Molecular Basis of Age-Dependent Vernalization in Cardamine flexuosa

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Science  31 May 2013:
Vol. 340, Issue 6136, pp. 1097-1100
DOI: 10.1126/science.1234340

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.


Plants flower in response to many varied cues, such as temperature, photoperiod, and age. The floral transition of Cardamine flexuosa, a herbaceous biennial-to-perennial plant, requires exposure to cold temperature, a treatment known as vernalization. C. flexuosa younger than 5 weeks old are not fully responsive to cold treatment. We demonstrate that the levels of two age-regulated microRNAs, miR156 and miR172, regulate the timing of sensitivity in response to vernalization. Age and vernalization pathways coordinately regulate flowering through modulating the expression of CfSOC1, a flower-promoting MADS-box gene. The related annual Arabidopsis thaliana, which has both vernalization and age pathways, does not possess an age-dependent vernalization response. Thus, the recruitment of age cue in response to environmental signals contributes to the evolution of life cycle in plants.

Plants show diverse life-cycle strategies. Annual plants go from germination to death within 1 year. Biennial and perennial plants take 2 or more years to complete their life cycles. Monocarpic perennials, such as bamboo, bloom once then die, whereas polycarpic perennials preserve some meristems in the vegetative state to sustain flowering in subsequent years (13). In Arabidopsis thaliana, an annual plant, the floral transition is controlled by various exogenous and endogenous cues, such as photoperiod or day length, transient exposure to low or high temperature, hormone, and age (47).

We studied the molecular mechanisms that regulate flowering in the biennial-to-perennial wavy bittercress (Cardamine flexuosa), a herbaceous Brassicaceae widely distributed in Europe (8) (supplementary text). Under constant temperature (23°C) and humidity (50%) conditions, C. flexuosa exhibited a typical polycarpic perennial growth habit. Plants did not flower when grown continuously in long (16 hours of light) or short (8 hours of light) days (Fig. 1A and fig. S1). Exposure of 8-week-old long-day plants to short days did not induce flowering (fig. S1I). C. flexuosa plants flowered only when exposed to cold temperature (4°C) for 2 months (Fig. 1B and fig. S1I), a treatment called vernalization. After seed maturation, all inflorescences senesced and died, whereas the rosette and axillary shoots (branches) initiated after vernalization remained alive and vegetative (Fig. 1C). A second round of flowering could be induced by another 2-month cold treatment (fig. S1H).

Fig. 1

Age-dependent vernalization in C. flexuosa. (A) Wild-type (Wt) C. flexuosa was grown in long days for 10 weeks without vernalization (–V). The plant did not flower. (B) Vernalization induced flowering. A 10-week-old wild-type plant grown in long days was vernalized (+V) for 8 weeks and returned to 23°C in long days. The plant started bolting and flowering. (C) C. flexuosa did not die after flowering. The bolts turned yellow and died. The rosette kept green and stayed in the vegetative phase. (D and E) CfFLC regulated vernalization. Wild type (D) and 35S::CfFLC-migs (E) were grown in long days for 5 weeks. The 35S::CfFLC-migs flowered without vernalization. Scale bars in (A) to (E), 5 cm. (F) Flowering time of wild-type and 35S::CfFLC-migs plants. The number of leaves when the plants started to flower was scored. NF indicates never flowering; error bar indicates SD. n = 13. (G) Age-dependent vernalization in C. flexuosa. Plants of different ages (from 1 to 8 weeks old) were vernalized for 2 months and returned to 23°C long days. Number of flowered plants was scored. n = 20. 1w+V, plants grown for 1 week in long days and then exposed to vernalization. (H and I) Temporal expression pattern of CfFLC (H) and CfSOC1 (I). Wild-type plants were grown in long days for 5 weeks before cold treatment. BV, before vernalization; AV, after a 2-month vernalization treatment; AV+2w, plants grown in long days for 2 weeks after vernalization. RNA levels in leaves were measured by real-time quantitative polymerase chain reaction (qPCR). The expression levels were normalized to that of CfTUB. The data presented are the means of two technical replicates; error bars indicate SD. (J and K) Expression of CfFLC (J) and CfSOC1 (K) in response to vernalization and age. One- or 5-week-old C. flexuosa plants were subjected to vernalization treatment for 2 months. **P < 0.001, Student's t test.

The vernalization response in the perennial Brassicaceae Arabis alpina is controlled by the MADS-box gene PERPETUAL FLOWERING 1 (PEP1), an ortholog of A. thaliana FLOWERING LOCUS C (AtFLC) (7, 9). We isolated CfFLC, an AtFLC homolog, in C. flexuosa (fig. S2 and supplementary text). When we overexpressed CfFLC in A. thaliana, flowering was delayed in long days (fig. S3A), indicating that CfFLC acts as a flowering repressor. To analyze the function of CfFLC in C. flexuosa, we generated CfFLC knockdown plants with the microRNA-induced gene silencing (MIGS) technology (35S::CfFLC-migs) (10). These plants had about fivefold less CfFLC expression than wild type (fig. S3B) and were able to flower without vernalization (Fig. 1, D to F). All subsequent axillary shoots were floral (fig. S3C). Thus, CfFLC encodes an ortholog of A. alpina PEP1and regulates perennial flowering in C. flexuosa.

Wild-type plants were grown in long days for 5 weeks and then exposed to cold temperature for 8 weeks. Cold treatment decreased the transcript levels of CfFLC by three-fourths (Fig. 1H). After return to 23°C long days, CfFLC mRNA levels gradually increased, reaching the same level as that before vernalization within 1 month (Fig. 1H). Therefore, in the perennials C. flexuosa and A. alpina, FLC expression is only transiently suppressed by vernalization, whereas vernalization stably represses FLC expression in the annual A. thaliana (7, 11).

In A. thaliana, flowering time pathways are integrated by several integrator genes, including SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) (12). Overexpression of C. flexuosa SOC1 (CfSOC1) in C. flexuosa bypassed the vernalization requirement (figs. S4 and S5), suggesting that CfSOC1 acts downstream of the vernalization pathway. The expression pattern of CfSOC1 was opposite to that of CfFLC, with the expression transiently increased upon cold treatment (Fig. 1I).

We found that the ability of cold exposure to cause vernalization of C. flexuosa plants was age-dependent. Plants less than 5 weeks old were not fully responsive to cold treatment (Fig. 1G). The expression of CfFLC was repressed by vernalization in both young (1-week-old) and old (5-week-old) plants (Fig. 1J). However, cold treatment induced an increase of CfSOC1 expression in old but not young plants (Fig. 1K). We infer that the insensitivity of young plants to cold treatment is due to a defect in activation of CfSOC1 rather than a change in the repression of CfFLC.

MicroRNA156 (miR156), which targets a group of SQUAMOSA PROMOTER BINDING–LIKE (SPL) transcription factors, regulates the age-dependent developmental transitions (1316). As in A. thaliana, maize, and poplar, miR156 levels correlated with age in C. flexuosa (13, 14, 17, 18), being most abundant in seedlings (Fig. 2A). The miR156 target gene CfSPL9 showed an opposite expression pattern (Fig. 2B and figs. S6 and S7). miR156 level did not decline in 1-week-old seedlings during a 2-month cold treatment (Fig. 2C).

Fig. 2 miR156 contributes to age-dependent vernalization.

(A and B) Expression of miR156 (A) and CfSPL9 (B) during development. Wild-type plants of different ages (2 to 5 weeks old) were used for expression analyses. miR156 levels were monitored by small RNA blots. The level of U6 was monitored as the loading control. Error bars, SD. (C) Expression of miR156 during vernalization. One-week-old wild type grown in long days was vernalized for 2 months. The expression of miR156 was examined every 2 weeks during vernalization. (D to G) Vernalization response of transgenic plants. One-week-old wild-type (D), 1-week-old 35S::MIM156 (E), 5-week-old wild-type (F), and 5-week-old 35S::MIR156 (G) plants were vernalized for 2 months and returned to 23°C long days. Scale bars in (D) to (G), 5 cm. (H) Percentage of flowered plants after vernalization treatment. Plants of different ages (from 1 to 10 weeks old) were vernalized for 2 months and returned to 23°C long days. n = 20.

To determine whether miR156 contributes to the age-dependent vernalization response, we generated transgenic C. flexuosa plants that overexpressed either miR156 (35S::MIR156) or a target mimicry of miR156 (35S::MIM156), in which miR156 activity is attenuated (13) (figs. S8 and S9). Neither 35S::MIM156 nor 35S::MIR156 flowered without vernalization (fig. S8D). Wild-type, 35S::MIR156, and 35S::MIM156 plants were grown in long days for 1 week or 5 weeks and then exposed to 8 weeks of cold temperature. One-week-old wild-type plants were insensitive to cold treatment, whereas flowering was readily induced in 1-week-old 35S::MIM156 plants (Fig. 2, D, E, and H). The 35S::MIR156 plants became less insensitive to vernalization (Fig. 2, F to H), even when much older. At 10 weeks of age, only 20% of 35S::MIR156 plants responded to vernalization (Fig. 2H). These results illustrate a correlation between miR156 abundance and the sensitivity in response to vernalization.

To understand the molecular mechanism by which miR156 contributes to the process to make plants susceptible to vernalization, we examined the expression of CfFLC and CfSOC1. The response of CfFLC to cold treatment was independent of miR156 levels (fig. S10A). Upon cold treatment, CfSOC1 expression was elevated in 1-week-old 35S::MIM156 plants but not in wild type at the same age (Fig. 3, A and E). CfSOC1 transcripts in the shoot apical meristems were detected when 5-week-old wild type had been vernalized (Fig. 3, B and C). In 35S::MIR156 plants, CfSOC1 was underexpressed even when 8-week-old plants were vernalized (Fig. 3D). A. alpina TERMINAL FLOWER1 (AaTFL1) regulates the perennial flowering in A. alpina (19). Expression of CfTFL1, a homolog of AaTFL1 in C. flexuosa (fig. S11), was independent of age and miR156 levels (fig. S12).

Fig. 3 CfSOC1 acts downstream of vernalization and age pathways.

(A) Expression of CfSOC1 in the shoot apices. Wild type, 35S::MIM156, and 35S::MIR156 plants were grown in long days for 1 or 5 weeks. The plants were then vernalized for 2 months. **P < 0.001; Student's t test; error bars, SD. (B to E) Expression of CfSOC1 in the shoot apices of 5-week-old wild type before vernalization (B), 5-week-old wild type after vernalization (C), 8-week-old 35S::MIR156 after vernalization (D), and 1-week-old 35S::MIM156 after vernalization (E). Scale bars, 50 μm.

miR156 promotes the juvenile-to-adult transition through miR172, which targets APETALA2 (AP2)–like genes in A. thaliana (14, 20). The level of miR172 increased as C. flexuosa aged (Fig. 4A). miR172 accumulation was reduced in 35S::MIR156 and enhanced in 35S::MIM156 plants (fig. S9). Thus, as in A. thaliana, maize, rice, and poplar (14, 17, 21, 22), miR172 is regulated by miR156 in C. flexuosa.

Fig. 4 Integration of age and vernalization pathways in C. flexuosa.

(A) Expression of miR172 during development. Wild-type plants of different ages (from 2 to 4 weeks old) were used for real-time qPCR analyses. Error bars, SD. (B) Flowering time measurement. Plants were grown in long days. n = 12. (C) Vernalization response of wild-type and 35S::MIR172 plants (line 5). Plants of different ages (from 1 to 5 weeks old) were vernalized for 2 months and returned to 23°C long days. Wild type, n = 10. 35S::MIR172 (line 5), n = 12. (D) Schematic of CfSOC1 genomic region. Solid lines, promoter or introns. Black boxes, exons. The first four exons are shown. Three regions (a to c) were chosen for qPCR analyses. The black triangle stands for CArG boxes. (E) ChIP analyses of 35S::CfTOE1-3xHA and 35S::CfFLC-3xHA plants. Crude chromatin extracts were immunoprecipated with antibody against hemagglutinin. Purified ChIP and input DNAs were used for qPCR analyses. Error bars, SD.

To understand how miR172 regulates flowering in C. flexuosa, we generated the transgenic plants overexpressing miR172 (35S::MIR172). These 35S::MIR172 plants exhibited varying degrees of vernalization response in accordance with their miR172 levels (fig. S13). Wild-type plants did not fully respond to vernalization until they were 5 weeks old. However, 1-week-old transgenic plants with a moderate miR172 level (line 5) flowered when vernalized (Fig. 4, B and C). The transgenic plants with the highest miR172 level (line 4) flowered without exposure to cold temperature (Fig. 4B and fig. S14). Expression analyses indicated that miR172 level did not change in response to cold treatment and that CfFLC expression was not affected by miR172 overexpression (fig. S10, B and C). Epistasis analysis further showed that miR172 promotes flowering in parallel with CfFLC (Fig. 4B and fig. S14).

In A. thaliana, FLC and miR172-targeted AP2-like proteins repress flowering through SOC1 (2326). We identified a homolog of A. thaliana miR172 target TARGET OF EAT1 (TOE1), CfTOE1, in C. flexuosa (fig. S15) (27). The transgenic C. flexuosa plants that overexpressed in-frame fusions of CfFLC or CfTOE1 to triple hemagglutinin epitope tags (3xHA) (35S::CfFLC-3xHA or 35S::CfTOE1-3xHA) did not flower in long days after vernalization (fig. S16). Chromatin immunoprecipitation (ChIP) assays indicated that CfFLC and CfTOE1 bound to the promoter region of CfSOC1 (Fig. 4, D and E, and supplementary text), indicating that CfFLC and CfTOE1 regulate C. flexuosa flowering through repressing CfSOC1.

We suggest that age and vernalization cues coordinate to regulate floral induction in C. flexuosa by removal of two repressors, CfFLC, which is repressed by vernalization, and CfTOE1, which is down-regulated by the miR156-SPL-miR172 cascade (fig. S17). In the young seedling, high levels of miR156 lead to the accumulation of CfTOE1. CfTOE1 represses CfSOC1 expression regardless of vernalization; as the plant grows, the endogenous sugar content is elevated, resulting in a decreased level of miR156 and the concomitant increase in miR172 (2830). As a consequence, the occupation of CfTOE1 at CfSOC1 promoter was reduced, leading to a cold-sensitive state. Flowering can be successfully induced when CfFLC expression is reduced by vernalization.

The integration of age and vernalization pathways offers an advantage for the perennial growth habit by ensuring that plants do not flower until they develop axillary vegetative shoots and sufficient biomass. Although both vernalization and age pathways operate in the annual A. thaliana, this species does not have a pronounced age-dependent vernalization response. The relative stronger contribution of the age pathway in C. flexuosa suggests a scenario in which the species-specific imbalance of repressive versus inductive signals determines the life-cycle strategy of flowering plants.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S20

Table S1

References (3142)

References and Notes

  1. Acknowledgments: We thank I. R. Somoza and D. Weigel (MPI, Tübingen) for commenting on the manuscript, S. Bergonzi and G. Coupland (MPI, Kӧln) for exchanging unpublished results, and Y.-L. Guo (Institute of Botany, CAS, Beijing) for suggestion on phylogenetic analyses. The early phase of the work was conducted at the MPI for Developmental Biology, where it was supported by Max Planck Society funds to D. Weigel. The majority of this work was conducted at SIPPE and supported by grants from National Natural Science Foundation of China (31222029 and 912173023), State Key Basic Research Program of China (2013CB127000), Shanghai Pujiang Program (12PJ1409900), Recruitment Program of Global Experts (China), and NKLPMG (SIPPE, SIBS) to J.-W.W., and Open Project Program of NKLPMG (SIPPE, SIBS) to X.W.
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