THE JOURNAL OF BIO~GICAL CHEMISTRY Vol. 240, No. 4, April 1965

Printed in u.s.A+

Biosynthesis of Gibberellins

I. THE BIOSYNTHESIS OF (-)-KAURENE, (-)-KAUREN-19-OL, AND TRANX-GERANYL- GERANIOL IN ENDOSPERM NUCELLUS OF ECHINOCYSTIS MACROCARPA GREENE*

JAN E. GRAEBE,~ DAVID T. DENNIS, CHRISTEN D. UPPER, AND CHARLES A. WEST

From the Biochemistry Division, Department of Chemistry, University of California, Los Angeles, California 900,%$

(Received for publication, August 10, 1964)

The gibberellins are an important class of natural growth regulators in flowering plants as indicated by a considerable body of evidence showing their natural occurrence in a wide variety of plants and their activity in regulating many aspects of plant growth and development (1, 2). Thus, the regulation of the biosynthesis of gibberellins in plants may be an important means by which various developmental processes are controlled. A more detailed knowledge of this biosynthetic pathway in flowering plants is required as a basis for investigations in this area of plant physiology.

Cross et al. first suggested the structural and presumably bio- genetic relationship of gibberellic acid (Ia) to diterpenes (3). The number and positions of i4C atoms in gibberellic acid pro- duced from 2-I%-mevalonate or 1-i4C-acetate in cultures of

including gibberellin Ai (IIa), Aa (gibberellic acid, Ia), A4 (IIb) A7 (Ib), and several unidentified gibberellin-like materials (12, 13).x This paper describes the formation of the diterpenes (-)- kaurene, ( -)-kauren-19-01, and trans-geranylgeraniol, along with unidentified components from 2-14C-mevalonate in cell-free homogenates of the endosperm nucellus of E. macrocarpa. Evi- dence is also presented for the intermediary role of ( -)-kaurene and ( -)-kauren-19-01 in gibberellin biosynthesis.

EXPERIMENTAL PROCEDURE

Materials

The N ,N’-dibenzylethylenediammonium salt of 2-14C-u~- mevalonic acid was purchased from Volk Radiochemical Com-

Fusarium monilijorme supported this view (4, 5). Further, a large number of neutral and acidic diterpenoid compounds in- cluding (-)-kaurene (III,) have been identified in addition to gibberellins by Cross et al. in culture filtrates of this fungus (6, 7). Conversion of 14C-labeled (-)-kaurene to gibberellic acid (8), and the discovery of biological activities of ( -)-kaurene, (-)-kauren-19-01 (IIIb) and closely related diterpenoid com- pounds in gibberellin assay systems (9, 10) have suggested an intermediate role for t.hese substances in gibberellin biosynthesis.

In flowering plants the endosperm nucellus tissue of immature seed of Echinocystis macrocarpa Greene (“wild cucumber”) has been shown to be a relatively rich source of gibberellins (ll),

* This work was supported by Public Health Service Research Grant GM-07065 of the National Institutes of Health. Part of this work was included in the dissertation of Jan E. Graebe submitted in partial satisfaction of the requirements for the Doctoral Degree in Botanv and Plant Biochemistrv. Universitv of California. Los Angeles, “l961. A preliminary report was presented at the annual meeting of the American Society of Plant Physiologists, Boulder, Colorado, August 24 to 27, 1964.

t Present address, Medieinische Forschungsanstalt der Max- Planck-Gesellschaft, Gottingen, Germany.

III a. R=CH3 b.R=CH20H

pany. A reference sample of (-)-kaurene was kindly supplied by Dr. B. E. Cross of the Imperial Chemical Industries’ Akers Research Laboratories, Welwyn, England. A second sample of (-)-kaurene and a sample of kaurenoP were supplied by Dr. P. R. Jefferies of the University of Western Australia, Nedlands. A generous sample of trans-farnesylacetone and a reference sample of lycopersene were gifts of Dr. Otto Isler, Hoffmann- La Roche, Basel, Switzerland. Methyl trans-geranylgeranate was synthesized from trczns-farnesylacetone and methyl diethyl phos- phonoacetate by the modified Wittig reaction of Wadsworth and Emmons (14); methyl trans-Geranylgeranate was reduced with lithium aluminum hydride to trans-geranylgeraniol. A detailed description of the chemical synthesis of trans-geranylgeraniol will be published elsewhere. Gibberellic acid was a highly purified sample kindly supplied by Abbott Laboratories, North Chicago, Illinois. Samples of 4-(4’-nitrophenylazo)benzoyl chloride and geranyllinalool were obtained from Dr. Feodor

1 Also supported by unpublished information from T. R. Reilly in this laboratory.

2 The abbreviations used are: MVA, mevalonate; kaurenol, (-)-kauren-19-01.

1847

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

1848 Biosynthesis of Gibberellins. I Vol. 240, No. 4

TABLE I Mobilities of terpenoids on silica gel thin layer

chromatograms, 0.86 mm thick

I Developing solvent

System B (benzene-

ethyl ace- tate, 9:l)

Compound System A

(hexane)

RF

(-)-Kaurene ................ 0.89 Squalene. ................... 0.35 Lycopersene ................ 0.15 &Carotene. ................. 0.095 Geraniol.................... Farnesol. ................... trans-Geranylgeraniol ....... Phytol...................... Linalool .................... Geranyllinalool ............. Isophytol. .................. Kaurenol ................... Cholesterol. ................ Ergosterol. .................

RphtltOZ*

0.66 0.78 0.90 1.00 1.07 1.36 1.46 1.00 0.62 0.63

system c (hexane-ethyl

acetate-l- propanol, 82: 15:3)

QWZ*

0.74

0.85 1.00

1.03

* Phytol RF = 0.475 in System B; 0.43 in System C. &,hYtOl is calculated as the ratio of the RF of the compound to the RF of phytol run on the same plate.

Lynen, Max-Planck Institut fiir Zellchemie, Munich, Germany. Squalene, isophytol, phytol, farnesol, and p-carotene were pur- chased from California Corporation for Biochemical Research.

Chromatographic Procedures

Paper Chromatography-One-inch strips of Whatman No. 1 filter paper were developed by descending flow of l-butanol- I.5 M ammonia (3 : 1; upper phase). The lipid fraction was eluted from the region between RF 0.6 to 1.0 by a descending flow of acetone containing a small amount of water in an elution cham- ber until about 5 ml of eluate were collected.

Thin Layer Chromatography-Silica gel G plates (0.25 mm thick) activated in an oven at 110-120” for at least 30 minutes were employed in thin layer chromatography. Compounds contain- ing ethylenic double bonds were detected as yellow spots on a pink background by exposure of developed plates containing 0.1 To sodium fluoresceinate to bromine vapor. Relative mobili- ties of a variety of terpenoid compounds on thin layer chromato- grams are presented in Table I.

Vapor Phase Chromatography-Samples were injected onto a column (1.5 meters x 5 mm) of 5a/, SE-30 on Chromosorb W which was developed at a column temperature of 181” and a helium flow rate of 40 ml per minute in an Aerograph A-90-P instrument equipped with a thermistor detector. Average reten- tion times for several reference compounds under these conditions were as follows: farnesol, 3.5 minutes; trans-geranylgeraniol, 11 to 21 minutes; (-)-kaurene, 17 minutes; and kaurenol, 55 minutes. It is believed that the allylic alcohols underwent, decomposition by dehydration on the column under these conditions. For ex- ample, trans-geranylgeraniol gave a relatively broad peak with an average retention time very close to that of (-)-kaurene, a C-20 hydrocarbon. Furthermore, the effluent sample collected at the detection port, migrated to the solvent front in thin layer

System A on a silica gel G plate, a behavior expected for a hydro- carbon and not for a C-20 alcohol.

Radioassay Techniques

Measurements of radioactivity were made in the Nuclear- Chicago liquid scintillation spectrometer, model 720. Each sample was dissolved in 15 ml of toluene containing 60 mg of 2,5-diphenyloxazole and 0.75 mg of p-bis-2’-(5’-phenyloxazolyl)- benzene. mere necessary, quench corrections were made by internal standardization. Radioactive compounds on paper chromatograms and thin layer plates were located with a Nuclear- Chicago A&graph II strip scanner. Where quantitative meas- urement of the 14C on thin layer chromatograms was desired, the gel in the appropriate region was either transferred directly into the scintillation solution and counted, or was placed in a Pasteur pipette, and the soluble material was eluted with acetone, benzene, or ethanol, a portion being counted as above. Samples from vapor phase chromatograms for radioassay were collected by passing the effluent gas through 0.2 g of glass wool coated with benzene in a scintillation vial.

Preparation of Seed Tissues

Endosperm nucellus-Immature fruit of E. macrocarpa was collected in the Santa Monica mountains near Los Angeles. Fruit, containing seeds in which the embryo did not completely surround the endosperm were stored at -20”. Seeds at this stage have been shown to contain maximal concentrations of gibberellins (11). Just prior to use several seeds were cut open, the endosperm nucellus was removed with a spatula, gently homogenized in a glass homogenizer, and centrifuged or filtered through glass wool to remove cell debris. This extract, which was used as the source of endosperm nucellus enzymes, had a pH of 6.9 and contained 1 to 2 mg per ml of trichloroacetic acid- precipitable protein measured by the biuret procedure (15).

Embryo-Embryos dissected from seeds obtained and stored as described above were washed three times by suspending them in 0.01 M Tris buffer, pH 7.0. A volume of 0.1 M Tris buffer, pH 7.0, equivalent to the volume of endosperm nucellus removed from these embryos was added, and the suspension was ground manually for a short time in a glass homogenizer. The resulting homogenate was taken as the source of embryo enzymes.

Preparation of the J-(4’-Nitrophenylazo)benzoyl Esters of Kaurenol and trans-Geranylgeraniol

4-(4’-Nitrophenylazo)benzoyl esters of kaurenol and trans- geranylgeraniol were prepared according to the procedure of Hecker (16) by dissolving the sample containing 1% and 8 mg of 4-(4’Xtrophenylazo)benzoyl chloride in benzene. A sample of the appropriate alcohol (2 mg) and 0.03 ml of anhydrous pyridine were added, and the volume was made to 3 ml with benzene. After standing overnight the reaction mixture was washed with 2 ml of 0.5 M sulfuric acid and twice with 1 ml of water. The product was evaporated to dryness from benzene solution in a stream of nitrogen three times to remove the last traces of water and finally dried overnight in a vacuum. The residue, dissolved in a small volume of benzene, was applied to a column, 1.7 X 10.5 cm, of neutral alumina (20 g, Woelm, activity grade II) which was developed with benzene. The second 25-ml fraction collected contained the orange-red ester derivative. A bright, red band of 4-(4’-nitrophenylazo)benzoic

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

April 1965 J. E. Graebe, D. T. Dennis, C. D. Upper, and C. A. West

acid remained at the top of the column. The sample containing the ester was concentrated to a volume of 5.0 ml and the ab- sorbance at 465 rnp was determined. The total amount of derivative present was calculated from the experimentally de- termined molar extinction coefficient (e) of 631 M-I cm-l. A portion was removed for the determination of radioactivity, and the remaining sample was taken to dryness for recrystalliza- tion from warm benzene in the case of the kaurenol derivative or hexane in the case of the geranylgeraniol derivative. This process was repeated until the sample showed constant specific radioactivity during several successive crystallizations. The melting point of the 4-(4’-nitrophenylazo)benzoyl ester of truns-geranylgeraniol after the final recrystallization was 78.5 79” (Kirschner (17) reported a value of 77-78”).

Evaluation of Y-Labeled Substrates as Precursors of Gibberellic Acid in F. monilijorme Suspensions

The mycelia in an g-day-old culture of F. monilijorme (strain L.M-119), grown in 250 ml of a defined medium (glucose-am- monium tartrate-minerals medium of Borrow et al. (18)) in a l-liter culture flask with shaking at 28”, were harvested by centrifugation. The cell mass was washed once with 0.02 M

potassium phosphate, pH 6.9, and then suspended in 250 ml of this buffer. The 14C-labeled substrate to be tested was added in a small volume of ethanol (0.1 to 0.8 ml) to 20 ml of this cell suspension in a sterile 125-ml flask. Another sample of the I%-labeled substrate was added to a control flask containing 20 ml of cell suspension which had been heated for 5 minutes in a boiling water bath. The flasks were incubated at 28” for 114 hours on a rotary shaker.

At the end of the incubation period the cells were removed by centrifugation, and the acidic lipid components including the gibberellins were extracted from the supernatant solution with ethyl acetate. The ethyl acetate extract was applied to the origin of a Kieselguhr thin layer plate, 2 x 8 inches, which was developed in the manner described by MacMillan and Suter (19) in the upper phase of a mixture of benzene-propionic acid- water (80:30:50) after equilibration with the lower phase. The plate was scanned for radioactivity, and the gel from the region containing suspected gibberellic acid (approximately 1 to 5 cm from the origin on plates developed to 15 cm) as judged from control plates was scraped from the plate except for a narrow longitudinal strip of 0.5 cm width which was left in the center. This strip was sprayed with ethanol-sulfuric acid (95:5), whereupon the presence of gibberellic acid was indicated by a green fluorescent zone after gentle heating (19). The material in the Kieselguhr scraped from this region was eluted by stirring it several times with acetone, and the com- bined acetone eluates were mixed with several milligrams of gibberellic acid. This sample was carried through several successive crystallizations from ethyl acetate-petroleum ether mixtures (approximately 1: I), the specific radioactivity being determined at each stage.

RESULTS

Preliminary Studies of .%-‘4C-MVA Metabolism in Endosperm Nucellus

In a typical experiment 2-‘4C-MVA (0.1 PC; 7.5 x 10-5 M) was mixed with endosperm (1.0 ml) and ATP (lo-4 M) (total volume 1.1 ml), and the mixture was shaken in a water bath

at 33”. The reaction was stopped after 1 hour by heating the tube in a boiling water bath, and the resulting precipitate was removed by low speed centrifugation. Paper chromatography of the supernatant fraction in the butanol-ammonia solvent system showed a peak of radioactivity at the origin (5-phospho- MVA and probably other phosphorylated compounds) as well as one at RF 0.10 to 0.15 which corresponds to unchanged 2-I%-MVA. A smaller peak was sometimes observed at Rv 0.62 to 0.70, although the occurrence of this peak was quite variable. An acetone extract of the precipitate also contained radioactive material. A single peak of radioactivity near the solvent front was apparent when a chromatogram of this extract developed in the butanol-ammonia system was scanned. A second, smaller peak running behind this became evident only in longer incubation mixtures (8 to 20 hours). The material in the region above Rp 0.60 is referred to in this paper as the lipid fraction. An incubation with heat-inactivated endosperm showed only the radioactive peak corresponding to unchanged 2-14C-MVA in the supernatant fraction.

The metabolism of 2J4C-MVA in stored endosperm was completely dependent on added ATP. The optimal concen- tration of added ATP for the formation of the lipid fraction was lo+ M, while 10e2 M was highly inhibitory. The formation of the lipid fraction was maximal at 33”.

The pooled endosperm nucellus from young seeds in which the cotyledons were not yet visible and the seed cavity was filled mostly with nucellus was the most active in metabolizing MVA. Approximately 1200 c.p.m. were incorporated into the lipid fraction from 2J4C-MVA by this tissue under standard assay conditions, while the endosperm from medium age seeds, in which the cotyledons had developed to approximately half the over-all length of the seed and the remaining seed cavity contained primarily endosperm, showed incorporation of approxi- mately 800 c.p.m. into lipid under comparable conditions. Endosperm from older seed in which the cotyledons nearly filled the seed cavity did not metabolize 2J4C-MVA.

Identification of Radioactive Components of Lipid Fraction

The %-lipid fraction remained soluble in ethyl acetate and gave no 14C-digitonin precipitate by the procedure of Sperry and Webb (20) after it was heated 2 hours at 75” in the presence of 2% ethanolic KOH. Thus, the fraction appeared to contain no free or esterified sterols.

A thin layer chromatographic system was developed for the separation of the lipid fraction into several components as shown in Fig. 1. This was used as the basis for a general pro- cedure for the production and assay of the Y?-labeled lipid fraction. Magnesium chloride (1 pmole) and ATP (1 kmole) were mixed with 1.0 ml of endosperm homogenate in a 25-ml flask. The resulting solution was shaken for 10 minutes in a bath at 32”, and 2-14C-MVA (2.04 X lo5 c.p.m.; 0.021 pmole) was added (final volume 1.2 ml). The contents were trans- ferred to a centrifuge tube after the desired incubation period, and the reaction was stopped by heating the tube in a boiling water bath for 5 minutes. The precipitate was separated by centrifugation and washed three times with approximately 1 ml of water. The residue was extracted three times with l.O-, 0.5-, and 0.5-ml portions of acetone. The combined extracts were evaporated to a small volume in a stream of nitrogen and applied to a silica gel thin layer plate. The plate was developed to 15 cm in System A, the zone between 12 cm and the solvent

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

1850 Biosynthesis of Gibberellins. 1 Vol. 240, No. 4

FIG. 1. Resolution of the ‘%-lipid fraction produced from 2-l%- MVA in endosperm nucellus on silica gel G plates. The thin layer plate was scanned for radioactivity in System A. The gel con- taining Fraction 1 was removed (12 to 15 cm) and the plate was redeveloped in System B and again scanned for radioactivity. It should be noted that the sensitivity setting of the scanner in the latter scan was 3 times that of the first scan.

TABLE II

Co-crystallization of Fraction 1 and (-)-kaurene

A weighed mixture of authentic (-)-kaurene and Fraction 1 was dissolved in a very small volume of benzene and then made up to 1.00 ml with ethanol. A 0.05-ml portion was removed for radioassay. The remaining mixture was evaporated to 0.2 ml under a stream of nitrogen while maintained at approximately 60”. Kaurene crystallized as needles while the solution was held at -20” overnight. The supernatant was removed, and the crystals were dried under a stream of nitrogen and then in a vacuum for 2 hours. The crystals were weighed, and the process was repeated through three recrystallizations.

Sample Weight Total “C

Initial mixture.. First crystals. . Second crystals. . Third crystals. .

w c.p.m. c.p.m./mg

3.75 1376 376 1.91 772 404 1.46 644 441 1.16 450 388

Specific radioactivity

front (Fraction 1, Fig. 1) was scraped from the plate, and the

radioactivity it contained was measured. The plate was then developed a second time to 12 cm in the same direction in System

B. The gel in the region from the origin to 4 cm (Fraction 2,

Fig. 1) was scraped from the plate and the radioactivity meas- ured directly. The gel in the regions from 4 to 9 cm (Fraction

3, Fig. 1) and 9 to 12 cm (Fraction 4, Fig. 1) were separately

treated with 2 ml of acetone, and the amount of radioactivity in each eluate so obtained was measured.

Fraction I-Fraction 1 co-chromatographed with ( -)-kaurene in System A. It ran immediately behind a zone of unsaturated material near the solvent front which was not radioactive. The retention times on a vapor phase chromatogram of Fraction 1 and authentic (-)-kaurene, with which it was mixed prior to injection, were identical. Further confirmation of the iden- tity of Fraction 1 with ( -)-kaurene was obtained when a mixture of these t.wo components was recrystallized from ethanol to constant specific radioactivity (Table II).

Fraction S-Comparative chromatography of Fraction 3 with truns-geranylgeraniol and kaurenol in thin layer System B showed that the radioactivity of Fraction 3 overlapped both reference compounds. Solvent System C, which would more clearly separate these two compounds (Table I), also resolved Fraction 3 into two radioactive peaks (Fig. ‘2). Fraction 3A, the slower moving component at Rphytol 0.85, corresponded in position to truns-geranylgeraniol, whereas Fraction 3B, the faster moving component at &!phytol 1.03 corresponded to kaurenol. The gel containing Fraction 3A and that containing Fraction 3B were separately scraped from the plate and the radioactive material present in each was eluted. Fraction 3A and authentic trans-geranylgeraniol corresponded exactly when chromatographed together in thin layer Systems B and C. Also vapor phase chromatography of Fraction 3A showed exact correspondence of the time curve of radioactivity eluted and the thermistor response due to added authentic trans-geranyl- geraniol. The co-crystallization of the 4-(4’-nitrophenylazo)- benzoyl ester of a mixture of Fraction 3A and authentic trans- geranylgeraniol (Table III) demonstrated that at least 70% of the radioactivity of this fraction is associated with trans- geranylgeraniol. This is a minimal estimate since the exact amount of geranylgeraniol introduced with Fraction 3A was not known.

Mixtures of Fraction 3B and authentic kaurenol were chro- matographed to the same position in thin layer Systems B and C as evidenced by the coincidence of the radioactive peak and unsaturated compound detected on the developed plates. Also the retention times (55 minutes) and elution patterns of the radioactive component of this fraction and authentic kaurenol with which it was mixed prior to vapor phase chromatography were identical. The 4-(4’-nitrophenylazo)benzoyl ester deriva- tive of a mixture of this fraction and authentic kaurenol was

FIG. 2. Resolution of Fraction 3 by thin layer chromatography on silica gel G plates 0.25 mm t,hick, developed to 12 cm in System C (hexane-ethyl acetate-propanol, 82: 15:3).

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

April 1965 J. E. Graebe, D. T. Dennis, C. D. Upper, and C. A. West 1851

recrystallized to constant specific radioactivity as shown in Table IV. These results indicate that at least 70 y0 of the radio- activity of this fraction was associated with kaurenol.

Fractions Z? and d-Fractions 2 and 4 each contain at least two components which have not been identified.

The incorporation of radioactivity from 2-W-MVA into the various lipid fractions as a function of time is shown in Fig. 3.

Studies of d-W-MVA Metabolism in Embryo Extracts

A comparison was made of the metabolism of 2-l*C-MVA in the embryo extracts with that in the endosperm nucellus de- scribed above. In a typical incubation 1.0 ml of embryo homog- enate was mixed with ATP (1 pmole), magnesium chloride (1 pmole), and 2-l*C-MVA (2.50 X lo5 c.p.m.; 0.025 pmole) (final volume 1.2 ml). The incubation was carried out for 2 hours at 32”, and the lipid was extracted from the precipitate with acetone as described for the endosperm nucellus incuba- tions. The acetone extract, which contained 8.90 x 104 c.p.m. or 36% of the added radioactivity, was evaporated to a small volume in a stream of nitrogen and spotted on a silica gel thin layer plate. The plate was developed to 15 cm in System B and scanned for radioactivity. A small radioactive peak was observed at the origin (8y0 of the radioactivity), another peak was detected at RF 0.45 (15% of the radioactivity), and a large peak was found at the solvent front (77% of the radioactivity). The material in the gel from the latter two peak regions was eluted separately with acetone-benzene (1: 1) for further char- acterization.

The radioactive material eluted from the region of RF 0.45

was rechromatographed in System B in comparison with reference samples of farnesol, trans-geranylgeraniol, phytol, and ergosterol (see Table I). A peak of radioactivity was found at Rphytol

0.77 which agrees most closely with that of farnesol among the reference materials tested. Vapor phase chromatography of another portion of this extract under conditions described in “Experimental Procedure” showed an identical retention time for the radioactivity and a reference sample of farnesol (4 min- utes). On the basis of this information it was tentatively con- cluded that farnesol was the major radioactive component in this fraction. Neither thin layer nor vapor phase chromato- graphic analyses gave any evidence for the presence of geranyl- geraniol or kaurenol in this fraction.

The radioactive material eluted from the zone near the solvent front in System B was chromatographed on a silica gel plate in

TABLE III

Co-crystallization of the 4-(4’-nitrophenylazo)benzoyl esters of Fraction z?A and trans-geranylgeraniol

Fraction 3A (3380 c.p.m.) was mixed with approximately 2 mg of trans-geranylgeraniol. The derivative was prepared, purified, and recrystallized as described in “Experimental Procedure.”

Sample Weight Total 14C Specific radioactivity

Column eluate First crystals. Second crystals. . Third crystals. . Fourth crystals. . Fifth crystals.

w C.).?n. c.p.m./~mole

1.48 1675 611 1.30 1445 600 1.13 1325 636 0.97 1131 632 0.86 966 608 0.74 840 612

TABLE IV

Co-crystallization of 4-(4’-nitrophenylazo)benzoyl ester

of Fraction SB and kaurenol

Fraction 3B (3360 c.p.m.) was mixed with 2 mg (6.94 pmoles) of kaurenol. The derivative was prepared, purified, and re- crystallized as described in “Experimental Procedure.”

Sample Weight Total “C

Initial. . Column eluate First crystals. Second crystals. Third crystals.. . Fourth crystals.

w

2.16 1.75 1.31 Lost 1.11

c.p.m.

1380 1170 831

708

I specific

radioactivity

C.P.?n./jmh?

484 346 360 343

343

0 1 2 3 4

TIME (HOURS)

FIG. 3. Incorporation of MVA into lipid fractions as a function of time. Incubations were carried out as described in the text. Fractions 2,3A, and 3B were counted directly and Fractions 1 and 4 were eluted prior to determination of radioactivity. Fractions 3A and 3B were separated by rechromatography of Fraction 3 in System C.

System A. Two radioactive zones were resolved under these conditions, one at the origin and a larger one at RF 0.35. The latter radioactive peak was coincident with an unsaturated compound detectable on a fluorescein-treated plate. It also co-chromatographed with authentic all-trans squalene in this system. No radioactivity was found in the region near the solvent front in the position where (-)-kaurene would be expected. The suspected radioactive squalene was mixed with a sample of carrier squalene and treated with HCl in acetone to form the hexahydrochloride according to the procedure of Heilbron, Kamm, and Owens (21). Two isomers which melted at 108 and 143” respectively were recovered, the latter in a very small amount. The specific radioactivity of the isomer melting at 108” was determined after each of several recrystallizations as shown in Table V. The data confirm that at least a portion of the radioactivity of this fraction is associated with squalene.

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

1852 Biosynthesis of Gibberellins. I Vol. 240, No. 4

TABLE V cell suspensions were either synthesizing gibberellins from Co-crystallization of squalene hexahydrochloride endogenous reserve materials or releasing preformed gibberellins

The isomerides of the hexahydrochloride of a mixture of sus- into the surrounding medium during the incubations. When petted squalene extracted from thin layer plate (5760 c.p.m.) and either I%-kaurene or 14C-labeled kaurenol was present as a carrier squalene (45.6 mg) were prepared according to the proce- substrate during the incubation, peaks of radioactivity were dure of Heilbron et al. (21). The isomeride melting at 108” was detectable on the thin layer plate which corresponded in posi- recrystallized from small volumes of acetone.

Salllple Weight Total “C

First crystals. Second crystals. Third crystals............. Fourth crystals.

w c.p.?&

20.7 642 17.5 543 15.3 474

6.0 234

Specific radioactivity

c.p.m./mg 31 31 31 29

tion to the observed fluorescent zones. The suspected gib- berellic acid zone from each of these two incubation mixtures was extracted, the extract mixed with authentic gibberellic acid carrier, and the mixture recrystallized to constant specific radioactivity. The results, summarized in Table VI, show that 14C from both substrates was incorporated into gibberellic acid to a significant extent. The per cent conversions of added radioactivity found in these experiments are minimal estimates since no information is available on the yields during extraction

TABLE VI

Incorporation of 14C-labeled diterpenoid substrates into gibberellic acid in Fusarium moniliforme

The incubations and recovery of gibberellic acid for the determination of specific radioactivities were performed as described in “Experimental Procedure.”

‘4C substrate

Fraction from which gibberellic acid was recovered Kaurene, 67.4 X 102 c.p.m.

I Weight SphtiC radioactivity

Crude................................ First crystals......................... Second crystals.. Third crystals. Fourth crystals..

mg c.p.m./mg w c.p.m.fmg w c.p.n.fmg

10.2* 44.3t 10.4* 47.6t 10.8* 0 9.2 42.8 8.7 47.3 8.5 1.4 5.5 50.0 6.9 43.8 6.4 0 4.5 47.7 5.9 42.5 4.5 0 2.8 47.9 4.1 43.9 3.5 0

yO added 14C recovered in gibberellic acid................................ 1.03 1.55 0

* Milligrams of carrier gibberellic acid added.

Kaurenol, 41.5 X 108 c.p.m.

Weight specilic radioactivity

trans.Geranylgeraniol, 76.7 X 10” C.P.lll.

Weight Specific radioactivity

t Calculated on the basis of total counts per minute eluted in the gibberellic acid fraction per mg of carrier gibberellic acid added.

Conversion of W-Diterpenoid Substrates to

Gibberellic Acid in F. monilijwme

In order to evaluate the possible intermediary role of (-)- kaurene, kaurenol, and trans-geranylgeraniol in gibberellin biosynthesis, suspensions of F. moniliforme cells were incubated with each of these substrates labeled with W, and the incorpora- tion of radioactivity into gibberellic acid was assessed in the manner described in “Experimental Procedure.” The radio- active (-)-kaurene was obtained by pooling Fraction 1 from 10 incubations of 2W-MVA with endosperm nucellus. Radio- active kaurenol was prepared in a similar manner. The sample of WC-geranylgeraniol (2.41 X IO* c.p.m. per mmole) was prepared synthetically.

and purification of gibberellic acid prior to the addition of un- labeled gibberellic acid carrier. No radioactivity was incor- porated into the gibberellic acid fraction from either of these substrates when the cell suspensions were boiled before assay.

In contrast to the above experiments with kaurene and kaurenol, no radioactivity was detected in the gibberelhc acid produced in the presence of 2-14C-trans-geranylgeraniol (Table VI).

DISCUSSION

Suspensions of F. moniliforme cells in phosphate buffer incu- bated in the absence of added substrate but otherwise as de- scribed in “Experimental Procedure” were analyzed for the presence of gibberellins in the incubation medium by means of thin layer chromatography. An intense green fluorescence of the type and in the position expected of gibberellic acid was detected after spraying the plate with sulfuric acid-ethanol and heating at 50” for 2 minutes. Less intense fluorescence was also observed in the regions expected for gibberellins A4 or A?, or both, and gibberellin As. These findings indicated that the

The formation of (-)-kaurene and kaurenol in this cell-free system from a higher plant is of particular significance in relation to gibberellin biosynthesis. Cross et al. (6, 7) have isolated (-)-kaurene as one of several neutral diterpenoid compounds found in culture filtrates of F. monilijorme which also contain gibberellins. Furthermore, it was established in the same laboratory that (-)-kaurene labeled in the exocyclic methylene position with 14C was converted to gibberellic acid labeled in the same position when incubated with cultures of F. monilijorme

(8). The present investigation has confirmed that the radio- activity of W-kaurene is incorporated to a significant extent into gibberellic acid and probably other gibberellms in suspen- sions of F. moniliforme. Although neither the kaurene nor the

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

April 1965 J. E. Graebe, D. T. Dennis, C. D. Upper, and C. A. West

gibberellic acid was chemically degraded in this case to establish MEVALONATE the location of the radioactivity, it seems reasonable to assume, in view of the results of Cross et al. (6, 7), that a direct conver- $

* sion was involved. Recently Phinney et al. (10) have demon- ISOPENTENYL - DIMETHYLALLYL

strated that (-)-kaurene will stimulate leaf sheath elongation FYROPHOSPHATE - PYROPHOSPHATE /

in seedlings of the dwarf-5 and anther-l mutants of Zea mays, CAROTENO~DS _ PHYTOL

1853

an effect qualitatively indistinguishable from that produced by exogenous gibberellins. These findings, taken together, point to (-)-kaurene as an intermediate in gibberellin biosynthesis both in the fungus and in higher plants.

The evidence also indicates a similar position for kaurenol as an intermediate in gibberellin biosynthesis. Katsumi et al. (9) have shown that the dwarf-5 and anther-l mutants of maize respond to treatment with kaurenol in the same fashion as they do to treatment with (-)-kaurene and gibberellins. The present investigation has shown that the radioactivity of 14C- kaurenol produced in endosperm nucellus from 2-W-MVA was incorporated into gibberellic acid in F. monilijorme sus- pensions.

The evidence presently available suggests that free trans- geranylgeraniol is not an intermediate in gibberellin biosynthesis. This compound has been found to be inactive in dwad-5 maize assays under conditions where kaurene and kaurenol stimulate a growth response.3 Attempts to show the incorporation of radioactivity of 2-14C-trans-geranylgeraniol into gibberellic acid in suspensions of F. monilijorme were negative under conditions where added W-kaurene and W-kaurenol both served as precursors.

The rapid formation of the likely gibberellin intermediates, (-)-kaurene and kaurenol, from MVA in cell-free homogenates of endosperm nucellus has been described in this paper; thus, it seems reasonable to propose that gibberellins themselves can be formed in the endosperm nucellus of this dicotyledonous plant. Synthesis of (-)-kaurene and kaurenol were found in the endosperm nucellus of E. macrocarpa at the same stages of seed development where Corcoran and Phinney (11) showed the presence of high concentrations of gibberellin-like substances in the endosperm nucellus relative to other seed parts. Ob- viously, the direct demonstration of the conversion of (-)- kaurene or kaurenol to gibberellins in the endosperm nucellus is needed to verify this hypothesis.

Paleg (22) has suggested for monocotyledonous plants such as barley that gibberellins may be produced by the embryo and translocated to the endosperm where they act as “endo- sperm-mobilizing hormones.” However, (-)-kaurene or kau- renol were not found as products of ‘4C-MVA metabolism in homogenates of embryo from the E. macrocarpa seed under the same general conditions which permitted the formation of these diterpenoids in endosperm. Thus, this finding does not support the idea that gibberellin synthesis occurs in the embryo in this seed.

Mevalonate was metabolized extensively in the embryo homogenates even though the diterpenoids were not among the products. Squalene and farnesol were tentatively identified among the products in the embryo, even though there was no suggestion of the accumulation of significant quantities of these substances in the endosperm. Thus, it appears that MVA

8 These tests with trams-geranylgeraniol were performed by Dr. B. 0. Phinney of the Department of Botany and Plant Biochemis- try, University of California, Los Angeles, and the results com- municated to us prior to publication.

GERANYLGERANYL PYROPHOSPHATE GERANYLGERANIOL

KAURENE GIBBERELLINS FIG. 4. Suggested metabolic conversions involving diterpenoid

and related compounds.

metabolism in the endosperm nucellus may be directed to diterpenoid biosynthesis while that of the embryo from the same seed may be directed to the synthesis of triterpenoid materials. Such a differentiation, if confirmed, could have interesting implications for development. The seed may prove to be a very useful system for the study of regulation of terpene synthesis.

The scheme shown in Fig. 4 serves as a working hypothesis for the formation and further conversions of the diterpenoid compounds identified in the endosperm nucellus. The forma- tion of these substances from MVA was dependent on the addi- tion of ATP, which is presumably required for the reactions leading to the formation of isopentenyl pyrophosphate as has been well documented in the case of sterol synthesis from MVA (23). Evidence has been obtained for the occurrence of a mevalonate kinase in the endosperm nucellus with properties similar to those of this enzyme from other plant tissues (24). trans.Geranylgeranyl pyrophosphate is a logical intermediate in the formation of diterpenes from MVA, although no direct evidence has been obtained to date for its presence in this system. Kandutsch et al. (25) have recently described the properties of an enzyme from Micrococcus lysoo!eikticus which catalyzes the synthesis of trans-geranylgeranyl pyrophosphate from isopen- tenyl pyrophosphate and dimethylallyl pyrophosphate. A crude enzyme from carrot tissue has recently been reported by Nandi and Porter (26) to catalyze the formation of geranyl- geranyl pyrophosphate from isopentenyl pyrophosphate and trans-farnesyl pyrophosphate. This reaction was originally studied in yeast by Kirschner (17). The accumulation of trans- geranylgeraniol in the endosperm nucellus homogenate may well have resulted from the hydrolytic cleavage of trans-geranyl- geranyl pyrophosphate catalyzed by a phosphatase. Evidence that such a reaction does occur in the endosperm nucellus ho- mogenate as well as evidence that trans-geranylgeranyl pyrophos- phate can be cyclized to (-)-kaurene has been obtained with the use of synthetic substrate and will be discussed in a forth-

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

1854 Biosynthesis of Gibberellins. I Vol. 240, No. 4

coming publication. Co&es (27) has shown that geranylgeraniol dilutes incorporation of acetate-X into phytol, suggesting that free geranylgeraniol is the precursor of the phytyl moiety of chlorophyll. Incorporation of 14C from geranylgeranyl pyro- phosphate into lycopersene (28) and phytoene (29) has been reported, although the nature of the condensation steps and the immediate C4,, product is still uncertain. As is apparent from a consideration of Fig. 4, geranylgeranyl pyrophosphate plays a central role according to this hypothesis.

Encyclopedia of plant physiology, Vol. XIV, Springer-Verlag, Berlin, 1961, p. 1185.

3.

4.

5.

CROSS, B. E.! GROVE, J. F., MACMILLAN, J., AND MULHOL- LAND, T. P. C., Chem. and Ind. (London), 954 (1956).

BIRCH, A. J., RICKAFLDS, R. W., AND SMITH, H., Proc. Chem. sot., 192 (1958).

Demonstration of the synthesis of diterpenoid compounds in a cell-free system readily obtained from a higher plant should greatly facilitate studies of the nature of the enzymatic reactions involved in diterpenoid interconversions and their regulation, in particular, those reactions related to the biosynthesis of t,he growth-regulating gibberellins.

BIRCH, A. J., AND SMITH, H., in G. E. W. WOLSTENHOLME (Editor), Biosynthesis of terpenes and s!erols, Little, Brown and Company, Boston, 1959, p. 245.

CROSS, B. E., GALT, R. H. B., HANSON, J. R., AND KLYNE, W., Tetrahedron Letters, 145 (1962).

CROSS, B. E., GALT, R. H. B., HANSON, J. R., CURTIS, P. J., GROVE, J. F., AND MORRISON, A., J. Chem. Sot., 2937, (1963).

CROSS, B. E., GALT, R. H. B., AND HANSON, J. R., J. Chem. Sot., 295, 1964.

SUMMARY

The formation of the diterpenoid compounds (-)-kaurene, ( -)-kauren-19-01, and Wns-geranylgeraniol from mevalonate in the presence of ATP was demonstrated in cell-free homoge- nates of the endcsperm nucellus of the seed of Echinocystis

macrocurpa Greene (wild cucumber). Homogenates of embryos of these seeds did not catalyze the formation of these diterpenoid compounds from mevalonate in the presence of ATP under the same conditions which supported synthesis in the endosperm nucellus. However, mevalonate was metabolized in the embryo extracts to several products among which squalene and farnesol were tentatively identified.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

KATSUMI, M., PHINNEY, B. O., JEFFERIES, P. R., AND HEN- RICK, C. A., Science, 144,849 (1964).

PHINNEY, B. O., JEFFERIES, P. R., KATSUMI, M., AND HEN- RICK, C. A., Plant Physiol., 39 (suppl.), xxvii (1964).

CORCORAN, M. R., AND PHINNEY, B. O., Physiol. Plantarum, 15, 252 (1962).

ELSON, G. W.,. JONES, D. F., MACMILLAN, J., AND SUTER, P. J., Phytochemistry, 3, 93 (1964).

WEST, C. A., AND REILLY, T. R., Advances in Chem. Ser., 28, 37 (1961).

WADSWORTH, W. S., AND EMMONS, W. D., J. Am. Chem. Sot., 83, 1733 (1961).

16. 17.

18.

BEISENHERZ, G., BOTTZE, H. J., BUTCHER, T., CZOK, R., GAR- BADE, K. H., MEYER-ARENDT, E., AND PFLEIDERER, G., Z. Naturforsch., Pt. B, 8,555 (1953).

HECKER, E., Chem. Ber., 88, 1666 (1955). KIRSCHNER, K., Doctoral dissertation, University of Munich,

1961.

I%-Kaurene and 14C-kaurenol were incorporated into gib- berellic acid in washed suspensions of Fusarium monilijorme

cells, a finding which was interpreted as confirmatory evidence for the intermediary role of these substances in gibberellin biosynthesis. 2-14~~trans.Geranylgeraniol did not contribute radioactivity to gibberellic acid under similar conditions and was judged not a likely intermediate in gibberellin biosynthesis.

BORROW, A., BRIAN, P. W., CHESTER, I’. E., CURTIS, P. J., HEMMING, H. G., HENEHAN, C., JEFFREYS, E. CT., LLOYD, P. B., NIXON, I. S., NORRIS, G. L. F., A~GD RADLEY, M., J. Sci. Food Agr., 6, 340 (1955).

19. MACMILLAN, J.. AND SUTER, P. J., Nature, 197. 790 (1963). 20. SPERRY, W. M., AND WEBB: M., J.’ Biol. Chem., 187, 97 (1950). 21. HEILBRON, I. M., KAMM, E. D., AND OWENS, W. M., J. Chem.

Sot., 1630 (1926).

Acknowledgments-We wish to express appreciation to Mr.

Donald R. Abram, Miss Hester Almon, and Mrs. Alice McLure for able and enthusiastic technical assistance in various aspects of this work.

REFERENCES

1. PHINNEY, B. O., AND WEST, C. A., Ann. Rev. Plant Physiol., 11, 411 (1960).

22. 23.

24.

25.

26.

27. 28.

29.

PALEG, L. G., Plant Physiol., 36, 829 (1961). POPJAK. G.. AND CORNFORTH. J. W.. Advances in Enzumol.. 22.

281 (i96Oj. ” I,

LOOMIS, W. D., AND BATTAILE, J., Biochim. et Biophys. Acta, 67, 54 (1963).

KANDUTSCH, A. A., PAUL~~S, H., LEVIN, E., AND BLOCH, K., J. Biol. Chem., 239, 2507 (1964).

NANDI, D. L., AND PORTER, J. W., Arch. Biochem. Biophys., 105, 7 (1964).

COSTES, C., Compt. rend., 266,355 (1962). GROB, E. C., KIRSCHNER, K., AND LYNEN, F., Chimia (Switz.),

16, 308 (1961).

2. PHINNEY, B. O., AND WEST, C. A., in W. RUHLAND (Editor), ANDERSON, D. G., AND PORTER, J. W., Arch. Biochem. Bio-

phys., 97, 509 (1962).

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Jan E. Graebe, David T. Dennis, Christen D. Upper and Charles A. WestNUCELLUS OF ECHINOCYSTIS MACROCARPA GREENE

ENDOSPERM(-)-KAUREN-19-OL, AND TRANS-GERANYLGERANIOL IN Biosynthesis of Gibberellins: I. THE BIOSYNTHESIS OF (-)-KAURENE,

1965, 240:1847-1854.J. Biol. Chem. 

  http://www.jbc.org/content/240/4/1847.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/240/4/1847.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from