Available online at www.sciencedirect.com
ScienceDirect Flowering responses to seasonal cues: what’s new? Maida Romera-Branchat, Fernando Andre´s and George Coupland Seasonal cues of day length or winter cold trigger flowering of many species. Forward and reverse genetic approaches are revealing the mechanisms by which these responses are conferred. Homologues of the Arabidopsis thaliana protein FLOWERING LOCUS T (FT) are widely used to mediate seasonal responses to day length and act as grafttransmissible promoters or repressors of flowering. Winter cold in A. thaliana promotes flowering by repressing transcription of the MADS box gene FLOWERING LOCUS C (FLC). The mechanism by which this occurs involves a complex interplay of different forms of long noncoding RNAs induced at the FLC locus during cold and changes in the chromatin of FLC. In perennial relatives of A. thaliana, flowering also requires the age-dependent downregulation of miRNA156 before winter. Addresses Max Planck Institute for Plant Breeding Research, D-50829 Cologne, Germany Corresponding author: Coupland, George ([email protected])
Current Opinion in Plant Biology 2014, 21:120–127 This review comes from a themed issue on Cell signalling and gene regulation 2014 Edited by Xiangdong Fu and Junko Kyozuka
http://dx.doi.org/10.1016/j.pbi.2014.07.006 1369-5266/Published by Elsevier Ltd.
Seasonal responses in the regulation of flowering Flowering is precisely controlled in many species by seasonal cues of day length (photoperiod) and winter temperatures (vernalization). These responses often exhibit quantitative variation among individuals of a single species, ensuring that flowering occurs at the optimal time to maximize seed production in specific environments. Such natural genetic variation supplemented with induced mutations in model species has allowed isolation of genes controlling these complex responses. Such studies have provided regulatory frameworks for photoperiod and vernalization responses and suggested the underlying regulatory logic. They have also identified conserved mechanisms between distantly related species such as Arabidopsis thaliana and cereal Current Opinion in Plant Biology 2014, 21:120–127
crops, however perhaps as expected for such fast-evolving adaptive traits important differences are found even between closely related species. The inherent fascination of these seasonal patterns as well as their importance in adaptation to local environments and to yield in agriculture have led to an extensive literature including several recent comprehensive reviews [1–5]. Here we focus on highlights of the recent literature mainly from the last two years that have deepened our understanding of photoperiodic and vernalization responses and the open questions that these publications pose.
Photoperiodic response: transcriptional regulation of FT homologues The canonical photoperiodic flowering pathway of A. thaliana promotes early flowering under long days (LDs) of spring and early summer, but not under short days (SDs) of winter [1,4]. Two genes, FLOWERING LOCUS T and TWIN SISTER OF FT are increased in transcription in the phloem companion cells under LDs. These proteins, related in sequence to phosphatidylethanolamine binding proteins (PEBPs) of animals, move to the apex where they induce flowering. Transcriptional activation of FT, which is expressed at higher levels than TSF, in response to LDs is the limiting step in photoperiodic induction of A. thaliana, and similar data have been obtained for FT homologues in many other species. In A. thaliana this activation of FT occurs through the CONSTANS (CO) transcription factor, which accumulates specifically under LDs and promotes FT and TSF transcription. The CO protein contains B-box zinc fingers at the amino terminus and a CO COL TOC1 (CCT) domain at the C-terminus. The CCT domain has sequence similarities to the NUCLEAR FACTOR-YA (NF-YA) protein and acts as a DNA binding domain [6– 8]. CO binds to the proximal region of the FT promoter recognizing two CO Responsive Elements (CORE) that are required for FT activation [7,9]. Deletion analysis of the FT promoter showed that a distal region is also required for CO response [9]. Recently this region was shown to be recognized by NF-Y that binds to a CCAAT element around 5.3 kb upstream of the transcriptional start site [10]. Previous genetic analysis showed that certain NF-Y subunit paralogues are required for CO to activate FT transcription [11], and mutational analysis demonstrated that this CCAAT box is required for COmediated activation of FT [10]. Looping of the FT promoter has now been detected between the distal site to which NF-Y binds and the proximal site at which CO binds [10], and since CO and NF-Y were previously www.sciencedirect.com
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shown to interact [6,12], this looping is proposed to be caused by interactions between CO and NF-Y bound to DNA [10] (Figure 1). As both complexes and their binding sites are required for FT activation, and loops are only detected at the times of day at which CO and FT are expressed, this looping may be required for the photoperiodic dependent activation of FT transcription. These data help clarify the structure of the FT promoter and the mechanism of its transcriptional activation in A. thaliana in response to photoperiod. The significance and basis of promoter looping must now be tested using precise mutations in FT promoter motifs.
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photoperiodic flowering is strongly conserved. However, typically these contain at their N-terminus a pseudoresponse regulator receiver domain rather than the Bboxes found in CO [13,15–17]. These PSEUDO RESPONSE REGULATOR (PRR) proteins are members of small gene families and encode components of the circadian clock in A. thaliana, but members of the family have been shown to have specific roles in FT activation and flowering in several other species. Most recently, the BOLTING gene from sugar beet was added to this list [16]. This species includes annual accessions that flower rapidly without a requirement for winter cold, and others that are biennial and flower only if vernalized. These varieties differ at the BOLTING (B) locus, so that dominant alleles at B confer the early flowering annual growth habit, whereas biennials carry the recessive allele. Isolation of this gene showed that it encodes a PRR protein
Genetic analysis in a range of species using naturally occurring alleles identified proteins containing CCT domains that are important in FT photoperiodic regulation [13–17], suggesting that the role of these proteins in Figure 1
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Features of FT transcriptional activation and protein structure. (a) Model of recruitment of CO to the CO Response Elements (CORE, labeled C1 and C2) (adapted from [10]). NF-Y complexes bind to two CCAAT sequences located at 2 and 5.5 kb away from the FT transcription start site (TSS). A chromatin loop, whose formation is favored at the end of a long day, maintains the NF-Y complex in close proximity to the CORE sequences (at 220 and 161 bp away from the TSS). This allows NF-Y complex to interact with and stabilize CO when it binds to the CORE sequences and thereby to activate FT transcription. (b) Mutations introduced in certain amino acids (colored blue in the sequence) convert FT into a TFL1-like floral repressor. Some of these changes are predicted to change also the protein surface charge (blue with orange boxes). Mutations in Asp-71 (in yellow), which is embedded in the binding pocket, do not result in activity changes. Residues in Segment B also differ in sugar beet BvFT2 and cause the protein to act as a repressor of flowering. www.sciencedirect.com
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called BOLTING TIME CONTROL 1 (BvBTC1), and that in biennial cultivated sugar beet the gene carries a 28 kb insertion in the promoter region, that is absent in annuals [16]. This insertion is proposed to reduce BvBTC1 expression and delay flowering. In biennials vernalization increases BvBTC1 expression leading to earlier flowering. BvBTC1 activates expression of the BvFT2 gene-triggering flowering. This is a further striking example of allelic variation at a CCT domain influencing seasonal control of flowering time through influencing FT expression and contributing to domestication of crops. Seasonal responses to photoperiod do not only affect flowering but other developmental programs such as bud break in trees or tuberization in potato. Both of these processes have been shown to be regulated through transcriptional regulation of FT-like genes [18,19]. The regulation of potato tuberization appears to be closely related to flowering of A. thaliana, involving CO-like and FT-like genes [18,20]. Recently this similarity was extended by showing that CYCLING DOF (CDF) transcription factors that repress CO transcription in A. thaliana [21] are also important in tuberization [22]. A potato allele of one of the genes (StCDF1) encoding CDF proteins was found to colocalize with a maturity locus that allows tubers to be formed under LDs, and was therefore important in the spread of potato as a cultivated crop [22]. This allele of StCDF1 carries a deletion that stabilizes the protein throughout the day allowing it to repress CO homologues leading to tuberization under LDs. This is an interesting example of common regulatory mechanisms being used in different plant families within the photoperiodic pathway at higher levels in the hierarchy than CCT domain and FT-like proteins. As more such studies are performed it will be interesting to determine whether this is an example of a conserved mechanism or of convergent evolution of the utilization of CDF transcription factors which might have been recruited to this process independently in the Brassicaceae and Solanaceae families.
Photoperiodic responses: function of FT proteins FT proteins appear to be universal regulators of flowering in angiosperms and are transported from the leaves to the apex to induce flowering [1,5,23]. They form protein complexes with bZIP transcription factors to regulate transcription of target genes. In A. thaliana their effect is mediated by two closely related bZIPs, FD and FD PARALOGUE (FDP) [24–26]. Biochemical analyses in vitro as well as crystallization studies using rice proteins indicated that this interaction between FD and FT is indirect and is mediated by a 14-3-3 protein [27]. Recently the activity of FT proteins has been expanded to include repressors of flowering that mediate environmental responses to photoperiod and vernalization. Current Opinion in Plant Biology 2014, 21:120–127
In sugar beet two FT-like genes, BvFT1 and BvFT2, were isolated [28]. BvFT2 is transcriptionally activated by BvBTC1 and is essential for flowering [16,28]. Reduction of BvFT2 expression by RNA interference prevents flowering whereas overexpression of BvFT2 strongly accelerates flowering both in transgenic sugar beet and transgenic A. thaliana. BvFT2 therefore behaves as a promoter of flowering, as expected from analysis of FT-like genes from many other species. Surprisingly, BvFT1 had the opposite effect. BvFT1 was repressed by BvBTC1, while its overexpression in transgenic sugar beet delayed flowering and reduced expression of the BvFT2 mRNA [28]. Similarly its overexpression in transgenic A. thaliana delayed flowering. Therefore BvFT1 is a repressor of flowering acting partly through transcriptional repression of BvFT2, although it likely has additional roles in the repression of bolting and flowering [28]. Other members of the plant family of proteins related to PEBPs were previously described to act as repressors of flowering, particularly TERMINAL FLOWER 1 (TFL1) of A. thaliana and its homologues. However FT and TFL1 differ at well characterized residues (Figure 1). For example, Tyr85 and Gln140 are indicative of FT activity, while His88 and Asp144 are diagnostic of TFL1 [29,30–32]. At these positions, BvFT1 is recognizably an FT-like protein, despite acting as a repressor of flowering [28]. Elegant domain swapping experiments showed that 2 amino acid changes in the fourth exon, which encodes an external loop in the crystal structure of FT, are responsible for the change in activity between BvFT1 and BvFT2 [28,30]. The BvFT1 protein appears to have evolved recently to act as a repressor of flowering that contributes to the vernalization response. The importance of antagonistic interactions between proteins in this family as part of the flowering response to environment also recently emerged in Chrysanthemum [33]. Here the authors characterized two proteins expressed in leaves that contribute to the photoperiodic response. The first of these proteins is encoded by Chrysanthemum seticuspe FT-like 3 (CsFTL3), which is transcriptionally induced during SDs that trigger flowering in this species [34], its overexpression drives early flowering and an inductive signal produced in the over-expressing plants is graft transmissible [33]. Therefore CsFTL3 behaves as a classical FT-floral promoting gene and its protein product contains the signature amino acids characteristic of FT among other members of the family. By contrast, C. seticuspe ANTI-FLORIGENIC FT/TFL1 FAMILY PROTEIN (CsAFT) is expressed under noninductive LDs, causes late flowering when constitutively expressed and can delay flowering when grafted roots of overexpressing plants are grafted onto wild-type shoots [33]. CsAFT contains the characteristic His88 and Asp140 residues of TFL1. Therefore CsAFT is a graft transmissible TFL1-like repressor of flowering that antagonizes the activity of FTL3 in the photoperiodic induction of flowering [33]. www.sciencedirect.com
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As mentioned above, plant PEBP-like proteins act by interacting with bZIP transcription factors to trigger transcriptional reprogramming of the shoot meristem. However, other binding partners of FT were described recently, suggesting that the protein has additional functions. FT was reported to interact with transcription factors of the TEOSINTE BRANCHED, CYCLOIDEA, PCF (TCP) family [29,35,36]. One of these BRANCHED 1 (BRC1) is expressed in axillary meristems and is proposed to act as a repressor of axillary meristem differentiation [37]. BRC1 also delays flowering of axillary branches as these flower early in brc1 mutants [36]. FT protein fusions to fluorescent proteins were shown to move into the axillary bud after expression in the leaf blade, suggesting that they could interact with BRC1 in wild-type plants [36]. These and further genetic data support the idea that BRC1 acts to repress flowering in axillary meristems by interacting with FT. The mechanism of interaction would be different than with FD, as BRC1 does not require 14-3-3 proteins to mediate the interaction. In addition to proteins, FT was found to bind to lipids in vitro [38]. Animal PEBPs contain a binding pocket identified in their crystal structure in which ligands such as the lipid phosphatidylethanolamine (PE) are presumed to bind [39]. Crystallization of the plant proteins demonstrated that they also contain this pocket [32,40]. However, neither FT nor TFL1 were shown to bind lipids and the functional significance of the pocket in the plant proteins is unclear. Furthermore, mutation of the FT protein at Asp71, which is located deep in the pocket, had no effect on protein activity in vivo [29]. Nevertheless, Tyr85 is located at the entrance of the pocket, and this residue is not only invariant in FT proteins but conversion of the analogous residue in TFL1 from His88 to Tyr is sufficient to switch TFL1 from a repressor to an activator of flowering [31]. These observations have now been extended by showing that FT binds the lipid phosphatidylcholine (PC) in vitro, but that in similar assays binding to the related lipid PE could not be detected [38]. In transgenic plants PC levels were elevated by increasing the ratio of PC:PE, and this was associated with early flowering that at least partially required active FT and TSF genes [38]. PC is a lipid found at high levels in all cellular membranes, and how this might influence FT activity at the biochemical level remains unclear. However, these data demonstrate for the first time that plant PEBP-like proteins such as FT have the capacity to bind lipids and that this might contribute to their activity in vivo.
Vernalization response: age-related vernalization response In addition to day length, vernalization is another cue that strongly affects seasonal flowering patterns. Genetic www.sciencedirect.com
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analysis of vernalization response in different plant families identified distinct regulatory pathways suggesting that it has evolved independently in each case [1]. In the Brassicaceae the MADS box transcription factor FLOWERING LOCUS C is the central node in the vernalization pathway [41]. FLC represses flowering until the plant is exposed to cold for several weeks. Under these conditions FLC transcription is progressively repressed. After return to warm temperatures, FLC transcript remains at low levels and the plant proceeds to flowering [41]. This progressive repression of FLC transcription is an epigenetic phenomenon that requires accumulation of trimethylation of lysine 27 on histone 3 (H3K27me3) [42–44]. Recently the complexity of FLC regulation increased further by the identification of long noncoding RNAs (lncRNAs) expressed at the FLC locus [45]. An antisense RNA, called COOLAIR, is expressed from a promoter at the 30 end of FLC [46], whereas a sense RNA, COLDAIR, is expressed in the first intron [47] (Figure 2). Both of these lncRNAs are expressed at higher levels during vernalization (Figure 2). COLDAIR is not polyadenylated, but is bound directly by components of the enzymic machinery required to deposit the H3K27me3 mark on the FLC gene and is proposed therefore to recruit these to the FLC gene contributing to its repression [47]. By contrast, COOLAIR exists in several forms due to use of different polyadenylation sites [46,48]. One form is polyadenylated at a proximal site (close to the transcriptional start site of COOLAIR) but other forms are spliced and polyadenylated at distal sites close to the transcriptional start site of FLC. Those polyadenylated at distal positions close to the FLC transcriptional start site might be related to FLC activation. For example, mutations in trans-acting proteins such as FPA that reduce the efficiency of detection of the proximal polyadenylation site and cause more antisense transcription around the FLC promoter are also associated with increased expression of FLC sense transcript and late flowering [48,49,50]. Such conclusions were strengthened recently by identifying mutations that impair the spliceosome leading to alterations in COOLAIR splicing and by mutating particular splice sites in COOLAIR [51]. Altering the abundance of specific forms of COOLAIR in this way also influenced FLC transcript levels [51]. Therefore, altering levels of specific COOLAIR forms appears to influence FLC mRNA levels. However, how these observations relate to the increased transcription of COOLAIR during vernalization, the ratios of specific forms that are present in cold and their relationship to FLC repression during vernalization remains to be described in detail. The recent demonstration that COOLAIR structure and expression patterns are conserved in distantly related Brassicaceae species further suggests a function in vernalization [52], although mutations that impair COOLAIR expression in A. thaliana did not have a dramatic effect on FLC repression by vernalization [53]. The age at which plants can respond to vernalization and induce flowering is highly variable. A. thaliana flowers in Current Opinion in Plant Biology 2014, 21:120–127
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Figure 2
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Noncoding RNAs encoded at the FLC locus. (a) Diagram of the different forms of transcripts expressed at the FLC locus in Arabidopsis thaliana. Exons in FLC mRNA are shown as rectangles in gray. Red asterisk (left) is transcription start site of FLC sense transcript. The Poly(A) site on FLC locus is also shown. Blue asterisk (right) is transcription start of antisense RNA transcripts known as COOLAIR (in blue). The transcription of COOLAIR increases as plant is exposed to cold temperatures and appears in different spliced isoforms [46,48,49]. Different components of the autonomous pathway and splicing patterns have been recently shown to regulate the abundance of these COOLAIR RNA transcripts [49,50,51]. A sense RNA called COLDAIR expressed in the first intron when plants are exposed to cold is shown in green. (b) Timing of expression of FLC, COOLAIR and COLDAIR during vernalization. NV, non-vernalized; T0 corresponds to the starting point of cold treatment; T10 is 10 days of vernalization treatment; T20, 20 days of vernalization treatment; T30, 30 days of vernalization treatment. BV, before vernalization; DV, during vernalization; AV, after vernalization.
response to vernalization treatments given from germination. By contrast several of its perennial relatives only flower in response to vernalization if these treatments are given when the plants are at least 5–6-weeks old [54,55,56]. The mechanism by which this age-dependent response to vernalization is controlled was recently examined in Cardamine flexuosa and Arabis alpina, two perennial relatives of A. thaliana [55,56]. Both species flower in response to vernalization if they are exposed to cold when around 6-weeks old, but not when they are treated at 3-weeks old. Nevertheless, FLC orthologues are reduced in expression in young or old plants exposed to vernalization. Therefore flowering of young plants must be blocked at a step downstream of transcriptional repression of FLC. In many plants species, Current Opinion in Plant Biology 2014, 21:120–127
microRNA156 (miR156), which targets the mRNAs of a subclass of SPL transcription factors, is reduced in abundance as the plant ages and this is associated with vegetative phase change [57–59]. Also in A. thaliana miR156 influences flowering time, as shown using miRNA156 mimics that reduce activity of the miRNA (mim156) as well as miR156 overexpression lines [60]. In A. alpina and C. flexuosa the time at which plants become sensitive to vernalization correlates with the time at which miR156 reaches trough levels. Strikingly overexpressing MIM156, which reduced miR156 activity, in both species reduced the age at which the plants became sensitive to vernalization, so that they responded to vernalization when 3–4 weeks younger than wild-type. These experiments indicate that in these perennial species flowering www.sciencedirect.com
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in response to vernalization requires both downregulation of FLC mRNA and reduced miR156 levels. By contrast in annual A. thaliana, vernalization can trigger flowering prior to downregulation of miR156. These results suggest that rapidly flowering annual species such as A. thaliana have evolved mechanisms for bypassing the requirement for miR156 downregulation and SPL transcription factor expression to flower. Deciphering these mechanisms will provide an avenue for understanding how age-related flowering evolves and how this is related to the divergence of annual and perennial life history.
Conclusion Our understanding of the mechanisms controlling seasonal flowering responses and how these have diversified among plant families is increasing rapidly. Identification of genes controlling these responses and how they interact at the genetic level has been carried out in many species. However, fundamental questions remain. The basis of temperature perception in the vernalization pathway has not been defined. The biochemical functions of FT proteins seem to have become more complex with the demonstration that they bind different transcription factors in distinct ways and the possibility that they also bind lipids in vivo. The wide conservation of these proteins and their role as the primary trigger of the transition from vegetative to reproductive development at the shoot meristem makes understanding their biochemical function a priority for the field. Furthermore, many genes are activated rapidly in response to FT at the shoot apical meristem [61]. Defining these pathways, how they interact and how they relate to growth regulators such as gibberellins [62,63–66] are challenges that are still at an early stage. Finally the complexity of seasonal responses described here must be integrated with those to ambient cues such as high daily temperatures [67,68,69], drought or light quality. Some of the same steps are used, such as transcriptional activation of FT-like genes, but how seasonal and ambient responses are prioritized remains to be defined in detail. In addition to these emerging issues, study of seasonal flowering responses is likely to generate as many exciting surprises in the future as it has in the past.
Acknowledgements The lab of George Coupland is funded by the European Research Council (339113-HyLife), the Cluster of Excellence in Plant Sciences (CEPLAS) and a core grant from the Max Planck Society.
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28. Pin PA, Benlloch R, Bonnet D, Wremerth-Weich E, Kraft T, Gielen JJ, Nilsson O: An antagonistic pair of FT homologs mediates the control of flowering time in sugar beet. Science 2010, 330:1397-1400. 29. Ho WW, Weigel D: Structural features determining flower promoting activity of Arabidopsis FLOWERING LOCUS T. Plant Cell 2014, 26:552-564. This paper employs systematic mutagenesis of FT and screening for mutant forms in vivo to identify residues required for FT activity. Important amino acids in the external loop region are confirmed. 30. Klintenas M, Pin PA, Benlloch R, Ingvarsson PK, Nilsson O: Analysis of conifer FLOWERING LOCUS T/TERMINAL FLOWER1-like genes provides evidence for dramatic biochemical evolution in the angiosperm FT lineage. N Phytol 2012, 196:1260-1273. 31. Hanzawa Y, Money T, Bradley D: A single amino acid converts a repressor to an activator of flowering. Proc Natl Acad Sci U S A 2005, 102:7748-7753. 32. Ahn JH, Miller D, Winter VJ, Banfield MJ, Lee JH, Yoo SY, Henz SR, Brady RL, Weigel D: A divergent external loop confers antagonistic activity on floral regulators FT and TFL1. EMBO J 2006, 25:605-614. 33. Higuchi Y, Narumi T, Oda A, Nakano Y, Sumitomo K, Fukai S, Hisamatsu T: The gated induction system of a systemic floral inhibitor, antiflorigen, determines obligate short-day flowering in chrysanthemums. Proc Natl Acad Sci U S A 2013, 110:17137-17142. This paper provides evidence for a TFL1-like inhibitor of flowering, that is transported from the leaves to the apex to repress flowering under noninductive conditions in chrysanthemum.
43. Sung S, Amasino RM: Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3. Nature 2004, 427:159164. 44. Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C: Vernalization requires epigenetic silencing of FLC by histone methylation. Nature 2004, 427:164-167. 45. Rataj K, Simpson GG: Message ends: RNA 30 processing and flowering time control. J Exp Bot 2014, 65:353-363. 46. Swiezewski S, Liu FQ, Magusin A, Dean C: Cold-induced silencing by long antisense transcripts of an Arabidopsis Polycomb target. Nature 2009, 462:799-802. 47. Heo JB, Sung S: Vernalization-mediated epigenetic silencing by a long intronic noncoding RNA. Science 2011, 331:76-79. 48. Hornyik C, Terzi LC, Simpson GG: The spen family protein FPA controls alternative cleavage and polyadenylation of RNA. Dev Cell 2010, 18:203-213. 49. Liu F, Marquardt S, Lister C, Swiezewski S, Dean C: Targeted 30 processing of antisense transcripts triggers Arabidopsis FLC chromatin silencing. Science 2010, 327:94-97. 50. Duc C, Sherstnev A, Cole C, Barton GJ, Simpson GG: Transcription termination and chimeric RNA formation controlled by Arabidopsis thaliana FPA. PLoS Genet 2013, 9:e1003867. This paper uses direct RNA sequencing to quantitatively compare the structure and frequency of COOLAIR transcripts at the FLC locus in FPA mutant and wild-type plants.
34. Oda A, Narumi T, Li T, Kando T, Higuchi Y, Sumitomo K, Fukai S, Hisamatsu T: CsFTL3, a chrysanthemum FLOWERING LOCUS T-like gene, is a key regulator of photoperiodic flowering in chrysanthemums. J Exp Bot 2012, 63:1461-1477.
51. Marquardt S, Raitskin O, Wu Z, Liu F, Sun Q, Dean C: Functional consequences of splicing of the antisense transcript COOLAIR on FLC transcription. Mol Cell 2014, 54:156-165. Trans-acting and cis-acting mutations that impair splicing of COOLAIR alter levels of FLC mRNA and flowering time. This work emphasizes the importance of different forms of COOLAIR in FLC regulation.
35. Mimida N, Kidou S, Iwanami H, Moriya S, Abe K, Voogd C, Varkonyi-Gasic E, Kotoda N: Apple FLOWERING LOCUS T proteins interact with transcription factors implicated in cell growth and organ development. Tree Physiol 2011, 31:555-566.
52. Castaings L, Bergonzi S, Albani MC, Kemi U, Savolainen O, Coupland G: Evolutionary conservation of cold-induced antisense RNAs of FLOWERING LOCUS C in A. thaliana perennial relatives. Nat Commun 2014, 5 Article number: 4457.
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53. Helliwell CA, Robertson M, Finnegan EJ, Buzas DM, Dennis ES: Vernalization-repression of Arabidopsis FLC requires promoter sequences but not antisense transcripts. PLOS ONE 2011, 6:e21513. 54. Wang RH, Albani MC, Vincent C, Bergonzi S, Luan M, Bai Y, Kiefer C, Castillo R, Coupland G: Aa TFL1 confers an agedependent response to vernalization in perennial Arabis alpina. Plant Cell 2011, 23:1307-1321. 55. Bergonzi S, Albani MC, Ver Loren van Themaat E, Nordstrom KJ, Wang R, Schneeberger K, Moerland PD, Coupland G: Mechanisms of age-dependent response to winter temperature in perennial flowering of Arabis alpina. Science 2013, 340:1094-1097. Arabis alpina, a perennial relative of A. thaliana only shows a flowering response to vernalization 5 or more weeks after germination. This agerelated response is shown to be timed by downregulation of mR156 and upregulation of its target mRNAs encoding SPL transcription factors. 56. Zhou CM, Zhang TQ, Wang X, Yu S, Lian H, Tang H, Feng ZY, Zozomova-Lihova J, Wang JW: Molecular basis of agedependent vernalization in Cardamine flexuosa. Science 2013, 340:1097-1100. Cardamine flexuosa, a second perennial relative of A. thaliana, times sensitivity to vernalization in a similar way to described in Ref [55] for A. alpina. 57. Wu G, Park MY, Conway SR, Wang JW, Weigel D, Poethig RS: The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 2009, 138:750-759. 58. Wu G, Poethig RS: Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development 2006, 133:3539-3547. 59. Chuck G, Cigan AM, Saeteurn K, Hake S: The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA. Nat Genet 2007, 39:544-549. 60. Wang JW, Czech B, Weigel D: miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana. Cell 2009, 138:738-749. 61. Torti S, Fornara F, Vincent C, Andre´s F, Nordstro¨m K, Go¨bel U, Knoll D, Schoof H, Coupland G: Analysis of the Arabidopsis shoot meristem transcriptome during floral transition identifies distinct regulatory patterns and a Leucine-rich repeat protein that promotes flowering. Plant Cell 2012, 24: 444-462. 62. Yu S, Galvao VC, Zhang YC, Horrer D, Zhang TQ, Hao YH, Feng YQ, Wang S, Schmid M, Wang JW: Gibberellin regulates the Arabidopsis floral transition through miR156-targeted
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SQUAMOSA promoter binding-like transcription factors. Plant Cell 2012, 24:3320-3332. DELLA proteins are shown to interact directly with SPL transcription factors, demonstrating how these proteins could be regulated at the posttranslational level by GA. 63. Porri A, Torti S, Romera-Branchat M, Coupland G: Spatially distinct regulatory roles for gibberellins in the promotion of flowering of Arabidopsis under long photoperiods. Development 2012, 139:2198-2209. 64. Galvao VC, Horrer D, Kuttner F, Schmid M: Spatial control of flowering by DELLA proteins in Arabidopsis thaliana. Development 2012, 139:4072-4082. 65. Pearce S, Vanzetti LS, Dubcovsky J: Exogenous gibberellins induce wheat spike development under short days only in the presence of VERNALIZATION1. Plant Physiol 2013, 163:14331445. 66. Andre´s F, Porri A, Torti S, Mateos J, Romera-Branchat M, Garcı´aMartı´nez JL, Fornara F, Gregis V, Kater MM, Coupland G: SHORT VEGETATIVE PHASE reduces gibberellin biosynthesis at the Arabidopsis shoot apex to regulate the floral transition. Proc Natl Acad Sci U S A 2014, 111:E2760-E2769. 67. Kumar SV, Lucyshyn D, Jaeger KE, Alos E, Alvey E, Harberd NP, Wigge PA: Transcription factor PIF4 controls the thermosensory activation of flowering. Nature 2012, 484:242245. Exposure to high temperatures induces flowering of A. thaliana under short days. This paper shows that the PIF4 transcription factor binds to the FT promoter specifically under high temperatures and causes FT transcription. 68. Lee JH, Ryu HS, Chung KS, Pose D, Kim S, Schmid M, Ahn JH: Regulation of temperature-responsive flowering by MADSbox transcription factor repressors. Science 2013, 342:628632. The floral repressor SVP, a MADS box transcription factor, is shown to be unstable at high temperature and that is, argued to contribute to upregulation of FT and early flowering under those conditions. 69. Pose D, Verhage L, Ott F, Yant L, Mathieu J, Angenent GC, Immink RG, Schmid M: Temperature-dependent regulation of flowering by antagonistic FLM variants. Nature 2013, 503:414-417. Differential splicing of the mRNA encoding the MADS box transcription factor FLM is shown to occur in a temperature dependent way. Forms of the protein made at high temperature are proposed to interact with SVP and prevent it binding to DNA. Therefore, reduced SVP activity at high temperature through interaction with inhibitory forms of FLM is proposed to cause more FT transcription and earlier flowering.
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ScienceDirect Flowering responses to seasonal cues: what’s new? Maida Romera-Branchat, Fernando Andre´s and George Coupland Seasonal cues of day length or winter cold trigger flowering of many species. Forward and reverse genetic approaches are revealing the mechanisms by which these responses are conferred. Homologues of the Arabidopsis thaliana protein FLOWERING LOCUS T (FT) are widely used to mediate seasonal responses to day length and act as grafttransmissible promoters or repressors of flowering. Winter cold in A. thaliana promotes flowering by repressing transcription of the MADS box gene FLOWERING LOCUS C (FLC). The mechanism by which this occurs involves a complex interplay of different forms of long noncoding RNAs induced at the FLC locus during cold and changes in the chromatin of FLC. In perennial relatives of A. thaliana, flowering also requires the age-dependent downregulation of miRNA156 before winter. Addresses Max Planck Institute for Plant Breeding Research, D-50829 Cologne, Germany Corresponding author: Coupland, George ([email protected])
Current Opinion in Plant Biology 2014, 21:120–127 This review comes from a themed issue on Cell signalling and gene regulation 2014 Edited by Xiangdong Fu and Junko Kyozuka
http://dx.doi.org/10.1016/j.pbi.2014.07.006 1369-5266/Published by Elsevier Ltd.
Seasonal responses in the regulation of flowering Flowering is precisely controlled in many species by seasonal cues of day length (photoperiod) and winter temperatures (vernalization). These responses often exhibit quantitative variation among individuals of a single species, ensuring that flowering occurs at the optimal time to maximize seed production in specific environments. Such natural genetic variation supplemented with induced mutations in model species has allowed isolation of genes controlling these complex responses. Such studies have provided regulatory frameworks for photoperiod and vernalization responses and suggested the underlying regulatory logic. They have also identified conserved mechanisms between distantly related species such as Arabidopsis thaliana and cereal Current Opinion in Plant Biology 2014, 21:120–127
crops, however perhaps as expected for such fast-evolving adaptive traits important differences are found even between closely related species. The inherent fascination of these seasonal patterns as well as their importance in adaptation to local environments and to yield in agriculture have led to an extensive literature including several recent comprehensive reviews [1–5]. Here we focus on highlights of the recent literature mainly from the last two years that have deepened our understanding of photoperiodic and vernalization responses and the open questions that these publications pose.
Photoperiodic response: transcriptional regulation of FT homologues The canonical photoperiodic flowering pathway of A. thaliana promotes early flowering under long days (LDs) of spring and early summer, but not under short days (SDs) of winter [1,4]. Two genes, FLOWERING LOCUS T and TWIN SISTER OF FT are increased in transcription in the phloem companion cells under LDs. These proteins, related in sequence to phosphatidylethanolamine binding proteins (PEBPs) of animals, move to the apex where they induce flowering. Transcriptional activation of FT, which is expressed at higher levels than TSF, in response to LDs is the limiting step in photoperiodic induction of A. thaliana, and similar data have been obtained for FT homologues in many other species. In A. thaliana this activation of FT occurs through the CONSTANS (CO) transcription factor, which accumulates specifically under LDs and promotes FT and TSF transcription. The CO protein contains B-box zinc fingers at the amino terminus and a CO COL TOC1 (CCT) domain at the C-terminus. The CCT domain has sequence similarities to the NUCLEAR FACTOR-YA (NF-YA) protein and acts as a DNA binding domain [6– 8]. CO binds to the proximal region of the FT promoter recognizing two CO Responsive Elements (CORE) that are required for FT activation [7,9]. Deletion analysis of the FT promoter showed that a distal region is also required for CO response [9]. Recently this region was shown to be recognized by NF-Y that binds to a CCAAT element around 5.3 kb upstream of the transcriptional start site [10]. Previous genetic analysis showed that certain NF-Y subunit paralogues are required for CO to activate FT transcription [11], and mutational analysis demonstrated that this CCAAT box is required for COmediated activation of FT [10]. Looping of the FT promoter has now been detected between the distal site to which NF-Y binds and the proximal site at which CO binds [10], and since CO and NF-Y were previously www.sciencedirect.com
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shown to interact [6,12], this looping is proposed to be caused by interactions between CO and NF-Y bound to DNA [10] (Figure 1). As both complexes and their binding sites are required for FT activation, and loops are only detected at the times of day at which CO and FT are expressed, this looping may be required for the photoperiodic dependent activation of FT transcription. These data help clarify the structure of the FT promoter and the mechanism of its transcriptional activation in A. thaliana in response to photoperiod. The significance and basis of promoter looping must now be tested using precise mutations in FT promoter motifs.
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photoperiodic flowering is strongly conserved. However, typically these contain at their N-terminus a pseudoresponse regulator receiver domain rather than the Bboxes found in CO [13,15–17]. These PSEUDO RESPONSE REGULATOR (PRR) proteins are members of small gene families and encode components of the circadian clock in A. thaliana, but members of the family have been shown to have specific roles in FT activation and flowering in several other species. Most recently, the BOLTING gene from sugar beet was added to this list [16]. This species includes annual accessions that flower rapidly without a requirement for winter cold, and others that are biennial and flower only if vernalized. These varieties differ at the BOLTING (B) locus, so that dominant alleles at B confer the early flowering annual growth habit, whereas biennials carry the recessive allele. Isolation of this gene showed that it encodes a PRR protein
Genetic analysis in a range of species using naturally occurring alleles identified proteins containing CCT domains that are important in FT photoperiodic regulation [13–17], suggesting that the role of these proteins in Figure 1
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Features of FT transcriptional activation and protein structure. (a) Model of recruitment of CO to the CO Response Elements (CORE, labeled C1 and C2) (adapted from [10]). NF-Y complexes bind to two CCAAT sequences located at 2 and 5.5 kb away from the FT transcription start site (TSS). A chromatin loop, whose formation is favored at the end of a long day, maintains the NF-Y complex in close proximity to the CORE sequences (at 220 and 161 bp away from the TSS). This allows NF-Y complex to interact with and stabilize CO when it binds to the CORE sequences and thereby to activate FT transcription. (b) Mutations introduced in certain amino acids (colored blue in the sequence) convert FT into a TFL1-like floral repressor. Some of these changes are predicted to change also the protein surface charge (blue with orange boxes). Mutations in Asp-71 (in yellow), which is embedded in the binding pocket, do not result in activity changes. Residues in Segment B also differ in sugar beet BvFT2 and cause the protein to act as a repressor of flowering. www.sciencedirect.com
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called BOLTING TIME CONTROL 1 (BvBTC1), and that in biennial cultivated sugar beet the gene carries a 28 kb insertion in the promoter region, that is absent in annuals [16]. This insertion is proposed to reduce BvBTC1 expression and delay flowering. In biennials vernalization increases BvBTC1 expression leading to earlier flowering. BvBTC1 activates expression of the BvFT2 gene-triggering flowering. This is a further striking example of allelic variation at a CCT domain influencing seasonal control of flowering time through influencing FT expression and contributing to domestication of crops. Seasonal responses to photoperiod do not only affect flowering but other developmental programs such as bud break in trees or tuberization in potato. Both of these processes have been shown to be regulated through transcriptional regulation of FT-like genes [18,19]. The regulation of potato tuberization appears to be closely related to flowering of A. thaliana, involving CO-like and FT-like genes [18,20]. Recently this similarity was extended by showing that CYCLING DOF (CDF) transcription factors that repress CO transcription in A. thaliana [21] are also important in tuberization [22]. A potato allele of one of the genes (StCDF1) encoding CDF proteins was found to colocalize with a maturity locus that allows tubers to be formed under LDs, and was therefore important in the spread of potato as a cultivated crop [22]. This allele of StCDF1 carries a deletion that stabilizes the protein throughout the day allowing it to repress CO homologues leading to tuberization under LDs. This is an interesting example of common regulatory mechanisms being used in different plant families within the photoperiodic pathway at higher levels in the hierarchy than CCT domain and FT-like proteins. As more such studies are performed it will be interesting to determine whether this is an example of a conserved mechanism or of convergent evolution of the utilization of CDF transcription factors which might have been recruited to this process independently in the Brassicaceae and Solanaceae families.
Photoperiodic responses: function of FT proteins FT proteins appear to be universal regulators of flowering in angiosperms and are transported from the leaves to the apex to induce flowering [1,5,23]. They form protein complexes with bZIP transcription factors to regulate transcription of target genes. In A. thaliana their effect is mediated by two closely related bZIPs, FD and FD PARALOGUE (FDP) [24–26]. Biochemical analyses in vitro as well as crystallization studies using rice proteins indicated that this interaction between FD and FT is indirect and is mediated by a 14-3-3 protein [27]. Recently the activity of FT proteins has been expanded to include repressors of flowering that mediate environmental responses to photoperiod and vernalization. Current Opinion in Plant Biology 2014, 21:120–127
In sugar beet two FT-like genes, BvFT1 and BvFT2, were isolated [28]. BvFT2 is transcriptionally activated by BvBTC1 and is essential for flowering [16,28]. Reduction of BvFT2 expression by RNA interference prevents flowering whereas overexpression of BvFT2 strongly accelerates flowering both in transgenic sugar beet and transgenic A. thaliana. BvFT2 therefore behaves as a promoter of flowering, as expected from analysis of FT-like genes from many other species. Surprisingly, BvFT1 had the opposite effect. BvFT1 was repressed by BvBTC1, while its overexpression in transgenic sugar beet delayed flowering and reduced expression of the BvFT2 mRNA [28]. Similarly its overexpression in transgenic A. thaliana delayed flowering. Therefore BvFT1 is a repressor of flowering acting partly through transcriptional repression of BvFT2, although it likely has additional roles in the repression of bolting and flowering [28]. Other members of the plant family of proteins related to PEBPs were previously described to act as repressors of flowering, particularly TERMINAL FLOWER 1 (TFL1) of A. thaliana and its homologues. However FT and TFL1 differ at well characterized residues (Figure 1). For example, Tyr85 and Gln140 are indicative of FT activity, while His88 and Asp144 are diagnostic of TFL1 [29,30–32]. At these positions, BvFT1 is recognizably an FT-like protein, despite acting as a repressor of flowering [28]. Elegant domain swapping experiments showed that 2 amino acid changes in the fourth exon, which encodes an external loop in the crystal structure of FT, are responsible for the change in activity between BvFT1 and BvFT2 [28,30]. The BvFT1 protein appears to have evolved recently to act as a repressor of flowering that contributes to the vernalization response. The importance of antagonistic interactions between proteins in this family as part of the flowering response to environment also recently emerged in Chrysanthemum [33]. Here the authors characterized two proteins expressed in leaves that contribute to the photoperiodic response. The first of these proteins is encoded by Chrysanthemum seticuspe FT-like 3 (CsFTL3), which is transcriptionally induced during SDs that trigger flowering in this species [34], its overexpression drives early flowering and an inductive signal produced in the over-expressing plants is graft transmissible [33]. Therefore CsFTL3 behaves as a classical FT-floral promoting gene and its protein product contains the signature amino acids characteristic of FT among other members of the family. By contrast, C. seticuspe ANTI-FLORIGENIC FT/TFL1 FAMILY PROTEIN (CsAFT) is expressed under noninductive LDs, causes late flowering when constitutively expressed and can delay flowering when grafted roots of overexpressing plants are grafted onto wild-type shoots [33]. CsAFT contains the characteristic His88 and Asp140 residues of TFL1. Therefore CsAFT is a graft transmissible TFL1-like repressor of flowering that antagonizes the activity of FTL3 in the photoperiodic induction of flowering [33]. www.sciencedirect.com
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As mentioned above, plant PEBP-like proteins act by interacting with bZIP transcription factors to trigger transcriptional reprogramming of the shoot meristem. However, other binding partners of FT were described recently, suggesting that the protein has additional functions. FT was reported to interact with transcription factors of the TEOSINTE BRANCHED, CYCLOIDEA, PCF (TCP) family [29,35,36]. One of these BRANCHED 1 (BRC1) is expressed in axillary meristems and is proposed to act as a repressor of axillary meristem differentiation [37]. BRC1 also delays flowering of axillary branches as these flower early in brc1 mutants [36]. FT protein fusions to fluorescent proteins were shown to move into the axillary bud after expression in the leaf blade, suggesting that they could interact with BRC1 in wild-type plants [36]. These and further genetic data support the idea that BRC1 acts to repress flowering in axillary meristems by interacting with FT. The mechanism of interaction would be different than with FD, as BRC1 does not require 14-3-3 proteins to mediate the interaction. In addition to proteins, FT was found to bind to lipids in vitro [38]. Animal PEBPs contain a binding pocket identified in their crystal structure in which ligands such as the lipid phosphatidylethanolamine (PE) are presumed to bind [39]. Crystallization of the plant proteins demonstrated that they also contain this pocket [32,40]. However, neither FT nor TFL1 were shown to bind lipids and the functional significance of the pocket in the plant proteins is unclear. Furthermore, mutation of the FT protein at Asp71, which is located deep in the pocket, had no effect on protein activity in vivo [29]. Nevertheless, Tyr85 is located at the entrance of the pocket, and this residue is not only invariant in FT proteins but conversion of the analogous residue in TFL1 from His88 to Tyr is sufficient to switch TFL1 from a repressor to an activator of flowering [31]. These observations have now been extended by showing that FT binds the lipid phosphatidylcholine (PC) in vitro, but that in similar assays binding to the related lipid PE could not be detected [38]. In transgenic plants PC levels were elevated by increasing the ratio of PC:PE, and this was associated with early flowering that at least partially required active FT and TSF genes [38]. PC is a lipid found at high levels in all cellular membranes, and how this might influence FT activity at the biochemical level remains unclear. However, these data demonstrate for the first time that plant PEBP-like proteins such as FT have the capacity to bind lipids and that this might contribute to their activity in vivo.
Vernalization response: age-related vernalization response In addition to day length, vernalization is another cue that strongly affects seasonal flowering patterns. Genetic www.sciencedirect.com
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analysis of vernalization response in different plant families identified distinct regulatory pathways suggesting that it has evolved independently in each case [1]. In the Brassicaceae the MADS box transcription factor FLOWERING LOCUS C is the central node in the vernalization pathway [41]. FLC represses flowering until the plant is exposed to cold for several weeks. Under these conditions FLC transcription is progressively repressed. After return to warm temperatures, FLC transcript remains at low levels and the plant proceeds to flowering [41]. This progressive repression of FLC transcription is an epigenetic phenomenon that requires accumulation of trimethylation of lysine 27 on histone 3 (H3K27me3) [42–44]. Recently the complexity of FLC regulation increased further by the identification of long noncoding RNAs (lncRNAs) expressed at the FLC locus [45]. An antisense RNA, called COOLAIR, is expressed from a promoter at the 30 end of FLC [46], whereas a sense RNA, COLDAIR, is expressed in the first intron [47] (Figure 2). Both of these lncRNAs are expressed at higher levels during vernalization (Figure 2). COLDAIR is not polyadenylated, but is bound directly by components of the enzymic machinery required to deposit the H3K27me3 mark on the FLC gene and is proposed therefore to recruit these to the FLC gene contributing to its repression [47]. By contrast, COOLAIR exists in several forms due to use of different polyadenylation sites [46,48]. One form is polyadenylated at a proximal site (close to the transcriptional start site of COOLAIR) but other forms are spliced and polyadenylated at distal sites close to the transcriptional start site of FLC. Those polyadenylated at distal positions close to the FLC transcriptional start site might be related to FLC activation. For example, mutations in trans-acting proteins such as FPA that reduce the efficiency of detection of the proximal polyadenylation site and cause more antisense transcription around the FLC promoter are also associated with increased expression of FLC sense transcript and late flowering [48,49,50]. Such conclusions were strengthened recently by identifying mutations that impair the spliceosome leading to alterations in COOLAIR splicing and by mutating particular splice sites in COOLAIR [51]. Altering the abundance of specific forms of COOLAIR in this way also influenced FLC transcript levels [51]. Therefore, altering levels of specific COOLAIR forms appears to influence FLC mRNA levels. However, how these observations relate to the increased transcription of COOLAIR during vernalization, the ratios of specific forms that are present in cold and their relationship to FLC repression during vernalization remains to be described in detail. The recent demonstration that COOLAIR structure and expression patterns are conserved in distantly related Brassicaceae species further suggests a function in vernalization [52], although mutations that impair COOLAIR expression in A. thaliana did not have a dramatic effect on FLC repression by vernalization [53]. The age at which plants can respond to vernalization and induce flowering is highly variable. A. thaliana flowers in Current Opinion in Plant Biology 2014, 21:120–127
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Figure 2
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Noncoding RNAs encoded at the FLC locus. (a) Diagram of the different forms of transcripts expressed at the FLC locus in Arabidopsis thaliana. Exons in FLC mRNA are shown as rectangles in gray. Red asterisk (left) is transcription start site of FLC sense transcript. The Poly(A) site on FLC locus is also shown. Blue asterisk (right) is transcription start of antisense RNA transcripts known as COOLAIR (in blue). The transcription of COOLAIR increases as plant is exposed to cold temperatures and appears in different spliced isoforms [46,48,49]. Different components of the autonomous pathway and splicing patterns have been recently shown to regulate the abundance of these COOLAIR RNA transcripts [49,50,51]. A sense RNA called COLDAIR expressed in the first intron when plants are exposed to cold is shown in green. (b) Timing of expression of FLC, COOLAIR and COLDAIR during vernalization. NV, non-vernalized; T0 corresponds to the starting point of cold treatment; T10 is 10 days of vernalization treatment; T20, 20 days of vernalization treatment; T30, 30 days of vernalization treatment. BV, before vernalization; DV, during vernalization; AV, after vernalization.
response to vernalization treatments given from germination. By contrast several of its perennial relatives only flower in response to vernalization if these treatments are given when the plants are at least 5–6-weeks old [54,55,56]. The mechanism by which this age-dependent response to vernalization is controlled was recently examined in Cardamine flexuosa and Arabis alpina, two perennial relatives of A. thaliana [55,56]. Both species flower in response to vernalization if they are exposed to cold when around 6-weeks old, but not when they are treated at 3-weeks old. Nevertheless, FLC orthologues are reduced in expression in young or old plants exposed to vernalization. Therefore flowering of young plants must be blocked at a step downstream of transcriptional repression of FLC. In many plants species, Current Opinion in Plant Biology 2014, 21:120–127
microRNA156 (miR156), which targets the mRNAs of a subclass of SPL transcription factors, is reduced in abundance as the plant ages and this is associated with vegetative phase change [57–59]. Also in A. thaliana miR156 influences flowering time, as shown using miRNA156 mimics that reduce activity of the miRNA (mim156) as well as miR156 overexpression lines [60]. In A. alpina and C. flexuosa the time at which plants become sensitive to vernalization correlates with the time at which miR156 reaches trough levels. Strikingly overexpressing MIM156, which reduced miR156 activity, in both species reduced the age at which the plants became sensitive to vernalization, so that they responded to vernalization when 3–4 weeks younger than wild-type. These experiments indicate that in these perennial species flowering www.sciencedirect.com
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in response to vernalization requires both downregulation of FLC mRNA and reduced miR156 levels. By contrast in annual A. thaliana, vernalization can trigger flowering prior to downregulation of miR156. These results suggest that rapidly flowering annual species such as A. thaliana have evolved mechanisms for bypassing the requirement for miR156 downregulation and SPL transcription factor expression to flower. Deciphering these mechanisms will provide an avenue for understanding how age-related flowering evolves and how this is related to the divergence of annual and perennial life history.
Conclusion Our understanding of the mechanisms controlling seasonal flowering responses and how these have diversified among plant families is increasing rapidly. Identification of genes controlling these responses and how they interact at the genetic level has been carried out in many species. However, fundamental questions remain. The basis of temperature perception in the vernalization pathway has not been defined. The biochemical functions of FT proteins seem to have become more complex with the demonstration that they bind different transcription factors in distinct ways and the possibility that they also bind lipids in vivo. The wide conservation of these proteins and their role as the primary trigger of the transition from vegetative to reproductive development at the shoot meristem makes understanding their biochemical function a priority for the field. Furthermore, many genes are activated rapidly in response to FT at the shoot apical meristem [61]. Defining these pathways, how they interact and how they relate to growth regulators such as gibberellins [62,63–66] are challenges that are still at an early stage. Finally the complexity of seasonal responses described here must be integrated with those to ambient cues such as high daily temperatures [67,68,69], drought or light quality. Some of the same steps are used, such as transcriptional activation of FT-like genes, but how seasonal and ambient responses are prioritized remains to be defined in detail. In addition to these emerging issues, study of seasonal flowering responses is likely to generate as many exciting surprises in the future as it has in the past.
Acknowledgements The lab of George Coupland is funded by the European Research Council (339113-HyLife), the Cluster of Excellence in Plant Sciences (CEPLAS) and a core grant from the Max Planck Society.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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