Response of plant development to environment: control of flowering by daylength and temperature Paul H Reeves* and George Coupland

Transcription

1 37 Response of plant development to environment: control of flowering by daylength and temperature Paul H Reeves* and George Coupland The transition from vegetative growth to flowering is often controlled by environmental conditions and influenced by the age of the plant. Intensive genetic analysis has identified pathways that regulate flowering time of Arabidopsis in response to daylength or low temperature (vernalization). These pathways are proposed to converge to regulate the expression of genes that act within the floral primordium and promote floral development. In the past year, genes that confer the responses to daylength or vernalization have been cloned and have enabled aspects of the genetic models to be tested at the molecular level. Addresses John Innes Centre, Colney Lane, Norwich NR4 7UH, UK * paul.reeves@bbsrc.ac.uk george.coupland@bbsrc.ac.uk Current Opinion in Plant Biology 2000, 3: /00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. Abbreviations AG AGAMOUS AP APETALA CAB2 CHLOROPHYLL A/B BINDING PROTEIN 2 CCA1 CIRCADIAN CLOCK ASSOCIATED 1 CRY2 CRYPTOCHROME 2 CVI Cape Verde Islands EDI EARLY DAYLENGTH INSENSITIVE elf3 early flowering 3 FLC FLOWERING LOCUS C FRI FRIGIDA GI GIGANTEA ld luminidependens LFY LEAFY lhy late elongated hypocotyl vrn vernalization Introduction The time of year at which plants flower varies widely and is closely adapted to the environment in which they grow. Temperature and daylength are the principal environmental conditions that synchronise flowering to the changing seasons. Many papers published from the 1920s onward described variations in the flowering response of varieties of the same species isolated from different latitudes and, in some cases, identified genetic loci that confer these responses. The use of genetic approaches to compare the flowering responses of naturally occurring varieties of Arabidopsis and to isolate induced mutations that disrupt these responses has enabled the isolation of genes that underlie the control of flowering by photoperiod and vernalization. Models that were initially based on genetic interactions are being confirmed or adapted on the basis of the analysis of gene expression in different genetic backgrounds. In this short review we concentrate on progress that has been made in understanding the control of flowering by both photoperiod and vernalization since publication of the most recent longer reviews [1 3]. Vernalization Many Arabidopsis ecotypes collected at high latitudes or from mountainous regions are winter annuals that flower in the spring after exposure to winter conditions. In the laboratory, these strains flower very late but will flower much earlier if exposed to low temperatures for several weeks. This process, called vernalization, prevents flowering late in the summer when seed maturation may be curtailed by onset of winter conditions. Genetic analysis has identified two classes of late-flowering Arabidopsis plants that will respond to vernalization. First, naturally occurring late-flowering varieties collected from many locations across Europe carry dominant alleles at the FRIGIDA (FRI) locus that delay flowering, and these plants will flower early if given low temperature treatments [4 6]. Second, a group of induced recessive mutations delay flowering in early flowering varieties such as Landsberg erecta and Columbia and this phenotype can be corrected by low temperature treatments [7,8]. These mutations were assigned to the autonomous flowering pathway, because they delay flowering in all photoperiods. The observation that these mutants respond to low temperature treatment suggests that vernalization can overcome the requirement for the autonomous pathway. During the past year it has become clear that the flowering-time gene FLOWERING LOCUS C (FLC) plays a central role in the vernalization response (Figure 1). The FLC gene was identified genetically because the widely used laboratory strain Landsberg erecta carries an inactive FLC allele. This was recognised when dominant FRI alleles or the luminidependens (ld) mutation that affects the autonomous pathway were shown not to delay flowering in Landsberg erecta, because for FRI and ld to cause late flowering an active FLC allele is required [9,10]. On the basis of these genetic interactions it was proposed that FLC inhibits flowering and that this inhibition is enhanced by FRI and repressed by LD. These interpretations have been supported by the molecular analysis of FLC. The FLC gene was cloned independently by activation T- DNA tagging [11 ] and chromosome walking [12 ]. Both experiments identified the same MADS-box containing gene, and proposed that it acts to repress flowering. Analysis of FLC expression provided a means to test genetic models for the control of vernalization. Late-flowering plants that can be induced to flower early if given a low temperature treatment were shown to have high FLC mrna levels, mainly in roots and at the shoot apex, that

2 38 Growth and development Figure 1 Vernalization (vrn) mutations are likely to define genes that are required for the vernalization process and at least the vrn2 mutation prevents the reduction of FLC mrna abundance that is observed on low temperature treatment [11 ]. Vegetative growth FRI LD Autonomous pathway FLC FCA VRN2 Flowering Current Opinion in Plant Biology Genetic and molecular interactions involved in vernalization in Arabidopsis. The FLC gene encodes a MADS-box transcription factor that represses the transition from vegetative growth to flowering. The abundance of FLC mrna is reduced by low temperature treatment and is proposed to play a central role in vernalization. The FRI, FCA, LD and VRN2 genes regulate the abundance of FLC mrna [11,12 ]. FCA acts within the autonomous flowering pathway and other genes within this pathway have similar effects to those of FCA. The FRI gene increases FLC expression. The VRN2 gene acts during vernalization to reduce FLC expression in response to low temperature treatments. The FCA and LD genes reduce FLC mrna abundance, so that fca and ld mutants are late flowering but this can be corrected by low temperature treatments. are reduced on vernalization [11,12 ]. Dominant alleles at the FRI locus, ld mutations and other mutations that affect the autonomous pathway increase FLC mrna abundance (Figure 1). Thus genotypes with a vernalization requirement have high FLC mrna levels and vernalization acts to reduce FLC mrna abundance [11,12 ]. The analysis of FLC has extended the genetic models and more clearly demonstrated a link between the autonomous pathway and vernalization. The mechanism by which low temperatures reduce FLC mrna levels now becomes a key question. The reduced level of FLC mrna caused by treating seedlings with low temperatures is maintained throughout the development of the treated plant but their progeny again show high levels of FLC mrna and the acceleration of flowering caused by low temperatures is progressive with a six week exposure having a more severe effect than a two week exposure [7,11,12 ]. There are similarities between this and the regulation of gene expression by methylation, and some early flowering plants in which methyl transferase activity was reduced using anti-sense technology show reduced levels of FLC mrna, suggesting that methylation might play a role in FLC regulation [11 ]. However, genes required for vernalization have been identified genetically by the isolation of mutations that reduce the acceleration of flowering caused by low temperature treatments [13]. These vernalization Photoperiod response Flowering of Arabidopsis is also regulated by daylength. Flowering occurs much earlier under long days of 16 hours light than under short days of 8 hours, and daylengths between these two extremes give an intermediate response. Mutations that disrupt the effect of photoperiod on flowering have been described, and one gene has been identified in an analysis of allelic differences between ecotypes. The daylength-insensitive mutants fall into two classes: late-flowering mutants, which compared to wildtype flower late under long days but are unaffected under short days, and early-flowering mutants, which flower much earlier under short days. The late-flowering mutants are believed to affect a single genetic pathway that promotes flowering in response to long days [10,14 ]. In addition, an extensive analysis of the allelic differences in flowering time genes between the Cape Verde Islands (CVI) and Landsberg erecta ecotypes identified a locus EARLY DAYLENGTH INSENSITIVE (EDI) [15 ]. When introgressed into Landsberg erecta the dominant CVI allele of EDI caused early flowering and these plants were almost daylength insensitive. No induced dominant mutation has been isolated that causes early flowering, highlighting the benefits of studying natural variation in flowering time. Classic experiments implicated the circadian clock as the timer in daylength measurement, and analysis of daylengthinsensitive flowering-time mutants has provided genetic support for this. The first of these was early flowering 3 (elf3), which in addition to causing early flowering that is insensitive to daylength also shows alterations in circadian clock regulation [16,17]. In elf3 plants grown under light/dark cycles and then shifted to continuous light, the regulation of genes such as CHLOROPHYLL A/B BINDING PROTEIN 2 (CAB2) by the circadian clock is disrupted as is the rhythm in leaf movements [17]; the effect of the elf3 mutation is, however, conditional and does not affect circadian clock function under continuous darkness. Two other genotypes were recently described that have dramatic effects on flowering time and disrupt circadian clock function [18,19 ]. One of these is the late elongated hypocotyl (lhy) mutation, which was identified by activation tagging and falls into the group of late-flowering mutations causing delays in flowering only under long days [18 ]. The LHY gene encodes a protein containing a single MYB repeat and its expression is regulated by the circadian clock with a peak in expression just after dawn. In the mutant, the LHY gene is expressed at an elevated and constant level. In entrained mutant plants shifted to either continuous dark or continuous light, all circadian clock controlled rhythms that have been tested are disrupted. This pheno-

3 Control of flowering by daylength and temperature Reeves and Coupland 39 type together with the daylength-insensitive flowering time of the mutant suggests a relationship between daylength responses and circadian clock function [18 ]. Transgenic plants in which the CIRCADIAN CLOCK ASSO- CIATED 1 (CCA1) gene is expressed from the cauliflower mosaic virus 35S promoter show a similar phenotype to lhy mutants [19 ]. CCA1 was initially identified because its protein product binds to the CAB promoter [20]. The CCA1 protein is 43% identical to LHY overall, and 87% identical in the DNA-binding domain [18 ]. The initial publications on the effects of CCA1 and LHY on circadianclock regulation relied on the phenotypes of plants in which the genes were overexpressed. The recent observation that inactivation of the CCA1 gene also affects circadian-clock control of gene expression was therefore an important step in establishing the importance of these genes in circadian-clock regulation [21 ]. A T-DNA insertion within an intron of the CCA1 gene prevents synthesis of CCA1 protein, and causes the circadian clock to run approximately three hours faster than in wild-type plants under continuous light [21 ]. The CCA1 knockout allele may have a less severe effect on circadian-clock control than CCA1 overexpression, because of the presence of genes such as LHY that may be able to partially compensate for the loss of CCA1 function. No effect of the cca1 mutation on flowering time was reported; another mutation that shortens the period of the circadian clock, toc 1, was however, previously shown to cause early flowering under short days in the Landsberg erecta ecotype, but not in the C24 ecotype [22,23 ]. It is possible, therefore, that introgression of cca1 into other ecotypes may be required to observe any effect on flowering time and that allelic differences between ecotypes can mask the effects of period mutations on flowering time. GIGANTEA (GI) is also implicated in the control of flowering in response to daylength [8,24 26], and the gene was isolated during the past year [27,28 ]. The characterisation of GI again emphasises the role of the circadian clock in daylength responses. The gi mutation was first described as causing late flowering under long days and daylength insensitivity [8,24]. GI encodes a large protein that is predicted to be located in the plasma membrane and to contain at least five membrane-spanning domains in the first 660 amino acid residues. The carboxy-terminal region is hydrophilic and has no homology to any protein of known function but is probably important for GI function because several mutant alleles affect this region of the protein [27 ]. The abundance of GI mrna is also regulated by the circadian clock and peaks around 10 hours after dawn. Furthermore, expression of GI does not show its normal peak in expression in the lhy mutant nor in plants overexpressing CCA1 [27 ], which may be expected as the expression of all other circadian-clock regulated genes tested is also disrupted in these genotypes. More surprisingly, however, gi mutations also reduce the expression of LHY and CCA1 in long-day grown plants and in Figure 2 Photoperiod (b) FT/FWA (a) (h) CO/GI AP1 (g) (d)? LFY FLC The activation of floral-meristem identity genes by floral promotion pathways. Photoperiod and vernalization control the timing of floral induction through independent genetic pathways [1,2]. Eventually these pathways must converge to activate genes, such as LFY and AP1, that confer floral identity upon the shoot apical meristem [34 ]; however, little is known about how signals from separate floral promotion pathways become integrated. (a) The main response to photoperiod in Arabidopsis is the promotion of flowering under long days. Both the circadian clock and light receptors are involved in this response, which is mediated through genes such as CO and GI [14,26,27,38]. (b) Genetic analysis suggests that the FT and FWA genes are also involved in the promotion of flowering in response to long days [14 ]. (c) One role of vernalization is to reduce the mrna abundance of the floral repressor FLC [11,12 ]. The autonomous floral promotion pathway also interacts with FLC (see Figure 1). (d) The photoperiodic and autonomous floral promotion pathways act additively to promote flowering and are involved in the regulation of the same floral meristem identity genes, but the stage at which these pathways converge is unknown. (e) Genetic and molecular evidence illustrates that genes from the photoperiodic and autonomous floral promotion pathways are involved in the regulation of the LFY gene [37,38,39,40,41 ]. (f,g) Although flowering time genes have been shown to be involved in the regulation of LFY, they are also likely to regulate other floral meristem identity genes [37,38,40,41 ]. AP1 is a possible candidate, but other floral meristem identity genes are also likely to be involved. For example, ld ; ap1 ; cal triple mutants show a more severe shoot phenotype than lfy; ap1; cal mutant plants, suggesting that LD could act on floral meristem identity genes such as APETALA2 or UNUSUAL FLORAL ORGANS [40 ]. (h) The main function of FT and FWA is not the transcriptional activation of LFY [37 ]. One function of these genes is likely to be the activation of AP1, as AP1 expression is abolished in the floral structures formed on ft ; lfy and fwa ; lfy double mutants [39]. (i) Floral meristem identity genes establish and regulate the expression patterns of floral homeotic genes [34 ]. (e) (i) AP3/PI AP1/AP2 AG Floral patterning (f) Vernalization (c) Current Opinion in Plant Biology

4 40 Growth and development plants shifted to continuous light indicating that LHY/CCA1 and GI do not act in a simple linear pathway but that they affect each other s expression [27,28 ]. But gi does not, however, affect LHY expression in plants shifted to continuous darkness indicating that GI may play a role in the control of expression of circadian-clock regulated genes in response to light [28 ]. There are complications in establishing a connection between the effects of gi on flowering time and on the period of the circadian clock because two alleles that showed different effects on the period of CAB2 expression had very similar effects on flowering time [28 ]. The delay in flowering caused by gi may be due to a general reduction in the amplitude of expression of circadian clock regulated genes, as observed for LHY and CCA1 [27,28 ]. In order to detect and respond to daylength, light receptors must act within the long-day pathway. Classically, phytochrome was implicated as the photoreceptor that controls photoperiodic responses. This was supported by the demonstration that mutations in the gene encoding phytochrome A abolish the photoperiodic control of flowering in pea plants [29]. In Arabidopsis and other crucifers, however, both far red and blue light have long been known to promote flowering [30], and mutations in the gene encoding the blue light receptor CRYPTOCHROME (CRY) 2 both delay flowering and reduce the photoperiodic response [31 ]. As described above, the circadian clock regulates the expression of genes that act within the long-day pathway, and the circadian clock is itself entrained (or synchronised) to the daily cycle of light and dark by light. Perhaps surprisingly, however, cry2 mutations have a weak effect on circadian clock entrainment. This has become clear from a detailed analysis of the effect of mutations in different light receptor genes on circadian clock controlled expression of the CAB2 gene. Using a fusion of the CAB2 promoter to luciferase it was shown that under high fluence light mutations in the PHYB or CRY1 genes lengthen the period of the circadian clock but that mutations in PHYA and CRY2 only do so under specialised conditions of low fluence light [32 ]. This indicates that the phya and cry2 mutations do not affect the photoperiodic response by affecting circadian clock control but may do so by more directly affecting the expression or function of genes that control flowering time. This is supported by recent observations that the promotion of flowering by CRY2 occurs through antagonising an inhibitory effect on flowering that is mediated by PHYB, and may act by increasing the expression of the flowering time gene CONSTANS [31,33 ]. The interaction between flowering time and floral meristem identity Several of the genes that act during the early stages of flower development to confer floral identity on the developing floral meristem have been isolated and their expression analysed in detail. LEAFY (LFY) is the earliest acting of these genes and encodes a transcription factor that acts within the developing primordium to promote the expression of another floral meristem identity gene, APETALA (AP) 1, or together with co-activators that are expressed within particular domains of the floral meristem to activate expression of genes such as AP3 and AGAMOUS (AG) that specify the identity of particular floral organs [34 36 ]. The role flowering-time genes play in the activation of these floral meristem identity genes is also being uncovered, although to date the greatest progress has been made in assessing the interactions between flowering time genes and LFY. One approach has been to analyse the effect of mutations causing late flowering on both LFY gene expression and on early-flowering transgenic plants that overexpress the LFY gene from the 35S promoter [37 ]. In general, two classes of flowering-time genes have been identified. The first class of gene appears to be involved in the transcriptional upregulation of LFY: mutations in genes from this class cause relatively weak upregulation of LFY promoter activity during development and do not severely attenuate the early flowering of 35S::LFY plants. Genes from several genetically distinct flowering time pathways including FCA/FVE (autonomous pathway), CO/GI (photoperiod pathway), and GIBBERELLIN RESPONSIVE 1/GIBBERELLIN INSENSITIVE (gibberellin-dependent pathway) fall into this group, suggesting that LFY is a common target for these flowering pathways (Figure 2; [37,38]). The second group is mainly defined by a subgroup of genes in the photoperiod pathway, namely FT and FWA. This class of gene does not appear to be involved in the transcriptional upregulation of LFY but is instead required for the response to LFY activity: mutations in these genes have only a small effect on the activity of the LFY promoter and the late-flowering phenotype of ft and fwa is epistatic to the early flowering of 35S::LFY plants (Figure 2; [37 ]). It is likely that one function of this second group is the activation of the AP1 gene [39]. More detailed genetic analysis combining individual lateflowering mutants with a range of meristem-identity mutants supports the conclusions reached from the analysis of the LFY gene [40 42 ]. These studies have also revealed that although some genes are involved in the transcriptional upregulation of LFY this is unlikely to be their sole function. For example, double fca ; lfy mutants show an enhancement of the lfy phenotype, suggesting that FCA also acts to promote flowering and flower development in a pathway parallel to LFY [41 ]. The analysis of the interaction of the FCA gene with meristem-identity genes also highlights an overlooked role of the AP1 gene in the regulation of flowering [41 ]. Overexpression of the AP1 gene causes early flowering [43], suggesting that one function of AP1 is to promote flowering, and the appearance of AP1 gene expression in primordia is generally considered to be a good marker for the commitment to flowering [44]. However, ap1 mutant

5 Control of flowering by daylength and temperature Reeves and Coupland 41 plants also flower slightly earlier than wild-type plants, suggesting that the AP1 gene may also repress flowering [41 ]. As the late flowering of the fca mutant is epistatic to the early flowering of ap1 plants, AP1 may repress flowering by antagonising the action of the autonomous promotion pathway [41 ]. Conclusions Flowering time is regulated by balancing the promotive or inhibitory effects of different environmental conditions. For example, the responses to vernalization and photoperiod are regulated by parallel genetic pathways that promote flowering in Arabidopsis. Recent progress has demonstrated the importance of the circadian clock in controlling the photoperiod pathway and shown that vernalization acts at least in part to reduce the abundance of the FLC mrna. These two pathways somehow converge to regulate the expression of LFY a gene that acts within the floral primordium to promote the expression of genes that specify floral organ identity (Figure 2). Future experiments will describe the molecular mechanism of this convergence and whether it occurs at the LFY gene itself or at an earlier stage in the transition from vegetative growth to flowering. Acknowledgements Our work on flowering time is supported by grants from the European Commission and the Human Frontiers Science Program. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Koornneef M, Alonso-Blanco C, Peeters AJM, Soppe W: Genetic control of flowering time in Arabidopsis. Annu Rev Plant Physiol Plant Mol Biol 1998, 49: Levy YY, Dean C: The transition to flowering. 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Mol Gen Genet 1991, 229: Lee I, Michaels SD, Masshardt AS, Amasino RM: The late-flowering phenotype of FRIGIDA and mutations in LUMINIDEPENDENS is suppressed in the Landsberg erecta strain of Arabidopsis. Plant J 1994, 6: Koornneef M, Blankestijn-de Vries H, Hanhart C, Soppe W, Peeters T: 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 1994, 6: Sheldon CC, Burn JE, Perez PP, Metzger J, Edwards JA, Peacock WJ, Dennis ES: The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 1999, 11: The authors describe the T-DNA tagging and isolation of the FLF gene, which was subsequently shown to be FLC. Together with results from [12 ] they provide strong evidence that FLC plays a central role in coordinating the promotion of flowering by both the autonomous and vernalization pathways. The flf-1 mutation, an allele of FLC that dominantly delays flowering, also causes a reduced response to gibberellic acid, suggesting that FLC may repress the promotion of flowering by this plant growth regulator. 12. Michaels SD, Amasino RM: FLOWERING LOCUS C encodes a novel MADS-domain protein that acts as a repressor of flowering. Plant Cell 1999, 11: This paper reports the isolation of the FLC gene using a map-based cloning strategy. The isolation of FLC null alleles by mutagenesis of plants carrying late FRI alleles or ld mutations confirms the role of FLC as a floral repressor, as does the phenotype of plants overexpressing the FLC gene. 13. Chandler J, Wilson A, Dean C: Arabidopsis mutants showing an altered response to vernalization. Plant J 1996, 10: Koornneef M, Alonso-Blanco C, Blankestijn-de Vries H, Hanhart CJ, Peeters AJM: Genetic interactions among late flowering mutants of Arabidopsis. Genetics 1998, 148: This paper describes genetic interactions between many of the late-flowering mutants. The exhaustive analysis of double mutants enables a detailed model of how these flowering time genes act to promote flowering to be proposed. 15. Alonso-Blanco C, El-Assal SE, Coupland G, Koornneef M: Analysis of natural allelic variation at flowering time loci in the Landsberg erecta and Cape Verde Islands ecotypes of Arabidopsis thaliana. Genetics 1998, 149: Allelic variation in flowering behavior between the Landsberg erecta and CVI ecotypes, which have similar responses to photoperiod and vernalization, can be mainly attributed to four quantitive trait loci (QTLs). The EDI locus appears to be involved in the photoperiodic response, whereas other loci have a similar behavior to genes in the autonomous and vernalization-dependent floral promotion pathways. Interestingly, the analysis of these other loci suggests that the activity of the autonomous pathway is also likely to be influenced by daylength, with short days inhibiting its activity. 16. Zagotta MT, Hicks KA, Jacobs CI, Young JC, Hangarter RP, Meeks- Wagner DR: The Arabidopsis ELF3 gene regulates vegetative photomorphogenesis and the photoperiodic induction of flowering. Plant J : Hicks KA, Millar AJ, Carré IA, Somers DE, Straume M, Meeks-Wagner DR, Kay SA: Conditional circadian dysfunction of the Arabidopsis early flowering 3 mutant. Science 1996, 274: Schaffer R, Ramsay N, Samach A, Corden S, Putterill J, Carré IA, Coupland G: The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering. Cell 1998, 93: The isolation of the LHY gene is described. The lhy mutation, which is due to a transposon insertion that results in overexpression of the LHY gene, causes photoperiod-insensitive flowering phenotype and disrupts circadian rhythms. Data are also presented showing that LHY participates in a negative-feedback loop that regulates its own expression. 19. Wang Z, Tobin EM: Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression. Cell 1998, 93: This paper demonstrates the circadian regulation of CCA1 gene expression and protein abundance. Overexpression of CCA1 causes arrhythmia in numerous circadian-clock-controlled processes and causes late flowering. In addition, overexpression of CCA1 suppresses the expression of the endogenous CCA1 and LHY genes, implicating CCA1 in a negative-feedback loop. Together with [18 ], the results suggest that CCA1 and LHY are closely associated with the circadian oscillator. 20. Wang Z, Kenigsbuch D, Sun L, Harel E, Ong MS, Tobin EM: A Mybrelated transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene. Plant Cell 1997, 9: Green RM, Tobin EM: Loss of circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression. Proc Nat Acad Sci USA 1999, 96: The isolation of a cca1 null allele is reported. The mutation shortens the period of circadian-clock regulated gene expression, indicating that LHY cannot fully compensate for the loss of CCA1. The cca1 mutation also affects the phytochrome induction of the Lhcb1*3 gene, suggesting that CCA1 may act at a point of integration between phytochrome and the clock.

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Mutations in this gene are shown to affect the period of the circadian clock, although the two gi alleles analysed have different effects. The effect of light fluence rate on the clock period in the gi-1 mutant, and the reduced effect gi mutations have on period length in continuous darkness, suggests that GI may be involved in controlling light signaling to the clock. 29. Weller JL, Murfet IC, Reid JB: Pea mutants with reduced sensitivity to far-red light define an important role for phytochrome A in daylength detection. Plant Physiol 1997, 114: Brown JAM, Klein WH: Photomorphogenesis in Arabidopsis thaliana (L.) Heyhn. Plant Physiol 1971, 47: Guo H, Yang W, Mockler TC, Lin C: Regulation of flowering time by Arabidopsis photoreceptors. Science 1998, 279: The late flowering of the fha mutant, which affects the photoperiodic floral promotion pathway, is shown to be due to a mutation in the blue light receptor CRYPTOCHROME Somers DE, Devlin PF, Kay SA: Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock. Science 1998, 282: The authors explore the effect of light fluence rate on the circadian period in mutants that are deficient in specific phytochromes and cryptochromes. The results show that several photoreceptors are involved in the regulation of the circadian clock. 33. Mockler TC, Guo H, Yang H, Duong H, Lin C: Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in the regulation of flowering. Development 1999, 126: Genetic analysis allows the authors to propose a detailed model for the interaction of cryptochromes and phytochrome B in the regulation of flowering time. Light quality is shown to be most important during a developmental stage between 1 and 7 days after germination. Interestingly, this also corresponds to the time required for long-day grown Arabidopsis plants to become committed to flower. 34. Parcy F, Nilsson O, Busch MA, Lee I, Weigel D: A genetic framework for floral patterning. Nature 1998, 395: The authors explore the role of the floral meristem identity gene LEAFY in the activation of floral homeotic genes, showing that it is involved in the regulation of APETALA1, APETALA3, and AGAMOUS. In addition, the LFY protein is shown to be nuclear localized and can bind to sequences present in the AP1 promoter. 35. Busch MA, Bomblies K, Weigel D: Activation of a floral homeotic gene in Arabidopsis. Science 1999, 285: This paper describes a LEAFY (LFY) responsive enhancer contained within the second intron of the AGAMOUS (AG) gene, and shows that LFY is an activator of AG. 36. 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