The recessive rga mutation is able to partially suppress phenotypic defects of the Arabidopsis gibberellin (GA) biosynthetic mutant ga1-3. Defects in stem elongation, flowering time, and leaf abaxial trichome initiation are suppressed by rga. This indicates that RGA is a negative regulator of the GA signal transduction pathway. We have identified 10 additional alleles of rga from a fast-neutron mutagenized ga1-3 population and used them to isolate the RGA gene by genomic subtraction. Our data suggest that RGA may be functioning as a transcriptional regulator. RGA was found to be a member of the VHIID regulatory family, which includes the radial root organizing gene SCARECROW and another GA signal transduction repressor, GAI. RGA and GAI proteins share a high degree of homology, but their N termini are more divergent. The presence of several structural features, including homopolymeric serine and threonine residues, a putative nuclear localization signal, leucine heptad repeats, and an LXXLL motif, indicates that the RGA protein may be a transcriptional regulator that represses the GA response. In support of the putative nuclear localization signal, we demonstrated that a transiently expressed green fluorescent protein-RGA fusion protein is localized to the nucleus in onion epidermal cells. Because the rga mutation abolished the high level of expression of the GA biosynthetic gene GA4 in the ga1-3 mutant background, we conclude that RGA may also play a role in controlling GA biosynthesis.

Despite extensive studies on the roles of phytochrome in photostimulated seed germination, the mechanisms downstream of the photoreceptor that promote germination are largely unknown. Previous studies have indicated that light-induced germination of Arabidopsis seeds is mediated by the hormone gibberellin (GA). Using RNA gel blot analyses, we studied the regulation of two Arabidopsis genes, GA4 and GA4H (for GA4 homolog), both of which encode GA 3 ␤ -hydroxylases that catalyze the final biosynthetic step to produce bioactive GAs. The newly isolated GA4H gene was expressed predominantly during seed germination. We show that expression of both GA4 and GA4H genes in imbibed seeds was induced within 1 hr after a brief red (R) light treatment. In the phytochrome B-deficient phyB-1 mutant, GA4H expression was not induced by R light, but GA4 expression still was, indicating that R light-induced GA4 and GA4H expression is mediated by different phytochromes. In contrast to the GA4 gene, the GA4H gene was not regulated by the feedback inhibition mechanism in germinating seeds. Our data demonstrate that expression of GA 3 ␤ -hydroxylase genes is elevated by R light, which may result in an increase in biosynthesis of active GAs to promote seed germination. Furthermore, our results suggest that each GA 3 ␤ -hydroxylase gene plays a unique physiological role during light-induced seed germination. INTRODUCTIONGibberellins (GAs) are a group of diterpenoids, some of which are growth regulators in higher plants. Studies using GA-deficient mutants have shown that active GAs control many aspects of plant development, including seed germination, stem elongation, flowering, and seed development (for a recent review, see Ross et al., 1997). In the major GA biosynthetic pathway in higher plants, GA 3 ␤ -hydroxylase catalyzes the conversion of both GA 9 to GA 4 and GA 20 to GA 1 (Hedden and Kamiya, 1997). Several GA-deficient dwarf mutants with reduced 3 ␤ -hydroxylase activities have been isolated. These include dwarf1 in maize (Fujioka et al., 1988), le in pea (Ingram et al., 1984), dy in rice (Kobayashi et al., 1989), and ga4 in Arabidopsis (Talon et al., 1990a). Biochemical studies using these mutants have shown that only GA 4 and GA 1 , but not their precursors GA 9 and GA 20 , are biologically active in stimulating stem elongation. Therefore, GA 3 ␤ -hydroxylase catalyzes the final step of the biosynthetic pathway to produce active GAs in these plant species.Based on their function in GA biosynthesis, expression of GA 3 ␤ -hydroxylase genes is likely to play a key regulatory role in controlling the appropriate levels of active GAs during plant growth. Recently, the Arabidopsis GA4 and pea LE genes have been cloned and shown to encode GA 3 ␤ -hydroxylase (Chiang et al., 1995;Lester et al., 1997;Martin et al., 1997;Williams et al., 1998). The transcript levels of both GA4 and LE are controlled by a feedback inhibition mechanism, that is, they are upregulated in the GA-deficient mutant background and downregulated by applied GAs (Chi...

RGA (for repressor of ga1-3 ) and SPINDLY ( SPY ) are likely repressors of gibberellin (GA) signaling in Arabidopsis because the recessive rga and spy mutations partially suppressed the phenotype of the GA-deficient mutant ga1-3 . We found that neither rga nor spy altered the GA levels in the wild-type or the ga1-3 background. However, expression of the GA biosynthetic gene GA4 was reduced 26% by the rga mutation, suggesting that partial derepression of the GA response pathway by rga resulted in the feedback inhibition of GA4 expression. The green fluorescent protein (GFP)-RGA fusion protein was localized to nuclei in transgenic Arabidopsis. This result supports the predicted function of RGA as a transcriptional regulator based on sequence analysis. Confocal microscopy and immunoblot analyses demonstrated that the levels of both the GFP-RGA fusion protein and endogenous RGA were reduced rapidly by GA treatment. Therefore, the GA signal appears to derepress the GA signaling pathway by degrading the repressor protein RGA. The effect of rga on GA4 gene expression and the effect of GA on RGA protein level allow us to identify part of the mechanism by which GA homeostasis is achieved. INTRODUCTIONGibberellins (GAs) are members of a large family of diterpenoid compounds, some of which are plant growth regulators that control such diverse processes as seed germination, stem growth, and flower development. Although the GA biosynthetic pathway has been elucidated (reviewed in Lange, 1998;Hedden and Proebsting, 1999;Hedden and Phillips, 2000;Yamaguchi and Kamiya, 2000), much less is known about its signal transduction pathway in plants. Recent molecular and pharmacological studies in cereal aleurone showed that Ca 2 ϩ , calmodulin, cyclic GMP, heterotrimeric G proteins, GAMYB, and protein kinases may play a role in GA signaling (reviewed in Bethke and Jones, 1998;Lovegrove and Hooley, 2000). Isolation of GA response mutants and molecular cloning of corresponding genes in Arabidopsis also have identified several novel components of the GA signal transduction pathway (reviewed in Thornton et al., 1999;Sun, 2000). The putative repressors include SPINDLY ( SPY ;Jacobsen et al., 1996), RGA (for repressor of ga1-3 ; Silverstone et al., 1998), GAI (for GA insensitive; Peng et al., 1997), and SHORT INTERNODES ( SHI ;Fridborg et al., 1999), and the potential activators are SLEEPY ( SLY ;Steber et al., 1998) and PICKLE ( PKL ; Ogas et al., 1999).SPY was identified originally because spy mutations allowed the seed to germinate in the presence of the GA biosynthesis inhibitor paclobutrazol (PAC; Jacobsen and Olszewski, 1993). The defect in the SPY function also was able to partially suppress the phenotype of the GA biosynthetic mutant ga1-3 , which is a nongerminating, male-sterile, extreme dwarf (Silverstone et al., 1997). Sequence analysis of SPY and in vitro enzyme assays using the recombinant SPY protein suggest that SPY probably is a Ser/Thr O -linked N -acetylglucosamine transferase (OGT; Thornton et al., 1999).We identified RG...

The first committed step in the gibberellin (GA) biosynthetic pathway is the conversion of geranylgeranyl pyrophosphate (GGPP) through copalyl pyrophosphate (CPP) to ent-kaurene catalyzed by ent-kaurene synthetases A and B. The ga1 mutants of Arabidopsis are gibberellin-responsive male-sterile dwarfs. Biochemical studies indicate that biosynthesis of GAs in the ga1 mutants is blocked prior to the synthesis of ent-kaurene. The GA1 locus was cloned previously using the technique of genomic subtraction. Here, we report the isolation of a nearly full-length GA1 cDNA clone from wild-type Arabidopsis. This cDNA clone encodes an active protein and is able to complement the dwarf phenotype in ga1-3 mutants by Agrobacterium-mediated transformation. In Escherichia coli cells that express both the Arabidopsis GA1 gene and the Erwinia uredovora gene encoding GGPP synthase, CPP was accumulated. This result indicates that the GA1 gene encodes the enzyme ent-kaurene synthetase A, which catalyzes the conversion of GGPP to CPP. Subcellular localization of the GA1 protein was studied using 35S-labeled GA1 protein and isolated pea chloroplasts. The results showed that the GA1 protein is imported into and processed in pea chloroplasts in vitro.

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