Planta (1998) 204: 409419

Review Molecular biology of gibberellin synthesis

Theo Lange
Albrecht-von-Haller-Institut fu t Go ttingen, Untere Karspu ttingen, Germany r Panzenwissenschaften der Universita le 2, D-37073 Go E-mail: tlange@gwdg.de Received: 28 July 1997 / Accepted: 8 October 1997

Key words: Development Gibberellin Gene expression (heterologous, functional) Growth Monooxygenase 2-Oxoglutarate dependent dioxygenase Terpene cyclase

Introduction Gibberellins (GAs) control multiple processes in the life cycle of higher plants, several of which are essential for normal plant growth and development (Crozier 1983; Pharis and King 1985; Graebe 1987). For example, internode growth of seedlings is usually retarded after treatment with LAB150978, an inhibitor of GA biosynthesis (Fig. 1, compare second plant from right with lefthand plant). Additional GA treatment results in plants with normal or even elevated internode growth, depending on the kind and dose of GAs supplied (Fig. 1, second plant from left and right-hand plant). Such overgrowth symptoms are typical GA eects and led to the discovery of GAs early this century by Japanese phytopathologists, who found that ``bakanae'' disease of rice was caused by the GA-producing ascomycete Gibberella fujikuroi. This example also gives an impression of the importance of ne adjustment and control of GA biosynthesis for normal plant growth. By deciphering the underlying molecular mechanisms, control and manipulation of plant developmental processes become possible. Such tempting prospects might have encouraged research into the molecular aspects of GA biosynthesis, the recent advances in which will be highlighted in this review. The reader is also referred to the comprehensive review by Hedden and Kamiya (1997) covering this topic. Gibberellin biosynthetic pathways are often described as being of considerable complexity (Graebe 1987;
Abbreviations: CPP ent-copalyl pyrophosphate; GA gibberellin; GGPP ent-geranylgeranyl pyrophosphate; IPP isopentenyl pyrophosphate; K synthase ent-kaurene synthase; MVA mevalonic acid

Sponsel 1995; MacMillan 1997). One hundred and sixteen dierent GA structures have been identied to date and there are more of these tetracyclic diterpenoid compounds likely to be found. However, only a few GAs are regarded as being biologically active per se. Their molecular structure consists of 19 carbon atoms (C19GAs) containing either a hydroxyl group at carbon-3b [GA1, gibberellic acid (GA3), GA4, GA7, and GA32] or an unsaturated carbon-3 atom (GA5). These GAs exhibit high activities in certain bioassays and are often described as plant hormones (Graebe and Ropers 1978). Furthermore, some of the other GAs with low or no activity in the known bioassays may have as yet undiscovered functions in other plant species, in certain tissues and/or at specic developmental stages. For instance, little is known about the role of GAs during seed development, even though they reach their highest concentrations in immature seeds of many plant species (Pharis and King 1985; Graebe 1987). First, the general course of the GA biosynthetic pathway will be outlined. Subsequently, the enzymes involved and their encoding genes in individual plant species will be discussed. Finally, some future prospects for molecular GA physiology will be presented. General principles of gibberellin biosynthetic pathways Gibberellin biosynthetic pathways are usually dissected into three parts according to the nature of the enzymes involved, a division which also reects their subcellular compartmentation (Fig. 2). Each of the three parts consists of several steps. Part I: biosynthesis of ent-kaurene. Cell-free systems from several plant species are well known to biosynthesize isopentenyl pyrophosphate (IPP), ent-kaurene, and even GAs from mevalonic acid (MVA; Graebe 1987; MacMillan 1997). However, the incorporation of MVA into GAs, or even into ent-kaurene, has never been achieved using intact plants or plant tissues. Recently, a second, MVA-independent pathway to IPP

410

T. Lange: Molecular biology of gibberellin biosynthesis

Fig. 1. Three-week-old pumpkin seedlings, treated with GA4 and/or the GA-biosynthesis inhibitor LAB150978 (Jung et al. 1986). Treatment from left to right: control, GA4 (10A6 M), LAB150978 (10A6 M), and GA4 plus LAB150978 (both 10A6 M)

via glyceraldehyde 3-phosphate/pyruvate was discovered in a green alga (Schwender et al. 1996). Both pathways have now been shown to occur in distinct compartments of the cell, one in the cytoplasm and the second in plastids of higher plants (Fig. 2; Lichtenthaler et al.

1997). Isopentenyl pyrophosphate is further converted to geranylgeranyl pyrophosphate (GGPP) by only two enzymes, IPP-isomerase and GGPP-synthase (Dogbo and Camara 1987). The latter enzyme catalyzes a threestep condensation and both enzymes were localized in plastids of higher plants (Dogbo and Camara 1987; Kuntz et al. 1992). Geranylgeranyl pyrophosphate is further cyclized to ent-copalyl pyrophosphate (CPP) and nally to ent-kaurene. In higher plants these reactions are catalyzed by two enzymes, CPP synthase (formerly

Fig. 2. General scheme of the GA biosynthetic pathway and its subcellular compartmentation in higher plants. ent-Kaurene is synthesized by soluble enzymes located, at least partly, in proplastids or plastids and is oxidised to GA12 by enzymes that are particulate and associated with the endoplasmic reticulum. Gibberellin A12 is further oxidized by soluble enzymes, and GAs with species- and tissuespecic hydroxylation patterns emerge. Some of these exhibit hormonal activity and eventually become inactivated by further oxidation

T. Lange: Molecular biology of gibberellin biosynthesis

411

Fig. 3. The central pathway of the third part of GA biosynthesis. Structures and metabolic relationships of GAs are discussed in the text

kaurene synthase A) and ent-kaurene (K) synthase (formerly kaurene synthase B; renaming was suggested by MacMillan 1997). Both were localized in isolated proplastids of meristematic shoot tissues, but not in mature chloroplasts of pea and wheat (Fig. 2; Aach et al. 1995, 1997) or of pumpkin endosperm (Simcox et al. 1975; Aach et al. 1995). Several genes encoding each of the two enzymes have now been cloned from higher plants (for a recent review, see Sun and Kamiya 1997). Part II: from ent-kaurene to GA12. The intermediate, second, part of the pathway is catalyzed by microsomal NADPH-dependent cytochrome P-450 monooxygenases at the endoplasmic reticulum (Fig. 2; Graebe 1987). Therefore, the very hydrophobic (and volatile) precursor ent-kaurene must be translocated from the proplastids to the endoplasmic reticulum, although nothing is known about the transport mechanism. ent-Kaurene is oxidized in six steps to GA12, via ent-kaurenol, ent-kaurenal, entkaurenoic acid, ent-7a-hydroxykaurenoic acid, and GA12-aldehyde. So far, only one gene for this part of the pathway, the Dwarf-3 gene of maize, has been cloned (see below). It is not known how many monooxygenases are involved in GA biosynthesis; a single enzyme might catalyze several reactions, as do some of the GA dioxygenases described below. Certain biosynthetic steps, such as 7-oxidation, 12a-hydroxylation, and 13-hydroxylation, are catalyzed by both particulate monooxygenases and soluble dioxygenases, which occasionally occur together within the same species or even within the same tissue (Lange and Graebe 1993). Particulate 3b-hydroxylation has only been found in the fungus Gibberella (Bearder 1983). Depending on the nature of the enzymes involved, these steps may be assigned to either the second or the third part of the pathway. Part III: steps after GA12-aldehyde. Some of the initial steps of this nal part of the pathway might overlap with the second part as described above. However, the third part starts here with GA12-aldehyde, because this is the rst intermediate in the pathway that is oxidized by soluble 2-oxoglutarate-dependent dioxygenases (Figs. 2, 3). The GA dioxygenases are often multifunctional with a broad substrate specicity, resulting in many side reactions and the numerous GAs found in higher plants

(Lange and Graebe 1993; Hedden and Kamiya 1997). These enzymes belong to the recently identied family of non-haem iron-containing oxygenases and oxidases (Prescott 1993; De Carolis and De Luca 1994; Prescott and John 1996; Barlow et al. 1997). From GA12aldehyde, a principal pathway to the GA plant hormones can be drawn that involves just three GA dioxygenases, 7-oxidase, 20-oxidase and 3b-hydroxylase (Fig. 3). First, 7-oxidation of GA12-aldehyde produces GA12. In the following steps, GA 20-oxidase catalyzes the whole series of oxidation reactions at carbon-20, leading to either C20-GAs (GA25), or, after loss of C20 and lactonisation, to C19-GAs (GA9, Fig. 3). Eventually, 3b-hydroxylation activates C19-GAs to plant hormones (GA4, Figs. 1, 3), which are subsequently inactivated by 2b-hydroxylation (GA34, Fig. 3). Moreover, C20-GAs are also 2b- and 3b-hydroxylated, but the resulting products (GA13 and GA43, respectively; Fig. 3) have no known function. Nevertheless, such ``side-activities'' might prevent enzymes with broad substrate specicity from producing GA hormones. The dioxygenases may be located within the cytosol, although location in another cellular compartment cannot be excluded. At least in pumpkin endosperm, only a little GA dioxygenase activity was found to be associated with the endoplasmic reticulum or with proplastid preparations (Lange et al. 1993a; data not shown). Gibberellin biosynthetic enzymes and their encoding genes Genes encoding GA enzymes were rst isolated by exploiting two plant species in particular, Arabidopsis thaliana and pumpkin. Subsequently, many homologous genes have been cloned from several other plant species, most successfully from pea. Recent progress made with these three species will be presented rst, followed by important ndings obtained from several other species. In Table 1, the presently cloned GA genes are listed. Arabidopsis thaliana Even though A. thaliana is the model plant for genetic analysis of plant growth and development, its GA biosynthetic pathways have not been studied extensively yet. The rst part of GA biosynthesis has been demonstrated in cell-free systems prepared from siliques

412

T. Lange: Molecular biology of gibberellin biosynthesis

Table 1. Genes encoding for GA biosynthetic enzymes and their GenBank accession numbers Plant species A. thaliana Gene GA1 GA5 GA4 C. maxima Encoded enzyme CPP synthase 20-oxidase 20-oxidases 3b-hydroxylase K synthase 7-oxidase 20-oxidases 2b-/3b-hydroxylase LS Le S. oleracea P. vulgaris M. macrocarpus Z. mays O. sativa Phaeosphaeria sp. AN1 D3 CPP synthase 20-oxidases 3b-hydroxylase 20-oxidase 20-oxidases 20-oxidase CPP synthase CYP88 20-oxidase CPP/K synthase GenBank accession nos. U11034 X83379, U20872, U20873, U20901 X83380, X83381 L37126 U43904 U61386 X73314, U61385 U63760 U63652 X91658, U58830, U70471 U93210, AF001219 U33330 U70530, U70531, U70532 Y09112 L37750 U32579 U50333 AB003395 References Sun et al. 1992 Phillips et al. 1995; Xu et al. 1995 Phillips et al. 1995 Chiang et al. 1995 Yamaguchi et al. 1996 Lange 1997 Lange et al. 1994b; Lange 1997 Lange et al. 1997b Ait-Ali et al. 1997a Martin et al. 1996; Lester et al. 1996; Garc a-Mart nez et al. 1997 Lester et al. 1997; Martin et al. 1997 Wu et al. 1996 Garc a-Mart nez et al. 1997 MacMillan et al. 1997 Bensen et al. 1995 Winkler and Helentjaris 1995 Toyomasu et al. 1997 Kawaide et al. 1997

P. sativum

(Barendse and Koornneef 1982) and the second part has not yet been analysed. Of the third part, numerous endogenous GAs have been identied in shoots, leading to the construction of GA biosynthetic pathways for this species (Talon et al. 1990) which have been partly conrmed by feeding studies with isotopically labelled GAs (Zeevaart and Talon 1992). Taken together, these results suggest a typical course of GA biosynthesis in this species. Presently cloned genes. The availability of GA-responsive mutants (Koornneef and Van der Veen 1980) has made A. thaliana extremely useful for studying molecular mechanisms of GA biosynthesis. The most prominent examples are the rst isolation of the GA1 locus by genomic subtraction (Sun et al. 1992) and the rst cloning of the GA4 locus by T-DNA tagging (Chiang et al. 1995). In addition, the existence of a multigene family encoding at least three GA 20-oxidases has been demonstrated (Phillips et al. 1995). Two of the three 20oxidase genes were identied by a polymerase chain reaction (PCR)-based cloning approach and the third was found in the EST data base at GenBank (Phillips et al. 1995). One of the 20-oxidase genes is identical to the GA5 locus (Xu et al. 1995). The GA1 gene is interrupted by 14 introns (Sun and Kamiya 1994), GA4 contains only one intron at base pair 490 from the starting codon (433 bp long; Chiang et al. 1995), and GA5 contains two introns, one at base pair 551 (191 bp long) and the other at base pair 873 (103 bp long; Xu et al. 1995). Properties of the enzymes obtained by cDNA expression in Escherichia coli. The functions of all GA genes isolated from Arabidopsis have been demonstrated by heterologous expression in E. coli. The GA1 locus

encodes CPP synthase, which catalyzes specically the last but one step of the rst part of GA biosynthesis, the conversion of GGPP to CPP (Fig. 2; Sun and Kamiya 1994). The authors demonstrated that in-vitro-translated, premature CPP synthase with a MW of 86 kDa is transported into isolated pea chloroplasts, where it is processed to a protein of 76 kDa. The three 20-oxidases produce mainly C19-GAs and the GA4-locus encodes GA 3b-hydroxylase. They are enzymes of the third part of GA biosynthesis that have broad substrate specicity and prefer non-hydroxylated to 13-hydroxylated GA substrates (J. Williams, A.L. Phillips and P. Hedden, IACR-Long Ashton Research Station, University of Bristol, Long Ashton, UK, personal communication; Phillips et al. 1995). Studies on recombinant Arabidopsis 20-oxidase showed that GA15 has to be in its open lactone form to be a substrate for this enzyme (Fig. 3; Ward et al. 1997). Similar results have also been obtained in cell-free systems from pumpkin endosperm (Hedden and Graebe 1982) and pea cotyledons (Kamiya et al. 1986). The most abundant endogenous GA plant hormone in Arabidopsis shoots is GA4 (Talon et al. 1990), a product of both 20-oxidase and 3b-hydroxylase activities (Fig. 3). However, considerable amounts of endogenous 13-hydroxylated GA intermediates have been detected (Talon et al. 1990). Their metabolism might be retarded, making them a reservoir of plant hormone precursors. Gene expression in vivo. In A. thaliana the highest transcript levels of the GA1 gene (CPP synthase), the GA4 gene (3b-hydroxylase) and two of the three 20oxidase genes are found in the silique, while the third 20oxidase gene (encoded by the GA5-locus) is mainly transcribed in the stem (Sun et al. 1992; Chiang et al.

T. Lange: Molecular biology of gibberellin biosynthesis

413

1995; Phillips et al. 1995; Xu et al. 1997). In addition, considerable GA1 promoter activity was determined not only in rapidly growing tissues like shoot apices, root tips, developing owers, and seeds, but also in nongrowing organs such as expanded leaves (Silverstone et al. 1997a). Feedback regulation has been reported for transcription of the three 20-oxidase and for the 3bhydroxylase genes: application of GA3 or GA4 to A. thaliana seedlings greatly reduces transcript levels, while in the ga1 and ga5 mutant plants elevated 20-oxidase and, in the ga4 mutant, elevated 3b-hydroxylase transcript levels were determined (Chiang et al. 1995; Phillips et al. 1995; Xu et al. 1995). Additionally, expression of the GA5 gene (20-oxidase), but not expression of the GA4 gene (3b-hydroxylase) has been demonstrated to be under photoperiodic control (Xu et al. 1997). Some of these Arabidopsis GA-responsive mutants are likely to contain null-mutations. In the ga1-3 mutant the CPP synthase gene is deleted to a large extent (Sun et al. 1992), the ga5 mutant produces only truncated protein due to a premature stop-codon (Xu et al. 1995), and ga4-2 is a T-DNA knockout mutant (Chiang et al. 1995). These genes are unlikely to produce protein with enzymatic activity. However, the ga1-3 mutant has been shown to express CPP activity and it still contains small amounts of GAs. In the ga5 mutant even substantial amounts of GAs were found (Talon et al. 1990; Zeevaart and Talon 1992). In addition, the ga5 and ga4-2 mutants have a semi-dwarf growth habit, which also indicates some GA hormone production. Therefore, these mutations are either `leaky' and/or Arabidopsis contains additional genes that can supplement the missing reactions. The simplest explanation for these observations is the existence of multigene families, as has been demonstrated already for GA 20-oxidases from several species (Phillips et al. 1995, see above and below). Cucurbita maxima For more than 25 years cell-free enzyme preparations from immature pumpkin seeds have been used extensively to demonstrate GA biosynthetic pathways in cellfree systems (Graebe 1987; Lange et al. 1993a,b). This plant material contains exceptionally high levels of GA enzymes and their encoding transcripts (Lange and Graebe 1993; Lange 1997). Furthermore, GA dioxygenases from immature pumpkin seeds have several unique catalytic properties leading to GAs of unknown function for plant development. Whether these enzymes are specically expressed during plant embryogenesis and/ or whether they are individual features of this species is not known yet. Presently cloned genes. Immature pumpkin seeds have been used for the rst isolation of GA enzymes and for the rst cloning of their encoding cDNA molecules by conventional immunoscreening strategies; these enzymes include K synthase (Saito et al. 1995; Yamaguchi et al. 1996), GA 20-oxidase (Lange 1994; Lange et al. 1994b)

and GA 2b-/3b-hydroxylase (Lange et al. 1994a, 1997b). Recently, a cDNA for a fourth GA enzyme from this source, GA 7-oxidase, has been isolated using a novel cloning strategy based on the heterologous expression of enzyme activity in recombinant E. coli (Lange 1997). Genomic DNA was isolated for each of the three dioxygenase genes and revealed intron(s) of approx. 200 bp in length (Lange et al. 1997b). However, the number and positions of the introns have not been determined yet. Properties of the enzymes obtained by cDNA expression in E. coli. The identity of all cDNA clones has been demonstrated by characterisation of the enzymatic properties of fusion proteins expressed in E. coli. entkaurene synthase catalyses the conversion of CPP to entkaurene, which is the last step of the rst part of GA biosynthesis (Fig. 2). In addition, the authors demonstrated that this enzyme is free of CPP synthase activity (Yamaguchi et al. 1996), in contrast to the bifunctional CPP/K synthase from Phaeosphaeria described below. Characterisation of the GA dioxygenases of the third part of the biosynthetic pathway revealed several surprising properties. Gibberellin 7-oxidase and GA 20-oxidase are both multifunctional enzymes (Lange 1994, 1997; Lange et al. 1994b) and GA 2b-/3bhydroxylase is a bifunctional enzyme with an exceptionally broad substrate specicity (Lange et al. 1997b). The GA 7-oxidase prefers the non-hydroxylated GA12-aldehyde as a substrate (Fig. 3), but also accepts the 3b-hydroxylated precursor GA14-aldehyde (not illustrated in Fig. 3; Lange 1997). After oxidation of GA12-aldehyde to GA12, the latter compound is converted to four other products by the GA 7-oxidase, two of which are monohydroxylated GAs (Lange 1997). These compounds, which have not yet been identied, might be the starting-point of unknown GA biosynthetic pathways. Gibberellin 7-oxidase is the smallest enzyme with the lowest pH-optimum of all GA dioxygenases cloned so far (Lange et al. 1994a; Lange 1997). This latter property might indicate some subcellular compartmentation of this step of the pathway (Fig. 2). Recombinant GA 20-oxidase is also a multifunctional enzyme with broad substrate specicity, preferring nonhydroxylated to 13-hydroxylated substrates. The enzyme catalyzes the whole series of oxidation at carbon-20, leading mainly to C20-GAs without known physiological function (GA25, Fig. 3; Lange 1994; Lange et al. 1994b). This property is in contrast to all other known 20oxidases which produce mainly C19-GAs. The molecular basis of this unique reaction has recently been studied by using chimeric proteins, constructed from closely related 20-oxidases of pumpkin and Marah macrocarpus; both sequences share 67% identical amino acids. The Cterminal end of the enzyme has been shown to be mainly responsible for directing the biosynthetic ow into either C19- or C20-GAs (Lange et al. 1997a). Gibberellin 2b-/3b-hydroxylase oxidises several GAs at the 3b-position, independently of their oxidation state at carbon-7 and carbon-20 (Lange et al. 1997b). However, GAs containing C-20 alcohol, aldehyde or

414

T. Lange: Molecular biology of gibberellin biosynthesis

carboxylic acid groups are preferred substrates. The presence of a 13-hydroxyl group has no inuence on the reaction. As for the second reaction of this bifunctional enzyme, only C20-GAs containing a C-20 carboxylic acid group are hydroxylated at the 2b-position (Lange et al. 1997b). In contrast, recombinant GA 3b-hydroxylases from Arabidopsis and from pea prefer non-hydroxylated substrates and 2b-hydroxylase activity has not been demonstrated with these enzymes (see above and below). The pumpkin 2b-,3b-hydroxylase sequence is only about 35% identical at the amino acid level to the 3bhydroxylases from pea or Arabidopsis, a degree of identity which is typical of dioxygenases with dierent functions (Prescott and John 1996). Many of the major endogenous GAs from immature pumpkin seeds (Blechschmidt et al. 1984; Lange et al. 1993a, b) are potential catalytic products of the three multifunctional GA dioxygenases described above. However, the occurrence of large amounts of C19-GAs, containing hydroxyl groups at the 12a- and 2b- positions, requires at least three additional enzyme activities that have not yet been identied. Gene expression in vivo. The K synthase and the three GA dioxygenases are mainly expressed in developing pumpkin seeds. Enzyme activity of one of the GA dioxygenases, the 2b,3b-hydroxylase, was exclusively found in cell-free enzyme preparations from the endosperm of developing pumpkin seeds, but not in developing embryos, indicating a tissue-specic expression pattern (Lange et al. 1993b, 1997b). Considerable transcript levels for K synthase and 7-oxidase were found in the shoot and root tips and, of K synthase, also in the hypocotyl of 7-d-old pumpkin seedlings (Yamaguchi et al. 1996; data not shown). However, transcript levels, as determined by quantitative reverse transcriptase PCR, for the three GA dioxygenase genes were at least 200-times lower in germinating seeds compared to developing seeds (Lange et al. 1997b). Furthermore, the transcript levels in germinating seeds did not correlate with the relatively high GA dioxygenase activities found in this tissue. Therefore, the high enzyme activities in germinating seeds are thought to be a legacy of the high gene-expression levels during seed development (Lange et al. 1997b). Pisum sativum For molecular analysis of GA biosynthetic pathways the pea system has some advantages over pumpkin and Arabidopsis. As with pumpkin, pea has been used extensively for studying GA-biosynthetic pathways and for partial purication and characterisation of the enzymes involved (Graebe 1987; Lange and Graebe 1993). Similar to Arabidopsis, many GA-responsive pea mutants are available (for a recent review, see Ross et al. 1997). Presently cloned genes. Recently, a number of structural genes controlling GA metabolism have been isolated from pea. A cDNA encoding the CPP synthase was

cloned and shown to be encoded by the LS locus (AitAli et al. 1997a). Three cDNA-encoding GA 20-oxidases have been isolated (Lester et al. 1996; Martin et al. 1996; Garc a-Mart nez et al. 1997). Finally, the isolation of the Le-locus of pea, originally described by Gregor Mendel, was independently accomplished in two laboratories (Lester et al. 1997; Martin et al. 1997). This gene encodes a GA 3b-hydroxylase, as had been predicted from biochemical and physiological studies (Potts et al. 1982; Ingram et al. 1984, 1986; Ross et al. 1989). Like the GA4-gene in Arabidopsis (see above) the Le-gene has only one intron at base pair 488 from the starting codon which is 544 bp long (Lester et al. 1997). The positions and sizes of introns of the other genes have not been reported yet. Properties of the enzymes obtained by cDNA expression in E. coli. Again, the function of all GA genes has been demonstrated by heterologous expression in E. coli. The results are similar to those obtained for the corresponding enzymes from Arabidopsis, but they are quite dierent from the ones obtained from pumpkin (see above). The CPP synthase specically catalyzes the synthesis of CPP from GGPP (Ait-Ali et al. 1997a). The three GA 20-oxidases produce mainly C19-GAs (Lester et al. 1996; Martin et al. 1996; Garc a-Mart nez et al. 1997). Contradictory results were obtained for the substrate preferences for two of the 20-oxidases. One enzyme prefers non-hydroxylated GAs (Martin et al. 1996), whereas a second very similar enzyme, which diers in only three amino acids, does not show this preference (Garc a-Mart nez et al. 1997). The third 20oxidase, which has an amino acid structure considerably dierent from those of the other two also has no preference for non-hydroxylated or 13-hydroxylated substrates (Lester et al. 1996). These results demonstrate the necessity for more detailed kinetic studies of the recombinant enzymes. However, the GA 3b-hydroxylase clearly favours the non-hydroxylated substrate GA9 over the 13-hydroxylated GA20 (Martin et al. 1997). Gene transcription in vivo. The LS-gene (CPP synthase) transcripts are mainly detected in developing pea seeds. Amounts rst increase in very young seeds (24 d from anthesis), whereafter they decline. A second increase occurs later at three weeks from anthesis (Ait-Ali et al. 1997a). Two 20-oxidase genes were expressed individually with a similar timing. Thus, one was transcribed mainly in very young developing seeds (and also in internodes and young leaves) and the other in threeweek-old developing seeds (Lester et al. 1996; Ait-Ali et al. 1997a; Garc a-Mart nez et al. 1997). This biphasic pattern explains corresponding changes in endogenous GA levels, which have been recognised for a long time (Sponsel 1985). High GA levels appear rst in very young and again in three-week-old seeds. However, 3bhydroxylated GAs are only found in the very young seeds (Sponsel 1983; Gaskin et al. 1985; Garc a-Mart nez et al. 1987, 1991; Swain et al. 1993, 1995; Santes et al. 1995; Rodrigo et al. 1997). These plant hormones are likely to be essential for early embryogenesis, because

T. Lange: Molecular biology of gibberellin biosynthesis

415

reduced GA levels in very young seeds of pea lhi mutants result in increased seed abortion (Swain et al. 1993, 1995). The Le gene (3b-hydroxylase) is mainly transcribed in roots; however, it is also transcribed in leaves and internodes, and, in contrast to the 2b-/3b-hydroxylase gene from pumpkin, it is not abundant in immature seeds (Martin et al. 1997). Transcription of both the 20-oxidase and the 3bhydroxylase genes is subject to feedback regulation (Martin et al. 1996, 1997) similar to the previous nding for the homologous genes from Arabidopsis (see above). Recently, it has been demonstrated that 20-oxidase transcription in pea, as in Arabidopsis (see above), is also regulated by light, possibly mediated by phytochrome (Ait-Ali et al. 1997b). In the pea led mutant the 3b-hydroxylase gene contains a single base deletion resulting in a shift in the reading frame which is likely to cause a null mutation. However, as with the Arabidopsis mutants, low levels of GA1 were detectable in these plants (Ingram et al. 1986, see above). Again, this observation suggests the existence of additional 3b-hydroxylase genes (Martin et al. 1997). Other systems Spinacia oleracea. Cell-free systems from spinach leaves were rst used to show that GA levels and GA dioxygenase activities change depending on photoperiod. It was also the rst system in which at least two dierent 20-oxidase activities were detected (Gilmour et al. 1986, 1987; Talon et al. 1991). Recently, the isolation of one 20-oxidase gene from this species was reported (Wu et al. 1996). The recombinant enzyme, as expressed in E. coli, catalyzes the three-step oxidation to C19-GAs, exhibiting multifunctional properties similar to those of most other 20-oxidases. This 20-oxidase gene is mainly transcribed in stems of plants growing under long-day conditions (Wu et al.1996). However, a second 20-oxidase activity, which is not under photoperiodic control and which catalyses the second step of 20oxidation, the formation of the C-20 aldehyde, was found in spinach leaves (Gilmour et al. 1986; cf. Fig. 3). The enzyme can utilise GAs with C-20 alcohol in its lactonic form as substrate (such as GA44, not illustrated in Fig. 3), and it is separable from the other spinach 20oxidase by anion-exchange chromatography (Gilmour et al. 1987). Similar activities have also been found in pea shoots and germinating barley embryos (Graebe 1987; Groelindemann et al. 1992), but not with recombinant Arabidopsis 20-oxidase nor in cell-free systems from developing pumpkin and pea seeds (see above). Phaseolus vulgaris. As in Arabidopsis and pea, French bean contains a multigene family for GA 20-oxidases, which, in contrast to the Arabidopsis and pumpkin enzymes, all convert 13-hydroxylated as eciently as non-hydroxylated GA substrates (Garc a-Mart nez et al. 1997). As in pea, one 20-oxidase gene is mainly transcribed in young leaves and very young developing

seeds, and a second is mainly transcribed in young leaves and two- to three-week-old developing seeds. Gene transcripts of the third 20-oxidase are most abundant in very young seeds but are also present at later stages of seed development (Garc a-Mart nez et al. 1997). Marah macrocarpus. From this species, which belongs to the cucurbit family, another gene encoding GA 20oxidase has been cloned (MacMillan et al. 1997). This enzyme is of particular interest because it is closely related to the pumpkin 20-oxidase, but produces mainly C19-GAs, in contrast to the pumpkin enzyme (Fig. 3, see above). Similar to the pumpkin 20-oxidase, transcripts of the Marah gene were found mainly in endosperm and embryos of immature seeds. However, substantial amounts of endogenous tricarboxylic C20-GAs as found in the endosperm of Marah seeds indicate the existence of another 20-oxidase with catalytic properties similar to the pumpkin 20-oxidase described above (MacMillan and Gaskin 1996). Zea mays. Most information on GA-enzyme-encoding genes from monocotyledons is available from studies with Zea mays. Two genes have been cloned already by transposon tagging using Robertson's Mutator (Bensen et al. 1995; Winkler and Helentjaris 1995). The Anther ear1 gene has considerable similarity to the GA1 gene from Arabidopsis, indicating that it also encodes a CPP synthase of the rst part of GA biosynthesis (cf. Fig. 2; Bensen et al. 1995). However, anther ear1 mutants exhibit only a semi-dwarf stature with reduced entkaurene levels, which might indicate the presence of further CPP synthases, as suggested for Arabidopsis (Bensen et al. 1995). The second gene that has been cloned from maize is the Dwarf-3 gene. It encodes a protein containing typical cytochrome P450-monooxygenase motifs (Winkler and Helentjaris 1995) and is the rst and only isolated gene encoding an enzyme involved in the second part of GA biosynthesis. The enzymatic properties of the encoded enzyme have not been characterised yet, and it is still unclear which step of the pathway is blocked in the dwarf-3 mutant plant (B.O. Phinney, cited in Hedden and Kamiya 1997). The Dwarf-3 gene has been shown to be expressed in all organs and tissues from maize, as demonstrated by reverse-transcriptase PCR, but, without quantitative analysis of the transcript levels (Winkler and Helentjaris 1995). Another interesting observation comes from GA20feeding studies using the dwarf-1 mutant of maize. The gene locus is responsible for several biosynthetic steps including GA 3b-hydroxylation (as occurred in GA1, GA3) and 2,3-desaturation (GA5, Spray et al. 1996). This might indicate the existence of another multifunctional 3b-hydroxylase in maize in addition to the one from pumpkin (see above). Oryza sativa. The rst reported GA 20-oxidase-encoding gene from a monocotyledonous plant was isolated from rice and also expressed in E. coli (Toyomasu et al. 1997). Again, the recombinant enzyme produces mainly

416

T. Lange: Molecular biology of gibberellin biosynthesis

C19-GAs, but prefers 13-hydroxylated substrates to nonhydroxylated substrates, which is in contrast to all other 20-oxidases. These catalytic properties are consistent with the endogenous GAs found in rice, which contain relatively high levels of 13-hydroxylased GAs (Takahashi and Kobayashi 1991; Toyomasu et al. 1997). Elevated GA 20-oxidase transcript levels were found in the GA-decient dwarf mutants, Tanginbozu and Waito-C, and were also increased by treatment with uniconazole-P, an inhibitor of GA biosynthesis, and decreased by treatment with GA3. These results indicate feedback regulation of transcription by mechanisms similar to those in Arabidopsis and pea (see above). Phaeosphaeria sp. Although fungi have played an important role in GA biosynthetic research (for review, see Bearder 1983), only scarce information is available on the genes encoding GA enzymes in these organisms. Recently, GA biosynthetic pathways have been elucidated in the fungus Phaeosphaeria sp. L487 that have signicant similarities to those of higher plants (Kawaide et al. 1995). Furthermore, an important gene from this species has now been isolated that shares only low sequence similarity with CPP synthases or K synthase from higher plants (Kawaide et al. 1997). Functional expression in E. coli conrmed that the recombinant fungal enzyme catalyses the last two steps of the rst part of GA biosynthesis, the conversion of GGPP to ent-kaurene via CPP (Fig. 2). This bifunctional property has not been found for corresponding enzymes from higher plants so far. Interestingly, Amo-1618, a specic inhibitor of CPP synthases from higher plants, also inhibits only the CPP synthase activity but not the K synthase activity of the bifunctional fungal enzyme, suggesting that the two reactions involve dierent residues at one active centre or that they are catalysed at dierent active centres within the enzyme (Kawaide et al. 1997). Conclusions Recently, exciting progress has been made in the eld of molecular biology of GA biosynthesis, and it is expected that within a few years isolated genes will be available for all steps of the pathway. In particular, the cloning and characterization of genes encoding the largely unknown GA monooxygenases will be one of the most challenging tasks of the near future. Molecular approaches have already added to a more profound understanding of GA biosynthesis and its control. A general scheme of the GA biosynthetic pathways can be drawn, catalysed by GA enzymes which often have multifunctional properties. Characterization of recombinant GA enzymes, which can often be obtained simply by heterologous expression in E. coli, will surely add to our understanding of GA biosynthesis and its regulation. Understanding of the molecular bases of regulatory principles of GA biosynthesis has just begun. Feedback regulation might occur generally for regulation of

20-oxidase and 3b-hydroylase activities to prevent overor underproduction of GA plant hormones. Light has been shown to be involved in regulation of transcription levels of certain 20-oxidases. Multigene families occur, as shown for several GA 20-oxidases, and even members of dierent enzyme families, such as monooxygenases and dioxygenases, catalyze identical steps of the pathway, even though they might be expressed at dierent developmental stages and in dierent parts of the plant. Such a redundancy of the pathway might be important for ne tuning of the GA plant hormone pool which is then less susceptible to unintended changes. However, it will make manipulated control of GA biosynthesis a dicult task. As presented above, GA-biosynthetic enzymes are expressed in a complex manner during the whole life cycle of higher plants. Expression of CPP synthase alternates during pea embryogenesis, as does expression of two dierent 20-oxidases from pea and bean. Also, two dierent 3b-hydroxylase activities are present in developing pumpkin seeds. These ndings indicate the operation of dierent GA biosynthetic pathways during the course of embryogenesis, some of which are likely to be essential for embryo and seed development. Investigations on the contribution of individual genes or articial gene constructs to specic GA-dependent developmental processes are no longer restricted by the availability of suitable mutant plants. The GA-biosynthesis genes can now be studied in situ in transformed plants. For this purpose Arabidopsis thaliana has several well-known advantages over other plant species which surely will facilitate future research in the eld of the molecular biology of GA synthesis. To date, little is known about the mechanism of GA plant hormone action and signal-transduction pathways (for reviews, see Hooley 1994; Swain and Olszewski 1996). Gibberellin synthesis and action may occur in dierent tissues, as discussed above for semi-dwarf GAresponsive mutants. In other cases, synthesis and action might occur within one and the same tissue but at dierent developmental stages, as suggested for GAmediated internode growth of wheat plants (Aach et al. 1997). However, nal clarication must await the identication of responsible GA-receptor molecules. Recently, genetic approaches have identied numerous genes essential for plant development, some of which appear to be coupled to GA signal-transduction pathways (Putterill et al. 1995; Weigel 1995; Okamuro et al. 1996, 1997; Kania et al. 1997; Silverstone et al. 1997b). It is expected that in the near future plant genetics will complement molecular GA physiology more frequently.
I thank Professor Jan E. Graebe for carefully reading the manuscript, my colleagues Drs Peter Hedden, John Ross (Department of Plant Science, University of Tasmania, Hobart, Australia), Hiroshi Kawaide, Yuji Kamiya (both Institute of Physical and Chemical Research, Wako, Saitama, Japan), Bill Proebsting (Department of Horticulture and Center for Gene Research, Oregon State University, Corvallis, USA) and Tai-ping Sun (Department of Botany, Duke University, Durham, N.C., USA) for making data available prior to publication, and Professor

T. Lange: Molecular biology of gibberellin biosynthesis Nobutaka Takahashi (Institute of Physical and Chemical Research, Wako, Saitama, Japan) for making space available at his RIKEN oce for me to write this review. Work in my laboratory was supported by the Deutsche Forschungsgemeinschaft.

417 Gilmour SJ, Bleecker AB, Zeevaart JAD (1987) Partial-purication of gibberellin oxidases from spinach leaves. Plant Physiol 85: 8790 Graebe JE (1987) Gibberellin biosynthesis and control. Annu Rev Plant Physiol 38: 419465 Graebe JE, Ropers HJ (1978) Gibberellins. In: Letham DS, Goodwin PB, Higgins TJV (eds) Phytohormones and related compounds a comprehensive treatise. vol 1. Elsevier Amsterdam, pp 107188 Grobelindemann E, Lewis MJ, Hedden P, Graebe JE (1992) Gibberellin biosynthesis from gibberellin A12-aldehyde in a cellfree system from germinating barley (Hordeum vulgare L. cv. Himalaya) embryos. Planta 188: 252257 Hedden P, Graebe JE (1982) Cofactor requirements for the soluble oxidases in the metabolism of the C20-gibberellins. J Plant Growth Regul 1: 105116 Hedden P, Kamiya Y (1997) Gibberellin biosynthesis: enzymes, genes and their regulation. Annu Rev Plant Physiol Plant Mol Biol 48: 431460 Hooley R (1994) Gibberellins: perception, transduction and responses. Plant Mol Biol 26: 15291555 Ingram TJ, Reid JB, Murfet IC, Gaskin P, Willis CL, MacMillan J (1984) Internode length in Pisum. The Le gene controls the 3bhydroxylation of gibberellin A20 to gibberellin A1. Planta 160: 455463 Ingram TJ, Reid JB, MacMillan J (1986) The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L. Planta 168: 414420 Jung J, Rentzea C, Rademacher W (1986) Plant growth regulation with triazoles of the dioxanyl type. J Plant Growth Regul 4: 181188 Kamiya Y, Takahashi N, Graebe JE (1986) The loss of carbon-20 in C19-gibberellin biosynthesis in a cell-free system from Pisum sativum L. Planta 169: 524528 Kania T, Russenberger D, Peng S, Apel K, Melzer S (1997) FPF1 promotes owering in Arabidopsis. Plant Cell 9: 1327 1338 Kawaide H, Sassa T, Kamiya Y (1995) Plant-like biosynthesis of gibberellin A1 in the fungus Phaeosphaeria sp L487. Phytochemistry 39: 305310 Kawaide H, Imai R, Sassa T, Kamiya Y (1997) Cloning of a cDNA encoding the gibberellin biosynthesis enzyme ent-kaurene synthase from a fungus Phaeosphaeria sp. L487. J Biol Chem 272: 2170621712 Koornneef M, Van der Veen JH (1980) Induction and analysis of gibberellin sensitive mutants in Arabidopsis thaliana (L.) Heynh. Theor Appl Genet 58: 257263 Kuntz M, Ro mer S, Suire C, Hugueney P, Weil JH, Schantz R, Camara B (1992) Identication of a cDNA for the plastidlocated geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening. Plant J 2: 2534 Lange T (1994) Purication and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima endosperm. Planta 195: 108115 Lange T (1997) Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli. Proc Natl Acad Sci USA 94: 6553 6558 Lange T, Graebe JE (1993) Enzymes of gibberellin synthesis. In: Lea PJ (ed) Methods in plant biochemistry, vol 9. Academic Press, London, pp 403430 Lange T, Hedden P, Graebe JE (1993a) Biosynthesis of 12a- and 13-hydroxylated gibberellins in a cell-free system from Cucurbita maxima endosperm and the identication of new endogenous gibberellins. Planta 189: 340349 Lange T, Hedden P, Graebe JE (1993b) Gibberellin biosynthesis in cell-free extracts from developing Cucurbita maxima embryos and the identication of new endogenous gibberellins. Planta 189: 350358

References
Aach H, Bo se G, Graebe JE (1995) ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species. Planta 197: 333342 Aach H, Bode H, Robinson DG, Graebe JE (1997) ent-Kaurene synthase is located in proplastids of meristematic shoot tissues. Planta 202: 211219 Ait-Ali T, Swain SM, Reid JB, Sun T-P, Kamiya Y (1997a) The LS locus of pea encodes the gibberellin biosynthesis enzyme entkaurene synthase A. Plant J 11: 443454 Ait-Ali T, Shannon F, Kendrick RE, Reid JB, Kamiya Y (1997b) Light regulates the expression of GA 20-oxidase in pea. Plant Physiol 114 [Suppl]: 55 Barendse GWM, Koornneef M (1982) Biosynthesis of the gibberellin precursor ent-kaurene in cell-free preparations from gibberellin-sensitive Arabidopsis mutants. Arabidopsis Inf Serv 19: 2528 Barlow JN, Baldwin JE, Clifton IJ, Gibson E, Hensgens CMH, Hajdu J, Hara T, Hassan A, John P, Lloyd MD, Roach PL, Prescott A, Robinson JK, Zhang Z-H, Schoeld CJ (1997) Studies on non-haem ferrous-dependent oxygenases and oxidases. Biochem Soc Trans 25: 8690 Bearder JR (1983) In vivo diterpenoid biosynthesis in Gibberella fujikuroi: The pathway after ent-kaurene. In: Crozier A (ed) The biochemistry and physiology of gibberellins, vol 1. Praeger, New York, pp 251387 Bensen RJ, Johal GS, Crane VC, Tossberg JT, Schnable PS, Meeley RB, Briggs RB (1995) Cloning and characterization of the maize An1 gene. Plant Cell 7: 7584 Blechschmidt S, Castel U, Gaskin P, Hedden P, Graebe JE, MacMillan J (1984) GC/MS analysis of the plant hormones in seeds of Cucurbita maxima. Phytochemistry 23: 553558 Chiang HH, Hwang I, Goodman HM (1995) Isolation of the Arabidopsis GA4 locus. Plant Cell 7: 195201 Crozier A, ed (1983) The biochemistry and physiology of gibberellins, vol 2. Praeger, New York De Carolis E, De Luca V (1994) 2-Oxoglutarate-dependent dioxygenases and related enzymes: Biochemical characterisation. Phytochemistry 36: 10931107 Dogbo O, Camara B (1987) Purication of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthase from Capsicum chromoplasts by anity chromatography. Biochim Biophys Acta 920: 140148 Garc a-Mart nez JL, Sponsel VM, Gaskin P (1987) Gibberellins in developing fruits of Pisum sativum cv. Alaska: studies on their role in pod growth and seed development. Planta 170: 130137 Garc a-Mart nez JL, Santes C, Croker SJ, Hedden P (1991) Identication, quantication, and distribution of gibberellins in fruits of Pisum sativum L. cv. Alaska during pod development. Planta 184: 5360 Garc a-Mart nez JL, Lopez-Diaz I, Sanchez-Beltran MJ, Phillips AL, Ward DA, Gaskin P, Hedden P (1997) Isolation and transcript analysis of gibberellin 20-oxidase genes in pea and bean in relation to fruit development. Plant Mol Biol 33: 1073 1084 Gaskin P, Gilmour SJ, MacMillan J, Sponsel VM (1985) Gibberellins in immature seeds and dark-grown shoots of Pisum sativum. Gibberellins identied in the tall cultivar Alaska in comparison with those in Progress No. 9. Planta 163: 283 289 Gilmour SJ, Zeevaart JAD, Schwenen L, Graebe JE (1986) Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod. Plant Physiol 83: 190195

418 Lange T, Schweimer A, Ward DA, Hedden P, Graebe JE (1994a) Separation and characterization of three 2-oxoglutarate-dependent dioxygenases from Cucurbita maxima L. endosperm involved in gibberellin biosynthesis. Planta 195: 98107 Lange T, Hedden P, Graebe JE (1994b) Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis. Proc Natl Acad Sci USA 91: 8552 8556 Lange T, Kegler C, Hedden P, Phillips AL, Graebe JE (1997a) Molecular characterization of gibberellin 20-oxidases: structure-function studies on recombinant enzymes and chimaeric proteins. Physiol Plant 100: 543549 Lange T, Robatzek S, Frisse A (1997b) Cloning and expression of a gibberellin 2b,3b-hydroxylase cDNA from pumpkin endosperm. Plant Cell 9: 14591467 Lester DR, Ross JJ, Ait-Ali T, Martin DN, Reid JB (1996) A gibberellin 20-oxidase cDNA (accession no. U58830) from pea (Pisum sativum L.) seed. Plant Physiol 111: 1353 Lester DR, Ross JJ, Davies PJ, Reid JB (1997) Mendel's stem length gene (Le) encodes a gibberellin 3b-hydroxylase. Plant Cell 9: 14351443 Lichtenthaler HK, Schwender J, Disch A, Rohmer M (1997) Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway. FEBS Lett 400: 271274 MacMillan J (1997) Biosynthesis of the gibberellin plant hormones. Nat Prod Rep 14: 221243 MacMillan J, Gaskin P (1996) Gibberellins in endosperm and embryos of Marah macrocarpus. Phytochemistry 42: 12631266 nchez-Beltra n MJ, Gaskin MacMillan J, Ward DA, Phillips AL, Sa P, Lange T, Hedden P (1997) Gibberellin biosynthesis from gibberellin A12-aldehyde in endosperm and embryos of Marah macrocarpus. Plant Physiol 113: 13691377 Martin DN, Proebsting WM, Parks TD, Dougherty WG, Lange T, Lewis MJ, Gaskin P, Hedden P (1996) Feed-back regulation of gibberellin biosynthesis and gene expression in Pisum sativum L. Planta 200: 159166 Martin DN, Proebsting WM, Hedden P (1997) Mendel's dwarng gene: cDNA from the Le alleles and the function of the expressed proteins. Proc Natl Acad Sci USA 94: 89078911 Okamuro JK, den Boer BGW, Lotys-Prass C, Szeto W, Jofuku KD (1996) Flowers into shoots: photo and hormonal control of a meristem identity switch in Arabidopsis. Proc Natl Acad Sci USA 93: 1383113836 Okamuro JK, Szeto W, Lotys-Prass C, Jofuku KD (1997) Photo and hormonal control of meristem identity in the Arabidopsis ower mutants apetala2 and apetala1. Plant Cell 9: 3747 Pharis RP, King RW (1985) Gibberellins and reproductive development in seed plants. Annu Rev Plant Physiol 36: 517568 Phillips AL, Ward DA, Uknes S, Appleford NEJ, Lange T, Huttly AK, Gaskin P, Graebe JE, Hedden P (1995) Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis. Plant Physiol 108: 10491057 Potts C, Reid JB, Murfet IC (1982) Internode length in Pisum . I. The eect of the Le/le gene dierence on endogenous gibberellin-like substances. Physiol Plant 55: 323328 Prescott AG (1993) A dilemma of dioxygenases (or where biochemistry and molecular-biology fail to meet). J Exp Bot 44: 849861 Prescott AG, John P (1996) Dioxygenases: molecular structure and role in plant metabolism. Annu Rev Plant Physiol Plant Mol Biol 47: 245271 Putterill I, Robson F, Lee K, Simon R, Coupland G (1995) The CONSTANS gene of Arabidopsis promotes owering and encodes a protein showing similarities to zinc-nger transcription factors. Cell 80: 847857 Rodrigo MJ, Garc a-Mart nez JL, Santes CM, Gaskin P, Hedden P (1997) The role of gibberellins A1 and A3 in fruit growth of Pisum sativum and the identication of gibberellins A4 and A7 in young seeds. Planta 201: 446455

T. Lange: Molecular biology of gibberellin biosynthesis Ross JJ, Reid JB, Gaskin P, MacMillan J (1989) Internode length in Pisum. Estimation of GA1 levels in genotypes Le, le, and led. Physiol Plant 76: 173176 Ross JJ, Murfet IC, Reid JB (1997) Gibberellin mutants. Physiol Plant 100: 550560 Saito T, Abe H, Yamane H, Sakurai A, Murofushi N, Takio K, Takahashi N, Kamiya Y (1995) Purication and properties of ent-kaurene synthase B from immature seeds of pumpkin. Plant Physiol 109: 12391245 Santes CM, Hedden P, Gaskin P, Garc a-Mart nez JL (1995) Gibberellins and related compounds in young fruits of pea and their relationship to fruit-set. Phytochemistry 40: 13471355 Schwender J, Seemann M, Lichtenthaler HK, Rohmer M (1996) Biosynthesis of isoprenoids (carotenoids, sterols, prenyl sidechains of chlorophylls and plastoquinone) via a novel pyruvate/ glyceraldehyde 3-phosphate non-mevalonate pathway in the green alga Scenedesmus obliquus. Biochem J 316: 7380 Silverstone AL, Chang C-W, Krol E, Sun T-P (1997a) Developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana. Plant J 12: 919 Silverstone AL, Mak PYA, Martinez EC, Sun T-P (1997b) The new RGA locus encodes a negative regulator of gibberellin response in Arabidopsis thaliana. Genetics 146: 10871099 Simcox PD, Dennis DT, West CA (1975) Kaurene synthetase from plastids of developing plant tissues. Biochem Biophys Res Commun 66: 166172 Sponsel VM (1983) The localization, metabolism and biological activity of gibberellins in maturing and germinating seeds of Pisum sativum cv. Progress No. 9. Planta 159: 454468 Sponsel VM (1985) Gibberellins in Pisum sativum their nature, distribution and involvement in growth and development of the plant. Physiol Plant 65: 533538 Sponsel VM (1995) The biosynthesis and metabolism of gibberellins in higher plants. In: Davies PJ (ed) Plant hormones. Kluwer Academic Publishers, Dordrecht, pp 6697 Spray CR, Kobayashi M, Suzuki Y, Phinney BO, Gaskin P, MacMillan J (1996) The dwarf-1 (d1) mutation of Zea mays blocks three steps in the gibberellin biosynthetic pathway. Proc Natl Acad Sci USA 93: 1051510518 Sun T-P, Kamiya Y (1994) The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis. Plant Cell 6: 15091518 Sun T-P, Kamiya Y (1997) Regulation and cellular localization of ent-kaurene synthesis. Physiol Plant 101: 701708 Sun T-P, Goodman HM, Ausubel FM (1992) Cloning the Arabidopsis thaliana GA1 locus by gene subtraction. Plant Cell 4: 119128 Swain SM, Olszewski NE (1996) Genetic analysis of gibberellin signal transduction. Plant Physiol 112: 1117 Swain SM, Reid JB, Ross JJ (1993) Seed development in Pisum The lhi allele reduces gibberellin levels in developing seeds, and increases seed abortion. Planta 191: 482488 Swain SM, Ross JJ, Reid JB, Kamiya Y (1995) Gibberellins and pea seed development Expression of the lhi, ls and le5839 mutations. Planta 195: 426433 Takahashi N, Kobayashi M (1991) Organ-specic gibberellins in rice: Roles and biosynthesis. In: Takahashi N, Phinney BO, MacMillan J (eds) Gibberellins. Springer, Berlin Heidelberg New York, pp 921 Talon M, Koornneef M, Zeevaart JAD (1990) Endogenous gibberellins in Arabidopsis thaliana and possible steps blocked in the biosynthetic pathways of the semidwarf ga4 and ga5 mutants. Proc Natl Acad Sci USA 87: 79837987 Talon M, Zeevaart JAD, Gage DA (1991) Identication of gibberellins in spinach and eects of light and darkness on their levels. Plant Physiol 97: 15211526 Toyomasu T, Kawaide H, Sekimoto C, Von Numers C, Phillips AL, Hedden P, Kamiya Y (1997) Cloning and characterization of a cDNA encoding gibberellin 20-oxidase from rice (Oryza sativa L.) seedlings. Physiol Plant 99: 111118

T. Lange: Molecular biology of gibberellin biosynthesis Ward JL, Jackson GJ, Beale MH, Gaskin P, Hedden P, Mander LN, Phillips AL, Seto H, Talon M, Willis CL, Wilson TM, Zeevaart JAD (1997) Stereochemistry of the oxidation of gibberellin 20-alcohols, GA15 and GA44, to 20-aldehydes by gibberellin 20-oxidases. Chem Commun: 1314 Weigel D (1995) The genetics of ower development: from oral induction to ovule morphogenesis. Annu Rev Genet 29: 1939 Winkler RG, Helentjaris T (1995) The maize Dwarf3 encodes a cytochrome P450-mediated step in gibberellin biosynthesis. Plant Cell 7: 13071317 Wu K, Li L, Gage DA, Zeevaart JAD (1996) Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach. Plant Physiol 110: 547554 Xu Y-L, Li L, Wu K, Peeters AJM, Gage DA, Zeevaart JAD (1995) The GA5 locus of Arabidopsis thaliana encodes a

419 multifunctional gibberellin 20-oxidase: molecular cloning and functional expression. Proc Natl Acad Sci USA 92: 66406644 Xu Y-L, Gage DG, Zeevaart JAD (1997) Gibberellins and stem growth in Arabidopsis thaliana. Eects of photoperiod on expression of the GA4 and GA5 loci. Plant Physiol 114: 1471 1476 Yamaguchi S, Saito T, Abe H, Yamane H, Murofushi N, Kamiya Y (1996) Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.). Plant J 10: 203 213 Zeevaart JAD, Talon M (1992) Gibberellin mutants in Arabidopsis thaliana. In: Karssen CM, Van Loon LC, Verugdenhil D (eds) Progress in plant growth regulation. Kluwer, Dordrecht, pp 3442