Planta

Planta (1992)188:252-257

9 Springer-Verlag1992

Gibberellin biosynthesis from gibberellin A12-aldehyde in a cell-free system from germinating barley (Hordeum vulgare L., cv. Himalaya) embryos Elke GroBelindemann:*, Mervyn J. Lewis 2, Peter Hedden 2, and Jan E. Graebe l* 1 Pflanzenphysiologisches Institut und Botanischer Garten der Universit~it, Untere Karspfile 2, W-3400 G6ttingen, Federal Republic of Germany 2 Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Long Ashton, Bristol BS18 9AF, UK Received 19 March; accepted 9 May 1992

Abstract. Gibberellin (GA) metabolism from GA12aldehyde was studied in cell-free systems from 2-d-old germinating embryos o f barley. [14C]- or [17-2H2]Gib berellins were used as substrates and all products were identified by combined gas chromatography-mass spectrometry. Stepwise analysis demonstrated the conversion of GAlz-aldehyde via the 13-deoxy pathway to GAs~ and via the 13-hydroxylation pathway to GA29, GA~ and G A s. In addition, GA3 was formed from GA2o via GAs. We conclude that the embryo is capable o f producing gibberellins that can induce a-amylase production in the aleurone layer. There was no evidence for 1213- or 18hydroxylation and GAg was neither synthesised nor metabolised by the system. All metabolically obtained GAs, with the exception of G A a, were also found as endogenous components o f the cell-free system in spite of ammonium-sulfate precipitation and desalting steps. Key words: Gibberellin biosynthesis - Hordeum (gibberellin synthesis) - Metabolism (cell-free system)

Introduction We have studied gibberellin (GA) metabolism in the embryos of germinating barley grain as part of an investigation into the role o f the embryo in the production of a-amylase in the aleurone layer. According to the wellestablished theory, G A is produced de novo in the embryo and diffuses to the aleurone layer, where it induces the formation o f a-amylase (Paleg 1960; Yomo 1960; for a modern presentation, see Jones and Jacobsen 1991). * To whom correspondence should be addressed; FAX (0) 551/ 397823 ** Present address: CSIRO, Division of Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia Abbreviations: GAn=gibberellin A.; GC-MS=combined gas chromatography-mass spectrometry; HPLC = high-performance liquid chromatography

Whereas the action and specificity of G A in barley germination is well established, the source of G A in the grain has not been demonstrated conclusively (Fincher 1989). Indeed, GroBelindemann et al. (1991) found that inhibiting ent-kaurene biosynthesis by the addition of plant growth retardants had little effect on u-amylase production, which would therefore appear to be independent of de-novo G A biosynthesis. Unless a-amylase production does not require the presence of biologically active GA, an alternative source of this hormone, such as a stored precursor, must be postulated, since neither free (GroBelindemann 1990) nor conjugated (Yomo and Iinuma 1966; Atzorn and Weiler 1983) active GAs are present in the mature dry grain. In order to evaluate the role of potential GA-precursors, it was first necessary to establish their biosynthetic pathway in barley embryos. Although it was not possible to demonstrate conversion of ent-kaurene to GA12aldehyde, we assume that this part o f the pathway follows the usual scheme as established in immature seeds (Graebe 1987) and recently also in homogenates and tissue from shoots of Zea mays (Suzuki et al. 1992). Murphy and Briggs (1973) also failed to show metabolism of ent-[14C]kaurene to GAs in barley grains, but demonstrated the stepwise conversion of ent-kaurenol to ent-7a-hydroxykaurenoic acid in microsomal preparations from embryos (Murphy and Briggs 1975). This paper demonstrates the complete biosynthetic pathway in the embryos from GA12-aldehyde, the intermediate immediately following ent-7a-hydroxykaurenoic acid, to GAs that are known to induce a-amylase in isolated aleurone layers. Material and methods Plant material. Barley (Hordeum vuloare L., cv. Himalaya, harvest 1985) grain was purchased from the Crops & Soils Club, Department of Agronomy and Soils, Washington State University, Pullman, USA. Grains were surface-sterilised in 1% (w/v) NaOC1 for 5 min and washed thoroughly with double-distilled H20. Seeds were germinated in large Petri dishes (19 cm diameter) on a moist filter paper (0.2 ml H20 per 100 seeds) for 2 d in a growth cabinet

E. Grol3elindemann et al. : Gibberellin pathways from GA12-aldehyde under a 14-h photoperiod at 22 ~ C (light) and at 18~ C (darkness). Light was supplied by L65 W/25 white-universal tubes (Osram, Miinchen, FRG), located 80 cm above the grains, giving a photon fluence rate o f 1 . 6 2 . 1 0 4 m o l . m 2 . s 1.

Preparation of radiolabelled substrates. [14C]Gibberellin A12-aldehyde (6.4.1012 Bq. tool- 1), [14C]GA12 (6.6" 1012 Bq. m o l - t) and [14C]GA15 (3.8-1012 B q ' m o 1 - 1 ) were prepared from basehydrolysed R-[2-14C]mevalonic acid lactone (1.96.1012 Bq- m o l - 1, Amersham-Buchler, Braunschweig, F R G ) using a cellfree system from Cucurbita maxima endosperm (Graebe et al. 1972, modified by Turnbull et al. 1985). [14C]Gibberellin Asa (6.7 9 1012 Bq" mol-1) was prepared from [14C]GA12 with a cell-free system from immature pea cotyledons according to the method of Lange and Graebe (1989), but with 2 mM N A D P H and the addition of 2 mM EDTA, the latter to prevent conversion of GA12 to GAs of the 13-deoxy pathway. [14C]Gibberellin A44 (5.3" 1012 B q ' m o l - 1 ) , [14C]GA19 (4.5- 1012 B q - m o l - 1 ) , [14C]GA2o (1.9' 1012 Bq-mo1-1) and [14C]GA29 (1.85-1011 B q ' mo1-1) were prepared from [14C]GA53 using the 200000"9 supernatant from an homogenate of immature (21 d after anthesis) pea cotyledons prepared in 0.1 M Tris-HC1 buffer (pH 7.0, 30 ~ C) containing 4 m M dithiothreitol (DTT). Incubations were for 2 h at 30 ~ C in the presence of 5 m M ascorbate, 0.5 mM FeSO4 and 5 mM 2-oxoglutarate. [14C]Gibberellin A24 (3.5"1012 B q ' m o 1 - 1 ) and [14C]GAs1 (3.3 9 1012 Bq. mo1-1) were prepared from [14C]GA15 (open lactone form; Kamiya and Graebe 1983) using the cell-free system from pea cotyledons described above. [14C]Gibberellin A1 (1.6.1012 Bq. tool -1) was prepared from [t4C]GAzo using a cellfree system from 2-d-old germinating barley embryos as described below. The labelled GAs were purified by high-performance liquid chromatography (HPLC) and their isotope contents determined by combined gas chromatography-mass spectrometry ( G C - M S ; Bowen et al. 1972). [17-2H2]Gibberellin A3, [17-2H2]GA4, [17-ZH2]GA5 and [17-2Hz]GA9 (each approx. 99% purity) were gifts from Prof. L. Mander, Australian National University, Canberra, Australia.

Preparation of cell-free extracts. All enzyme-preparation steps were carried out at 4 ~ C, unless otherwise stated. Embryos with attached scutella (approx. 20 g) were separated from the germinating grains, frozen in liquid N 2 and pulverised for 3 min at full speed in a precooled ( - 2 0 ~ C) Waring blendor. After addition of 0.2 M Tris-HC1 buffer (pH 7.0, 30 ~ C) containing 8 mM DTT (1 : 1, w/v), the mixture was allowed to thaw. Soluble enzymes were prepared by centrifuging the homogenate at 20000 - g for 20 min to remove starch and cell fragments, and collecting proteins that precipitated from the resulting supernatant between 20-50% saturated (NH4)2SO 4 by centrifuging at 15000-9 for 15 min. The precipitated proteins were resuspended in 2.5 ml 0.1 M Tris-HC1 buffer (pH 7.0, 30 ~ C) containing 4 m M DTT, filtered through a PD-10 column (Sephadex G-25, Pharmacia, Freiburg, FRG) which was equilibrated and eluted with the same buffer, and stored in liquid N 2. A microsomal fraction was prepared from the homogenate by centrifuging at 5000-9 for 5 min, and then recentrifuging the resulting supernatant at 140000 9g for 1 h. After carefully removing the supernatant, the pellet and the layer between pellet and supernatant were pooled and resuspended in 0.1 M Tris-HC1 buffer (pH 7.0, 30 ~ C) containing 4 m M DTT. The suspension was frozen and stored in liquid N 2. Protein estimation. Protein contents were estimated by the method of Sedmak and Grossberg (1977) as modified by Scopes (1982), using crystalline bovine serum albumin as standard.

Incubations. Enzyme preparations were diluted to the required protein concentrations with 0.1 M Tris-HC1 buffer (pH 7.0, 30 ~ C). Incubation mixtures (1 ml) for soluble enzyme preparations consisted of 96.8% (v/v) diluted enzyme preparation and 3% (v/v) of a solution containing (final concentrations) 2-oxoglutarate (5 mM), ascorbate (5 mM) and FeSO4 (0.5 mM). Incubation mixtures (1 ml)

253 for microsome preparations contained 93.8% (v/v) diluted microsomes and 6% (v/v) of a solution containing (final concentrations) N A D P H (2 mM), glucose-6-phosphate (3 mM), E D T A (2 mM) and glucose-6-phosphate dehydrogenase (0.85 units 100 per 100 ~tl assay). Reactions were started by adding 0.2% (v/v) substrate in methanol, allowed to proceed in a shaking water bath at 30 ~ C for 3 h, and stopped by adding 10% (v/v) acetic acid (96%) which lowered the pH to 3.2. Gibberellins were extracted by partitioning five times against equal volumes of ethyl acetate containing 1% water (v/v); the solvent was then removed with a stream of N2.

Application of [l~C]GA12-aldehyde to intact shoots. The substrate (925 Bq per plant) was applied in 10% acetone-1% Tween-80 (v/v; 10 lal) to shoots of 40 3-d-old seedlings. After 48 h the shoots were homogenised in 80% methanol, stirred for 20 h at 4 ~ C and centrifuged. After removing methanol from the resulting supernatant, the aqueous residue was adjusted to pH 3.2 with 1 N HC1, and partitioned against an equal volume petroleum ether to remove nonpolar components, including the substrate. Products were extracted with ethyl acetate containing 1% water (v/v) as described above. Possible GA-conjugates were hydrolysed by incubating the aqueous phase at pH 4.5 with cellulase from Aspergillus niger (Serva, Heidelberg, F R G ; technical grade; 10 m - g -1 fresh weight shoots) and four drops of toluene for 24 h at 31 ~ C. The hydrolysate was adjusted to pH 2.8 with 96% acetic acid and partitioned against ethyl acetate (3 times equal volume). Extracts were taken to dryness under N 2.

High-performance liquid chromatography. Products from incubations were separated by reverse-phase HPLC using a Waters dualpump HPLC with a model 680 gradient programmer (Waters, Eschborn, F R G ) and connected on-line to a Ramona D (Raytest, Essen, F R G ) radioactivity detector as described by Lange and Graebe (1989). Dried samples were dissolved in 55% methanol-10 m M aqueous acetic acid (110 Ixl) and injected via a Rheodyne sample injector (100 lal loop) onto a Waters Radial Pak Liquid Chromatography Cartridge (5 ~tm particle size, 15 cm long, 0.39 cm i.d.), which had been equilibrated with 40% (v/v) methanol in 10 mM aqueous acetic acid. Samples were eluted by a 10-min concave gradient (Waters gradient 8) from 40-65% methanol followed by an 8-min concave gradient (gradient 9) from 65-80% methanol, a 3-rain linear gradient to 100% methanol and finally isocratic elution with methanol for 9 min. The radioactivity monitor was calibrated with 14C-labelled standards, the radioactivity of which was measured in a liquid scintillation counter (PW 4700; Philips, Eindhoven, The Netherlands) after HPLC. Products from incubations with 2H-labelled substrates were collected according to the previously determined retention times of 14C standards, allowing wide margins.

Gas chromatography-mass spectrometry. Dried HPLC fractions were dissolved in methanol and methylated with ethereal diazomethane. Samples were then transferred to glass ampoules, dried and trimethylsilylated with 5 lal N-methyl-N-trimethylsilyltrifluoracetamide (MSTFA; Macherey and Nagel, Diiren, FRG) at 90 ~ C for 30 min. Derivatised samples were analysed using an MS80 R F A mass spectrometer (Kratos Analytical, Manchester, UK). The samples (1 lxl) were co-injected with 0.1 ~tl of a solution of Parafilm in hexane for determining Kovats retention indices (Gaskin et al. 1971) into a fused silica wall-coated open tubular (WCOT) OV-1 capillary column (25 m long, 0.32 mm diameter, 0.25 ~tm film thickness; Phase Separations, Deeside, UK) at an oven temperature of 50 ~ C with the injector split valve closed. After 0.5 min the split valve (50: 1) was opened and after I min the oven temperature was increased at 20 ~ C 9m i n - 1 to 240 ~ C and then at 4 ~ C 9m i n to 295 ~ C. The He inlet pressure was 0.04 MPa and injector and interface temperatures were 220 and 250 ~ C, respectively. After 12 min, positive ion electron impact mass spectra were acquired, scanning from 700-50 amu at 1 s per mass decade. The electron energy was 70 eV, the source temperature 200 ~ C.

254 Some analyses were carried out using an Incos 500 G C - M S system (Finnigan MAT, Bremen, FRG). The samples (2 lal) were injected into a DB-5 capillary column (29 m long, 0.25 mm diameter, 0.25 lam film thickness; J&W Scientific, Folsom, Calif., USA) at an oven temperature of 50 ~ C. The split valve (50: 1) was opened after 0.7 min; the temperature programme was the same as described above, except that the final temperature was 285 ~ C. The He flow rate was 1.3 ml 9 m i n - 1 , electron energy was 70 eV and injector, interface and source temperatures were 250, 275 and 135 ~ C, respectively.

E. GroBelindemann et al.: Gibberellin pathways from GA12-aldehyde

10-

"7

r-

6

9

-

-6 E C 0

Results

Preliminary experiments. Since it had been shown that ent-kaurene was turned over rapidly in germinating barley caryopses (Grol3elindemann et al. 1991), we attempted first to study the metabolism of this intermediate using microsomal preparations from 2-d-old embryos. However, neither ent-[14C]kaurene nor its known biosynthetic products ent-[~4C]kaurenol, ent-[14C]kaurenal and ent-7a-hydroxy[~4C]kaurenoic acid were metabolised, although several methods for preparing the microsomes and different incubation mixtures were tried (Grol3elindemann 1990). Therefore, attention was turned to the metabolism of GA12-aldehyde, a general intermediate in GA biosynthesis. The ~4C-labelled compound was applied to shoots of 3-d-old seedlings, which metabolised it to numerous products in 48 h. Although several of these products had similar retention times on HPLC to GA standards, none could be identified by GC-MS. The same pattern of unidentifiable products was obtained in several experiments and the approach of feeding [~4C]GA~2-aldehyde to the intact tissues was abandoned.

Metabolism of GA in cell-free systems. The activity of cell-free extracts of germinating embryos was determined by measuring the metabolism of [~4C]GA12 at different times. Highest enzyme activity was obtained with 2-d-old embryos (Fig. 1), yielding several products, which were not identified at this point. Extracts of aleurone-endosperm contained trace activity (0.87 pmol. h -~. g-~ fresh weight), but, unlike those from the embryos, they converted [~4C]GA~2 to [14C]GAa5 only (data not shown). Extracts of 3-d-old roots and older were inactive (data not shown). Two-day-old embryos were therefore used as the source of cell-free extracts for studying GA metabolism. The results are summarised in Tables 1 and 2 for incubations with microsomal and supernatant preparations, respectively. All products were identified by full-scan GC-MS and, with the exception of GA3, were diluted by endogenous GAs, which were present in the extracts despite ammonium-sulfate precipitation and desalting. Both the microsomal and the soluble preparations converted [~4C]GA~2-aldehyde, in the first case (Table 1) to [14C]GA12 (3%) and [14C]GA53 (7%) and in the second case (Table 2) to [14C]GAt2 only (12%). The figures in parentheses indicate the percentage of total recovered radioactivity, which was 68-87% of the substrate added in all experiments. [~4C]Gibberellin A~ 2 was also metabo-

~ 0-

I

I

0

~~ I

4

I

I

I

6

o I

I

8

9 I

10

Time from stort of imbibition [ d ]

Fig. 1. Conversion of GA12 by cell-free preparations from 1- to 10-d-old barley embryos and shoots. [14C]GA12 (925 Bq) was incubated with soluble enzyme preparations (6 mg protein) and cofactors as described in Material and methods but for 1 h only. For the first 2 d the whole embryos, including radicles and scutella, but without endosperm and aleurone, were used. Thereafter, the (now green) shoots with scutella but without roots were taken

Table 1. Metabolism of labelled GAs by a microsomal preparation from 2-d-old barley embryos (standard incubations) Substrate added

Product(s)

Protein

GA

pmol

GA

pmol

(mg 9ml - l)

GAlz-aldehyde

145 140 240 185 150 280

5 10 56 1 3 0 0

4.5

GA12 GA15 GA24 GA 9 GAs1

GA12 GAs3 GAs3 GA44 GA19 none none

4.5 7.5 7.5 7.0 7.5

lised by both enzyme preparations, yielding [ 1 4 C ] G A 5 3 (40%) with the microsomal preparation and [14C]GA15/ GA9 (46%), [14C]GA24 (18%) and [14C]GAs~ (13%) with the concentrated supernatant enzymes. Both preparations converted [a4C]GA12 at higher rates than [a4C]GA~2-aldehyde. However, [a4C]GA~5 (1%) and [14C]GA24 (3 %) were poorly 13-hydroxylated by the microsomal preparation and [17-2H2]GA9 and [14C]GA51 not at all (Table 1). In confirming the single steps of the 13-deoxy pathway (Table 2), the lactone form of [14C]GA15 was converted in good yield, but only to [a4C]GA24 (29%), which was metabolised in a separate incubation to [14C]GA9 and [~4C]GA51 in very good yields (32% and 45%, respectively). No labelled products were found when [17-2Hz]GA9, [~4C]GAsa or [17-2H2]GA4 were used as substrates, even when the incubation times were increased to 20 h. As for the 13-hydroxylation pathway, [~4C]GA53 was converted by the soluble enzyme preparation to [~4C]GA44 (11%), which, in the lactone form, was converted to [14C]GA19 (59%; Table 2). However, no metab-

E. GroBelindemannet al.: Gibberellin pathways from GA12-aldehyde Table 2. Metabolism of labelled GAs by a desalted 20-50% ammonium-sulfatefraction of the 20000 9g supernatant from a cellfree extract of 2-d-old barley embryos Substrate a d d e d GA pmol 13-Deoxy pathway GA 12-aldehyde 72 GA12 84

P r o d u c t ( s ) IncubationProtein GA pmol time (h) (mg"ml-i)

GA12 9 GA15/9 39 GA24 15 GAs1

GAi5 GA24

145 106

GA9 GAs1 GA4

300 450 240

13-Hydroxy pathway GAs3 55 GA44 175 GA44a 700 GA19 GA2ob

200 980

GAl GA29 GAs GA3

230 2000 240 100

3 3

10 20

3 3

20 20

20 20 20

20 20 20

3 3 20

20 20 10

3 20

20 25

3 3 20 3

20 13 20 13

11

GA24 42 GA9 GAs1 none none none

34 48 0 0 0

GA44

6

GAi9 103 GAi9 370 GA2o 63 none 0 GAs 17 GA1 47 GAs 10 GA29 12 GAs 5 none 0 GA3 nap none 0

8 ml incubation mixture b 2 ml incubation mixture not determined olism of [14C]GAi9 could be obtained under these incubation conditions. The [14C]GAI9 obtained as a product from [14C]GA44 was found from its mass spectrum to be highly diluted by endogenous Gml9, and this could account for its apparent lack of conversion. In a larger-scale incubation of [14C]GA44 in which the incubation volume was increased from 1 ml to 8 ml and the incubation time from 3 h to 20 h, [14C]GA2o (9%) was formed in addition to [~4C]GA~9 (53%). Metabolism of [14C]GA2o, which required high concentrations of protein (25 mg. ml -~) and substrate (490 nM), yielded [14C]GAi (5%), [~4C]GA5 (2%), [~'C]GAs (1%) and [iaC]GA29 (1%). [14C]Gibberellin A~ was converted to [14C]GAs in 2% yield and [17-2H2]GA5 was converted to [17-2Hz]GAa to an unknown extent. [17-2H2]Gib berellin A 3 was not converted by the system. Discussion

The results of the stepwise analysis of GA biosynthetic pathways from GA~2-aldehyde in germinating barley embryos reported in this paper are summarised in Fig. 2. It is one of few investigations of GA metabolism using cell-free extracts of vegetative plant tissues. Suzuki et al. (1992) established the five-step sequence from entkaurene to GA~ 2-aldehyde using homogenates and tissue cubes from maize, Gilmour et al. (1986) showed the

255 conversion of GA12 via the 13-hydroxylation pathway to GA2o using cell-flee extracts from spinach leaves and Zander (1990) found evidence for both the 13-deoxy and the 13-hydroxylation pathways from GA12 using cellfree preparations from germinating pea cotyledons. In the cell-free system of barley embryos described in the present work both pathways were also obtained and, in addition, the 2,3-dehydrogenation of GA2o to GAs with further 3[3-hydroxylation to GA3. These results correspond well to the situation in vivo, as defined by analyses of GAs in barley seedlings: Yamada (1982) identified GA1 and GA3 in shoot tissue of 2-d-old Betzes barley and Gaskin et al. (1984) identified GA~, GA 3 (with reservation), GAIT, GA19 and GA2o from 3-d-old barley shoots (cv. Maris Otter). Two recent papers reported the presence of GAs of the 13-hydroxylation pathway in 7-d-old leaf sheath segments (Croker et al. 1990) and 9-d-old light- or dark-grown barley shoots (Boother et al. 1991). Gibberellin As,, GA48 and 18 OH GA34, small amounts of which have also been found in 3-d-old barley shoots (Gaskin et al. 1984), were not obtained as products in the present study. These GAs are thought to be formed directly from GA4 in developing barley grains (Gilmour et al. 1984). Since this GA was not obtained from GA 9 or metabolised by the cell-free embryo system, 1213- and 18-hydroxylation, as well as GA4 synthesis, may occur in the developing grain only, from where the respective products may be carried over to the young seedling. Our results agree with the general view that 13-hydroxylation occurs early in the biosynthetic pathway, with GA12 the preferred substrate (e.g. Kamiya and Graebe 1983; Gilmour et al. 1984). Gibberellin A44 and GA~5 were converted as lactones to GAi9 and GA24, respectively, as was shown also by Gilmour et al. (1986) for GA44 in spinach and by Zander (1990) for both GAs in pea shoots. Oxidation of the 6-1actone at C-20 may be typical for shoot tissues, whereas cell-free systems from immature seeds convert GA15 to Ci9-GAs only after hydrolysis to the free alcohol (Hedden and Graebe 1982; Kamiya and Graebe 1983). The conversion of C20-GAs to the physiologically active Ci9-GAs is an important step in GA biosynthesis, which is also underlined by the fact that it is regulated by photoperiod in spinach leaves (Gilmour et al. 1986). Although we had no difficulty in showing the conversion of GA24 to GAs, the formation of GA2o succeeded only with long incubation times and by the use of high substrate and enzyme concentrations, whereby [a4C]GA44 had to be used as the substrate, because not enough [i4C]GAi9 was available. Zander (1990) encountered similar difficulties in demonstrating the conversion of [14C]GA19 to [14C]GA2o in cell-free extracts of pea seedlings. Gibberellin ml9 is a predominant GA in shoots of rice (Kurogochi et al. 1979; Kobayashi et al. 1988) and maize (Fujioka et al. 1988), in leaf sheaths of barley (Croker et al. 1990) and in shoots (Lin and Stafford 1987), leaf sheaths (Appleford and Lenton 1991) and germinating embryos (Lenton and Appleford 1991) of wheat. Kurogochi et al. (1979) suggested that GAi9 might be a storage GA, whereas Lin and Stafford (1987),

256

E.Grol3elindemann etal.:GibberellinpathwaysfromGAtz-aldehyde HO

-,,~,."~ -i C02H

C02H

~~HCHO(

G GA12 A

2 +4GA15+

OH

OH

GAi2-aldehyde~

___ii~ o+ . ,~, ~~'C . 1O2H

/0

"~'~..i ~'C02H C02H

+

GA 9

OH ~HOH+~

GA

P

HO

51

P

--t1.,-

T ~ "CO2H

GA53

GA44

GAt9

GA 20

GA

OH GA5

OH Fig. 2. Gibberellin biosyntheticpathwaysin cell-free systemsfrom 2-d-old barley embryos Fujioka et al. (1988) and Croker et al. (1990) concluded that the conversion of GA19 to GA20 may be a rate-limiting step for GA biosynthesis in monocotyledonous species. The lack of conversion of GA19 in the barley cellfree system may thus be due to a low turnover rate of this GA in vivo, compounded by dilution of the substrate with endogenous GA19 , which was not removed after ammonium-sulfate precipitation and gel filtration. It is also possible that newly formed [a+C]GA19 is preferentially converted to [14C]GA2o and that this is the reason why the incubation with [a+C]GAa4 was successful. The more rapid conversion of [14C]GA24 (Table 2) may indicate that it is a better substrate for the C-20 oxidase, or that different enzymes catalyse C-20 oxidation in the 13-hydroxylation and non- 13-hydroxylation pathways, besides the fact that less endogenous GA2+ is present. Gibberellin A2o lies at a branch point from which three pathways diverge. It is 213-hydroxylated to the physiologically inactive GA29, 313-hydroxylated to GA1, which is also subsequently deactivated by 2[3-hydroxylation to GA8, and it is desaturated at C-2,3 to produce GAs, which is 3[3-hydroxylated to GA 3. Boother et al. (T991) showed that [17-13C,3H2]GA2o was converted to labelled GA~, GAs and GA29 in 6-d-old barley shoots (cv. Triumph). Our demonstration of the formation of GA 3 in the cell-free barley system supports the findings of Fujioka et al. (1990), who showed that it was synthesised from GA2o via GAs in maize shoots. Although we found no dilution of [17-2H2]GA3 by endogenous compound in the barley system, GA3 has been detected in shoots of this species (see above), substantiating the view that the reactions found also occur in vivo. This study was not primarily designed to identify endogenous GAs and only such GAs were detected as remained with the large-molecule fraction after ammonium-sulfate pre-

GA 1

OH HO" ~ r.-i "C02H GA 8

GA 3

cipitation and desalting. These GAs may be associated in an unknown way with macromolecules or other cellular components and this may not be true for GA 3. Grol3elindemann et al. (1991) found that ent-kaurene biosynthesis starts 24-48 h after imbibition of barley grains and that ent-kaurene does not accumulate but is turned over continually. Taken together with the results of Murphy and Briggs (1975) and those presented here, this shows that there is the potential for de-novo synthesis of both GA1 and GA 3 at this stage of germination. These GAs are among the most active ones known for the induction of growth in various species and for the induction of a-amylase in barley aleurones (Crozier et al. 1970; Reeve and Crozier 1974). Since seedling growth, but not a-amylase production, was suppressed when ent-kaurene oxidation was inhibited by growth retardants (GroBelindemann et al. 1991), shoot growth is obviously dependent on de novo GA biosynthesis, whereas a-amylase formation is not. Theoretically, stored GA intermediates, such as GA19 or GA20, which do not themselves induce a-amylase or do so poorly (Crozier et at. 1970), could be converted by the embryo to GA1 and-or GA3, which are transported to the aleurone layer where they induce ~t-amylase formation in accord with the hypothesis originally formulated by Paleg (1960) and Yomo (1960). Several endogenous GAs were identified in the cell-free extract of 48-h-old embryos in this work and it remains to be seen which are also present in ungerminated seeds independently of entkaurene oxidation. Since shoot growth is suppressed at 48 h when entkaurene oxidation is inhibited, the limited supply of GA1 and-or GA 3 hypothetically formed from stored intermediate GAs may be preferentially used for the induction of a-amylase in this situation.

E. GroBelindemann et al. : Gibberellin pathways from GAlz-aldehyde We thank Mrs. G. Bodtke and Mrs. B. Schattenberg for preparing the barley embryos and the Deutsche Forschungsgemeinschaft for supporting this work.

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