Plant Physiol. (1999) 119: 1199-1208

Extragenic Suppressors of the Arabidopsis gai Mutation Alter the Dose-Response Relationship of Diverse Gibberellin Responses¹

Jinrong Peng, Donald E. Richards, Thomas Moritz, Ana Caño-Delgado, and Nicholas P. Harberd^*

Department of Molecular Genetics, John Innes Centre, Colney Lane, Norwich NR4 7UJ, United Kingdom (J.P., D.E.R., A.C.-D., N.P.H.); and Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, S-90183, Umeå, Sweden (T.M.)

	ABSTRACT

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Active gibberellins (GAs) are endogenous factors that regulate plant growth and development in a dose-dependent fashion. Mutant plants that are GA deficient, or exhibit reduced GA responses, display a characteristic dwarf phenotype. Extragenic suppressor analysis has resulted in the isolation of Arabidopsis mutations, which partially suppress the dwarf phenotype conferred by GA deficiency and reduced GA-response mutations. Here we describe detailed studies of the effects of two of these suppressors, spy-7 and gar2-1, on several different GA-responsive growth processes (seed germination, vegetative growth, stem elongation, chlorophyll accumulation, and flowering) and on the in planta amounts of active and inactive GA species. The results of these experiments show that spy-7 and gar2-1 affect the GA dose-response relationship for a wide range of GA responses and suggest that all GA-regulated processes are controlled through a negatively acting GA-signaling pathway.

	INTRODUCTION

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GA is essential for normal plant growth regulation (Hooley, 1994), and studies of GA-deficient and GA-response mutants have greatly advanced our understanding of GA action (Ross, 1994; Swain and Olszewski, 1996; Ross et al., 1997; Harberd et al., 1998). GA responses are dose dependent (Bethke and Jones, 1998). For example, GA-deficient mutant Arabidopsis hypocotyls are short in the absence of exogenous GA and become progressively longer with increasing doses of exogenous GA. As the GA dose increases further, the corresponding increase in hypocotyl length becomes less, and eventually the response reaches saturation, at which time further increases in GA dose cause little or no further increase in hypocotyl length (Jacobsen and Olszewski, 1993; Reed et al., 1996; Silverstone et al., 1997; Cowling et al., 1998).

The phenotype of the Arabidopsis gai mutant mimics the effects of GA deficiency: gai plants are dwarfed and have dark-green leaves (Koornneef et al., 1985; Peng and Harberd, 1993, 1997a; Wilson and Somerville, 1995; Peng et al., 1997; Harberd et al., 1998). However, unlike GA-deficient mutants, gai mutants cannot be restored to normal phenotype by application of GA (Koornneef et al., 1985; Wilson and Somerville, 1995; Peng and Harberd, 1997a) and accumulate bioactive GAs (GA₁ and GA₄) to a higher level than do wild-type plants (Talon et al., 1990a). Recent experiments have shown that in normal plants endogenous GA levels are homeostatically controlled via GA-mediated negative feedback regulation (Hedden and Croker, 1992). As a part of this process, the expression of a number of the genes encoding enzymes involved in GA biosynthesis is down-regulated by GA (Chiang et al., 1995; Phillips et al., 1995; Xu et al., 1995; Martin et al., 1996; Hedden and Kamiya, 1997; Cowling et al., 1998). This down-regulation is perturbed in gai (Talon et al., 1990a; Xu et al., 1995; Peng et al., 1997), thus explaining the increased endogenous GA levels in gai and indicating the involvement of GAI (the GAI gene product) in the homeostatic regulation of GA levels.

The GA responses of gai are saturated because the gai phenotype is unaffected by exogenous GA. However, the gai phenotype is affected by reductions in the endogenous GA level (Koornneef et al., 1985). ga1-1 mutants are severely dwarfed and GA deficient (Koornneef and van der Veen, 1980; Sun et al., 1992). gai ga1-1 double mutants are more severely dwarfed than gai and are similar in height to a ga1-1 single mutant (Koornneef et al., 1985). Treatment of gai ga1-1 plants with increasing doses of GA results in progressive increases in plant height, and a sufficiently large dose fully restores the height of gai ga1-1 plants to that of gai (Koornneef et al., 1985). Thus, gai plants are not completely GA insensitive and exhibit reduced GA responses (Wilson and Somerville, 1995; Cowling, 1997).

GAI encodes a protein that may act as a GA-controlled transcriptional regulator (Peng et al., 1997). gai encodes a mutant protein that lacks a 17-amino acid segment near the N terminus (Peng et al., 1997). In addition, RGA, another Arabidopsis gene involved in GA signaling, encodes a protein (RGA) whose amino acid sequence is closely related to that of GAI. GAI and RGA appear to have overlapping roles as negative regulators of GA signaling (Peng et al., 1997; Harberd et al., 1998; Silverstone et al., 1998).

The Arabidopsis spy mutations confer resistance to the plant-growth and GA-biosynthesis inhibitor PAC (Hedden and Graebe, 1985; Davis and Curry, 1991) and suppress the effects of GA deficiency (Jacobsen and Olszewski, 1993; Jacobsen et al., 1996). Thus, although the GA-deficient ga1-2 mutant requires exogenous GA for germination (Koornneef and van der Veen, 1980; Sun et al., 1992), spy-1 ga1-2 double-mutant seeds germinate in the absence of exogenous GA (Jacobsen and Olszewski, 1993). In addition, spy mutations can suppress the phenotype conferred by gai (Jacobsen et al., 1996). GA dose-response data suggest that spy mutants are not saturated in their GA responses (Jacobsen and Olszewski, 1993; Jacobsen et al., 1996). SPY encodes a tetratrichopeptide repeat protein (SPY) that has significant homology to animal O-linked N-acetylglucosamine transferase (Jacobsen et al., 1996; Robertson et al., 1998). The gar2-1 mutation also suppresses the gai phenotype and identifies a genetic locus (GAR2) that is distinct from SPY (Wilson and Somerville, 1995).

Previously, we described a model to explain the GA regulation of stem elongation (Peng et al., 1997; Harberd et al., 1998). According to this model, the GAI (wild-type gene), RGA, SPY, and GAR2 gene products are components or modulators of a signaling pathway that negatively regulates stem elongation and whose activity is opposed by GA. Here we describe further studies of the effects of the spy-7 and gar2-1 alleles on GA signaling. We wanted to (a) know whether spy-7 and gar2-1 affect the regulation of several different GA responses, in addition to their previously documented effects on the GA regulation of stem elongation; (b) test the effects of spy-7 and gar2-1 on endogenous GA levels; and (c) investigate the possibility of interactions between these two mutations by studying the effects of spy-7 and gar2-1 in single- and double-mutant combinations. The results of these experiments suggest that the negative regulation model of GA signaling, which was originally based on observations of stem elongation, is generally applicable to a wide range of GA responses. In addition, spy-7 and gar2-1 alter the GA dose-response relationship by reducing the amount of GA needed to elicit a given level of response.

	MATERIALS AND METHODS

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Plant Mutant Lines

To identify plants carrying gar2-1 in the absence of gai, gai gar2-1 plants were crossed with the wild type. F₂ plants that were visually indistinguishable from the wild type were identified and their F₃ progeny were further analyzed. F₃ families that segregated dwarf (gai) plants were discarded. The remaining 10 F₃ families contained only tall plants, which were all homozygous for GAI. Because GAR2 is unlinked to GAI, the plants in these families were all homozygous for GAR2, all homozygous for gar2-1, or segregating for GAR2/gar2-1 heterozygotes and homozygotes. Nine of these 10 F₃ families exhibited PAC-resistant seed germination (data not shown), presumably conferred by gar2-1.

Plant Maintenance

For plants grown in sterile conditions, seeds were surface sterilized and sown on germination medium (Valvekens et al., 1988) supplemented (where appropriate) with GA₃ or PAC, and then grown in an 18-h photoperiod (Peng and Harberd, 1993; Peng et al., 1997). Germination tests compared batches of seeds harvested at approximately the same time and were scored 7 d after sowing.

Measurement of Plant Growth and Chlorophyll Content

Determination of Endogenous GA Content

The HPLC system consisted of a pump (model 600, Waters) connected via an autosampler (model 717, Waters) to a 4-µm Nova-Pak C₁₈ column (150 × 3.9 mm i.d., Waters). The mobile phase was a 20-min linear gradient of 20% to 100% methanol in 1% aqueous acetic acid at a flow rate of 1 mL min¹. Five fractions corresponding to the GAs of interest were dried, methylated with ethereal diazomethane, and after evaporation trimethylsilylated in 20 µL of dry pyridine/N,O-bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (50:50:1, v/v) at 70°C for 30 min. The derivatization mixture was then reduced to dryness and dissolved in dichloromethane. Samples were injected in the splitless mode into an HP 5890 gas chromatograph (Hewlett-Packard), fitted with a fused silica glass capillary column (30 m long, 0.25 mm i.d.), with a chemically bonded 0.25-µm DB-5MS stationary phase (J & W Scientific, Folsom, CA). The injector temperature was 270°C. The column temperature program varied depending on which GA was being analyzed. The column effluent was introduced into the ion source of a JMS-SX/SX102A mass spectrometer (JEOL). The interface temperature was 280°C and the ion source temperature was 250°C. The acceleration voltage was 10 kV, and ions were generated with 70 eV at an emission current of 500 µA. For quantification, samples were analyzed in either high resolution selected-ion monitoring mode or selected-reaction monitoring mode (Moritz and Olsen, 1995). For each of the GAs, calibration curves were recorded from 0.5 to 20 pg of GA with 5 pg of [²H₂]GA as the internal standard. All data were processed by a JEOL MS-MP7010 data system.

	RESULTS

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spy-7 and gar2-1 Confer PAC-Resistant Seed Germination

gar2-1 is a dominant, partial suppressor of the gai phenotype and is unlinked to either gai or spy (Wilson and Somerville, 1995; J. Peng and N.P. Harberd, unpublished data). To determine whether gar2-1 also confers PAC-resistant seed germination, the PAC resistances of wild-type, gai, gai spy-7, gai gar2-1, and gai spy-7 gar2-1 seeds were compared (Fig. 1). Although the wild type and gai failed to germinate, approximately 50% of gai spy-7 seeds achieved germination, consistent with spy-7 being a relatively mild spy allele (Peng et al., 1997). It is interesting that gai gar2-1 seeds are more PAC resistant than are gai spy-7 seeds (approximately 90% of the gai gar2-1 seeds germinated). The PAC resistance of gai spy-7 gar2-1 seeds was similar to that of gai gar2-1 (Fig. 1).

View larger version (36K): [in this window] [in a new window]   Figure 1. Germination of wild-type (WT), gai, gai spy-7, gai gar2-1, and gai spy-7 gar2-1 seeds grown in the presence (gray bars) or absence (black bars) of 0.1 mM PAC. Germination was scored 7 d after moving the plates containing the seeds from the cold room. Results are presented as mean ± SE of three separate experiments (for each sample, n = 10-22).

To determine the phenotype conferred by gar2-1 in the absence of gai, gai gar2-1 was backcrossed to the wild type and F₂ and F₃ phenotypes were analyzed (see ``Materials and Methods''). These experiments showed that gar2-1 (in the absence of gai) confers PAC-resistant seed germination and a visible adult plant phenotype that is not obviously different from that conferred by the wild-type (GAR2) allele.

gai gar2-1 Plants Do Not Respond to Exogenous GA

We compared the effects of exogenous GA on the growth of gai gar2-1 plants with its effects on the growth of gai and other controls (Fig. 2). As expected, wild-type plants showed an effect of GA₃ on growth, ga4 (GA deficient; Talon et al., 1990b) plants exhibited a marked growth response, and gai plants exhibited no response. gai gar2-1 plants, although taller than gai plants, did not respond to exogenous GA. Thus, gai gar2-1, like gai spy-7 and gai, was saturated in its GA responses. As for gai spy-7, the plant height associated with the saturated GA responses of gai gar2-1 was greater than that of gai.

View larger version (29K): [in this window] [in a new window]   Figure 2. Comparison of GA response of the wild type (WT), gai, and gai gar2-1. Adult heights (47 d after sowing) of GA3-treated (gray bars; 0.1 mM GA3) and control (black bars) plants are shown. Wild-type and ga4 (GA-deficient mutant) plants were taller after GA treatments, whereas gai and gai gar2-1 were unaffected. Plants were grown in an 18-h photoperiod. Results are presented as means ± SE (n = 18-30).

gai spy-7 gar2-1 Plants Are Taller Than gai spy-7 or gai gar2-1

View larger version (35K): [in this window] [in a new window]   Figure 3. A, Photograph of (left to right) wild-type, gai, gai spy-7, gai gar2-1, and gai spy-7 gar2-1 plants. B, Comparison of adult plant heights (55 d after sowing). Plants were grown in an 18-h photoperiod. Results are presented as means ± SE (n = 19-26).

spy-7 Affects Chlorophyll Accumulation

View larger version (57K): [in this window] [in a new window]   Figure 4. Chlorophyll contents of leaves of wild type, gai, gai spy-7, gai gar2-1, and gai spy-7 gar2-1 (21-d-old plants, grown in an 18-h photoperiod). Results are presented as means ± SE of three separate experiments.

gai spy-7 gar2-1 Plants Flower Earlier Than gai spy-7 or gai gar2-1

View larger version (27K): [in this window] [in a new window]   Figure 5. A, Photograph of (left to right) wild type, gai, gai spy-7, gai gar2-1 and gai spy-7 gar2-1 grown (50 d after sowing) in SD. The wild-type and gai plants do not have open flowers, whereas suppressed gai plants, in particular gai gar2-1 and gai spy-7 gar2-1, have already flowered. B, Comparison of flowering times of the wild type (WT), gai, gai spy-7, gai gar2-1, and gai spy-7 gar2-1 under LD (black bars) and SD (gray bars). Numbers of rosette and cauline leaves that appeared prior to the opening of the first flower bud were counted. Results are presented as means ± SE (n = 29-35).

gai spy-7 gar2-1 Plants Are Strongly Resistant to the Growth-Retarding Effects of PAC

View larger version (30K): [in this window] [in a new window]   Figure 6. Vegetative rosette radii of wild-type (WT), gai, gai spy-7, gai gar2-1, and gai spy-7 gar2-1 plants (30 d old) grown in the presence (gray bars) or absence (black bars) of 1 nM PAC. Results are presented as means ± SE (n = 16-29).

PAC Treatments Reveal GA Responsiveness in gai spy-7 gar2-1 Plants

View larger version (56K): [in this window] [in a new window]   Figure 7. Growth of wild-type, gai, and gai spy-7 gar2-1 plants on germination medium (Valvekens et al., 1988) containing different concentrations of PAC (A, 0 nM PAC; B, 0.5 nM PAC; and C, 5 nM PAC) and GA3. Seeds (15-18 seeds for each sample) were sterilized, placed on appropriate medium, chilled for 7 d at 4°C, and then grown at 23°C with an 18-h photoperiod. For each of the three genotypes, three representative plants (22 d old) are shown for each treatment.

The first of these experiments tested the effects of increasing concentrations of GA₃ in the absence of PAC (Fig. 7A). The wild-type plants exhibited a typical dose-dependent GA response, with 50 nM GA₃ eliciting a stronger response (elongated petioles and pale-green leaf color) than 0.5 nM GA₃, whereas, as expected, the gai plants displayed no obvious response to exogenous GA (Fig. 7A; Koornneef et al., 1985; Carol et al., 1995). The gai spy-7 gar2-1 plants also displayed no obvious response to exogenous GA₃. At all three GA₃ doses, the gai spy-7 gar2-1 plants resembled wild-type plants treated with 50 nM GA₃, having elongated petioles and pale-green leaves (Fig. 7A). Thus, although the phenotypes of gai (dwarf) and gai spy-7 gar2-1 (tall) are different, they are both saturated in their GA responses.

Additional experiments used two concentrations of PAC (low: 0.5 nM PAC, Fig. 7B; and high: 5 nM PAC, Fig. 7C) to reduce the endogenous GA levels in the wild type, gai, and gai spy-7 gar2-1. On high PAC (without exogenous GA) wild-type plants were very small, compact, and dark green (Fig. 7C). However, exogenous GA₃ partially overcame the effects of high PAC on wild-type plants (Fig. 7C). For example, the mean rosette radius of wild-type plants on high PAC alone was 0.24 cm, whereas that of the wild type on high PAC, 50 nM GA, was 0.65 cm (the means of 8 and 10 plants, respectively, P < 0.01), a 1.7-fold increase in rosette radius. GA (0.5 nM) also partially overcame the dwarfism of wild-type plants grown on high PAC, but to a lesser extent than did 50 nM GA, demonstrating that GA reversed the effect of high PAC in a dose-dependent manner (Fig. 7C).

Wild-type plants grown on low PAC were also dwarfed compared with untreated controls (Fig. 7B), although not as dwarfed as the wild type grown (without exogenous GA) on high PAC. Again, exogenous GA overcame the effects of low PAC on wild-type plants in a dose-dependent fashion (Fig. 7B). For example, the petiole length of the wild type on low PAC, 50 nM GA (Fig. 7B), was very similar to that exhibited by the wild type on 50 nM GA alone (Fig. 7A) and much greater than that of wild-type plants on low PAC alone (Fig. 7B). GA (0.5 nM) also reduced the effect of low PAC on wild-type petiole length but to a lesser extent than did 50 nM GA (Fig. 7B).

On high PAC, gai plants were compact, dark green, and resembled the wild type grown in the same conditions (Fig. 7C). GA doses greatly reduced these effects (Fig. 7C). For example, the mean rosette radius of gai grown on high PAC alone was 0.25 cm, whereas that of gai grown on high PAC, 50 nM GA, was 0.40 cm (the means of two and seven plants respectively, P < 0.01), an approximate 70% increase in gai rosette radius. This is consistent with previous observations of GA responses in gai ga1-1 plants (see introduction; Koornneef et al., 1985). However, gai plants grown on low PAC showed no detectable growth response to exogenous GA (Fig. 7B). This is also consistent with observations that the gai phenotype is unaffected by exogenous GA and that GA responses are saturated in gai (Koornneef et al., 1985).

When grown on high PAC without exogenous GA, gai spy-7 gar2-1 plants were larger than comparably treated wild-type or gai plants (Fig. 7C), again demonstrating the relatively strong PAC resistance of gai spy-7 gar2-1. However, these plants were considerably more compact than were gai spy-7 gar2-1 plants grown in the absence of PAC (Fig. 7A). Furthermore, exogenous GA caused dose-dependent increases in the sizes of gai spy-7 gar2-1 plants grown on high PAC (Fig. 7C). The mean rosette radius of gai spy-7 gar2-1 plants grown on high PAC alone was 0.55 cm, whereas that of gai spy-7 gar2-1 plants on high PAC, 50 nM GA, was 0.70 cm (the means of 12 and 7 plants, respectively, P < 0.01), an approximate 30% increase in rosette radius. On low PAC, gai spy-7 gar2-1 plants were still compact relative to controls grown without PAC (Fig. 7, A and B). Again, exogenous GA caused dose-dependent promotion of the growth of gai spy-7 gar2-1 plants grown on low PAC. This is most clearly seen with respect to petiole elongation. Petioles of gai spy-7 gar2-1 plants on low PAC, 50 nM GA (Fig. 7B), were much longer than those of controls on low PAC, 0 nM GA (Fig. 7B), but are not obviously different from those of gai spy-7 gar2-1 plants grown without PAC or GA (Fig. 7A) or of wild-type plants on 0 nM PAC, 50 nM GA (Fig. 7A). GA (0.5 nM) also reduced the effect of low PAC on gai spy-7 gar2-1 petiole length but to a lesser extent than did 50 nM GA (Fig. 7B).

These observations show that the phenotype of gai spy-7 gar2-1 is GA dependent. When endogenous GA levels are reduced by PAC, gai spy-7 gar2-1 plants exhibit reduced growth. This effect of PAC is reversed in a dose-dependent manner by exogenous GA. In addition, these experiments indicate that gai and gai spy-7 gar2-1 plants, although having very visibly different plant phenotypes, are both saturated in their GA responses, whereas wild-type plants are not.

spy-7 and gar2-1 Suppress the Effects of gai on Endogenous GA Levels

gai plants accumulated higher than the wild-type levels of transcripts encoding the 20-oxidase enzyme of GA biosynthesis (Xu et al., 1995; Peng et al., 1997). spy-7 and gar2-1, in addition to suppressing the dwarfism conferred by gai, also suppressed the effects of gai on 20-oxidase transcript accumulation (Peng et al., 1997). Because gai had elevated C₁₉-GA levels and reduced C₂₀-GA levels, we were interested in seeing whether spy-7 or gar2-1 would also suppress the effects of gai on endogenous GA levels. As shown in Table II, GA levels in gai spy-7, gai gar2-1, and gai spy-7 gar2-1 were indeed different from those in gai. C₁₉-GA levels (especially GA₁, GA₄, GA₉, and GA₃₄) were elevated in gai, partially elevated in gai spy-7 and gai gar2-1, and returned to approximately wild-type levels in gai spy-7 gar2-1. Similarly, C₂₀-GA levels were reduced in gai, partially restored in gai spy-7 and gai gar2-1, and fully restored to wild-type levels in gai spy-7 gar2-1. Thus, endogenous GA levels in gai spy-7 gar2-1 plants were similar to those of the wild type, whereas those in gai spy-7 and gai gar2-1 were between those of the wild type and gai.

	DISCUSSION

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spy-7 and gar2-1 partially suppressed the dwarfism conferred by gai (Carol et al., 1995; Wilson and Somerville, 1995). Here we have shown that spy-7 and gar2-1 affected several different GA responses and that, for many of these responses, the effects of both alleles combined are greater than that of either allele alone (Peng et al., 1997). gai spy-7 gar2-1 plants are taller than gai spy-7 or gai gar2-1 and resemble wild-type plants that have received a large exogenous GA dose. These properties are common to some other GA-signaling mutations. For example, the pea la and cry^s alleles have stronger effects in combination than either allele has alone. la cry^s double mutants are tall irrespective of endogenous GA level and resemble GA-treated wild-type plants (Potts et al., 1985). Also, combinations of Arabidopsis rga and spy alleles suppressed the ga1-3 phenotype more effectively than either allele alone (Silverstone et al., 1997).

Our observations can be understood if spy-7 and gar2-1 both alter the GA dose-response relationship, as has already been established for other spy alleles. Dose-response experiments showed that spy ga1-2 requires a smaller GA dose than SPY ga1-2 to achieve equal hypocotyl length (Jacobsen and Olszewski, 1993; Jacobsen et al., 1996). We propose that the mutant alleles described in this paper (spy-7 and gar2-1) reduce the repression of GA signaling and alter the GA dose-response relationship in such a way that less GA (or GA signal) is required to elicit a given level of response in spy-7 and gar2-1 than in the wild type. Thus, the GAR2 gene product (like the SPY gene product; Jacobsen et al., 1996) may also be a negative regulator of GA signaling.

The GA dose-response concept can now be used to explain why gai spy-7 and gai gar2-1 plants are taller than gai plants. gai mutant plants are thought to be dwarfed because gai (the gai mutant gene product) represses GA signaling (Peng et al., 1997). Because spy-7 and gar2-1 mutants require less GA signal than SPY or GAR2 plants for a given level of response, gai spy-7 and gai gar2-1 plants are taller than gai. Furthermore, spy-7 gar2-1 double mutants require an even smaller signal for a given level of response than does either single mutant. Thus, gai spy-7 gar2-1 plants are taller than gai spy-7 or gai gar2-1.

Under normal conditions gai spy-7 gar2-1 plants do not respond to exogenous GA and grow as tall as wild-type plants. However, growth of gai spy-7 gar2-1 plants is inhibited by PAC, which is relieved by exogenous GA. Thus, growth of gai spy-7 gar2-1 plants is not completely independent of GA.

The gai mutant has elevated GA levels (Talon et al., 1990a; see ``Results''). An alternative explanation for the action of spy-7 and gar2-1 in suppressing the visible phenotype of gai could have been that these mutations somehow restored responses to the elevated GA. However, we found that spy-7 and gar2-1 also suppressed the effects of gai on the GA level. gai spy-7 and gai gar2-1 had GA levels intermediate between gai and the wild type, whereas gai spy-7 gar2-1 had wild-type GA levels. Thus, spy-7 and gar2-1 do not increase the responses of gai to an elevated GA level.

The gai mutation affects all known Arabidopsis GA responses (Wilson and Somerville, 1995). Here we have shown that spy-7 and gar2-1 suppress the effects of gai on a wide range of these responses. Previously, we had outlined a model to explain the role of GAI and RGA (the GAI and RGA gene products) and associated signal transduction components in mediating GA responses (Peng et al., 1997; Harberd et al., 1998). This model proposed that GAI and RGA act as repressors of GA-regulated stem elongation and that the action of these repressors is opposed by GA. According to this model, the gai protein is a mutant repressor with reduced sensitivity to GA. The model also proposed that the SPY gene product either acts downstream of the GAI and RGA repressors or modulates their activities (Peng et al., 1997; Harberd et al., 1998). This model was initially elaborated on the basis of stem-elongation phenotypes. The data presented in this paper show that the model can be extended to explain GA responses in addition to stem elongation and is perhaps applicable to GA responses in general.

	FOOTNOTES

   Received October 26, 1998; accepted December 18, 1998.

	ABBREVIATIONS

Abbreviations: LD, long-day photoperiod. PAC, paclobutrazol. SD, short-day photoperiod.

	ACKNOWLEDGMENTS

We thank Inga-Britt Carlsson for technical assistance and Pierre Carol, George Coupland, Rachel Cowling, Caroline Dean, Thierry Desnos, Kati King, and Pilar Puente for helpful discussion.

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