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Epigenetics: The Flowers That Come In From The Cold
Current Biology, Vol. 12, R129–131, February 19, 2002, ©2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)00705-4
Epigenetics: The Flowers That Come In From The Cold Claudia Köhler* and Ueli Grossniklaus†
Plants can remember periods of cold several weeks later and respond by switching from vegetative to reproductive development. Recent findings shed light on this phenomenon by showing that a gene responsible for keeping this memory encodes a member of the Polycomb group of proteins.
In flowering plants, the transition from vegetative growth to flowering is controlled by both developmental and environmental signals. Using Arabidopsis thaliana as a model system, four major flowering promoting pathways have been defined genetically. The pathways that respond to photoperiod and ‘vernalization’ — a period of cold treatment that accelerates flowering — promote flowering by integrating environmental signals, whereas the autonomous and gibberellin pathways act largely independent of the environment, but depend on the developmental competence of the plant [1]. Many Arabidopsis ecotypes collected at high latitudes or alpine regions are winter annuals that flower in spring after exposure to winter conditions. In the laboratory, these ecotypes flower very late, but they flower much earlier when the seedlings are exposed to prolonged cold treatment. The requirement for vernalization ensures that flowering occurs in spring, providing the maximal opportunity for seed set. Vernalization often occurs at the seedling stage, with flowering occurring weeks later. The meristem thus has to remember this stimulus over several cycles of cell division, suggestive of an epigenetic process. It was hypothesized that DNA methylation is the epigenetic mark that enables the cell to recall previous cold conditions [2]. A recent study by Gendall et al. [3] has shed new light on the epigenetic basis of vernalization by showing that a Polycomb group protein is involved in the maintenance of the vernalization response. Epigenetic regulation of gene expression has been extensively studied in the fruitfly Drosophila. During embryogenesis in Drosophila, the expression of homeotic genes is established by segmentation genes, and the transcriptional repression that contributes to the patterning is maintained by the action of Polycomb group genes. Interestingly, plants seem to use the same protein machinery to stably maintain genes in a repressed state. But the targets of Polycomb group proteins in plants are MADS box genes, rather than homeobox genes as in Drosophila. This was first demonstrated for the Polycomb group protein CURLY Institute of Plant Biology, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland. E-mail: *[email protected]; †[email protected]
Dispatch
LEAF that regulates expression of the MADS box gene AGAMOUS [4]. Before the work of Gendall et al. [3] it had been established that FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) play a central role in the vernalization response [5,6]. FRI encodes a novel protein that increases the mRNA level of the MADS box gene FLC [7–9]. FLC acts as a strong floral repressor by negatively regulating the expression of genes that promote the floral transition. Vernalization promotes flowering by downregulating expression of FLC [8,9]. The duration of cold treatment correlates with the extent to which FLC mRNA is reduced and flowering is promoted. FLC expression is also downregulated by genes involved in the autonomous floral promotion pathway, including FCA, LUMINIDEPENDENS, FVE and FPA. Mutations in these genes result in upregulated FLC mRNA levels and a late flowering phenotype that can be reversed by vernalization, indicating the central role of FLC in floral transition [8,9]. Several vernalization (vrn) mutants have been isolated which are unable to reduce the FLC mRNA in response to cold temperature, indicating that they carry defects in regulators of FLC expression [10]. Gendall et al. [3] have now shown that vrn2-1 carries a mutation in a nuclear-localized zinc finger protein which is similar to the Drosophila Polycomb group protein Suppressor of zeste 12 [11] and two other proteins from Arabidopsis, FERTILIZATION-INDEPENDENT SEED 2 (FIS2) [12] and EMBRYONIC FLOWER 2 (EMF2) [13]. The vrn2-1 mutant was isolated in a genetic screen for mutations that cause an inability to respond to cold treatment [10] when combined with the fca-1 mutation, which alone causes a late-flowering but vernalizationresponsive phenotype. FCA is an RNA-binding protein which is required for downregulation of FLC expression [14]. The fca-1 vrn2-1 double mutant exhibits a clear reduction in its vernalization response that correlates with increased FLC mRNA levels [15]. Furthermore, the double mutant plant flowers later than the fca-1 single mutant, indicating that VRN2 promotes flowering in an fca-1 background. Similar observations were made with vrn2-1 in other mutant backgrounds causing FLC upregulation: fve-1 and a dominant FRI mutation. In a wild-type background, however, vrn2-1 causes no reduction in flowering time. VRN2 function is therefore only revealed in the presence of mutations leading to an upregulation of FLC expression [3]. This prompted Gendall et al. [3] to examine in detail the FLC mRNA profile in the vrn21 fca-1 double mutant after vernalization treatment during development. As expected, in fca-1 mutants, the FLC mRNA level decreased after the transfer of vernalized seedlings to normal growth conditions, and remained stably repressed during development. Similarly, immediately after shifting the fca-1 vrn2-1 double mutant from cold to normal conditions a
Dispatch R130
Without vernalization
VRN2 Promoter
Intron1
FLC on Floral transition genes
After vernalization
Figure 1. A model for the action of the Polycomb group protein VRN2 in vernalization, based on the results of Gendall et al. [3]. vrn2 mutants show an increased DNAse sensitivity of FLC after vernalization suggesting that VRN2 changes the structure at the FLC locus by recruiting a protein complex with chromatin remodeling activity. This could establish or maintain the epigenetic mark, which enables the plant to remember periods of cold temperature for several weeks. (Pictures courtesy of Caroline Dean.)
FLC off Promoter Intron1
Floral transition genes
VRN2 Current Biology
decrease in FLC mRNA level was observed. But this low FLC mRNA level is not maintained during development: FLC levels were seen to increase several days after the transfer from vernalization. These data indicate that VRN2 itself is required, not for the initial vernalization-induced decrease of FLC mRNA, but rather for the stable maintenance of FLC repression. This indicates that VRN2 behaves in a functionally similar way to Polycomb group proteins of Drosophila, and thus contributes to the epigenetic basis of vernalization. Polycomb group proteins most likely exert their function by remodeling the chromatin structure of their target genes [16]. Consequently, Gendall et al. [3] tested whether the FLC gene has a different DNAse sensitivity in fca-1 and vrn2-1 fca-1 seedlings after vernalization. They found that, indeed, the vrn2-1 mutation causes a higher DNAse sensitivity within the first intron of FLC after vernalization. VRN2 is a member of a small gene family in Arabidopsis. One of the homologs, FIS2, was identified in mutant screens that also identified the Polycomb group genes MEDEA (MEA) and FERTILISATION INDEPENDENT ENDOSPERM (FIE) [12,17,18]. These are homologs, respectively, of the Drosophila genes enhancer of zeste and extra sex combs. Although FIS2 has not been shown to directly interact with MEA and FIE, they most likely act together to regulate gene expression. Therefore, it will be interesting to study whether VRN2 is present in a protein complex with proteins homologous to MEA and FIE. As VRN2 expression was not altered by vernalization, VRN2 function may be regulation by recruiting a constitutively present VRN2 complex to target genes, such as FLC. The work of Gendall et al. [3] gives us an insight into the epigenetic basis of vernalization in plants. The fact that vernalization is regulated by a Polycomb group protein indicates that similar mechanisms for remembering transcriptional states are used in animals and plants. This work sets the stage for investigating new interesting questions — for example, what gives rise
to the initial downregulation of FLC, and is there a connection between VRN2 and DNA methylation? — and points the direction for further research. References 1. Simpson, G.G., Gendall, A.R. and Dean, C. (1999). When to switch to flowering. Annu. Rev. Cell. Dev. Biol. 15, 519–550. 2. Sheldon, C.C., Finnegan, E.J., Rouse, D.T., Tadege, M., Bagnall, D.J., Helliwell, C.A., Peacock, W.J. and Dennis, E.S. (2000). The control of flowering by vernalization. Curr. Opin. Plant. Biol. 3, 418–422. 3. Gendall, A.R., Levy, Y.Y., Wilson, A. and Dean, C. (2001). The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis. Cell. 107, 525–535. 4. Goodrich, J., Puangsomlee, P., Martin, M., Long, D., Meyerowitz, E.M. and Coupland, G. (1997). A Polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386, 44–51. 5. Koornneef, M., Blankestijn-deVries, H., Hanhart, C., Soppe, W. and Peeters, T. (1994). The phenotype of some late-flowering mutants is enhanced by a locus on chromosome 5 that is not effective in the Landsberg erecta wild-type. Plant J. 6, 911–919. 6. Lee, I., Michaels, S.D., Masshardt, A.S. and Amasino R.M. (1994). The late-flowering phenotype of FRIGIDA and mutations in LUMINIDEPENDENS is suppressed in the Landsberg erecta strain of Arabidopsis. Plant J. 6, 903–909. 7. Johanson, U., West, J., Lister, C., Michaels, S., Amasino, R. and Dean, C. (2000). Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time. Science 290, 344–347. 8. Michaels, S.D. and Amasino, R.M. (1999). FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11, 949–956. 9. Sheldon, C.C., Burn, J.E., Perez, P.P., Metzger, J., Edwards, J.A., Peacock, W.J. and Dennis, E.S. (1999). The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11, 445–458. 10. Chandler, J., Wilson, A. and Dean, C. (1996). Arabidopsis mutants showing an altered response to vernalization. Plant J. 10, 637–644. 11. Birve, A., Sengupta, A.K., Beuchle, D., Larsson, J., Kennison, J.A., Rasmuson-Lestander A, A. and Muller, J. (2001). Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants. Development 17, 3371–3379. 12. Luo, M., Bilodeau, P., Koltunow, A., Dennis, E.S., Peacock, W.J. and Chaudhury, A.M. (1999). Genes controlling fertilization-independent seed development in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 96, 296–301. 13. Yoshida, N., Yanai, Y., Chen, L., Kato, Y., Hiratsuka, J., Miwa, T., Sung, Z.R and Takahashi, S. (2001). EMBRYONIC FLOWER2, a novel Polycomb group protein homolog, mediates shoot development and flowering in Arabidopsis. Plant Cell 13, 2471–2481.
Current Biology R131
14.
15.
16. 17.
18.
Macknight, R., Bancroft, I., Page T., Lister, C., Schmidt, R., Love, K., Westphal, L., Murphy, G., Sherson, S., Cobbett, C. and Dean, C. (1997). FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains. Cell 89, 737–745. Sheldon, C.C., Rouse, D.T., Finnegan, E.J., Peacock, W.J. and Dennis, E.S. (2000). The molecular basis of vernalization: The central role of FLOWERING LOCUS C (FLC). Proc. Natl. Acad. Sci. USA 97, 3753–3758. Brock, H.W. and van Lohuizen, M. (2001). The Polycomb group-no longer an exclusive club? Curr. Opin. Genet. Dev. 11, 175–181. Grossniklaus, U., Vielle-Calzada, J.P., Hoeppner, M.A. and Gagliano, W.B. (1998). Maternal control of embryogenesis by MEDEA, a Polycomb group gene in Arabidopsis. Science 280, 446–450. Ohad, N., Margossian, L., Hsu, Y.-C., Williams, C., Repetti, P. and Fischer, R.L. (1996). A mutation that allows endosperm development without fertilization. Proc. Natl. Acad. Sci. USA 93, 5319–5324.
Epigenetics: The Flowers That Come In From The Cold Claudia Köhler* and Ueli Grossniklaus†
Plants can remember periods of cold several weeks later and respond by switching from vegetative to reproductive development. Recent findings shed light on this phenomenon by showing that a gene responsible for keeping this memory encodes a member of the Polycomb group of proteins.
In flowering plants, the transition from vegetative growth to flowering is controlled by both developmental and environmental signals. Using Arabidopsis thaliana as a model system, four major flowering promoting pathways have been defined genetically. The pathways that respond to photoperiod and ‘vernalization’ — a period of cold treatment that accelerates flowering — promote flowering by integrating environmental signals, whereas the autonomous and gibberellin pathways act largely independent of the environment, but depend on the developmental competence of the plant [1]. Many Arabidopsis ecotypes collected at high latitudes or alpine regions are winter annuals that flower in spring after exposure to winter conditions. In the laboratory, these ecotypes flower very late, but they flower much earlier when the seedlings are exposed to prolonged cold treatment. The requirement for vernalization ensures that flowering occurs in spring, providing the maximal opportunity for seed set. Vernalization often occurs at the seedling stage, with flowering occurring weeks later. The meristem thus has to remember this stimulus over several cycles of cell division, suggestive of an epigenetic process. It was hypothesized that DNA methylation is the epigenetic mark that enables the cell to recall previous cold conditions [2]. A recent study by Gendall et al. [3] has shed new light on the epigenetic basis of vernalization by showing that a Polycomb group protein is involved in the maintenance of the vernalization response. Epigenetic regulation of gene expression has been extensively studied in the fruitfly Drosophila. During embryogenesis in Drosophila, the expression of homeotic genes is established by segmentation genes, and the transcriptional repression that contributes to the patterning is maintained by the action of Polycomb group genes. Interestingly, plants seem to use the same protein machinery to stably maintain genes in a repressed state. But the targets of Polycomb group proteins in plants are MADS box genes, rather than homeobox genes as in Drosophila. This was first demonstrated for the Polycomb group protein CURLY Institute of Plant Biology, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland. E-mail: *[email protected]; †[email protected]
Dispatch
LEAF that regulates expression of the MADS box gene AGAMOUS [4]. Before the work of Gendall et al. [3] it had been established that FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) play a central role in the vernalization response [5,6]. FRI encodes a novel protein that increases the mRNA level of the MADS box gene FLC [7–9]. FLC acts as a strong floral repressor by negatively regulating the expression of genes that promote the floral transition. Vernalization promotes flowering by downregulating expression of FLC [8,9]. The duration of cold treatment correlates with the extent to which FLC mRNA is reduced and flowering is promoted. FLC expression is also downregulated by genes involved in the autonomous floral promotion pathway, including FCA, LUMINIDEPENDENS, FVE and FPA. Mutations in these genes result in upregulated FLC mRNA levels and a late flowering phenotype that can be reversed by vernalization, indicating the central role of FLC in floral transition [8,9]. Several vernalization (vrn) mutants have been isolated which are unable to reduce the FLC mRNA in response to cold temperature, indicating that they carry defects in regulators of FLC expression [10]. Gendall et al. [3] have now shown that vrn2-1 carries a mutation in a nuclear-localized zinc finger protein which is similar to the Drosophila Polycomb group protein Suppressor of zeste 12 [11] and two other proteins from Arabidopsis, FERTILIZATION-INDEPENDENT SEED 2 (FIS2) [12] and EMBRYONIC FLOWER 2 (EMF2) [13]. The vrn2-1 mutant was isolated in a genetic screen for mutations that cause an inability to respond to cold treatment [10] when combined with the fca-1 mutation, which alone causes a late-flowering but vernalizationresponsive phenotype. FCA is an RNA-binding protein which is required for downregulation of FLC expression [14]. The fca-1 vrn2-1 double mutant exhibits a clear reduction in its vernalization response that correlates with increased FLC mRNA levels [15]. Furthermore, the double mutant plant flowers later than the fca-1 single mutant, indicating that VRN2 promotes flowering in an fca-1 background. Similar observations were made with vrn2-1 in other mutant backgrounds causing FLC upregulation: fve-1 and a dominant FRI mutation. In a wild-type background, however, vrn2-1 causes no reduction in flowering time. VRN2 function is therefore only revealed in the presence of mutations leading to an upregulation of FLC expression [3]. This prompted Gendall et al. [3] to examine in detail the FLC mRNA profile in the vrn21 fca-1 double mutant after vernalization treatment during development. As expected, in fca-1 mutants, the FLC mRNA level decreased after the transfer of vernalized seedlings to normal growth conditions, and remained stably repressed during development. Similarly, immediately after shifting the fca-1 vrn2-1 double mutant from cold to normal conditions a
Dispatch R130
Without vernalization
VRN2 Promoter
Intron1
FLC on Floral transition genes
After vernalization
Figure 1. A model for the action of the Polycomb group protein VRN2 in vernalization, based on the results of Gendall et al. [3]. vrn2 mutants show an increased DNAse sensitivity of FLC after vernalization suggesting that VRN2 changes the structure at the FLC locus by recruiting a protein complex with chromatin remodeling activity. This could establish or maintain the epigenetic mark, which enables the plant to remember periods of cold temperature for several weeks. (Pictures courtesy of Caroline Dean.)
FLC off Promoter Intron1
Floral transition genes
VRN2 Current Biology
decrease in FLC mRNA level was observed. But this low FLC mRNA level is not maintained during development: FLC levels were seen to increase several days after the transfer from vernalization. These data indicate that VRN2 itself is required, not for the initial vernalization-induced decrease of FLC mRNA, but rather for the stable maintenance of FLC repression. This indicates that VRN2 behaves in a functionally similar way to Polycomb group proteins of Drosophila, and thus contributes to the epigenetic basis of vernalization. Polycomb group proteins most likely exert their function by remodeling the chromatin structure of their target genes [16]. Consequently, Gendall et al. [3] tested whether the FLC gene has a different DNAse sensitivity in fca-1 and vrn2-1 fca-1 seedlings after vernalization. They found that, indeed, the vrn2-1 mutation causes a higher DNAse sensitivity within the first intron of FLC after vernalization. VRN2 is a member of a small gene family in Arabidopsis. One of the homologs, FIS2, was identified in mutant screens that also identified the Polycomb group genes MEDEA (MEA) and FERTILISATION INDEPENDENT ENDOSPERM (FIE) [12,17,18]. These are homologs, respectively, of the Drosophila genes enhancer of zeste and extra sex combs. Although FIS2 has not been shown to directly interact with MEA and FIE, they most likely act together to regulate gene expression. Therefore, it will be interesting to study whether VRN2 is present in a protein complex with proteins homologous to MEA and FIE. As VRN2 expression was not altered by vernalization, VRN2 function may be regulation by recruiting a constitutively present VRN2 complex to target genes, such as FLC. The work of Gendall et al. [3] gives us an insight into the epigenetic basis of vernalization in plants. The fact that vernalization is regulated by a Polycomb group protein indicates that similar mechanisms for remembering transcriptional states are used in animals and plants. This work sets the stage for investigating new interesting questions — for example, what gives rise
to the initial downregulation of FLC, and is there a connection between VRN2 and DNA methylation? — and points the direction for further research. References 1. Simpson, G.G., Gendall, A.R. and Dean, C. (1999). When to switch to flowering. Annu. Rev. Cell. Dev. Biol. 15, 519–550. 2. Sheldon, C.C., Finnegan, E.J., Rouse, D.T., Tadege, M., Bagnall, D.J., Helliwell, C.A., Peacock, W.J. and Dennis, E.S. (2000). The control of flowering by vernalization. Curr. Opin. Plant. Biol. 3, 418–422. 3. Gendall, A.R., Levy, Y.Y., Wilson, A. and Dean, C. (2001). The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis. Cell. 107, 525–535. 4. Goodrich, J., Puangsomlee, P., Martin, M., Long, D., Meyerowitz, E.M. and Coupland, G. (1997). A Polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386, 44–51. 5. Koornneef, M., Blankestijn-deVries, H., Hanhart, C., Soppe, W. and Peeters, T. (1994). The phenotype of some late-flowering mutants is enhanced by a locus on chromosome 5 that is not effective in the Landsberg erecta wild-type. Plant J. 6, 911–919. 6. Lee, I., Michaels, S.D., Masshardt, A.S. and Amasino R.M. (1994). The late-flowering phenotype of FRIGIDA and mutations in LUMINIDEPENDENS is suppressed in the Landsberg erecta strain of Arabidopsis. Plant J. 6, 903–909. 7. Johanson, U., West, J., Lister, C., Michaels, S., Amasino, R. and Dean, C. (2000). Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time. Science 290, 344–347. 8. Michaels, S.D. and Amasino, R.M. (1999). FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11, 949–956. 9. Sheldon, C.C., Burn, J.E., Perez, P.P., Metzger, J., Edwards, J.A., Peacock, W.J. and Dennis, E.S. (1999). The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11, 445–458. 10. Chandler, J., Wilson, A. and Dean, C. (1996). Arabidopsis mutants showing an altered response to vernalization. Plant J. 10, 637–644. 11. Birve, A., Sengupta, A.K., Beuchle, D., Larsson, J., Kennison, J.A., Rasmuson-Lestander A, A. and Muller, J. (2001). Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants. Development 17, 3371–3379. 12. Luo, M., Bilodeau, P., Koltunow, A., Dennis, E.S., Peacock, W.J. and Chaudhury, A.M. (1999). Genes controlling fertilization-independent seed development in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 96, 296–301. 13. Yoshida, N., Yanai, Y., Chen, L., Kato, Y., Hiratsuka, J., Miwa, T., Sung, Z.R and Takahashi, S. (2001). EMBRYONIC FLOWER2, a novel Polycomb group protein homolog, mediates shoot development and flowering in Arabidopsis. Plant Cell 13, 2471–2481.
Current Biology R131
14.
15.
16. 17.
18.
Macknight, R., Bancroft, I., Page T., Lister, C., Schmidt, R., Love, K., Westphal, L., Murphy, G., Sherson, S., Cobbett, C. and Dean, C. (1997). FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains. Cell 89, 737–745. Sheldon, C.C., Rouse, D.T., Finnegan, E.J., Peacock, W.J. and Dennis, E.S. (2000). The molecular basis of vernalization: The central role of FLOWERING LOCUS C (FLC). Proc. Natl. Acad. Sci. USA 97, 3753–3758. Brock, H.W. and van Lohuizen, M. (2001). The Polycomb group-no longer an exclusive club? Curr. Opin. Genet. Dev. 11, 175–181. Grossniklaus, U., Vielle-Calzada, J.P., Hoeppner, M.A. and Gagliano, W.B. (1998). Maternal control of embryogenesis by MEDEA, a Polycomb group gene in Arabidopsis. Science 280, 446–450. Ohad, N., Margossian, L., Hsu, Y.-C., Williams, C., Repetti, P. and Fischer, R.L. (1996). A mutation that allows endosperm development without fertilization. Proc. Natl. Acad. Sci. USA 93, 5319–5324.