old plants remained stably high throughout the cold treatment and declined only after a return to warm conditions (Fig. 3E). This reduction of miR156 accumulation after cold also correlated with flowering when plants were exposed to a second vernalization (Fig. 3E). The levels of miR156 declined slowly in young plants exposed to cold for up to 60 weeks, suggesting that cold strongly delays but does not prevent the reduction in miR156 abundance (Fig. 3F). A similar but less extreme effect was observed in A. thaliana (fig. S5C). In addition, Aa pre-miR156a showed the same pattern of accumulation in cold-exposed plants as mature miR156 (fig. S5D), suggesting that transcription of MIR156 genes is regulated by cold. Thus, young A. alpina plants do not respond to vernalization, even if maintained in cold for more than a year, because reduction of MIR156 transcription is delayed.

The importance of reduced miR156 levels for the age-related response to vernalization was tested with the use of transgenic plants. First, MIR156b was expressed at high levels from the heterologous CaMV35S promoter in wild-type plants. Eight-week-old overexpressor plants failed to flower when exposed to 12 weeks of vernalization, in contrast to control wild-type plants (Fig. 4, A and B). Although these transgenic plants did not flower, PEP1 mRNA level was reduced during vernalization (Fig. 4C), so miR156 does not prevent flowering in response to vernalization through PEP1. The effect of miR156 overexpression was also tested in the pep1-1 mutant, which flowers in long days without vernalization and shows a temporal pattern of miR156 accumulation similar to that of the wild type (fig. S6A) (17). Expression of MIR156b from the CaMV35S promoter in pep1-1 strongly delayed flowering when the transgenic plants were grown under long days (fig. S6B). By contrast, in A. thaliana, MIR156b overexpression strongly delays flowering only in short days, whereas in long days it causes a moderate delay (4, 7, 26). To test whether lowering miR156 activity reduces the age at which plants become sensitive to vernalization, we overexpressed a mimicry construct (MIM156) in A. alpina (27). These transgenic plants did not flower without vernalization (Fig. 4D) but flowered when vernalized at the age of 3 weeks, whereas wild-type plants of this age did not (Fig. 4, E and F). Transcript levels of several miR156-regulated AaSPL genes were increased in apices of 3-week-old 35S:MIM156 lines compared to the wild type (Fig. 4G). Thus, AaSPL transcription factors are essential for the promotion of flowering in perennial A. alpina, and the antagonistic regulation of miR156 and SPL expression determines the age at which these plants can flower in response to vernalization.

Fig. 4

Reducing miR156 levels accelerates age-related sensitivity to vernalization. (A and B) Flowering of plants overexpressing MIR156b. Cuttings from T1 plants grown for 8 weeks, exposed to cold for 12 weeks, and transferred back to warm long days for 5 weeks (n = 20). WT, wild type; NF, never flowers. (C) PEP1 mRNA expression is down-regulated by vernalization in 35S:MIR156b plants. PEP1 mRNA levels after 8 weeks in long days (black) and after transfer to cold for 12 weeks (white). Plants as in (A). (D) Wild-type plants overexpressing MIM156 do not flower in long days. (E and F) Wild-type plants overexpressing MIM156 flower when exposed to cold 3 weeks after germination. n = 12. Bars, 12 cm. (G) mRNA levels of AaSPL genes 3 weeks after germination in WT and transgenic plants overexpressing MIM156. AaSPL genes targeted (red) and not targeted (blue) by miR156. Error bars in (A) and (C) indicate standard deviation.

Our findings indicate that miR156 and PEP2/PEP1 act in parallel repressive pathways to ensure that A. alpina meristems become competent to flower only if they have reached the appropriate age and have been exposed to winter temperatures (fig. S7). The floral repressor AaTFL1 also delays the acquisition of age-related response to cold (16), probably by setting a threshold for AaSPL activation of flowering (fig. S7). In A. thaliana and maize, the decrease in miR156 is coupled with an increase in miR172, but we did not observe this relationship in A. alpina. Maintaining miR172 at low levels during vegetative growth might allow the activity of its target PEP2 to remain high, ensuring that plants do not flower before they are exposed to cold. In the annual species A. thaliana flowering in response to environmental cues can occur in young plants before the decline in miR156 levels, and SPL activity is not essential for flowering under inductive photoperiods. Probably other flowering pathways can bypass the requirement for SPL factors in A. thaliana under long days, and this is likely important in conferring its rapid cycling life history. Our data illustrate how recruitment of the miR156 and miR172 regulatory modules to control environmental responses contributes to evolution of the plant life cycle and that this can occur rapidly, even among closely related species.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S9

Tables S1 to S4


References and Notes

  1. Supplementary materials are available on Science Online.
  2. Acknowledgments: We thank E. Willing, G. Velikkakam, and the "A. alpina genome consortium" for access to genomic information and Y. Zeng for technical support. We thank members of G.C.'s laboratory and P. Huijser for critical reading of the manuscript and D. Weigel for MIR156 constructs and comments on the manuscript. We acknowledge funding from Deutsche Forschungsgemeinschaft SPP1530 grants to G.C. and M.C.A., the Max Planck Genome Centre, and a core grant from the Max Planck Society. GenBank accession numbers are GSE40455 (microarray data), KC815948 to KC815963 (AaSPLs), KC831439 (PEP2), and KC848668 (Aapre-miR156a). Materials described here are available from G.C. subject to a Material Transfer Agreement with the Max Planck Institute for Plant Breeding Research.
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