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 Arabidopsisin 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 *e-mail: [email protected]
†e-mail: [email protected]
Current Opinion in Plant Biology2000, 3:37–42
1369-5266/00/$ — 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 envi-ronmental 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 natu-rally 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 labo-ratory, 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-flower-ing 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 erectaand 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 tem-perature treatment suggests that vernalization can overcome the requirement for the autonomous pathway.
During the past year it has become clear that the flower-ing-time gene FLOWERING LOCUS C (FLC) plays a central role in the vernalization response (Figure 1). The
FLCgene was identified genetically because the widely used laboratory strain Landsberg erectacarries an inactive
FLCallele. This was recognised when dominant FRI alle-les or the luminidependens (ld) mutation that affects the autonomous pathway were shown not to delay flowering in Landsberg erecta, because for FRI andldto cause late flow-ering an active FLCallele 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 FLCgene 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 FLCexpression provided a means to test genet-ic 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
Response of plant development to environment: control of
flowering by daylength and temperature
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 abun-dance (Figure 1). Thus genotypes with a vernalization requirement have high FLC mRNA levels and vernaliza-tion acts to reduce FLCmRNA abundance [11••,12••].
The analysis of FLChas 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 FLCmRNA 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
FLCregulation [11••]. However, genes required for vernal-ization have been identified genetically by the isolation of mutations that reduce the acceleration of flowering caused by low temperature treatments [13]. These vernalization
(vrn) mutations are likely to define genes that are required for the vernalization process and at least the vrn2mutation prevents the reduction of FLC mRNA abundance that is observed on low temperature treatment [11••].
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 eco-types. The daylength-insensitive mutants fall into two classes: late-flowering mutants, which compared to wild-type 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 pro-motes 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 erectathe dominant CVI allele of EDI caused early flowering and these plants were almost daylength insensitive. No induced dominant muta-tion 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 daylength-insensitive 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 insensi-tive 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 elf3mutation 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 LHYgene 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 continu-ous dark or continucontinu-ous light, all circadian clock controlled rhythms that have been tested are disrupted. This pheno-Figure 1
Genetic and molecular interactions involved in vernalization in Arabidopsis. The FLCgene encodes a MADS-box transcription factor that represses the transition from vegetative growth to flowering. The abundance of FLCmRNA is reduced by low temperature treatment and is proposed to play a central role in vernalization. The FRI, FCA, LDand VRN2genes regulate the abundance of FLC mRNA [11••,12••]. FCAacts within the autonomous flowering pathway and other genes within this pathway have similar effects to those of FCA. The FRIgene increases FLCexpression. The VRN2gene acts during vernalization to reduce FLCexpression in response to low temperature treatments. The FCAand LDgenes reduce FLCmRNA abundance, so that fcaand ldmutants are late flowering but this can be corrected by low temperature treatments.
FLC
FRI LD FCAVRN2
Vegetative growth Flowering
Autonomous pathway Vernalization
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 theCIRCADIAN 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 CCA1and LHYon circadian-clock regulation relied on the phenotypes of plants in which the genes were overexpressed. The recent observa-tion that inactivaobserva-tion 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 inser-tion within an intron of the CCA1gene 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 LHYthat may be able to partially compen-sate for the loss of CCA1 function. No effect of the cca1
mutation on flowering time was reported; another muta-tion that shortens the period of the circadian clock, toc 1, was however, previously shown to cause early flowering under short days in the Landsberg erectaecotype, but not in the C24 ecotype [22,23••]. It is possible, therefore, that introgression of cca1into other ecotypes may be required to observe any effect on flowering time and that allelic dif-ferences between ecotypes can mask the effects of period mutations on flowering time.
GIGANTEA(GI) is also implicated in the control of flower-ing in response to daylength [8,24–26], and the gene was isolated during the past year [27••,28••]. The characterisa-tion of GIagain 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 func-tion because several mutant alleles affect this region of the protein [27••]. The abundance of GI mRNA is also regu-lated by the circadian clock and peaks around 10 hours after dawn. Furthermore, expression of GIdoes not show its normal peak in expression in the lhy mutant nor in plants overexpressing CCA1 [27••], which may be expect-ed as the expression of all other circadian-clock regulatexpect-ed genes tested is also disrupted in these genotypes. More surprisingly, however, gimutations also reduce the expres-sion of LHY and CCA1 in long-day grown plants and in
Figure 2
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 LFYand 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 Arabidopsisis 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 COand GI[14••,26,27••,38]. (b)Genetic analysis suggests that the FTand FWAgenes 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 LFYgene [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•]. AP1is a possible candidate, but other floral meristem identity genes are also likely to be involved. For example, ld ; ap1 ; caltriple mutants show a more severe shoot phenotype than lfy; ap1; cal mutant plants, suggesting that LDcould act on floral meristem identity genes such as APETALA2or UNUSUAL FLORAL ORGANS[40•]. (h)The main function of FTand FWAis not the transcriptional activation of LFY[37••]. One function of these genes
is likely to be the activation of AP1, as AP1expression is abolished in the floral structures formed on ft ; lfyand fwa ; lfydouble mutants [39]. (i)Floral meristem identity genes establish and regulate the expression patterns of floral homeotic genes [34••].
FT/FWA
CO/GI FLC
?
AP1 LFY (d) (a)
(b)
(c)
(e) (f) (g)
(h)
(i) AP3/PI AP1/AP2 AG
Floral patterning
Current Opinion in Plant Biology
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
gidoes not, however, affect LHYexpression in plants shift-ed to continuous darkness indicating that GI may play a role in the control of expression of circadian-clock regulat-ed genes in response to light [28••]. There are complications in establishing a connection between the effects of gion flowering time and on the period of the cir-cadian clock because two alleles that showed different effects on the period of CAB2expression 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 LHYand CCA1[27••,28••].
In order to detect and respond to daylength, light receptors must act within the long-day pathway. Classically, phy-tochrome was implicated as the photoreceptor that controls photoperiodic responses. This was supported by the demonstration that mutations in the gene encoding phy-tochrome A abolish the photoperiodic control of flowering in pea plants [29]. In Arabidopsisand other crucifers, however, both far red and blue light have long been known to pro-mote 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 surpris-ingly, 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 CAB2gene. Using a fusion of the CAB2 promoter to luciferase it was shown that under high fluence light muta-tions in the PHYBor CRY1genes lengthen the period of the circadian clock but that mutations in PHYAand CRY2only do so under specialised conditions of low fluence light [32••]. This indicates that the phyAand 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 flo-ral 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 with-in the developwith-ing primordium to promote the expression of
another floral meristem identity gene, APETALA (AP)1, or together with co-activators that are expressed within partic-ular 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 uncov-ered, 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 LFYgene expres-sion and on early-flowering transgenic plants that overexpress the LFYgene from the 35Spromoter [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::LFYplants. Genes from several genetically distinct flowering time pathways including FCA/FVE (autonomous pathway),
CO/GI (photoperiod pathway), and GIBBERELLIN RESPONSIVE 1/GIBBERELLIN INSENSITIVE (gib-berellin-dependent pathway) fall into this group, suggesting that LFYis 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 FTand FWA. This class of gene does not appear to be involved in the transcriptional upregulation of LFYbut is instead required for the response to LFYactivity: mutations in these genes have only a small effect on the activity of the LFY promot-er and the late-flowpromot-ering 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 sec-ond group is the activation of the AP1gene [39].
More detailed genetic analysis combining individual late-flowering mutants with a range of meristem-identity mutants supports the conclusions reached from the analy-sis of the LFY gene [40•–42•]. These studies have also revealed that although some genes are involved in the transcriptional upregulation of LFYthis is unlikely to be their sole function. For example, double fca;lfymutants show an enhancement of the lfy phenotype, suggesting that FCAalso acts to promote flowering and flower devel-opment in a pathway parallel to LFY [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 fcamutant is epistatic to the early flowering of ap1plants, AP1may repress flower-ing by antagonisflower-ing 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 photope-riod are regulated by parallel genetic pathways that promote flowering in Arabidopsis. Recent progress has demonstrated the importance of the circadian clock in con-trolling the photoperiod pathway and shown that vernalization acts at least in part to reduce the abundance of the FLC mRNA. These two pathways somehow con-verge 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 LFYgene 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.
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A detailed analysis of the effect of late-flowering mutations on the expression of the LFYgene, and the phenotype of transgenic 35S::LFYplants. The sep-aration of genes into groups that affect LFYtranscription and those that affect the response to LFY, does not correspond to the classical groupings that were made on the basis of physiological responses. A model for how the different floral promotion pathways interact is also proposed.
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