The Transition to Flowering

Floral Repression Pathways

The identification of loss-of-function mutations that accelerate flowering in rapid-cycling ecotypes such as Ler reveals that even in early-flowering ecotypes, some genes act to repress flowering. Most early-flowering mutants have been categorized by their response to photoperiod (Table 1); some (e.g., clf, elf1, elf2, elg, esd4, pef1, pef2, pef3, phyB, speedy, tfl1, tfl2, and wlc) retain a response to photoperiod, whereas others (elf3, emf1, emf2, and pif) do not. Because this division is not absolute, the early-flowering mutants are considered here collectively, and the products of the corresponding wild-type genes are thought to act in repression of flowering.

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Figure 1.

Genetic Map Showing the Approximate Positions of the Genes and Quantitative Trait Loci That Affect Flowering Time in Arabidopsis.

This map, which has been updated from that shown in Koornneef et al. (1998b), shows the five chromosomes as vertical bars, with the centromeres indicated by gray ellipses. Mutant loci are given in lowercase, whereas loci identified in natural populations are given in uppercase. The QTLs were initially described in the following publications: QLN1-12, Jansen et al. (1995); QFT1-5 and QTL1-7, Koornneef et al. (1998b); FDR1-2, Mitchell-Olds (1996); RLN1-5, Clarke et al. (1995); and DFF1-2, Kowalski et al. (1994).

The EMF genes have been considered to play a major role in repression of flowering because emf1 and emf2 mutants flower with essentially no preceding vegetative phase (Sung et al., 1992; Yang et al., 1995). The EMF genes may mediate the repression of flowering via their interactions with certain floral meristem identity genes (Figure 2). For example, AP1 and AG are expressed very early in germinating emf seedlings, and constitutive expression of LFY enhances the phenotype of weak emf1 alleles, These observations suggest that the EMF genes and AP1 and AG1 reciprocally regulate each other in a negative fashion (Figure 2; Chen et al., 1997).

Some gene products that promote flowering may act, in part, by directly or indirectly repressing EMF function. For example, emf1 and emf2 are, respectively, epistatic to gi and co (two late-flowering mutants in the photoperiodic promotion pathway; Figure 2) (Yang et al., 1995). However, when the emf mutations are combined with fca and other mutations that result in late flowering, the double-mutant plants flower after they have produced an intermediate number of leaves (Haung and Yang, 1998), which suggests that the corresponding wild-type products of these genes do not act by repressing EMF function.

TFL1, another floral repressor (Table 2), was cloned recently on the basis of its similarity to its Antirrhinum ortholog CENTRORADIALIS (CEN) (Bradley et al., 1997) and by T-DNA tagging (Ohshima et al., 1997). The tfl1 mutant flowers early, and the normally indeterminate shoot apex terminates with a flower. Ordinarily, therefore, TFL1 must function to suppress flower formation at the apex and to delay the transition from vegetative to reproductive development. Consistent with this role, overexpression of TFL1 greatly extends the vegetative and inflorescence growth phases (Ratcliffe et al., 1998). It is likely that TFL1 exerts this delay in flowering by repressing the function of genes such as FCA, FVE, and FPA, which operate in the autonomous promotion pathway (Figure 2). This is because the late-flowering phenotype conferred by mutations in these genes is epistatic to tfl1 (Ruiz-García et al., 1997; T. Page and C. Dean, unpublished results).

CLF and WLC (Table 2) act to delay flowering by repressing certain floral meristem identity genes. The clf mutant expresses AG ectopically in leaves, inflorescence stems, and flowers (Goodrich et al., 1997), and wlc expresses AG and AP3 ectopically in leaves. Thus, the wild-type function of CLF and WLC is to prevent the expression of the floral meristem identity genes in vegetative tissue. The CLF gene shares sequence homology with the Drosophila polycomb group of genes, which are involved in maintaining the repression of homeotic genes (Goodrich et al., 1997). The wlc mutant displays hypomethylation of repetitive sequences associated with the centromeres (C. Hutchison and C. Dean, unpublished results); thus, reduced methylation may directly alleviate the repression of AG and AP3 expression in leaves. Similarly, induced hypomethylation resulting from constitutive expression of an antisense methyltransferase gene resulted in ectopic expression of AG and AP3 and early flowering (Finnegan, 1996). Thus, methylation may play an important role in the repression of the floral transition.

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Figure 2.

Genetic Pathways That Control Flowering Time in Arabidopsis and Proposed Interactions among Some of the Genes Involved.

The horizontal line symbolizes the vegetative (V) to floral (F) transition, with the promotive and repressive pathways exerting their influence on this switch. Four pathways are shown: repression (green), autonomous promotion (red), photoperiodic promotion under long days (LDs; dark blue) and short days (SDs; light blue), and vernalization promotion (pink). Genes that influence both floral meristem identity and flowering time are shown in black. Promotive (arrows) and repressive (T-bars) interactions are based on genetic epistasis experiments and analysis of gene expression in mutant and overexpressing lines. Not all interactions have been tested directly, and little is known about how the floral repressors interact with the various promotive pathways; thus, most of the repressors have simply been represented below the horizontal line. Therefore, this model, which is an updated combination of those published by Koornneef et al. (1998b) and Nilsson et al. (1998), does not fully represent the complexity of the interactions between genes and pathways that control flowering time in Arabidopsis.

Methylation appears to play a role in the regulation of flowering time by the FWA gene. Working with the ddm1 mutant, which has decreased DNA methylation but unaltered methyltransferase activity (Richards, 1997), Kakutani et al. (1996) noted late flowering as a frequently appearing phenotype in repeatedly self-pollinated ddm1 lines. FTS, the dominant locus conferring this late-flowering phenotype, was mapped genetically (Kakutani, 1997) and localized close to FWA, which was previously characterized by Koornneef et al. (1991) as a dominant mutation conferring late flowering. Subsequent analysis of the methylation status of the genomic region surrounding the FWA locus in ddm1 and in EMS-induced fwa alleles showed the region to be hypomethylated (Koornneef et al., 1998b). Therefore, the wild-type product of the FWA gene may encode a repressor of flowering that normally is downregulated by methylation. However, because there is precedence for local hypermethylated sites within a hypomethylated region of a gene (see, e.g., Jacobsen and Meyerowitz, 1997), it is difficult to predict whether or not FWA expression will be up- or downregulated in the fwa mutant. Ronemus et al. (1996) speculated that a general and gradual increase in methylation during development could serve to change meristem competency and determinacy as a plant ages. It will interesting to test whether such a gradient of methylation exists in Arabidopsis and whether alleviation of the autonomous repression of flowering depends, at least in part, on changes in methylation at specific loci such as FWA.

Analysis of the natural variation in flowering time has revealed that the early-flowering ecotypes such as Ler and Col can themselves be considered as mutants in genes conferring strong repression of the floral transition. Crosses between a number of winter and spring Arabidopsis ecotypes revealed that late flowering and a requirement for vernalization segregated as a dominant monogenic trait (Sanda et al., 1997) that mapped to the FRI locus (J.E. Burn et al., 1993; Lee et al., 1993; Clarke and Dean, 1994). The recent map-based cloning of FRI has revealed that Ler and Col are likely to carry loss-of-function FRI alleles (U. Johanson and C. Dean, unpublished data).

Dominant alleles at a second locus, FLC, are required for the full repression of flowering by FRI (Lee et al., 1994b; Aukerman and Amasino, 1996). Most ecotypes carry dominant alleles at FLC, but Ler and the C24 ecotype carry recessive alleles (Michaels and Amasino, 1995). Map-based cloning of FLC is nearing completion (S.D. Michaels and R.M. Amasino, personal communication), and therefore, the basis of this variation can soon be analyzed at the molecular level. Future studies will also be able to address how the vernalization promotion pathway (see below) is able to bypass the repression of flowering mediated by FRI and FLC (Figure 2).

Given that so many genes are involved in the regulation of flowering time in Arabidopsis, it is interesting that a major determinant of both the natural variation in flowering time and the requirement for vernalization is allelic variation at FRI. FRI maps close to one of the two major QTLs that confer a vernalization requirement in Brassica spp (Osborn et al., 1997). Thus, an important question to address in the future is whether FRI orthologs correspond to flowering-time loci in a number of plant species.

Autonomous Promotion Pathway

The identification of loss-of-function mutations that delay flowering of rapid-cycling ecotypes reveals genes that act to promote flowering. Many of these late-flowering mutants have been categorized by their response to vernalization and photoperiod and in epistasis experiments (Table 1; Koornneef et al., 1991, 1998a). One group of mutants (co, fd, fe, fha, ft, fwa, and gi) show little response to photoperiod or vernalization, and the corresponding genes are thought to act in the photoperiodic promotion pathway (Figure 2). A second group of mutants (fca, fpa, ld, fve, and fy) respond strongly to vernalization but flower even later under noninductive photoperiods. Because the products of the corresponding wild-type genes appear to promote flowering independently of photoperiod, these genes are considered to act in the autonomous promotion pathway (Figure 2). Moreover, the fact that these mutants respond to vernalization suggests that the vernalization promotive pathway acts redundantly with the autonomous promotion pathway in these early-flowering ecotypes.

Two genes of the autonomous promotion pathway encode proteins whose function may be to regulate the expression of other genes (Table 2). LD encodes a putative homeodomain protein, and although the LD transcript is expressed throughout the plant, it is most abundant in the shoot and root apices (Lee et al., 1994a; Aukerman and Amasino, 1996). FCA encodes a protein with RNA binding and protein–protein interaction domains (Macknight et al., 1997). The RNA binding domains of FCA are similar to those of the Drosophila proteins SX-1 and ELAV, which regulate alternative splicing of pre-mRNA transcripts important for sex determination and neuronal differentiation (Macknight et al., 1997). The FCA transcript is itself alternatively spliced, and increasing the levels of specific FCA transcripts results in earlier flowering (R. Macknight and C. Dean, unpublished results).

Analysis of the interaction of FCA with meristem identity genes indicates that FCA function is required for both activation and competence to respond to LFY and AP1 (T. Page and C. Dean, unpublished results). FCA, or downstream gene products, appear to act in a cell non-autonomous manner, because even in plants in which a large proportion of the two inner layers of the SAM (i.e., L2 and L3) are genotypically fca, bolting and flowering are normal (Furner et al., 1996).

Transmissible signals that promote flowering are also the focus of recent work by Colasanti et al. (1998). The maize id1 mutation confers late flowering and altered floral development. ID1 encodes a protein with zinc finger motifs, suggesting that it acts as a transcriptional regulator. Several observations led Colasanti et al. (1998) to propose that ID1 may be involved in the production or transport of a transmissible signal. For example, id1 plants do not flower under field conditions, and plants containing an increasing proportion of transposon-induced wild-type ID1 sectors in a mutant id1 background flower progressively earlier (Colasanti et al., 1998). Taken together, these experiments suggest that ID1 is required to produce and/or modulate the activity of a signal that originates in immature leaves and influences reproductive development in the SAM.

That leaves are required to determine the developmental potential of the apex has also been established using cultured maize apices. Excised apices revert to producing a full set of leaves before they produce flowers, irrespective of how many leaves had been produced before they were placed in culture (Irish and Jegla, 1997). However, leaving the four to six youngest leaf primordia on the excised apices prevents the resetting of the developmental program, indicating that some signal from the leaves influences development of the apex.

Photoperiodic Promotion Pathway

Plants detect light in at least five regions of the visible spectrum by using at least three classes of photoreceptors. Blue light and ultraviolet-A are detected by the cryptochromes, red (R) and far-red (FR) light are detected by the phytochromes, and ultraviolet-B is detected by an as-yet-unidentified photoreceptor (Thomas and Vince-Prue, 1997). In Arabidopsis, there are at least five phytochromes (PHYA to PHYE) and two cryptochromes (CRY1 and CRY2) (Thomas and Vince-Prue, 1997). These photoreceptors typically have been characterized by the effect they have on seedling morphogenesis under different light conditions. Several Arabidopsis mutants that were originally isolated based on abnormal seedling photomorphogenesis are also affected in flowering time. These include cop1, det1, det2, hy1, hy2, hy4, phyA, phyB, pef1, pef2, and pef3 (Table 1). Conversely, several mutants isolated based on their flowering-time phenotypes were subsequently found to exhibit abnormal seedling photomorphogenesis. These include elf3, elg, fha, and lhy (Table 1).

The role of photoperiod in flowering was conclusively demonstrated by Garner and Allard in the 1920s in their classic experiments with the Maryland Mammoth mutant of tobacco and the Biloxi variety of soybean (reviewed in Thomas and Vince-Prue, 1997). Recent genetic studies have begun to identify molecular components of the photoperiodic promotion pathway (Figure 2), and an overall picture of how Arabidopsis perceives and responds to inductive photoperiods is beginning to emerge.

The pathway begins with photoreceptors (such as PHYA and CRY2), which initiate signals that interact with a circadian clock and entrain the circadian rhythm. Somehow, day length is measured, and when the length of the dark period decreases below a critical length, genes that promote flowering (such as CO) are activated. This activation leads, in turn, to the upregulation of floral meristem identity genes and, thereafter, flowering.

In Arabidopsis, light quality affects flowering time, with R light inhibiting and FR light promoting flowering (Martínez-Zapater et al., 1994). The phenotype of phyB mutants (Table 1) suggests that PHYB normally plays a role in inhibiting flowering under high R to FR conditions but is not involved in day-length perception (Koornneef and Peeters, 1997). Physiological studies on multiple mutant combinations suggest that in addition to PHYB, other light-stable phytochromes also regulate flowering in response to light quality (Koornneef and Peeters, 1997). In contrast, mutations in PHYA, which encodes a light-labile photoreceptor, prevent perception of low-fluence-rate, FR-enriched day-length extensions that promote flowering. These observations suggest that PHYA is involved in both day-length perception and promotion of flowering by inductive photoperiods (Figure 2; Koornneef and Peeters, 1997).

Blue light alone promotes flowering in Arabidopsis, and the product of the FHA gene has recently been shown to encode CRY2, one of the two cryptochromes thus far identified in Arabidopsis (Guo et al., 1998). Transgenic plants overexpressing CRY2 flowered earlier than did the wild type and had increased levels of CO mRNA (Guo et al., 1998), suggesting that blue light promotes flowering via CRY2 and CO (see below). Furthermore, the level of CO mRNA was found to be reduced in cry2 mutants grown under long days but not under short days (Guo et al., 1998), thereby providing a possible explanation for the basis of the original fha late-flowering phenotype. Because the levels of both PHYA and CRY2 proteins drop rapidly and dramatically in the light (Thomas and Vince-Prue, 1997), they could fulfill the role of providing information about light/dark transitions to the circadian clock.

CRY1, the other cryptochrome in Arabidopsis, was originally identified as the hy4 mutant, which has a long hypocotyl under blue light (Table 1). hy4 is sensitive to photoperiod and is not delayed in flowering in a Ler background under white light and inductive photoperiods. However, in the presence of non-Ler alleles of FLC and in blue-enriched light, hy4 is late flowering and exhibits photoperiodic sensitivity (Bagnall et al., 1996; Koornneef and Peeters, 1997). Therefore, CRY1 is involved in the promotion of flowering, but its interaction with floral promotion pathways is unclear.

Several genes that affect photoperiodic sensitivity and that may encode components of the circadian clock itself have been identified. CCA1 and LHY RNA levels oscillate in a rhythmic fashion, and overexpression of either gene results in long hypocotyls and late flowering (Schaffer et al., 1998; Wang and Tobin, 1998). Constitutive expression of either CCA1 or LHY also abolishes or alters the circadian expression of their own transcripts as well as several other genes, which suggests that CCA1 and LHY negatively regulate their own expression (Wang and Tobin, 1998).

Another likely component of the circadian clock is TOC1, which was identified as a semi-dominant mutation that shortened the period length of the circadian clock by 2 to 3 hr (Somers et al., 1998). The toc1 mutation reduces the sensitivity of plants to photoperiod and causes early flowering under short days, indicating that quantitative changes in the pace of the circadian clock, not rhythmicity/arhythmicity alone, can alter flowering time.

ELF3 may mediate the interaction of light signals generated by the photoreceptors with the circadian clock (Figure 2). The phenotype of the elf3 mutant (Table 1) suggests that the wild-type product of this gene is involved in repressing flowering under noninductive photoperiods. However, the conditional arhythmicity of the elf3 mutant suggests that ELF3, which has recently been cloned (Table 2), does not function in the circadian clock itself (Hicks et al., 1996; Koornneef and Peeters, 1997).

The circadian clock is believed to affect the expression of downstream genes that operate in the photoperiodic promotion pathway, including CO (Table 2) (Putterill et al., 1995). CO mRNA is expressed throughout the plant and is more abundant in plants grown under long days compared with short days (Piñeiro and Coupland, 1998). GI, which has recently been cloned (Table 2), probably acts upstream of CO (Figure 2), because the phenotype of plants that overexpress CO is epistatic to the gi mutation (Piñeiro and Coupland, 1998).

Several lines of evidence suggest that the level of CO activity in Ler plants is directly correlated with flowering time (reviewed in Piñeiro and Coupland, 1998). Using a glucocorticoid-inducible system, Simon et al. (1996) demonstrated that induction of CO activity is sufficient to rapidly cause flowering under short days and to initiate transcription of LFY and TFL1 as rapidly as when these genes are induced by transfer to inductive photoperiods. However, levels of AP1 mRNA increase more slowly after CO activation than they do in response to inductive photoperiods (Simon et al., 1996). These data suggest that CO acts in a pathway that is sufficient to activate LFY and TFL1 transcription but that rapid activation of AP1 requires an additional pathway (Figure 2). Interestingly, genetic analyses by Ruiz-García et al. (1997) have placed CO and TFL1 in different genetic pathways, so the rapid activation of TFL1 transcription remains to be explained.

Vernalization Promotion Pathway

Another seasonal cue in temperate zones is a winter period, and many species require exposure of imbibed seeds or vegetative plants to a period of cold temperature (typically 2 to 8 weeks at ~4°C) in order to flower. This process, known as vernalization, is slow and quantitative but requires active metabolism (reviewed in Chouard, 1960; Vince-Prue, 1975). The site of perception of vernalization is the shoot apex (e.g., Curtis and Chang, 1930; Metzger, 1988), but all actively dividing cells, not only those at the shoot apex, may be capable of responding to vernalization (Wellensiek, 1964). Unlike photoperiodic induction, vernalization prepares the plant to flower but does not itself evoke flowering. That is, there is a clear temporal separation between cold treatment and flowering, which commonly occurs after a period of growth at warmer temperatures. Vernalization is required in each generation for winter annuals and biennials and each growth year for perennials, which suggests that meiosis or some other aspect of reproductive growth resets the requirement for vernalization.

The features of vernalization suggest that an epigenetic mechanism may be responsible for the establishment, persistence, and resetting of whatever self-perpetuating changes occur during or subsequent to exposure to cold. The observations that the flowering of late-flowering, vernalization-sensitive Arabidopsis mutants is accelerated by azacytidine treatment (J.B. Burn et al., 1993) and that cold treatment leads to specific changes in gibberellin (GA) metabolism (Hazebroek and Metzger, 1990; Hazebroek et al., 1993) led J.B. Burn et al. (1993) to propose that vernalization causes a specific reduction in cytosine methylation. This reduction, J.B. Burn et al. (1993) hypothesized, results in the activation of the gene encoding kaurenoic acid hydroxylase, an enzyme that catalyzes an early step in GA biosynthesis. Indeed, when general levels of methylation were reduced in wild-type plants by introducing a transgene expressing an antisense version of a methyltransferase gene (antisense-MET1), developmental abnormalities and early flowering were observed (Finnegan, 1996; Finnegan et al., 1998). However, the role of methylation in vernalization is still unclear, because substantial demethylation did not prevent vernalization from fully accelerating flowering in these lines, nor did it prevent resetting of the vernalization requirement in the progeny of antisense-MET1 plants (Finnegan et al., 1998).

One approach to understanding the molecular basis of vernalization has been to isolate mutants of Arabidopsis that are specifically impaired in their response to cold treatment (Chandler et al., 1996). The starting point for this genetic screen was fca, a late-flowering mutant whose phenotype can be completely corrected by a period of vernalization. fca plants were mutagenized, and a population of progeny plants were vernalized and screened for individual plants that flowered late, that is, which no longer exhibited a strong response to vernalization. Of these candidate double mutants, those that flowered no later than fca itself without cold treatment were selected for further characterization (Chandler et al., 1996). Such vrn mutants may be defective either in the perception of cold temperature or in the transduction of the cold signal by the vernalization promotion pathway (Figure 2). An initial screen identified five independent recessive vrn mutations in at least three complementation groups (Chandler et al., 1996), and a second screen identified five additional mutants, which have not yet been assigned to complementation groups (Y.Y. Levy and C. Dean, unpublished results). Two mutants, vrn1 and vrn2 (Table 1), have been characterized in some detail and are being cloned by chromosome walking. Both vrn1 and vrn2 have a normal acclimation response, indicating either that they are downstream of a cold-perception pathway common to acclimation and vernalization or that cold perception occurs via independent pathways in these two responses (Chandler et al., 1996). Analysis of the VRN genes should reveal some of the molecular components involved in promotion of flowering by vernalization.

The Role of Carbohydrates in Flowering

Compelling evidence that sucrose may function in long-distance signaling during floral induction comes from studies of Sinapis alba, a long-day plant in the mustard family. After induction of flowering in S. alba by either a single long day or a displaced short day, the concentration of sucrose in the phloem reaching the apex increases rapidly and transiently (Bernier et al., 1993). Furthermore, this pulse of sucrose precedes the increase in cell division that is normally observed in the SAM upon floral induction. The sucrose reaching the apex appears to be derived from the mobilization of stored carbohydrates, most likely starch in the leaves and stems, because plants induced by a displaced short day receive the same photosynthetic input as plants maintained under noninductive photoperiods (Bernier et al., 1993).

In Arabidopsis, Ler plants grown in darkness with their apices in contact with sucrose-containing medium flower with the same number of leaves as do plants grown under long days (Roldán et al., 1997). In contrast, sucrose has a significant effect on the flowering of vernalization-requiring ecotypes Leiden and Stockholm, which flower early when grown under these conditions and with approximately the same number of leaves as Ler (Roldán et al., 1997). Furthermore, sucrose alone, whether supplied in the dark or in the light, is responsible for most of this acceleration. Therefore, supplying sucrose to these late-flowering ecotypes bypasses the inhibition of flowering normally conferred by the existence of dominant alleles at FRI and FLC (Table 1). Sucrose also accelerates the flowering of fve, fpa, fca, co, and gi but not of ft and fwa (Roldán et al., 1997). This result implies that FVE, FPA, FCA, CO, and GI function in processes that are either upstream of or separate from control of sucrose availability to the vegetative apex, whereas FT and FWA function in processes downstream of this control point.

Further genetic evidence connecting carbohydrate metabolism with control of flowering is available, but the nature of this connection is unclear. For example, there are at least five Arabidopsis mutants, adg1, cam1, gi, pgm, and sex1, which are altered in starch synthesis, accumulation, or mobilization and which flower late under some conditions (Table 1). The flowering time of cam1 and gi is not influenced by photoperiod, and therefore, both are likely to act in the photoperiodic promotion pathway (Eimert et al., 1995). pgm and sex1 mutants flower later in short days than they do in long days and so fall into the autonomous promotion pathway. Flowering of these mutants is accelerated by cold treatment, suggesting that vernalization does not depend on normal starch metabolism (Bernier et al., 1993).

Phytohormones

The role of GAs in the transition to flowering has been difficult to establish. On the one hand, there are many examples in which the abundance or composition of endogenous GAs changes under conditions that induce flowering (Pharis and King, 1985). Furthermore, because applying certain GAs can induce flowering in some species, there has been an emphasis on the study of GAs in floral initiation and in the search for florigen (reviewed in Chouard, 1960; Evans, 1971; Zeevaart, 1983; Thomas and Vince-Prue, 1997). On the other hand, applied GAs are rarely effective at inducing flowering in short-day plants. Moreover, they generally inhibit flowering of woody angiosperms, although they do promote flowering of conifers (Pharis and King, 1985). Even within long-day plants, the same GA can have a different effect in different species. For example, 2,2-dimethyl GA₄ has potent florigenic activity when applied to Lolium temulentum but has no effect on flowering in S. alba (Bernier et al., 1993).

In Arabidopsis, signaling mediated by GAs appears to play a promotive role in flowering, particularly under noninductive photoperiods (Figure 2). Application of GAs accelerates flowering of wild-type plants under short days (Langridge, 1957) and of the late-flowering mutants fb, fca, fd, fe, co, fpa, ft, fve, and fwa (Table 1) under long days (Chandler and Dean, 1994). Under noninductive photoperiods, the ga1 mutant (Table 1) does not flower unless provided with GAs (Wilson et al., 1992), and the gai mutant (Table 1) flowers very late. Furthermore, spy (Table 1), a mutant considered to exhibit constitutive GA-mediated signal transduction, flowers early (Jacobsen and Olszewski, 1993), as do plants constitutively expressing FPF1, a gene that appears to be involved in GA-mediated signal transduction or responsiveness to GAs (Table 1; Kania et al., 1997).

The role of GAs in activation of the LFY promoter has recently been analyzed (Blázquez et al., 1998). The basal level of LFY promoter activity is lower in ga1 mutants, and the upregulation by long days is delayed. In contrast, LFY activity is slightly higher in a spy mutant grown in short days, correlating with an acceleration of flowering. A cauliflower mosaic virus 35S–LFY transgene was also found to rescue flowering in ga1 mutant plants in short days. Thus, GAs promote flowering in Arabidopsis at least in part by activating LFY expression. Blázquez et al. (1998) also analyzed the direct effect of GA₃ with and without sucrose on LFY promoter activity. GA₃ alone had no effect, sucrose produced a small increase, and both together had a synergistic effect. This requirement for two activation signals for maximal effect may account for observations with excised Lolium apices (McDaniel and Hartnett, 1996). In this study, photoperiodic induction was found to result from two signals acting at the apex. One of these signals has not been identified (but from this analysis, it is possibly sucrose), and the other is GA (McDaniel and Hartnett, 1996).

The role of GAs in vernalization has received particular attention because in some species, application of GAs to vegetatively growing plants can substitute for cold treatment (see Chouard, 1960; Lang, 1965; Evans, 1971; Zeevaart, 1983; Martínez-Zapater et al., 1994). However, in the majority of species examined, including most cereals and nonrosette plants, application of GAs is not sufficient to overcome a requirement for vernalization (Chouard, 1960; Lang, 1965; Evans, 1971; Zeevaart, 1983). Because GAs are involved in flowering processes such as floral evocation (McDaniel and Hartnett, 1996) and bolting (Metzger, 1990), which occur well after the cold treatment, it is possible that application of GAs can simply bypass vernalization completely. Consistent with this possibility is the notion that vernalization may increase the sensitivity of plants to GAs but that GAs have no direct role in the process of vernalization itself (Chouard, 1960).

Further indication that GAs may not play a role in vernalization in Arabidopsis comes from experiments with ga1-3 (Table 1), a mutant severely impaired in GA biosynthesis (Sun and Kamiya, 1994). When combined with fca, which responds strongly to vernalization, the ga1-3 fca double mutants still exhibit a robust vernalization response (J. Chandler and C. Dean, unpublished data). However, because ga1-3 plants still contain residual GAs (T.-p. Sun, personal communication; Zeevaart and Talón, 1992), this result must be interpreted with caution. In summary, the precise role of GAs in the transition to flowering is unclear. Potential tissue-specific changes in GA biosynthesis and sensitivity need to be addressed, as does the potential existence of as-yet-undiscovered florigenic GAs (for a discussion of this possibility, see Evans, 1971; Zeevaart, 1983).

GAs are not the only class of phytohormones that has been implicated in affecting the floral transition. For example, there is evidence from studies on S. alba that long-distance signaling by cytokinins might play a role in the transition to flowering in response to inductive photoperiods (reviewed in Bernier et al., 1993). As discussed above, inductive photoperiods cause the rapid and transient export of sucrose from the leaves to both the shoot and root meristems. In the root, this sucrose leads to the export of cytokinin, primarily zeatin riboside, to the shoot and leaves, presumably via the xylem. Subsequently, another cytokinin, isopentenyladenine riboside, moves out of the leaves, and some makes its way to the shoot apex, where its levels increase within 16 hr of induction (Bernier et al., 1993).

The relative importance of the cytokinin and sucrose fluxes to the floral transition in Arabidopsis remains to be established. Application of cytokinins provokes a phenotype similar to that of deetiolated 1 mutants—early flowering and severe pleiotropic effects on growth (Chory et al., 1994). emf2 has been shown to be allelic (Z.R. Sung, personal communication) to the cytokinin resistance mutant cyr1 (Deikman and Ulrich, 1995), but the apparent lack of mutations that implicate cytokinins in flowering may be due to a high degree of redundancy in the genes involved. Alternatively, the mutant phenotypes may be so pleiotropic that such mutants have not been classified as cytokinin mutants.

In addition to GAs and cytokinins, other phytohormones, such as abscisic acid (ABA), ethylene, and polyamines, may be involved in flowering under certain circumstances and in some species (Martínez-Zapater et al., 1994). The ethylene-insensitive mutant ein2 is slightly delayed in flowering, and ABA-deficient mutants flower somewhat early under noninductive photoperiods (Martínez-Zapater et al., 1994), suggesting a role for ethylene and ABA in floral promotion and repression, respectively.