Molecular Biology of Gibberellin Synthesis

7/27/2019 Molecular Biology of Gibberellin Synthesis

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Key words: Development Gibberellin Gene expres-sion (heterologous, functional) Growth Monooxy-genase 2-Oxoglutarate dependent dioxygenase Terpene cyclase

Introduction

Gibberellins (GAs) control multiple processes in the life

cycle of higher plants, several of which are essential fornormal plant growth and development (Crozier 1983;Pharis and King 1985; Graebe 1987). For example,internode growth of seedlings is usually retarded aftertreatment with LAB150978, an inhibitor of GA biosyn-thesis (Fig. 1, compare second plant from right with left-hand plant). Additional GA treatment results in plantswith normal or even elevated internode growth, depend-ing on the kind and dose of GAs supplied (Fig. 1, secondplant from left and right-hand plant). Such overgrowthsymptoms are typical GA eects and led to the discoveryof GAs early this century by Japanese phytopathologists,who found that ``bakanae'' disease of rice was caused by

the GA-producing ascomycete Gibberella fujikuroi. Thisexample also gives an impression of the importance ofne adjustment and control of GA biosynthesis fornormal plant growth. By deciphering the underlyingmolecular mechanisms, control and manipulation ofplant developmental processes become possible. Suchtempting prospects might have encouraged research intothe molecular aspects of GA biosynthesis, the recentadvances in which will be highlighted in this review. Thereader is also referred to the comprehensive review byHedden and Kamiya (1997) covering this topic.

Gibberellin biosynthetic pathways are often describedas being of considerable complexity (Graebe 1987;

Sponsel 1995; MacMillan 1997). One hundred andsixteen dierent GA structures have been identied todate and there are more of these tetracyclic diterpenoidcompounds likely to be found. However, only a few GAsare regarded as being biologically active per se. Theirmolecular structure consists of 19 carbon atoms (C19-GAs) containing either a hydroxyl group at carbon-3b[GA1, gibberellic acid (GA3), GA4, GA7, and GA32] oran unsaturated carbon-3 atom (GA5). These GAsexhibit high activities in certain bioassays and are oftendescribed as plant hormones (Graebe and Ropers 1978).

Furthermore, some of the other GAs with low or noactivity in the known bioassays may have as yetundiscovered functions in other plant species, in certaintissues and/or at specic developmental stages. Forinstance, little is known about the role of GAs duringseed development, even though they reach their highestconcentrations in immature seeds of many plant species(Pharis and King 1985; Graebe 1987).

First, the general course of the GA biosyntheticpathway will be outlined. Subsequently, the enzymesinvolved and their encoding genes in individual plantspecies will be discussed. Finally, some future prospectsfor molecular GA physiology will be presented.

General principles of gibberellin biosynthetic pathways

Gibberellin biosynthetic pathways are usually dissectedinto three parts according to the nature of the enzymesinvolved, a division which also reects their subcellularcompartmentation (Fig. 2). Each of the three partsconsists of several steps.

Part I: biosynthesis of ent-kaurene. Cell-free systemsfrom several plant species are well known to bio-synthesize isopentenyl pyrophosphate (IPP), ent-kaure-ne, and even GAs from mevalonic acid (MVA; Graebe1987; MacMillan 1997). However, the incorporation ofMVA into GAs, or even into ent-kaurene, has neverbeen achieved using intact plants or plant tissues.Recently, a second, MVA-independent pathway to IPP

Abbreviations: CPP ent-copalyl pyrophosphate; GA gib-berellin; GGPP ent-geranylgeranyl pyrophosphate; IPP iso-pentenyl pyrophosphate; K synthase ent-kaurene synthase;MVA mevalonic acid

Planta (1998) 204: 409419

Review

Molecular biology of gibberellin synthesis

Theo Lange

Albrecht-von-Haller-Institut fu r Panzenwissenschaften der Universita t Go ttingen, Untere Karspu le 2, D-37073 Go ttingen, GermanyE-mail: [email protected]

Received: 28 July 1997 / Accepted: 8 October 1997


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

1997). Isopentenyl pyrophosphate is further converted

to geranylgeranyl pyrophosphate (GGPP) by only twoenzymes, IPP-isomerase and GGPP-synthase (Dogboand Camara 1987). The latter enzyme catalyzes a three-step condensation and both enzymes were localized inplastids of higher plants (Dogbo and Camara 1987;Kuntz et al. 1992). Geranylgeranyl pyrophosphate isfurther cyclized to ent-copalyl pyrophosphate (CPP) andnally to ent-kaurene. In higher plants these reactionsare catalyzed by two enzymes, CPP synthase (formerly

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

6 M), LAB150978(10A6 M), and GA4 plus LAB150978 (both 10

A6 M)

Fig. 2. General scheme of theGA biosynthetic pathway and itssubcellular compartmentation inhigher plants. ent-Kaurene issynthesized by soluble enzymeslocated, at least partly, in pro-plastids or plastids and is ox-idised to GA12 by enzymes thatare particulate and associatedwith the endoplasmic reticulum.Gibberellin A12 is further oxi-dized by soluble enzymes, andGAs with species- and tissue-

specic hydroxylation patternsemerge. Some of these exhibithormonal activity and eventuallybecome inactivated by furtheroxidation

410 T. Lange: Molecular biology of gibberellin biosynthesis


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kaurene synthase A) and ent-kaurene (K) synthase(formerly kaurene synthase B; renaming was suggestedby MacMillan 1997). Both were localized in isolatedproplastids of meristematic shoot tissues, but not inmature 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 ofthe two enzymes have now been cloned from higherplants (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 microsomalNADPH-dependent cytochrome P-450 monooxygenasesat the endoplasmic reticulum (Fig. 2; Graebe 1987).Therefore, the very hydrophobic (and volatile) precursorent-kaurene must be translocated from the proplastids tothe endoplasmic reticulum, although nothing is knownabout the transport mechanism. ent-Kaurene is oxidizedin six steps to GA12, via ent-kaurenol, ent-kaurenal, ent-kaurenoic acid, ent-7a-hydroxykaurenoic acid, andGA12-aldehyde. So far, only one gene for this part ofthe pathway, the Dwarf-3 gene of maize, has been cloned(see below). It is not known how many monooxygenasesare involved in GA biosynthesis; a single enzyme mightcatalyze several reactions, as do some of the GAdioxygenases described below. Certain biosynthetic steps,such as 7-oxidation, 12a-hydroxylation, and 13-hydrox-

ylation, are catalyzed by both particulate monooxygen-ases and soluble dioxygenases, which occasionally occurtogether within the same species or even within the sametissue (Lange and Graebe 1993). Particulate 3b-hydrox-ylation has only been found in the fungus Gibberella(Bearder 1983). Depending on the nature of the enzymesinvolved, these steps may be assigned to either the secondor the third part of the pathway.

Part III: steps after GA12-aldehyde. Some of the initialsteps of this nal part of the pathway might overlap withthe second part as described above. However, the thirdpart starts here with GA12-aldehyde, because this is the

rst intermediate in the pathway that is oxidized bysoluble 2-oxoglutarate-dependent dioxygenases (Figs. 2,3). The GA dioxygenases are often multifunctional witha broad substrate specicity, resulting in many sidereactions and the numerous GAs found in higher plants

(Lange and Graebe 1993; Hedden and Kamiya 1997).These enzymes belong to the recently identied family ofnon-haem iron-containing oxygenases and oxidases

(Prescott 1993; De Carolis and De Luca 1994; Prescottand John 1996; Barlow et al. 1997). From GA12-aldehyde, a principal pathway to the GA plant hor-mones can be drawn that involves just three GAdioxygenases, 7-oxidase, 20-oxidase and 3b-hydroxylase(Fig. 3). First, 7-oxidation of GA12-aldehyde producesGA12. In the following steps, GA 20-oxidase catalyzesthe whole series of oxidation reactions at carbon-20,leading to either C20-GAs (GA25), or, after loss of C20and lactonisation, to C19-GAs (GA9, Fig. 3). Eventually,3b-hydroxylation activates C19-GAs to plant hormones(GA4, Figs. 1, 3), which are subsequently inactivated by2b-hydroxylation (GA34, Fig. 3). Moreover, C20-GAs

are also 2b- a n d 3b-hydroxylated, but the resultingproducts (GA13 and GA43, respectively; Fig. 3) have noknown function. Nevertheless, such ``side-activities''might prevent enzymes with broad substrate specicityfrom producing GA hormones. The dioxygenases maybe located within the cytosol, although location inanother cellular compartment cannot be excluded. Atleast in pumpkin endosperm, only a little GA dioxygen-ase activity was found to be associated with theendoplasmic 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 byexploiting two plant species in particular, Arabidopsisthaliana and pumpkin. Subsequently, many homologousgenes have been cloned from several other plant species,most successfully from pea. Recent progress made withthese three species will be presented rst, followed byimportant 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 geneticanalysis of plant growth and development, its GAbiosynthetic pathways have not been studied extensivelyyet. The rst part of GA biosynthesis has beendemonstrated in cell-free systems prepared from siliques

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

T. Lange: Molecular biology of gibberellin biosynthesis 411


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(Barendse and Koornneef 1982) and the second part hasnot yet been analysed. Of the third part, numerousendogenous GAs have been identied in shoots, leadingto the construction of GA biosynthetic pathways for this

species (Talon et al. 1990) which have been partlyconrmed by feeding studies with isotopically labelledGAs (Zeevaart and Talon 1992). Taken together, theseresults suggest a typical course of GA biosynthesis inthis species.

Presently cloned genes. The availability of GA-respon-sive mutants (Koornneef and Van der Veen 1980) hasmade A. thaliana extremely useful for studying molec-ular mechanisms of GA biosynthesis. The most prom-inent examples are the rst isolation of the GA1 locus bygenomic subtraction (Sun et al. 1992) and the rstcloning of the GA4 locus by T-DNA tagging (Chiang

et al. 1995). In addition, the existence of a multigenefamily encoding at least three GA 20-oxidases has beendemonstrated (Phillips et al. 1995). Two of the three 20-oxidase genes were identied by a polymerase chainreaction (PCR)-based cloning approach and the thirdwas found in the EST data base at GenBank (Phillipset al. 1995). One of the 20-oxidase genes is identical to theGA5 locus (Xu et al. 1995). The GA1 gene is interruptedby 14 introns (Sun and Kamiya 1994), GA4 contains onlyone intron at base pair 490 from the starting codon(433 bp long; Chiang et al. 1995), and GA5 contains twointrons, one at base pair 551 (191 bp long) and the otherat base pair 873 (103 bp long; Xu et al. 1995).

Properties of the enzymes obtained by cDNA expressionin Escherichia coli. The functions of all GA genesisolated from Arabidopsis have been demonstrated byheterologous expression in E. coli. The GA1 locus

encodes CPP synthase, which catalyzes specically thelast but one step of the rst part of GA biosynthesis, theconversion of GGPP to CPP (Fig. 2; Sun and Kamiya1994). The authors demonstrated that in-vitro-translat-

ed, premature CPP synthase with a MW of 86 kDa istransported into isolated pea chloroplasts, where it isprocessed to a protein of 76 kDa. The three 20-oxidasesproduce mainly C19-GAs and the GA4-locus encodesGA 3b-hydroxylase. They are enzymes of the third partof GA biosynthesis that have broad substrate specicityand prefer non-hydroxylated to 13-hydroxylated GAsubstrates (J. Williams, A.L. Phillips and P. Hedden,IACR-Long Ashton Research Station, University ofBristol, Long Ashton, UK, personal communication;Phillips et al. 1995). Studies on recombinant Arabidopsis20-oxidase showed that GA15 has to be in its openlactone form to be a substrate for this enzyme (Fig. 3;

Ward et al. 1997). Similar results have also beenobtained in cell-free systems from pumpkin endosperm(Hedden and Graebe 1982) and pea cotyledons (Kamiyaet al. 1986). The most abundant endogenous GA planthormone in Arabidopsis shoots is GA4 (Talon et al.1990), a product of both 20-oxidase and 3b-hydroxylaseactivities (Fig. 3). However, considerable amounts ofendogenous 13-hydroxylated GA intermediates havebeen detected (Talon et al. 1990). Their metabolismmight be retarded, making them a reservoir of planthormone precursors.

Gene expression in vivo. In A. thaliana the highesttranscript levels of the GA1 gene (CPP synthase), theGA4 gene (3b-hydroxylase) and two of the three 20-oxidase genes are found in the silique, while the third 20-oxidase gene (encoded by the GA5-locus) is mainlytranscribed in the stem (Sun et al. 1992; Chiang et al.

Table 1. Genes encoding for GA biosynthetic enzymes and their GenBank accession numbers

Plant species Gene Encoded enzyme GenBank accession nos. References

A. thaliana GA1 CPP synthase U11034 Sun et al. 1992GA5 20-oxidase X83379, U20872,

U20873, U20901Phillips et al. 1995; Xu et al. 1995

20-oxidases X83380, X83381 Phillips et al. 1995GA4 3b-hydroxylase L37126 Chiang et al. 1995

C. maxima K synthase U43904 Yamaguchi et al. 19967-oxidase U61386 Lange 199720-oxidases X73314, U61385 Lange et al. 1994b; Lange 19972b-/3b-hydroxylase U63760 Lange et al. 1997b

P. sativum LS CPP synthase U63652 Ait-Ali et al. 1997a20-oxidases X91658, U58830, U70471 Martin et al. 1996; Lester et al. 1996;

Garca-Martnez et al. 1997Le 3b-hydroxylase U93210, AF001219 Lester et al. 1997; Martin et al. 1997

S. oleracea 20-oxidase U33330 Wu et al. 1996

P. vulgaris 20-oxidases U70530, U70531, U70532 Garca-Martnez et al. 1997

M. macrocarpus 20-oxidase Y09112 MacMillan et al. 1997

Z. mays AN1 CPP synthase L37750 Bensen et al. 1995D3 CYP88 U32579 Winkler and Helentjaris 1995

O. sativa 20-oxidase U50333 Toyomasu et al. 1997

Phaeosphaeria sp. CPP/K synthase AB003395 Kawaide et al. 1997



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1995; Phillips et al. 1995; Xu et al. 1997). In addition,considerable GA1 promoter activity was determined notonly in rapidly growing tissues like shoot apices, root

tips, developing owers, and seeds, but also in non-growing organs such as expanded leaves (Silverstoneet al. 1997a). Feedback regulation has been reported fortranscription of the three 20-oxidase and for the 3b-hydroxylase genes: application of GA3 or GA4 toA. thaliana seedlings greatly reduces transcript levels,while in the ga1 and ga5 mutant plants elevated20-oxidase and, in the ga4 mutant, elevated 3b-hydrox-ylase 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 notexpression of the GA4 gene (3b-hydroxylase) has beendemonstrated to be under photoperiodic control (Xuet al. 1997).

Some of these Arabidopsis GA-responsive mutantsare likely to contain null-mutations. In the ga1-3 mutantthe CPP synthase gene is deleted to a large extent (Sunet al. 1992), the ga5 mutant produces only truncatedprotein 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 withenzymatic activity. However, the ga1-3 mutant has beenshown to express CPP activity and it still contains smallamounts of GAs. In the ga5 mutant even substantialamounts of GAs were found (Talon et al. 1990; Zeevaartand Talon 1992). In addition, the ga5 and ga4-2 mutantshave a semi-dwarf growth habit, which also indicates

some GA hormone production. Therefore, these muta-tions are either `leaky' and/or Arabidopsis containsadditional genes that can supplement the missing reac-tions. The simplest explanation for these observations isthe existence of multigene families, as has been demon-strated 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 preparationsfrom immature pumpkin seeds have been used exten-

sively to demonstrate GA biosynthetic pathways in cell-free systems (Graebe 1987; Lange et al. 1993a,b). Thisplant material contains exceptionally high levels of GAenzymes and their encoding transcripts (Lange andGraebe 1993; Lange 1997). Furthermore, GA dioxygen-ases from immature pumpkin seeds have several uniquecatalytic properties leading to GAs of unknown functionfor plant development. Whether these enzymes arespecically expressed during plant embryogenesis and/or whether they are individual features of this species isnot known yet.

Presently cloned genes. Immature pumpkin seeds havebeen used for the rst isolation of GA enzymes and forthe rst cloning of their encoding cDNA molecules byconventional immunoscreening strategies; these enzymesinclude 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 thissource, GA 7-oxidase, has been isolated using a novel

cloning strategy based on the heterologous expression ofenzyme activity in recombinant E. coli (Lange 1997).Genomic DNA was isolated for each of the threedioxygenase genes and revealed intron(s) of approx.200 bp in length (Lange et al. 1997b). However, thenumber and positions of the introns have not beendetermined yet.

Properties of the enzymes obtained by cDNA expressionin E. coli. The identity of all cDNA clones has beendemonstrated by characterisation of the enzymaticproperties of fusion proteins expressed in E. coli. ent-kaurene synthase catalyses the conversion of CPP to ent-kaurene, which is the last step of the rst part of GAbiosynthesis (Fig. 2). In addition, the authors demon-strated that this enzyme is free of CPP synthase activity(Yamaguchi et al. 1996), in contrast to the bifunctionalCPP/K synthase from Phaeosphaeria described below.Characterisation of the GA dioxygenases of the thirdpart of the biosynthetic pathway revealed severalsurprising properties. Gibberellin 7-oxidase and GA20-oxidase are both multifunctional enzymes (Lange1994, 1997; Lange et al. 1994b) and GA 2b-/3b-hydroxylase is a bifunctional enzyme with an exception-ally broad substrate specicity (Lange et al. 1997b).

The GA 7-oxidase prefers the non-hydroxylatedGA12-aldehyde as a substrate (Fig. 3), but also accepts

the 3b-hydroxylated precursor GA14-aldehyde (not il-lustrated in Fig. 3; Lange 1997). After oxidation ofGA12-aldehyde to GA12, the latter compound is con-verted to four other products by the GA 7-oxidase, twoof which are monohydroxylated GAs (Lange 1997).These compounds, which have not yet been identied,might be the starting-point of unknown GA biosyntheticpathways. Gibberellin 7-oxidase is the smallest enzymewith the lowest pH-optimum of all GA dioxygenasescloned so far (Lange et al. 1994a; Lange 1997). Thislatter property might indicate some subcellular com-partmentation of this step of the pathway (Fig. 2).

Recombinant GA 20-oxidase is also a multifunctional

enzyme with broad substrate specicity, preferring non-hydroxylated to 13-hydroxylated substrates. The enzymecatalyzes the whole series of oxidation at carbon-20,leading mainly to C20-GAs without known physiologicalfunction (GA25, Fig. 3; Lange 1994; Lange et al. 1994b).This property is in contrast to all other known 20-oxidases which produce mainly C19-GAs. The molecularbasis of this unique reaction has recently been studied byusing chimeric proteins, constructed from closely related20-oxidases of pumpkin and Marah macrocarpus; bothsequences share 67% identical amino acids. The C-terminal end of the enzyme has been shown to be mainlyresponsible for directing the biosynthetic ow into eitherC19- or C20-GAs (Lange et al. 1997a).

Gibberellin 2b-/3b-hydroxylase oxidises several GAsat the 3b-position, independently of their oxidationstate at carbon-7 and carbon-20 (Lange et al. 1997b).However, GAs containing C-20 alcohol, aldehyde or



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carboxylic acid groups are preferred substrates. Thepresence of a 13-hydroxyl group has no inuence on thereaction. As for the second reaction of this bifunctional

enzyme, only C20-GAs containing a C-20 carboxylic acidgroup are hydroxylated at the 2b-position (Lange et al.1997b). In contrast, recombinant GA 3b-hydroxylasesfrom Arabidopsis and from pea prefer non-hydroxylatedsubstrates and 2b-hydroxylase activity has not beendemonstrated with these enzymes (see above and below).The pumpkin 2b-,3b-hydroxylase sequence is only about35% identical at the amino acid level to the 3b-hydroxylases from pea or Arabidopsis, a degree ofidentity which is typical of dioxygenases with dierentfunctions (Prescott and John 1996).

Many of the major endogenous GAs from immaturepumpkin seeds (Blechschmidt et al. 1984; Lange et al.1993a, b) are potential catalytic products of the threemultifunctional GA dioxygenases described above.However, the occurrence of large amounts of C19-GAs,containing hydroxyl groups at the 12a- and 2b- posi-tions, requires at least three additional enzyme activitiesthat have not yet been identied.

Gene expression in vivo. The K synthase and the threeGA dioxygenases are mainly expressed in developingpumpkin seeds. Enzyme activity of one of the GAdioxygenases, the 2b,3b-hydroxylase, was exclusivelyfound in cell-free enzyme preparations from the endo-sperm of developing pumpkin seeds, but not in devel-oping embryos, indicating a tissue-specic expression

pattern (Lange et al. 1993b, 1997b). Considerabletranscript levels for K synthase and 7-oxidase werefound in the shoot and root tips and, of K synthase, alsoin the hypocotyl of 7-d-old pumpkin seedlings (Yam-aguchi et al. 1996; data not shown). However, transcriptlevels, as determined by quantitative reverse transcrip-tase PCR, for the three GA dioxygenase genes were atleast 200-times lower in germinating seeds compared todeveloping seeds (Lange et al. 1997b). Furthermore, thetranscript levels in germinating seeds did not correlatewith the relatively high GA dioxygenase activities foundin this tissue. Therefore, the high enzyme activities ingerminating seeds are thought to be a legacy of the highgene-expression levels during seed development (Langeet al. 1997b).

Pisum sativum

For molecular analysis of GA biosynthetic pathways thepea system has some advantages over pumpkin andArabidopsis. As with pumpkin, pea has been usedextensively for studying GA-biosynthetic pathways andfor partial purication and characterisation of theenzymes involved (Graebe 1987; Lange and Graebe1993). Similar to Arabidopsis, many GA-responsive peamutants are available (for a recent review, see Ross et al.

1997).

Presently cloned genes. Recently, a number of structuralgenes controlling GA metabolism have been isolatedfrom pea. A cDNA encoding the CPP synthase was

cloned and shown to be encoded by the LS locus (Ait-Ali et al. 1997a). Three cDNA-encoding GA 20-oxidaseshave been isolated (Lester et al. 1996; Martin et al. 1996;

Garca-Martnez et al. 1997). Finally, the isolation of theLe-locus of pea, originally described by Gregor Mendel,was independently accomplished in two laboratories(Lester et al. 1997; Martin et al. 1997). This gene encodesa G A 3b-hydroxylase, as had been predicted frombiochemical and physiological studies (Potts et al.1982; Ingram et al. 1984, 1986; Ross et al. 1989). Likethe GA4-gene in Arabidopsis (see above) the Le-gene hasonly one intron at base pair 488 from the starting codonwhich is 544 bp long (Lester et al. 1997). The positionsand sizes of introns of the other genes have not beenreported yet.

Properties of the enzymes obtained by cDNA expressionin E. coli. Again, the function of all GA genes has beendemonstrated by heterologous expression in E. coli. Theresults are similar to those obtained for the correspond-ing enzymes from Arabidopsis, but they are quitedierent from the ones obtained from pumpkin (seeabove). The CPP synthase specically catalyzes thesynthesis of CPP from GGPP (Ait-Ali et al. 1997a). Thethree GA 20-oxidases produce mainly C19-GAs (Lesteret al. 1996; Martin et al. 1996; Garca-Martnez et al.1997). Contradictory results were obtained for thesubstrate preferences for two of the 20-oxidases. Oneenzyme prefers non-hydroxylated GAs (Martin et al.1996), whereas a second very similar enzyme, which

diers in only three amino acids, does not show thispreference (Garca-Martnez et al. 1997). The third 20-oxidase, which has an amino acid structure considerablydierent from those of the other two also has nopreference for non-hydroxylated or 13-hydroxylatedsubstrates (Lester et al. 1996). These results demonstratethe necessity for more detailed kinetic studies of therecombinant enzymes. However, the GA 3b-hydroxylaseclearly favours the non-hydroxylated substrate GA9 overthe 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 fromanthesis), whereafter they decline. A second increaseoccurs later at three weeks from anthesis (Ait-Ali et al.1997a). Two 20-oxidase genes were expressed individu-ally with a similar timing. Thus, one was transcribedmainly in very young developing seeds (and also ininternodes and young leaves) and the other in three-week-old developing seeds (Lester et al. 1996; Ait-Aliet al. 1997a; Garca-Martnez et al. 1997). This biphasicpattern explains corresponding changes in endogenousGA levels, which have been recognised for a long time(Sponsel 1985). High GA levels appear rst in veryyoung and again in three-week-old seeds. However, 3b-hydroxylated GAs are only found in the very youngseeds (Sponsel 1983; Gaskin et al. 1985; Garca-Martnezet al. 1987, 1991; Swain et al. 1993, 1995; Santes et al.1995; Rodrigo et al. 1997). These plant hormones arelikely to be essential for early embryogenesis, because



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reduced GA levels in very young seeds of pea lhi mutantsresult 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 andinternodes, and, in contrast to the 2b-/3b-hydroxylasegene from pumpkin, it is not abundant in immatureseeds (Martin et al. 1997).

Transcription of both the 20-oxidase and the 3b-hydroxylase genes is subject to feedback regulation(Martin et al. 1996, 1997) similar to the previous ndingfor the homologous genes from Arabidopsis (see above).Recently, it has been demonstrated that 20-oxidasetranscription in pea, as in Arabidopsis (see above), is alsoregulated by light, possibly mediated by phytochrome(Ait-Ali et al. 1997b).

In the pea led mutant the 3b-hydroxylase genecontains a single base deletion resulting in a shift inthe reading frame which is likely to cause a nullmutation. However, as with the Arabidopsis mutants,low levels of GA1 were detectable in these plants(Ingram et al. 1986, see above). Again, this observationsuggests the existence of additional 3b-hydroxylase genes(Martin et al. 1997).

Other systems

Spinacia oleracea. Cell-free systems from spinach leaveswere rst used to show that GA levels and GAdioxygenase activities change depending on photoperi-

od. It was also the rst system in which at least twodierent 20-oxidase activities were detected (Gilmouret al. 1986, 1987; Talon et al. 1991). Recently, theisolation of one 20-oxidase gene from this species wasreported (Wu et al. 1996). The recombinant enzyme, asexpressed in E. coli, catalyzes the three-step oxidation toC19-GAs, exhibiting multifunctional properties similarto those of most other 20-oxidases. This 20-oxidase geneis mainly transcribed in stems of plants growing underlong-day conditions (Wu et al.1996). However, a second20-oxidase activity, which is not under photoperiodiccontrol and which catalyses the second step of 20-oxidation, 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 itslactonic form as substrate (such as GA44, not illustratedin Fig. 3), and it is separable from the other spinach 20-oxidase by anion-exchange chromatography (Gilmouret al. 1987). Similar activities have also been found inpea shoots and germinating barley embryos (Graebe1987; Groelindemann et al. 1992), but not with recom-binant Arabidopsis 20-oxidase nor in cell-free systemsfrom developing pumpkin and pea seeds (see above).

Phaseolus vulgaris. As in Arabidopsis and pea, Frenchbean contains a multigene family for GA 20-oxidases,which, in contrast to the Arabidopsis and pumpkinenzymes, all convert 13-hydroxylated as eciently asnon-hydroxylated GA substrates (Garca-Martnez et al.1997). As in pea, one 20-oxidase gene is mainlytranscribed in young leaves and very young developing

seeds, and a second is mainly transcribed in young leavesand two- to three-week-old developing seeds. Genetranscripts of the third 20-oxidase are most abundant in

very young seeds but are also present at later stages ofseed development (Garca-Martnez et al. 1997).

Marah macrocarpus. From this species, which belongs tothe cucurbit family, another gene encoding GA 20-oxidase has been cloned (MacMillan et al. 1997). Thisenzyme is of particular interest because it is closelyrelated to the pumpkin 20-oxidase, but produces mainlyC19-GAs, in contrast to the pumpkin enzyme (Fig. 3, seeabove). Similar to the pumpkin 20-oxidase, transcriptsof the Marah gene were found mainly in endosperm andembryos of immature seeds. However, substantialamounts of endogenous tricarboxylic C20-GAs as foundin the endosperm of Marah seeds indicate the existenceof another 20-oxidase with catalytic properties similar tothe pumpkin 20-oxidase described above (MacMillanand Gaskin 1996).

Zea mays. Most information on GA-enzyme-encodinggenes from monocotyledons is available from studieswith Zea mays. Two genes have been cloned already bytransposon tagging using Robertson's Mutator (Bensenet al. 1995; Winkler and Helentjaris 1995). The Antherear1 gene has considerable similarity to the GA1 genefrom Arabidopsis, indicating that it also encodes a CPPsynthase 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 ent-kaurene levels, which might indicate the presence offurther CPP synthases, as suggested for Arabidopsis(Bensen et al. 1995).

The second gene that has been cloned from maize isthe Dwarf-3 gene. It encodes a protein containing typicalcytochrome P450-monooxygenase motifs (Winkler andHelentjaris 1995) and is the rst and only isolated geneencoding an enzyme involved in the second part of GAbiosynthesis. The enzymatic properties of the encodedenzyme have not been characterised yet, and it is stillunclear which step of the pathway is blocked in thedwarf-3 mutant plant (B.O. Phinney, cited in Hedden

and Kamiya 1997). The Dwarf-3 gene has been shown tobe expressed in all organs and tissues from maize, asdemonstrated by reverse-transcriptase PCR, but, with-out quantitative analysis of the transcript levels (Winklerand Helentjaris 1995).

Another interesting observation comes from GA20-feeding studies using the dwarf-1 mutant of maize. Thegene locus is responsible for several biosynthetic stepsincluding GA 3b-hydroxylation (as occurred in GA1,GA3) and 2,3-desaturation (GA5, Spray et al. 1996).This might indicate the existence of another multifunc-tional 3b-hydroxylase in maize in addition to the onefrom pumpkin (see above).

Oryza sativa. The rst reported GA 20-oxidase-encodinggene from a monocotyledonous plant was isolated fromrice and also expressed in E. coli (Toyomasu et al.1997). Again, the recombinant enzyme produces mainly



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C19-GAs, but prefers 13-hydroxylated substrates to non-hydroxylated substrates, which is in contrast to all other20-oxidases. These catalytic properties are consistent

with the endogenous GAs found in rice, which containrelatively high levels of 13-hydroxylased GAs (Taka-hashi and Kobayashi 1991; Toyomasu et al. 1997).Elevated GA 20-oxidase transcript levels were found inthe GA-decient dwarf mutants, Tanginbozu andWaito-C, and were also increased by treatment withuniconazole-P, an inhibitor of GA biosynthesis, anddecreased by treatment with GA3. These results indicatefeedback regulation of transcription by mechanismssimilar to those in Arabidopsis and pea (see above).

Phaeosphaeria sp. Although fungi have played animportant role in GA biosynthetic research (for review,see Bearder 1983), only scarce information is availableon the genes encoding GA enzymes in these organisms.Recently, GA biosynthetic pathways have been eluci-dated in the fungus Phaeosphaeria sp. L487 that havesignicant similarities to those of higher plants (Kawaideet al. 1995). Furthermore, an important gene from thisspecies has now been isolated that shares only lowsequence similarity with CPP synthases or K synthasefrom higher plants (Kawaide et al. 1997). Functionalexpression in E. coli conrmed that the recombinantfungal enzyme catalyses the last two steps of the rstpart of GA biosynthesis, the conversion of GGPP toent-kaurene via CPP (Fig. 2). This bifunctional prop-erty has not been found for corresponding enzymes

from higher plants so far. Interestingly, Amo-1618, aspecic inhibitor of CPP synthases from higher plants,also inhibits only the CPP synthase activity but not theK synthase activity of the bifunctional fungal enzyme,suggesting that the two reactions involve dierentresidues at one active centre or that they are catalysedat dierent active centres within the enzyme (Kawaideet al. 1997).

Conclusions

Recently, exciting progress has been made in the eld of

molecular biology of GA biosynthesis, and it is expectedthat within a few years isolated genes will be availablefor all steps of the pathway. In particular, the cloningand characterization of genes encoding the largelyunknown GA monooxygenases will be one of the mostchallenging tasks of the near future.

Molecular approaches have already added to a moreprofound understanding of GA biosynthesis and itscontrol. A general scheme of the GA biosyntheticpathways can be drawn, catalysed by GA enzymeswhich often have multifunctional properties. Character-ization of recombinant GA enzymes, which can often beobtained simply by heterologous expression in E. coli,

will surely add to our understanding of GA biosynthesisand its regulation.Understanding of the molecular bases of regulatory

principles of GA biosynthesis has just begun. Feedbackregulation might occur generally for regulation of

20-oxidase and 3b-hydroylase activities to prevent over-or underproduction of GA plant hormones. Light hasbeen 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 membersof dierent enzyme families, such as monooxygenasesand dioxygenases, catalyze identical steps of the path-way, even though they might be expressed at dierentdevelopmental stages and in dierent parts of the plant.Such a redundancy of the pathway might be importantfor ne tuning of the GA plant hormone pool which isthen less susceptible to unintended changes. However, itwill make manipulated control of GA biosynthesis adicult task.

As presented above, GA-biosynthetic enzymes areexpressed in a complex manner during the whole lifecycle of higher plants. Expression of CPP synthasealternates during pea embryogenesis, as does expressionof two dierent 20-oxidases from pea and bean. Also,two dierent 3b-hydroxylase activities are present indeveloping pumpkin seeds. These ndings indicate theoperation of dierent GA biosynthetic pathways duringthe course of embryogenesis, some of which are likely tobe essential for embryo and seed development.

Investigations on the contribution of individual genesor articial gene constructs to specic GA-dependentdevelopmental processes are no longer restricted by theavailability of suitable mutant plants. The GA-biosyn-thesis genes can now be studied in situ in transformedplants. For this purpose Arabidopsis thaliana has several

well-known advantages over other plant species whichsurely will facilitate future research in the eld of themolecular biology of GA synthesis.

To date, little is known about the mechanism of GAplant hormone action and signal-transduction pathways(for reviews, see Hooley 1994; Swain and Olszewski1996). Gibberellin synthesis and action may occur indierent tissues, as discussed above for semi-dwarf GA-responsive mutants. In other cases, synthesis and actionmight occur within one and the same tissue but atdierent developmental stages, as suggested for GA-mediated 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 numerousgenes essential for plant development, some of whichappear to be coupled to GA signal-transduction path-ways (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 plantgenetics will complement molecular GA physiologymore frequently.

I thank Professor Jan E. Graebe for carefully reading themanuscript, my colleagues Drs Peter Hedden, John Ross (Depart-ment 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



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Nobutaka Takahashi (Institute of Physical and Chemical Research,Wako, Saitama, Japan) for making space available at his RIKENoce for me to write this review. Work in my laboratory wassupported by the Deutsche Forschungsgemeinschaft.

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