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. 1999 Aug;120(4):1025–1032. doi: 10.1104/pp.120.4.1025

Independent Regulation of Flowering by Phytochrome B and Gibberellins in Arabidopsis1

Miguel A Blázquez 1, Detlef Weigel 1,*
PMCID: PMC59335  PMID: 10444085

Abstract

Phytochromes and gibberellins (GAs) coordinately regulate multiple aspects of Arabidopsis development. Phytochrome B (PHYB) promotes seed germination by increasing GA biosynthesis, but inhibits hypocotyl elongation by decreasing the responsiveness to GAs. Later in the life cycle of the plant, PHYB and GAs have opposite effects on flowering. PHYB delays flowering, while GAs promote flowering, particularly under noninductive photoperiods. To learn how PHYB and GAs interact in the control of flowering, we have analyzed the effect of a phyB mutation on flowering time and on the expression of the floral meristem-identity gene LFY (LEAFY). We show that the early flowering caused by phyB correlated with an increase in LFY expression, which complements our previous finding that GAs are required for activation of LFY under noninductive photoperiods (M.A. Blázquez, R. Green, O. Nilsson, M.R. Sussman, D. Weigel [1998] Plant Cell 10: 791−800). Since phyB did not change the GA responsiveness of the LFY promoter and suppressed the lack of flowering of severe GA-deficient mutants under short days, we propose that PHYB modulates flowering time at least partially through a GA-independent pathway. Interestingly, the effects of PHYB on flowering do not seem to be mediated by transcriptional up-regulation of genes such as CO (CONSTANS) and FT (Flowering locus T), which are known to mediate the effects of the photoperiod-dependent floral-induction pathway.

The control of plant development by light is exerted through the activity of photoreceptors. Among these, phytochromes mediate the responses to red and far-red light (Fankhauser and Chory, 1997). In Arabidopsis, phytochromes are encoded by five different genes, PHYA through PHYE (Sharrock and Quail, 1989; Quail et al., 1995). While each phytochrome seems to have specific roles, there is also considerable overlap in the function of individual phytochromes (Reed et al., 1994; Aukerman et al., 1997; Devlin et al., 1998). For example, PHYA seems to have a primary role in germination and in the regulation of seedling morphogenesis by far-red light (Nagatani et al., 1993; Parks and Quail, 1993; Whitelam et al., 1993). Later in development, PHYA is involved in sensing photoperiod, which is reflected in the insensitivity of phyA mutants to night breaks (Reed et al., 1994). PHYB, on the other hand, is an essential component of the shade-avoidance mechanism, and modulates the expression of genes in response to red light. Null mutations in PHYB cause increased elongation of hypocotyls, leaf petioles, and stems, as well as decreased chlorophyll accumulation and earlier flowering under both long and short photoperiods (Reed et al., 1993).

Phytochromes interact with plant hormones of the GA class to regulate certain aspects of plant development (Chory and Li, 1997). The phenotype of mutants defective in GA biosynthesis has confirmed that GAs regulate processes such as seed germination, cell expansion, and flowering, all of which are also under the control of phytochromes. For example, the Arabidopsis spy (spindly) mutant has a slender stature, is pale green, and flowers early, thus resembling phyB mutants, or wild-type plants treated with GA3 (Jacobsen and Olszewski, 1993). An opposite phenotype is seen in GA-deficient mutants of Arabidopsis that are dark-green dwarves that flower late (Koornneef and van der Veen, 1980). This phenotype is particularly severe in the ga1-3 mutant, which is blocked in a very early step of GA biosynthesis, and never flowers under short days (Wilson et al., 1992). Reduced expression of LFY (LEAFY), a floral meristem-identity gene, seems to be a main cause of the flowering defect in ga1-3 mutants, since constitutive expression of LFY from a transgene is sufficient to restore flowering of ga1-3 mutants under short days (Blázquez et al., 1998).

There are at least two possible mechanisms by which phytochromes and GAs may interact. The finding of elevated GA levels in the phyB-deficient mutants ein of Brassica rapa and ma3R of sorghum has suggested that PHYB regulates GA biosynthesis in certain species (Rood et al., 1990; Beall et al., 1991). However, the relationship between GA biosynthesis and PHYB activity in sorghum is complex, as GA biosynthesis follows a circadian regulation, and the ma3R mutant shows a phase shift in GA accumulation (Lee et al., 1998). An interaction between phytochromes and GA biosynthesis is also supported by the finding that GA-biosynthetic genes in Arabidopsis and spinach are regulated by light. In both species, GA 20-oxidase mRNA levels are higher under long than under short photoperiods (Xu et al., 1995; Wu et al., 1996). It has been suggested that GA 20-oxidase activity is limiting for stem elongation in long days, and that long photoperiods raise the concentration of active GAs above a certain threshold (Talón et al., 1991).

Similarly, GA 3β-hydroxylation is promoted by pulses of far-red light in cowpea (Martínez-García and García-Martínez, 1992) and by red light in Arabidopsis seeds (Yamaguchi et al., 1998), indicating that the synthesis of active GA species is under phytochrome control. In different situations, however, phytochromes have been shown to affect the responsiveness to GAs, rather than their biosynthesis. Putative phyB mutants of pea and cucumber, lv and lh, as well as the phyB mutant of Arabidopsis have wild-type levels of endogenous GAs but show enhanced hypocotyl elongation in response to exogenous GAs (Weller et al., 1994; López-Juez et al., 1995; Reed et al., 1996). At least in Arabidopsis, PHYB thus appears to control GA-dependent hypocotyl elongation by modulating GA sensitivity as opposed to regulating GA biosynthesis.

In this study, we have addressed the question of whether PHYB and GAs interact in the regulation of flowering in a way similar to what is observed for other responses, such as hypocotyl elongation, in Arabidopsis. Using promoter activity of the floral regulator LFY as an indicator, we show that PHYB and GAs regulate LFY expression independently. This finding is corroborated by the observation that the loss of PHYB function allowed GA-deficient mutants to flower under short days.

MATERIALS AND METHODS

Plant Material

LFY::GUS lines (DW150-304 and 304G1) in the Landsberg erecta background of Arabidopsis have been previously described (Blázquez et al., 1997, 1998; Hempel et al., 1997). Lines 304B5 (phyB-5 LFY::GUS) and 304G1B5 (ga1-3 phyB-5 LFY::GUS) were constructed by crossing line 304G1 (ga1-3 LFY::GUS) to plants carrying the phyB-5 mutations, a null allele in the Landsberg erecta background (Reed et al., 1993). Transgenic plants homozygous for ga1-3 were initially identified by their short stature and dark-green color, and were then confirmed by PCR (Silverstone et al., 1997). The presence of the phyB-5 allele was also monitored by PCR using a dCAPS marker (Neff et al., 1998). Lines homozygous for the transgene were identified by testing F3 progeny.

Growth Conditions

For experiments on soil, seeds were stratified for 2 to 3 d at 4°C before sowing. Plants were grown at 23°C in long (16 h of light and 8 h of dark) or short days (9 h of light and 15 h of dark) under a mixture of 3:1 cool-white and Gro-Lux fluorescent lights (Osram Sylvania, Danvers, MA). The spectral quality of the light received by the plants under these conditions was determined with a portable spectroradiometer (model LI-1800, LI-COR) and is shown in Figure 1. ga1-3 mutants required exogenous GAs to germinate (Koornneef and van der Veen, 1980) and were incubated with 50 μm GA3 (Sigma) during stratification. Seeds were rinsed thoroughly with water before sowing. Vegetatively growing plants were sprayed twice weekly with a solution of 100 μm GA3 and 0.02% (v/v) Tween 20 (Bio-Rad).

Figure 1.

Figure 1

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Light spectrum inside the growth chambers used in this work. The solid black line represents the spectrum provided by a 3:1 mixture of cool-white to fluorescent light, while the dotted line represents the spectrum provided by cool-white light alone.

The dose-response experiments with GA3 or paclobutrazol (Zeneca Ag Products, Wilmington, DE) were performed with seedlings growing on MS plates (Murashige and Skoog, 1962) without Suc under the light conditions described above. The fluence rate was around 66 μmol m−2 s−1. In experiments with soil-grown plants, paclobutrazol was applied by watering with a 37 mg/L solution.

Hypocotyl and GUS Activity Measurements

Hypocotyls were measured using a digitized image of 12 to 18 seedlings that had been placed between transparent acetate sheets (Neff and Chory, 1998). The image was analyzed with the public domain NIH Image program (developed at the United States National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). For quantitative measurements of GUS activity using 4-methylumbelliferyl-β-d-glucopyranoside as a substrate, samples of plants grown on soil or MS plates were collected and treated as previously described (Blázquez et al., 1997).

RNA Extraction and Analysis

Total RNA was extracted with TRIzol reagent as indicated by the manufacturer (GIBCO-BRL). RT-PCR was conducted on 1 μg of total RNA. cDNA synthesis was performed with a reverse transcription kit (Promega). A fragment of the CO (CONSTANS) gene was amplified using oligonucleotides 5′-ACG CCA TCA GCG AGT TCC-3′ and 5′-AAA TGT ATG CGT TAT GGT TAA TGG-3′ as primers (P. Reeves and G. Coupland, personal communication). FT was amplified using 5′-ACT ATA TAG GCA TCA TCA CCG TTC GTT ACT CG-3′ and 5′-ACA ACT GGA ACA ACC TTT GGC AAT G-3′ (J.H. Ahn and D. Weigel, unpublished data). As a control we used oligos 5′-GAT CTT TGC CGG AAA ACA ATT GGA GGA TGG T-3′ and 5′-CGA CTT GTC ATT AGA AAG AAA GAG ATA ACA GG-3′, which amplify two polyubiquitin gene fragments in the Landsberg erecta ecotype (Callis et al., 1995). The amplified fragments were separated on an agarose gel, blotted onto a membrane, and hybridized with radiolabeled CO, FT, and UBQ10 probes. Signal intensities were determined with a phosphor imager (Molecular Dynamics), and the values in the exponential range of amplification were compared.

RESULTS

Enhanced LFY Up-Regulation in phyB Mutants

Mutations at the PHYB locus cause early flowering, especially under short photoperiods (Goto et al., 1991; Reed et al., 1993) (Table I). Since several other mutations that affect flowering time also change the expression level of LFY (Blázquez et al., 1998; Nilsson et al., 1998), we investigated the effect of the phyB-5 null mutation on the activity of the LFY promoter using a fusion of the LFY promoter to the GUS reporter, which faithfully reflects endogenous LFY expression (Blázquez et al., 1997). Plants homozygous for the phyB-5 mutation and a LFY::GUS transgene (304B5) and isogenic PHYB+ plants (DW150-304) were grown on soil under long and short photoperiods, and GUS activity in the apices was determined at different ages during the vegetative phase. As shown in Figure 2, the phyB-5 mutation caused an increase in the expression of LFY::GUS under both photoperiods, although this effect was more pronounced under short days. This result paralleled the acceleration of flowering observed in these plants (Table I), and indicates that PHYB represses LFY expression.

Table I.

Flowering time of the ga1-3 and phyB-5 lines used in this study

Line Genotype Long Days
Short Days
Short Days + GA3
RL CL TL RL CL TL RL CL TL
150-304 Wild type 8.8  ± 0.2 3.1  ± 0.4 11.9  ± 0.3 25.1  ± 0.9 7.5  ± 0.3 32.6  ± 0.8 16.2  ± 0.9 10.0  ± 0.5 26.2  ± 0.8
304B5 phyB-5 7.4  ± 0.5 4.9  ± 0.7 12.1  ± 0.8 9.1  ± 0.6 4.6  ± 0.4 13.7  ± 0.7 7.0  ± 0.3 5.6  ± 0.4 12.6  ± 0.8
304G1 ga1-3 –a –a 15.3  ± 0.7 >58 –b >58 18.0  ± 0.9 8.3  ± 0.8 26.3  ± 0.9
304G1B5 ga1-3 –a –a 17  ± 1 –a –a 26  ± 1 8.1  ± 0.6 5.2  ± 0.3 13.3  ± 0.7
phyB-5
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All lines are in the Landsberg erecta background and homozygous for a LFY::GUS transgene. RL, Rosette leaves; CL, cauline leaves; TL, total number of leaves. Values are the means ± 2 se (i.e. with a 95% confidence interval). n ≥ 12 plants.

a

– indicates that these plants did not bolt in long days and that rosette and cauline leaves could not be distinguished. 

b

– indicates that ga1-3 plants did not flower in short days and that rosette and cauline leaves could not be distinguished. 

Figure 2.

Figure 2

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LFY::GUS expression during vegetative growth of phyB-5 mutants. Plants homozygous for the LFY::GUS transgene either in a PHYB+ (DW150-304; white symbols) or phyB-5 background (307B5; black symbols) were grown in long days (squares) or short days (circles) until flower buds were visible to the naked eye. Values are expressed as means ± 2 se (n = 12). Time represents days after sowing. Error bars that are not visible are contained within the symbol. MUG, 4-Methylumbelliferyl-β-d-glucopyranoside.

Application of GA3 from germination on accelerated flowering of wild-type plants, but not of phyB-5 mutants under short days (Table I). Although the number of rosette leaves was lower in GA3-treated phyB-5 plants than in untreated plants, the total number of leaves, including cauline leaves, was not significantly different between these populations (Table I). Consistent with the absence of an effect on flowering time, we observed no further increase in LFY::GUS expression when phyB-5 plants were treated with GA3 (results not shown).

Suppression of the Flowering Defect of ga1 Mutants by phyB

The increased expression level of LFY::GUS in phyB-5 mutants resembles what is seen upon application of GAs to wild-type plants (Blázquez et al., 1997) or in mutants with enhanced GA-signaling, such as spy (Blázquez et al., 1998). Since several other effects of PHYB signaling, such as hypocotyl elongation and seed germination, appear to be mediated by GAs, we wanted to know whether the early flowering of phyB mutants depended on the activity of GAs. Therefore, we constructed ga1-3 phyB-5 mutant plants and cultivated them under long and short photoperiods. Under long days, the double mutants were similar in size to ga1-3 plants, and much smaller than phyB-5 or wild-type plants, as previously described (Peng and Harberd, 1997). While flowering time of ga1-3 phyB-5 mutants under long days was not different from that of ga1-3 plants, the phyB-5 mutation suppressed the flowering defect of ga1-3 mutants under short days (Table I; Fig. 3). When plants were grown in a mixture of fluorescent and cool-white light, suppression was observed in over 90% of the double mutants after 7 weeks. Among these plants, the number of leaves produced before flowering did not deviate much from the mean, and the value was intermediate between that of wild-type and phyB-5 plants (Table I).

Figure 3.

Figure 3

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Suppression of the ga1-3 flowering defect in short days by phyB-5. Representative plants were photographed 50 d after sowing. Arrowhead indicates flowers.

Decreased LFY expression appears to be a main cause of the late-flowering phenotype of ga1 mutants under long days, and of the inability of ga1-3 mutants to flower at all under short days (Blázquez et al., 1998). To determine whether phyB suppressed the ga1 mutant flowering defect by restoring more normal levels of LFY promoter activity, we examined LFY::GUS expression in ga1-3 phyB-5 double mutants. As shown in Figure 4A, LFY::GUS expression under long days followed a similar pattern in both ga1-3 and ga1-3 phyB-5 mutants. The expression levels were very low during the first 15 d of growth, and although LFY::GUS expression eventually increased, it never reached the levels seen in wild-type plants. Application of GA3 restored the expression pattern seen in wild-type plants and phyB-5 single mutants. In contrast to long days, LFY::GUS expression remained very low in short-day-grown ga1-3 single mutants during the entire experimental period (Fig. 4B) (Blázquez et al., 1998). Although LFY::GUS expression in ga1-3 phyB-5 double mutants increased shortly before flowering occurred, it was not different from the ga1-3 single mutant during the first 4 weeks. Upon application of GA3, ga1-3 and ga1-3 phyB-5 mutants flowered as early as wild-type and phyB-5 plants treated with GA3 (Table I), which was paralleled by a similar increase in LFY::GUS expression (Fig. 4B).

Figure 4.

Figure 4

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LFY::GUS expression during vegetative growth of ga1-3 and ga1-3 phyB-5 mutants. Plants homozygous for the LFY::GUS transgene were grown in long (A) or short (B) days until flower buds were visible to the naked eye, except in the case of the ga1-3 mutant without GA3 treatment under short days (□), which had not flowered at the end of the experiment. 304G1 (ga1-3, white symbols) and 304G1B5 (ga1-3 phyB-5, black symbols) were treated with GA3 (circles) or left untreated (squares). Values are expressed as means ± 2 se (n = 12). Time represents days after sowing. Error bars that are not visible are contained within the symbol. MUG, 4-Methylumbelliferyl-β-d-glucopyranoside.

Interaction between PHYB and GAs

The suppression by phyB-5 of the flowering defect of ga1-3 mutants, along with the weak up-regulation of LFY::GUS expression in the ga1-3 phyB-5 double mutants are compatible with the idea that PHYB modulates flowering and LFY expression independently of GAs. However, the ga1-3 mutation does not completely abolish GA biosynthesis, and several GA species are still detectable in the ga1-3 mutant (Zeevaart and Talón, 1992; A. Silverstone, and T.-P. Sun, personal communication). In addition, there is the possibility that physiologically relevant levels of exogenous GAs are carried over from the parental generation or from the seed treatment required for germination. Thus, PHYB could act by increasing the low levels of GA biosynthesis or by enhancing the responsiveness toward the small amount of GAs present in ga1-3 mutants. An effect of phyB on GA biosynthesis is unlikely, since overall levels of several GA intermediates are unchanged in phyB mutants compared with the wild type (Reed et al., 1996). phyB mutants show enhanced responsiveness to GAs, as monitored by the dose response of hypocotyl elongation (Reed et al., 1996). We have found that continuous watering of plants with paclobutrazol, an inhibitor of the early steps of GA biosynthesis (Rademacher, 1991), did not prevent the flowering of ga1 phyB or phyB mutants under short days, while it abolished flowering of wild-type plants (results not shown).

To resolve the question of whether PHYB affects flowering by regulating GA biosynthesis or GA response, we determined whether increased responsiveness to GAs could account for the increased LFY::GUS expression in phyB mutants. ga1-3 and ga1-3 phyB-5 seedlings carrying a LFY::GUS transgene were grown on plates containing increasing concentrations of GA3. The hypocotyl length of each seedling was determined before measuring LFY::GUS activity. As previously reported (Reed et al., 1996), the hypocotyl of phyB mutants was longer than that of PHYB+ plants at all GA3 concentrations. More importantly, phyB mutants were also more responsive to exogenous GA3 than wild-type plants (Fig. 5A). In contrast, levels of LFY::GUS activity in phyB mutants did not show increased responsiveness to exogenous GA3 over the range of concentrations tested (Fig. 5B). The results were the same under long or short days.

Figure 5.

Figure 5

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Responses of ga1-3 and ga1-3 phyB-5 seedlings to exogenous GA3. Seeds of the lines 304G1 (LFY::GUS ga1-3, ○) and 304G1B5 (LFY::GUS ga1-3 phyB-5, •) were sown on MS plates containing the indicated concentrations of GA3. Hypocotyl length (A) and GUS activity (B) were determined 7 d after sowing. Values are expressed as means ± 2 se (n = 12). Time represents days after sowing. Error bars that are not visible are contained within the symbol. MUG, 4-Methylumbelliferyl-β-d-glucopyranoside.

To confirm that the responsiveness to exogenous GA3 reflects the behavior toward endogenous GAs, we analyzed both hypocotyl length and LFY::GUS activity in wild-type and phyB-5 seedlings growing on plates with increasing concentrations of paclobutrazol. As expected, paclobutrazol reduced the elongation of wild-type and phyB hypocotyls starting at concentrations as low as 0.03 μm (Fig. 6A) (Reed et al., 1996). At higher concentrations, paclobutrazol inhibited hypocotyl elongation faster in phyB-5 mutants than in wild-type plants. Although paclobutrazol reduced LFY::GUS expression in both wild-type and phyB-5 plants, there was no difference in responsiveness between the two lines (Fig. 6B).

Figure 6.

Figure 6

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Responses of ga1-3 and ga1-3 phyB-5 seedlings to the GA-biosynthesis-inhibitor paclobutrazol. Seeds of the lines DW150-304 (LFY::GUS, ○) and 304B5 (LFY::GUS phyB-5, •) were sown on MS plates containing the indicated concentrations of paclobutrazol. Hypocotyl length (A) and GUS activity (B) were determined 7 d after sowing. Values are expressed as means ± 2 se (n = 12). Time represents days after sowing. Error bars that are not visible are contained within the symbol. MUG, 4-Methylumbelliferyl-β-d-glucopyranoside.

Effect of phyB Mutation on Expression of CO and FT

GAs act redundantly with the long-day-dependent pathway of floral induction, as GA deficiency has much weaker effects on flowering in long than in short days (Wilson et al., 1992). In addition, double mutants carrying both the ga1-3 mutation and a mutation in CO, an essential element of the long-day pathway, often do not flower at all in long days (Putterill et al., 1995). Since all of our other data pointed to a GA-independent effect of PHYB on flowering, we wanted to determine whether the early flowering of phyB mutants was caused by an increase in the expression of genes known to be involved in the photoperiod-dependent pathway that promotes flowering. The expression of two genes in this pathway has been shown to be limiting for flowering, since their overexpression causes very early flowering under both long and short days. These genes are CO (Simon et al., 1996) and FT (I. Kardailsky and D. Weigel, unpublished data).

When we analyzed the expression of CO and FT by RT-PCR, we found that the expression levels of CO and FT did not differ dramatically between wild-type and phyB-5 plants (Fig. 7), suggesting that the early-flowering phenotype of phyB mutants under short days is not caused by overexpression of genes in the long-day pathway.

Figure 7.

Figure 7

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CO and FT RNA expression in phyB mutants. Seedlings of the lines DW150-304 (wild type [WT], white bars) and 304B5 (phyB-5, black bars) were grown on MS plates under short days (SD), and harvested during the 8th h of light on the indicated days. Expression was analyzed by RT-PCR (bottom panel) and the signals quantified and normalized using UBQ expression as a control (top panel, arbitrary units).

DISCUSSION

The level of LFY expression is an important determinant for the identity of the primordia that arise at the flanks of the shoot apical meristem during the transition to flowering (Blázquez et al., 1997). This idea has been corroborated by the observation that certain mutations that delay flowering, such as co or gi (gigantea), also reduce the level of LFY expression during the time that the transition to flowering occurs in wild type (Nilsson et al., 1998). In a similar way, the acceleration of flowering caused by mutations such as spy is paralleled by increased LFY expression (Blázquez et al., 1998). Hence, it is not surprising that PHYB, which represses flowering, functions as a negative regulator of the LFY promoter, although it has been difficult to assess whether phyB mutants flower early exclusively because of increased LFY expression, or also because of an increased response to LFY activity (Nilsson et al., 1998).

In our growth conditions (Fig. 1), PHYB affected flowering time mainly under short photoperiods. Although the total number of leaves produced was similar under long and short days (Table I), the number of days needed to produce the first flower was higher under short days, as previously reported (Koornneef et al., 1995). Accordingly, the increase of LFY promoter activity caused by the phyB mutation was more clearly observed under short than under long days. It has been previously observed that the contribution of different photoreceptors to the control of flowering time changes with photoperiod (Bagnall et al., 1995). For example, a specific role for the blue/UVA photoreceptor encoded by CRY2 is the promotion of flowering under long days (Guo et al., 1998; Lin et al., 1998).

The observation that a phyB mutation has a more pronounced effect on LFY expression under short days suggests an interaction with GAs, since GAs are essential for flowering under noninductive conditions (Martínez-Zapater et al., 1994). However, we present evidence that PHYB and GAs regulate LFY expression through independent pathways, since a phyB mutation did not enhance the response of the LFY promoter to GAs. An independent action was confirmed by the observation that phyB suppressed the flowering defect of ga1 mutants under short days, even when any GA1-independent synthesis of ent-kaurene was inhibited by paclobutrazol treatment. The simplest scenario for this suppression would be activation of the long-day-dependent pathway of flowering. However, the observation that the levels of RNA expression of CO and FT, the two genes believed to act downstream in the long-day pathway (Simon et al., 1996; Koornneef et al., 1998), are not dramatically changed in phyB mutants suggests that this activation would not occur at the transcriptional level. Alternatively, the suppression could take place through the FCA-dependent autonomous pathway (Macknight et al., 1997).

An important conclusion from our study and previous studies is that the relationship between PHYB and GAs is complex. For certain responses, such as germination, the GA-deficient ga1 mutant is completely epistatic over phyB. However, most other characteristics of ga1 phyB double-mutant seedlings are intermediate between those observed for the single mutant parents (Peng and Harberd, 1997; this study). Finally, while PHYB regulates hypocotyl elongation by modulating responsiveness to GAs (Reed et al., 1996), PHYB acts independently of GAs in the control of flowering time (this study). That genes do not always interact in the same fashion, even when controlling the same targets, is not uncommon in development. For instance, PHYA and PHYB regulate common responses to light, but they do so differently depending on the particular response. While both phytochromes have an inhibitory effect on hypocotyl elongation and promote seed germination, flowering is repressed by PHYB but promoted by PHYA.

It is not easy to imagine the molecular mechanism that integrates the different interactions between GAs and PHYB. Based on the effects of application of different GAs to ryegrass, it has been proposed that certain GA species possess low florigenic activity but promote stem elongation very efficiently and vice versa (Evans et al., 1990). For instance, 3β-hydroxylation is required for the promotion of stem elongation, but not of flowering in this plant (Evans et al., 1994). If these findings reflect the presence of different receptors for the various active GA species, PHYB might regulate receptors specific for GA species involved in hypocotyl and stem elongation but not the ones involved in flowering.

ACKNOWLEDGMENTS

We thank Ove Nilsson, Michael Neff, and José Miguel Martínez-Zapater for discussion and critical reading of the manuscript; Trevor Phan for technical assistance with the hypocotyl length measurements; Ji Hoon Ahn, Paul Reeves, and George Coupland for advice concerning the RT-PCR experiments; and Aron Silverstone for communicating unpublished results. phyB-5 mutant seeds were obtained from Joanne Chory.

Abbreviation:

RT

reverse transcriptase

Footnotes

1

This work was supported by grants from the National Science Foundation (no. MCB–9723823 to D.W.) and the Human Frontiers Science Program Organization (no. RG 303/97 to D.W.). M.A.B. received fellowships from the Spanish Ministry of Education and the Human Frontiers Science Program Organization. D.W. is a National Science Foundation Young Investigator and receives support from Agritope (Oregon).

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