Planta (Berl.) 125, 181--195 (1975) 9 by Springer-Verlag 1975

The Metabolism of Gibberellins A9, A20 and A29 in Immature Seeds of Pisum sativum cv. Progress No. 9" Valerie IV[. F r y d m a n and J a k e MacMillan School of Chemistry, University of Bristol, Bristol BS8 1TS, U.K. Received 19 April; accepted 21 May 1975 Summary. Gibberellin A9, GA20and GA29,all known to be endogenous to immature seeds of Pisum sativum cv. Progress No. 9, were fed in tritiatcd form to seeds of this cultivar cultured in vitro. [3H]GA9 was metabolised to [3H]GA20, 2/~-OH-[3H]GAg,H~-[3H]GA31and a Hg-[3H]GA31conjugate. The percentage conversion to each metabolite varied from feed to feed bu~ in each instance [ZH]GAz0was, at most, a minor product, suggesting that in vivo GA9 may not be the principal precursor of GAz0. [aH]GA20 was converted in very high yield to [SH]GA~9 under conditions which suggest very strongly that this is a normal metabolic sequence in developing seeds of this cultivar. In addition, conjugates of [~H]GA20 and [SH] GAz9were formed in low yield. It was presumed that the latter was a metabolite of [3H]GA~9. All metabolites, in each instance, were identified by combined gas chromatography-radiocounting and combined gas chromatography-mass spectrometry, and criteria for the unequivocal identification of radioactive products in feeds to plant tissues arc put forward.

Introduction One of the sites of gibberellin biosynthesis in higher plants appears to be immature fruits and seeds. Baldev et al. (1965) showed t h a t fruits of P i s u m sativum cv. Progress No. 9 developing in sterile culture accumulated large amounts of GA-like substances. This accumulation could be prevented b y the growth retardant AMO 1618 which has been shown to inhibit GA biosynthesis in higher plants (see Lang, 1970). Furthermore, extracts of immature seeds of Echinocystis macrocar19a, Cucurbita m a x i m a and P. sativum contain enzymes which catalyse m a n y of the stages of GA biosynthesis. Thus Graebe et al. (1974) have recently shown t h a t cell-free extracts of C. m a x i m a endosperm contain all the enzymes needed to catalyse the conversion of mevalonic acid to a C19 gibberellin. I n addition several workers have applied radioactive GAs to seeds developing in vivo and in vitro and have observed metabolism (e.g. Sembdner et al., 1963; Barendse et al., 1968; Musgrave and Kende, 1970; Y a m a n e et al., 1975). I n our previous work we have identified six GAs endogenous to immature seeds of P. sativum cv. Progress No. 9, namely, GA 9 (1), GAI~, GA20 (2), GA29 (5), GA~s and GA44 (Frydman and MacMillan, 1973 ; F r y d m a n et al., 1974). The buildup of each of these GAs during maturation suggested t h a t they were actually being synthesised during seed development. Moreover, the qualitative and quantitative distribution of these GAs throughout pea seed maturation, considered * Abbreviations. dpm, disintegrations per minute; GAn, gibberellin An; GC, gas chromatography; GC-MS, combined gas chromatography-mass spectrometry; GC-RC, combined gas chromatography-radio counting; Me, methyl ester; R T, retention time; TLC, thin layer chromatography; TMS, trimethylsilyl ether.

182

V.M. Frydman and J. MacMillan

,R2 I

0 I ~ ~ R1 7 [~H2

R3~ I

-7-COOH

CH3 (1) (2) (3) (4) (5)

R1

R2

R~

H OH H H OH

H H OH tt tt

H H H OH OH

(GAg) (GA2o) (I-I~/GA31) (2fl-OHGAg) (GA2~)

in the light of knowledge of GA biosynthesis in Gibberella/u]ikuroi and cell-free systems of higher plants (for review articles see MacMillan, 1971 and Graebe, 1974), suggested t h a t some of the GAs detected in peas might be directly related in a metabolic sequence. :For instance, a route from GAs(1 ) through GA~0(2) to GA29(5) was inferred both from our data (Frydman et al., 1974) and from t h a t of Railton et al. (1974a and b). The latter showed t h a t in germinating seeds and seedlings of P . s a t i v u m ev. Meteor, [SH]GAg(1) is metabolised to [3H]GA~0(2), [aH]GAlo and H~-[aIt]GAal (3), and [aH]GA20 is mctabolised to [aIt]GA~8(5); none of these GAs are however known to be endogenous to germinating seeds or seedlings of pea. I n the present work GAg, GA20 and GA29, previously established as endogenous GAs of immature seeds of Progress No. 9, were fed in tritiated form to developing seeds of this cultivar at the particular stages of seed development at which they are known to be present. Also they were fed at concentrations approximating to the previously determined endogenous concentrations of these GAs at selected ages. Thus the conditions were such t h a t data obtained should give an adequate representation of the natural physiological transformations occurring in seeds developing i n vivo. Some of this work has been briefly reported elsewhere (Frydm a n and MacMillan, 1974). Materials and Methods Preparation o/Radioactive GAs. GA~ was isolated from strain TP70 of Gibberella ]ujikuroi

by J.R. Bearder (Bearder and MacMillan, 1973). It was treated with osmium tetroxide and sodium periodate to give GA9 norketone. This, on reaction with tritiated methyl triphenyl phosphonium bromide and potassium tertiary butoxide in tetrahydrofuran gave [aH]GA~. After isolation it was shown to be radio-chemically pure by TLC and GC-RC, and had a specific activity of 29 mCi/mM (J. R. Bearder, unpublished work). [aH]GA20was prepared as described by Bearder et al. (unpublished work) and had a specific activity of ca. 33 mCi/mM. [aHJGA2~,of unknown specific activity, was isolated from [aHJGA~0feeds to peas, as described later in this paper. Plant Material and Feed Procedure. Seeds of P. sativum cv. Progress No. 9 were obtained from Sutton and Sons Ltd., Reading, England. They were grown as described previously (Frydman and MacMillan, 1973) except that established plants were maintained in an an-

Metabolism of Gibberellins in Seeds

183

heated greenhouse under natural photoperiod throughout the months April to October. From October, plants received supplementary illumination and were maintained at 17 ~ 2 ~ Individual flowers were labelled at anthesis to ensure that fruits were of a known age. Pods of specific ages were harvested, sterilized in 4% Domestos (Lever Brothers Ltd., England) and rinsed in sterile water. Seeds were removed from the pods aseptically by severing the funicles and were weighed under aseptic conditions. They were then transferred to sterile 100 ml. Erlenmeyer flasks each containing 20 ml nutrient medium (Nitseh, 1951) in which ferric citrate had been replaced by anhydrous ferric chloride. Approximately 20 g seeds were placed in each flask. Radioactive GAs were added to the medium at this stage in a small quantity of acetone. Uptake of GAs was facilitated by evacuation of the flasks for approximately 1 rain. Incubation proceeded for 2 to 10 days at 25 ~ in light on an orbital shaker. Extraction Procedure. At the end of the incubation period seeds were removed from the medium, washed with distilled water and deep-frozen. Media and washings were retained for radioactivity measurements and, in general, contained between 10 and 30% of the radioactivity fed. Deepfrozen seeds were macerated and extracted in 80% methanol for three 12 h periods. Pooled methanolie extracts were reduced to a small volume under vacuum at 35 ~ and radioactivity was measured in the resulting aqueous extract. This value was assumed to be the amount of radioactivity taken up. The aqueous residue was then extracted three times with an equal volume of ethyl acetate at p H 8.5 and five times at p i t 3.0. On occasions it was re-extracted three times at pH 3.0 with water-saturated n-butanol. Radioactivity was measured in all fractions. Organic extracts were reduced in volume under vacuum and water was added when appropriate to get rid of residual acid. Extracts were taken to dryness after adding toluene and were weighed. Enzyme Hydrolysis. Extracts to be hydrolysed were dissolved in 50 mM ptI 4.0 phosphate buffer. "Pectolytie enzyme" (The Boots Co. Ltd., Nottingham, England) adsorbed on kieselguhr was added, the ratio being 1 g extract: 100 ml buffer: 20 g enzyme. On occasions the enzyme was removed from the kieselguhr support by shaking in the appropriate volume of buffer for 15 min and filtering. The filtrate was then added to dry extracts. Incubation proceeded for 2 days at 37 ~ with gentle shaking. The hydrolysatc was filtered, adjusted to pH 3.0 and extracted four times with ethyl acetate. Radioactivity was measured in both fractions. The organic fraction was finally dried and weighed. Thin Layer Chromatography. Extracts were strip-loaded onto 20 • 20 cm plates of silica gel H F (Merck), 0.4 mm thickness, which had been prewashed with ethyl acetate. Chromategrams were developed with ethyl acetate: chloroform: acetic acid (15:5:1) for 15 cm. When activity permitted, plates were scanned using a Berthold LB2723 TLC scanner. For accurate quantitation in these cases, and in other instances where activity was too low to permit plate scanning, a 1 cm strip of silica was scraped off in half-Rf bands and added directly to scintillation fluid for liquid scintillation counting. Material in the appropriate zones was eluted from the silica gel with water-saturated ethyl acetate. Column Chromatography. When extracts were of too large a mass for preparative TLC they were chromatographed on columns of silica gel (Hopkin and Williams, England) packed in petroleum ether (b.p. 60-80~ Columns were eluted with an increasing percentage of ethyl acetate in petroleum ether followed by an increasing percentage of methanol in ethyl acetate until all radioactivity had been eluted. Twenty-five ml fraetio.ns were collected and an aliquot of each measured for radioactivity. Gas Chromatography. Extracts were derivatized as described previously (Frydman and MacMillan, 1973). Derivatized extracts were gas chromatographed on 2% SE-33 and 2% QF-1, both stationary phases being coated on Gas Chrom Q (80-100 mesh) packed in 182.5 cm • 0.7 cm o.d. glass columns, fitted in a Pye 104 gas chromatograph. Temperatures are specified later and nitrogen flow rate throughout was 60 ml min -1. Gas Chromatography-Radio Counting. The procedure used has been described by Mac Millan and Wels (1974). Effluent from the flame ionisation detectors was collected into scintillation fluid over 0.5 min intervals, and radioactivity was measured by liquid scinillation counting. Preparative Gas Chromatography. Methylated extracts were gas chromategraphed on 5 % SE-30 coated on Gas Chrom Q (60-80 mesh) packed in a 182.5 cm • 0.7 cm o.d. glass column and fitted with a 17:1 splitter. Fractions were collected into narrow bore glass tubes. The

184

V. M. Frydman and J. MacMillan Table 1. [aH]GA9 feeds Feed number

Total GAg fed (~g) % uptake N/B ethyl acetate e Acidic ethyl acetate e Acidic butanol e Residual aqueous e Date fed

1

2

3

4

100 a 72 4 73 __ 21 July

428 b 81 7 77 __ 16 July

250 c 59 0 52 __ 17 October

250 d 69 3 66 36 2 October

a To 344 g 20 day old seeds. 0.3 ~g GAg/gfw, 0.2 ~g/seed. b To 62 g 20 day old seeds. 6.9 ~g GAJgfw, 5.1 ~g/seed. c To 25 g 23 day old seeds. 9.8 tzg GAg/gfw, 2.2 tzg/seed. To 28 g 23 day old seeds. 8.9 ~tg GA~/gfw, 2.2 ~zg/seed. e Expressed as a percentage of radioactivity taken up. Table 2. TLC data for [SH]G% feeds, expressed as a percentage of radioactivity recovered from each plate Feed number

Zone A a Zone Bb Zone C c

1

2

3

4

35 26 35

25 19 43

53 25 18

41 17 29

Solvent system--Ethyl acetate: chloroform: acetic acid 15: 5:1. Approximate Rf value for

GA,=O.8. a Approximate Rf value ~ 0-0.15. b Approximate Rf value = 0.2-0.4 (occasionally separable into two zones of equal proportion). c Approximate Rf value----0.55-0.7.

collected samples were measured for radioactivity, trimethylsilylated and gas chromatographed on 2 % SE-33. Gas Chromatography-Mass Spectrometry. The procedure was the same as that described by l%ydman et al. (1974). Results

[aH]GA9 Feeds. I n our previous work ( F r y d m a n et al., 1974) we d e t e r m i n e d t h a t seeds of Progress 5 / 0 . 9 t w e n t y days from anthesis c o n t a i n e d 0.2 ~g. GA~ per g fresh weight (0.121 ~g per seed). I n order to reproduce physiological conditions as a c c u r a t e l y as possible [3H]GA~was initially fed a t a n a m o u n t approxi m a t i n g to t h e endogenous level of GA 9 (Table 1, feed 1) to seeds of a n age a t which t h e endogenous level of GA 9 was greatest. I n c u b a t i o n was for two days since in vivo GA~0(2), t h e p u t a t i v e m e t a b o l i t e of GA 0 i n peas, reaches a m a x i m a l conc e n t r a t i o n 2 days after the c o n c e n t r a t i o n of GA 9 is m a x i m a l . E x t r a c t i o n of seeds a t the e n d of the i n c u b a t i o n period yielded three fractions (Table 1, feed 1). TLC of aliquots of the ethyl acetate-soluble acidic fraction showed three m a i o r zones

Metabolism of Gibberellins in Seeds

tO Cl

121

llJ

185

J ~GA2~

GA20

b

b

~ 300/0 12 I

cO

I

I

I

t

,

,

,

,

10 20 30 h0

10 20 30 40 GA20~

123

rr

&

36

6

38

%

Hs1% I

F

I

I

1}3 20 30 40

I

I

I

10 20 30 40

280H GA9 H2GA31

GA20\'Pk~/

c

0 n

GA20 Pkl 213OHGA9

~e

123

rr

8 9oO.. 12 20.61-

/ y2% RT (in half-min )

"~ ~J~H2GA31

~_,,~F~2 8 12 16.2F

f

~5/.%

, , u, I 10 20 30 40 RT {in half-rain )

Fig. 1 a--f. GC-RC traces of [3H]GA9 feeds. (a) and (b) Zone C from first feed (quantitatively equivalent to second feed). (a) SE-33, temp. programmed from 184~ at 3~ min-1, attenuation 2 • 10a. (b) QF-1, temp. 210~ attenuation 2 • 10a. (e) and (d) : Zone C from fourth feed (qualitatively equivalent to third feed). (c) SE-33, temp. 187~ for 6 min then programmed at 3~ min-1, attenuation changed to 2 • 102. (d) QF-1, temp. 186~ for 6 min then programmed at 3~ min-1, attenuation changed to 2 • 102. (e) and (f) Zone I from bulked second and third feeds after column chromatography. (e) SE-33, temp. 184~ for 6 min then programmed at 3~ min-1, attentuation 5 • 102. (f) QF-1, temp. 185~ for 6 min then programmed at 3~ min-1, attenuation 5 • 102. GC-RC peaks are labelled with the percentage of radioactivity recovered that they represent. P start of programme

of r a d i o a c t i v i t y (Table 2, feed 1). V i r t u a l l y all t h e [3H]GA9 t a k e n u p appeared to have been metabolised since there was evidence of only low r a d i o a c t i v i t y i n the n e u t r a l / b a s i c fraction (Table 1, feed 1) a n d n o a c t i v i t y at the a p p r o p r i a t e Rf o n TLC of the acidic fraction. Zone C h a d a n R f e q u i v a l e n t to t h a t of GA20 i n the solvent system used. After e l u t i o n from the silica a n d derivatization, GC-RC of MeTMS Zone C was carried o u t o n SE-33 (Fig. 1 a). A single radioactive peak was o b t a i n e d w i t h R T 0.5 rain

186

V.M. Frydman and J. MacMillan

later than that of a mass peak identified as MeGA~0 TMS by GC-MS. As the MeTMS derivative on QF-1 (Fig. l b) two zones of radioactivity were observed by GC-RC, one of which had an identical R T to that of the mass peak identified as MeGA~0TMS by GC-MS. As the Me ester on SE-33 (184 ~ a single radioactive peak (71%) was obtained with R T 2.0 min later than that of MeGA~0 (GC-MS identification). As the Me ester on QF-1 (203 ~ a single, though broad radioactive peak (59 %) was observed at the R T of MeGA~0 (GC-MS identification). Thus it was concluded that there were two metabolites of [3H]GA9 in this TLC zone, neither of which was GA~0 and neither of which had RTS equivalent to any of the known GAs. However, insufficient quantities of metabolites were present to allow GC-MS identification. Attempts were made to purify Zone C by preparative GC. Methylated extracts were chromatographed on SE-30 (203~ One fraction contained on average 8.2 % of recovered radioactivity and virtually all the endogenous MeGA20. Another contained an average 64.7 % of recovered radioactivity. GC-MS as the MeTMS derivative on SE-33 of this fraction bulked from 3 preparative GC runs, although containing 0.2 ~g of radioactive material, contained too many mass peaks to allow GC-MS identification of the metabolites. Zone B (Table 2, feed 1) was separated into two radioactive zones during GC-RC as the MeTMS derivative on SE-33 (184 ~ prog. at 3 ~ min-1). One (31%) had an RT equal to a mass peak identified as MeGA~gTMS by GC-MS. The presence of endogenous GA~9 precluded the unequivocal identification of traces of [3H] GA29 (see later). The second radioactive peak (39 %) of longer R T than MeGA~9 TMS was not identified. Repeated GC-RC of derivatized material from Zone A resulted in less than 2 % of recovered radioactivity. In order to identify metabolites of [3H]GA0 it was deemed necessary to feed considerably higher levels of substrate. A second feed using approximately 40 times the endogenous concentration was carried out (Table 1, feed 2). Fractionation (Table 1, feed 2), TLC (Table 2, feed 2) and GC-RC gave similar results to the first feed, indicating that metabolism of physiological and non-physiological concentrations of [3H]GA9 was the same. A third feed (Table 1, feed 3), however, although appearing equivalent to those already mentioned on the basis of TLC data (Table 2, feed 3), was clearly not so. GC-RC of Zone C as the MeTMS derivative on SE-33 (see Fig. 1 c) revealed two radioactive peaks, one with R T identical to MeGA~0TMS (GC-MS identification) and one with R T 1.5 rain longer. On QF-1 (see Fig. 1 d) two radioactive peaks were also observed, one having an identical R T to MeGA20TMS. In order to obtain sufficient mass of metabolites for GC-MS identification, second and third feeds were combined before further purification. The bulked acidic ethyl acetate extracts from the two feeds (48.6 • 106 dpm. in total) were chromatographed on a silica column. Ninety percent of the applied radioactivity was recovered, there being two major zones. Zone 1 (eluted with 60 % ethyl acetate in hexane) contained 27 % recovered radioactivity and Zone 2 (2 % methanol in ethyl acetate) contained 34 %. GC-RC of MeTMS Zone 1 on SE-33 (Fig. 1 e) and on QF-1 (Fig. If) revealed two zones of radioactivity, one in each case of identical R T to MeGA26TMS. However, it was evident from the relative proportions

Metabolism of Gibberellins in Seeds

187

of peaks on the two columns that there were three radioactive components. GC-MS of MeTMS Zone 1 on SE-33 confirmed peak 1 (Fig. l e) to be MeGA20 TMS. Peak 2 was partially resolved into two components. The major component gave a spectrum reminiscent of a monohydroxylated GA s. This compound was identified as Me2fi-OHGAgTMS by comparison with an authentic sample provided by Professor N. Takahashi [m/e (%) 418(M+, 1.0), 403(3.6), 386(13.5), 328(t3.7), 296(14.9), 284(61.3), 268(45.8), 241(16.2), 227(25.3), 225(61.1), 75(100), 73(57.6)]. The minor component of peak 2 was not identified at this stage. GC-MS on QF-1 showed that peak 1 (Fig. lf) corresponded to MeGA~0TMS and Me2/~-OHGA 9 TMS which were partially resolved. Peak 2 gave an MS similar to that of a MeTMS derivative of a dihydro GAs1 (P~ailton et al., 1974a). This compound has not been fully characterised by these workers nor was a sample available for direct comparison of MS and R T. However the close similarity of the two spectra suggests that the two compounds are identical. Unequivocal identification of the metabolites [SH]GAe0, 2fi-OH-[3H]GAg, It2-[3H]GAsl and [sIt]GA~9 (see later) was established by two criteria. Firstly, the radioactivity was not separable from the mass peak identified by GC-MS, under several different GC conditions. Secondly, there was sufficient metabolite (as estimated from radioactivity) to give a strong mass spectrum. Thus, in the case of GA20 and GA29, the tritiated metabolites were shown by GC-MS to be indistinguishable from the endogenously-derived non-tritiated GA. l~econsidering GC-RC data for the second and third feeds prior to column chromatography in the light of the subsequent GC-MS identifications in combined extracts, it appears that Zone C of the second feed contained approximately 80% 2/~-Ott-[3H]GAg(4) and 20% tt2-[SH]GA31(3 ) and that of the third feed contained approximately equal proportions of [SH]GA20(2) and H~-[3I-I]GA31(3). By analogy with the second feed the first feed probably contained 2fl-OH-[sH]GAg(4) and H~-[SH]GA31(3). All extracts contained endogenous GA20 too. GC-RC of MeTMS Zone 2 from the silica column yielded at best 11% recoverable radioactivity. Working on the assumption that this zone constituted a polar conjugate of [stt]GA9 or of a metabolite of [sit]GAg, it was subjected to enzyme hydrolysis. GC-RC of the hydrolysate (SE-33, MeTMS derivative) showed a major radioactive peak (44%) which on GC-MS was identified as H2GAsl. (Enzyme rather than chemical hydrolysis of plant extracts thought to contain GA-conjugates was preferred. Enzyme hydrolysates were low in mass making GC-MS identification of hydrolysis products easier--see Fig. 2). GC-I~C of the MeTMS derivative on QF-1 and of other derivatives on both columns failed to reveal the presence of [3H]GA~0 or 2fi-OH-[3H]GAs in the hydrolysate. Zones A from TLC of both second and third [aH]GA 9 feeds must therefore contain this conjugate of H,~[SH]GAsl. I t is =presumably a glucosyl ether rather than ester since base hydrolysis (10% potassium hydroxide at 100 ~ for 1 h) failed to release free H~- [SH]GA3r A fourth [SH]GA feed (Tables 1 and 2, feed 4) was carried out giving similar results to the third feed. GC-I~C of MeTMS Zone C from TLC gave two zones of activity identifiable as MeGA20TMS and MeH2GA31TMS by GC-MS. Zone A on enzyme hydro]ysis yielded insufficient material for GC-MS identification of radioactive components. TLC of the butanol fraction using ethyl acetate:chloro-

188

V.M. Frydman and J. MacMillan

{c O O..

-~

H2GA31

c~ I_L

--, 1"0t 2.0 &5I-

io

30

io

RT(inhctlf-min)

Fig. 2. GC-RC, as the MeTMS derivative, of the enzyme hydrolysate of the butanol-soluble fraction from the fourth [aH]GA9 feed. 2% SE-33 column, temp. 188~ for 6 min, then programmed at 3~ rain-t, attenuation 5 X 102

Table 3. Metabolites of [alZf]GA~feeds Feed number

Acidic ethyl acetate fraction

Acidic butanol fraction

l~f 0-0.1 Zone A

l~f 0.6-0.7 Zone C

2

H2GA31 conjugate 10%

2fl-OHGA 9 H aGA3x

26% 6%

Not analysed

3

H2GA31 conjugate 15% Insufficient for GC-MS

5% 5% 9% 10 %

Not analysed

4

GA20 H 2GAs1 GA~0 H~GA31

H2GAsl conjugate 9%

Figures are expressed as a percentage of GAg taken up and represent the amounts of metabolites conclusively identified by GC-RC and GC-MS, in extracts after fraetionation, TLC and, where appropriate, hydrolysis. As hydrolysis was between 30 and 50% efficient the levels of H2GA81were in particular underestimated.

f o r m : m e t h a n o l : acetic acid (15:5 : 1 : 1) showed two radioactive zones a t Rf 0.05 a n d t~f 0.15. E l u t i o n of m a t e r i a l from these zones, e n z y m e hydrolysis, GC-RC of derivatized m a t e r i a l (Fig. 2) a n d GC-MS showed the presence of H2-[aH]GA31 at R f 0 . 1 5 . Hydrolysis of the eluate from the lower Rf b a n d gave insuifficient m a t e r i a l for GC-MS identification of radioactive components. The metabolites of [all] GA 9 conclusively identified b y GC-MS are s u m m a r i z e d i n T a b l e 3 a n d will be discussed later. The percentages represent the a m o u n t s of metabolites conclusively identified a n d as such are m i n i m a l estimations of the absolute quantities.

Metabolism of Gibberellins in Seeds

189

Table 4. [SH]GA20 feeds Feed number 1

2

3

4

5

6

Total GA20 fed (~zg)

1000 a

600 b

1000 c

800 a

800 e

600 f

Age of seeds (days from anthesis)

22-24

12-17

21

28

39

39

Date fed

June

July

July

July

Dee.

Dec.

3

4

4

4

4

10

66

62

84

45

64

68

1

0

91

81

6

16

1

2

Incubation time (days) % uptake

6

3

2

1

Acidic ethyl acetateg

N/B ethyl acetateg

86

84

70

73

Acidic butanolg

--

--

--

Residual aqueous g

10

8

7

3

To 125 g seeds (8.0 ~zg/gfw, 4.5 ~zg/seed). b To 60 g seeds (10.0 ~zg/gfw, 1.2 ~zg/seed). c To 100 g seeds (10.0 Ezg/gfw, 7.4 [xg/seed). d To 80 g seeds (10.0 tzg/gfw, 8.6 ~zg/seed). e To 80 g seeds (10 ~zg/gfw, 5.4 ~xg/seed). To 60 g seeds (10 ~zg/gfw, 5.4 ~g/seed). g Expressed as a percentage of radioactivity taken up.

[aHJGA~0 Feeds. F r o m our previous w o r k ( F r y d m a n et al., 1974) 22 d a y old seeds of Progress No. 9 c o n t a i n a p p r o x i m a t e l y 9.5 ~g GA20 per g. fresh weight (7.3 ~g per seed) a n d five d a y s l a t e r t h e levels of GA29(5), t h e p u t a t i v e m e t a b o l i t e of GA20(2), are m a x i m a l . A n initial feed of [~H]GA20 (Table 4, feed 1) was carried o u t using 22-24 d a y old seeds a n d t h e i n c u b a t i o n p e r i o d was 3 days. E x t r a c t i o n y i e l d e d t h r e e fractions (Table 4, feed 1). TLC was carried o u t on t h e acidic e t h y l a c e t a t e f r a c t i o n a n d t h e t w o m a j o r r a d i o a c t i v e zones o b s e r v e d (Table 5, feed 1) were eluted. Zone B on GC-RC as t h e MeTMS d e r i v a t i v e on b o t h SE-33 a n d QF-1 showed a single r a d i o a c t i v e p e a k of i d e n t i c a l 1~T to t h a t of a mass p e a k identified as MeGA29TMS b y GC-MS (Fig. 3). GC-RC of Zone C as t h e MeTMS d e r i v a t i v e on b o t h SE-33 a n d QF-1 a n d GC-MS r e v e a l e d t h e presence of u n m e t a b o l i s e d [aIt]GA2o. No m e t a b o l i t e s of [SH]GA2o o t h e r t h a n [aI-I]GA29 were identified alt h o u g h of t h e 66.5% of [aH]GA~0metabolised 18.0% was n o t a c c o u n t e d for (see later). Several a d d i t i o n a l [~H]GA20 feeds were carried out. F e e d s 2 4 (Table 4) utilised seeds of 3 ages. TLC d a t a (Table 5) a n d GC-RC d a t a i n d i c a t e d t h a t [aH]GA20 was efficiently c o n v e r t e d to [sIt] GA~9 in each feed (identifications m a d e b y GC-MS in each instance), a l t h o u g h t h e h i g h e s t p e r c e n t a g e conversion to [SH]GA29 was o b s e r v e d in feed 3. I n this feed a level of [SH]GA20 a l m o s t e x a c t l y e q u i v a l e n t t o t h e endogenous level was used, a n d t h e seeds were of an age a t which t h e endogenous GA20 levels were m a x i m a l .

190

V . M . F r y d m a n and J. MacMillan

Table 5. TLC data for [aHJGA20 feeds, expressed as a percentage of radioactivity recovered from each plate Feed n u m b e r 1

2

3

4

5

6

Identity

Acidic E t h y l acetate Zone A a Zone :Bb Zone C c

3 50 35

6 65 24

5 80 8

8 53 30

4 73 24

7 90 3

? GA29 GA20

Acidic Butanol a Zone D b Zone E c

. .

40 35

71 11

GA29 GA20

. .

. .

. .

Solvent s y s t e m - - E t h y l acetate: chloroform: acetic acid 15: 5: I. a Approximate R f value = 0-0.1. b Approximate Rf value = 0.3-0.4. c Approximate Rf value ~ 0.55-0.66. d After enzyme hydrolysis.

O

GA29

It_

E

lb

2'o

2o

5b

RT (in half-rnin ) Fig. 3. GC-RC, as the MeTMS derivative, of Zone B from TLC of the acidic ethyl acetate fraction of [3HJGA20 feed. 2% SE-33 column, temp. programmed from 184 ~ a t 3 ~ min-1, a t t e n u a t i o n changed to 1 • 103

[3H]GA~9 Feeds. S i n c e t h e l e v e l s of GA29 i n s e e d s m a t u r i n g in vivo d r o p t o a n u n d e t e c t a b l e l e v e l 35 d a y s f r o m a n t h e s i s ( F r y d m a n et al., 1974) t h e m e t a b o l i s m of [3HJGA29 w a s s t u d i e d . I n o r d e r t o i s o l a t e [3H]GA29 f o r r e f e e d i n g t h e a c i d i c f r a c t i o n s of [3H]GA20 f e e d s 2 4 w e r e b u l k e d a n d w e r e c h r o m a t o g r a p h e d o n a c o l u m n of silica gel. T w o z o n e s of r a d i o a c t i v i t y w e r e e l u t e d c o r r e s p o n d i n g t o

Metabolism of Gibberellins in Seeds

191

[3H]GA20 and [3It]GA29. Preparative TLC of the [aH]GA~D zone yielded a gum which contained 600 ~g [aH]GAes and, on the basis of previous results, probably contained an equal 'amount of endogenously derived GA~s. GC-RC on several columns showed the presence of no other radioactive components and GC-MS showed MeGA29 TMS to be the major mass peak. Minor contaminants were shown b y GC-MS to be non-gibberellin in nature. Due to the presence of endogenous GA2D in the extracts of these feeds the [3H]GA~s isolated had a lower specific activity t h a n the [3tI]GA20 fed. Fifty-nine micrograms of this [aH]GA2s (diluted by an unknown quantity of untritiated GA2s) was fed to 39 g. of 27-28 day old seeds. After 4 days incubation extraction of the seeds showed t h a t the 38.2 % [3tt]GA29 taken up was distributed between the neutral/basic ethyl acetate extract (0.4 %), the acidic ethyl acetate extract (80.5%) and the residual aqueous extract (3.1%). TLC, GC-I~C and GC~ MS showed t h a t over 90% of the radioactivity in the acidic ethyl acetate extract was attributable to unmetabolised [3HI GAeg. An 8 day feed of 100 ~zg [SH]GA29 to 51.5 gn 27 day old seeds yielded similar results, with 86 % of the radioactivity in the ethyl acetate acidic fraction being attributed to [aH]GA29 (GC-MS identification). Our failure to observe the metabolism of applied [aH]GA2D to seeds cultured in vitro led us to test whether the [SH]GA29 was reaching its normal site within the seed. Since [aH]GA20 is rapidly and efficiently converted to [3H]GA29 in cultured seeds the metabolism of [3H]GAe9 synthesised in situ from [aIt]GA20 was studied. Two feeds of [aH]GA~0 were carried out (Table 4, feeds 5 and 6), the incubation periods being four and ten days. Results of fractionation are shown in Table 4. TLC results of the ethyl acetate acidic fractions from both feeds are shown in Table 5, feeds 5 and 6. Zones C from each feed contained unmetabolised [3H]GA20, and zones B contained [aH]GA~9 (GC-RC and GC-MS identification). Zones A were not analysed although it was presumed t h a t they contained similar radioactive components to the butanol fractions (el. GA 9 feeds where this was shown to be the case). As GC-RC of butanol fractions resulted in the recovery of virtually no radioactivity they were subjected to enzyme hydrolyses and TLC (Table 5, feeds 5 and 6). GC-I~C of zones D from both feeds showed single radioactive peaks of g T identical to t h a t of MeGA~gTMS. MeGA~gTMS was identified b y GC-MS in Zone D from the 10 day feed but insufficient material was present in the 4 day feed for GC-MS identification. GC-RC of zones E from both feeds contained single radioactive peaks of 1%T identical to t h a t of MeGA20TMS. MeGA20 TMS was identified b y GC-MS in Zone E from the 4 day feed, but insufficient material was present in the 10 day feed for GC-MS. I t is concluded t h a t [3H]GA20 and [3H]GA29 are present in 4 and 10 day GA~0 feeds as polar conjugates, perhaps as glueosyl ethers or esters. To summarize, in the 4 day feed of [3H]GA20 22% of the [SH]GA20 taken up was unmetabolized, 66% was accounted for as [3H]GA~9, 1% as a [~H]GA~9-eonjugate (tentative identification) and 1% as a [3H]GA20conjugate. I n the 10 day GA20 feed 2% of the [~H]GA20 taken up was unmetabolized, 73% was accounted for as [3H]GA29, 9% as a [3H]GA29-conjugate and 1% as a [aH]GA20-conjugate (tentative identification).

192

V.M. Frydman and J. MacMillan Discussion

[aH]Gibbcrellin A20(2), 2fl-OH-[3HJGAg(4), H2-[3H~GA31(3) and a highly polar conjugate of H~-[3H]GAsl were all conclusively identified as metabolites of [aHJGAg(1) in immature seeds of Progress No. 9. The results of four feeds of [3H]GA9 were however not reproducible. I n the second feed, and on the basis of GC-RC data in the first feed too, 2fl-OH-[3H]GAg(4), Hu-[~H]GAal(3) and H~-[aH]GA31-conjugate were observed whilst in the third and fourth feeds [3HI GA20(2), H2-[aH]GAal(3) and H~-[aHJGA31-eonjugate were identified as metabolites of [aHJGAg. The reasons for this inconsistency arc not known. The substratc, incubation conditions and extraction procedures were the same in all instances. However, the first two feeds were conducted using plant material harvested in June, the latter two using material harvested in October. The maturation period for our seeds is considerably longer in winter than in summer. Those developing in June and J u l y reach maximal fresh weight 35 days after anthesis (Frydman et al., 1974) whilst up to 20 days more are required in winter for final fresh weight to be obtained. Hence the 20 day old seeds harvested for the first two feeds weighed approximately 0.7 gm whereas the 23 day old seeds used in the last two feeds weighed 0.2 gin. We have no detailed data on endogenous GAs of winter grown seeds of Progress No. 9 but have some evidence t h a t the levels of endogenous GA20 and GA2s are lower than in summer grown material. At present therefore, we attribute the different results obtained in summer and winter feeds to the different "physiological s t a t u s " of the seeds at the beginning of feeds. We acknowledge the imprecision of this statement and are currently investigating the effect of light and temperature on [aHJGA9 metabolism. Our data, however, do point out the danger of extrapolating from positive identifications in initial experiments to presumed identifications from TLC and GC data in subsequent experiments. A relevant example is the related study by Railton et al. (1974a) on the metabolism of [aH]GA s in seedlings of P. sativum cv. Meteor. These workers unambiguously identified [aH]GA20 and H a- [SH]GAsl as metabolitcs of [3tI]GAs and provided evidence for a polar metabolite which they suggested was formed from [SHJGA~0 or H2-[3H]GAsr Their results are similar to our "winter feeds". However Railton (1974 a and b) extrapolated from this original data in subsequent studies on the effects of light and cy~okinins on [3HJGAs metabolism, TLC being the only basis for identification. GAs(1 ) and GA20(2) are endogenous GAs of immature seeds of Progress No. 9 and we suggested (Frydman et al., 1974) t h a t GA s m a y be the direct precursor of GA~o. I n our feeds conversion of [aHJGA s to [SH]GA~0 varied from nil to approximately 10 %, hence, it seems unlikely t h a t GA s is the principal precursor of GA~0 in immature seeds of this cultivar. Further work is required to establish if any other GA endogenous to pea can serve as a more efficient precursor of GA~0 than GA s. The major products of our [aH]GA9 feeds were 2fl-OH-[aH]GAs(4) and H 2- [3H]GAsl(3). These gibberellins arc not known to be endogenous to this or any other plant source. They m a y therefore be artefacts of the system despite the precautions taken to avoid induced metabolism and if this is the case then GA s m a y indeed be the major precursor of GA20 in vivo. I n this context it is interesting to note tha~ in a GA molecule the 2fl- and 12~-positions are equi-

Metabolism of Gibberellins in Seeds

193

distant from the carbon atom at position 7 and become equivalent when the molecule is rotated 180 ~ about the C-6--C-7 bond. On the basis of Jones' model for the hydroxylation of steroids (Jones, 1973) the 2fl- and 12~-positions may be alternative sites of hydroxylation for a single enzyme whose major point of attachment with the GA molecule is at the C-7 carboxylic acid group. Twelve to 28 day old seeds, as shown in the present work, contain a 2fl-hydroxylating enzyme which converts [SH]GA~0 to [att]GA29. I t is possible that [aH]GA9 applied exogenously to seeds may not reach its normal site of action and instead it may be hydroxylated by this enzyme, whose normal substrate is GA20. Orientation of the unnatural substrate with the enzyme may occur in two ways to give hydroxylation in the two equivalent positions--2fi-hydroxylation to give 2/~-OHGA 9 and 12~- to give H2-GA31. Until 2fl-OIt-GA s and H~-GA31 are shown to be endogenous to developing pea seeds conclusions on the physiological significance of a n y of the GA s metabolites (GA20 included) are not warranted. This is especially true of conclusions relating to comparable rates of formation of metabolites (Railton 1974a and b). Railton et al. (1974b) have shown that [3H]GA~0 applied to dark grown seedlings and germinating seeds of P i s u m sativum cv. Meteor is metabolised to [3H] GA29. In the present work the extremely efficient conversion of [3H]GA20 to [3H]GA29 in immature seeds of Progress No. 9 has been established under conditions which fulfil the following criteria: (1) the exogenously applied GA and its metabolite were previously established to be endogenous GAs, (2) the GA was applied at a level calculated to be equivalent to the endogenous level, (3) the GA was fed at the previously determined developmental stage at which the endogenous level was maximal, (4) the incubation period corresponded to the time interval between the maximal endogenous levels of the two GAs. Hence it seems reasonable to conclude that the conversion of GA~0 to GA29 is a normal metabolic process in developing seeds of Progress No. 9. The detection of a conjugate of [3H]GA29 was predictable, that of a conjugate of [~H]GA20 was less so. These coniugates are probably GA glucosyl ethers, judging by their behaviour on fractionation. GA29-2fl-glucosyl ether has been isolated from immature seeds of Pharbitis nil b y Y o k o t a et al. (1970). GAe0-13-glucosyl ether has not been characterized. We infer from results of long and short term [aH]GA20 feeds that a small quantity of [aH]GA20-conjugate is formed simultaneously with [~H]GA29 and is perhaps a result of slight substrate overloading. [3H]GA29-coniugate built up slowly with time (although even after ten days the percentage formation was remarkably low) and is probably formed directly from [aH]GA29. However a route from [aH]GA20-coniugate is not inconceivable if the GA-conjugate linkage is at the same position in both molecules. The metabolic sequences observed in immature seeds of Progress No. 9 are summarized in Fig. 4. Since the levels of free acidic GAs are very low in mature seeds (e.g. in Progress No. 9, Frydman et al., 1974) it has been suggested that these become conjugated during the final stages of maturation, to yield inactive storage products. The fate of the conjugates on subsequent germination has not been clarified Sembdner et al. (1968) obtained evidence for hydrolysis of conjugated GAs during

194

V.M. Frydman and J. MacMillan ?

\

/

\

NGA

*

GA20-c~

ugate \ \ \\ *

50-90%

GA29

2O

at least 9% ) GA29 conj ugate

/

? ......

~GAg*

O

/

H2GA31

at least 9%)

H2GA31_conjugat e

Fig. 4. Metabolic sequences observed in immature seeds. Figures represent the percentage conversion observed. -~ conversion conclusively shown, i.e. product identified by GC-MS. ---> hypothetical pathway. * endogenous gibberellin

g e r m i n a t i o n . I n a d e t a i l e d s t u d y of t h e m e t a b o l i s m of GAs endogenous to Phaseolus vulgaris Y a m a n e et al. (1975) h a v e shown t h a t [3H]GA1 fed to i m m a t u r e seeds is c o n v e r t e d to [3HJGAs-glucoside ( t e n t a t i v e identification) t h r o u g h o u t m a t u r a t i o n a n d g e r m i n a t i o n . I f t h e GAs synthesised d u r i n g seed d e v e l o p m e n t are i r r e v e r s i b l y c o n j u g a t e d d u r i n g t h e final stages of m a t u r a t i o n , t h e n t h e y can p l a y no role in seed germination. Hence, a s s u m i n g t h a t these gibberellins synthesised d u r i n g seed d e v e l o p m e n t i n d e e d h a v e a physiological function, t h e i r effects m u s t be confined to t h e m a t u r a t i o n process. We are indebted to Dr. L.C. Luckwill of Long Ashton Research Station for providing all the plant material; Dr. J.R. Bearder for preparing tritiated GAs and GAs0; Mr. P. Gaskin for obtaining the mass spectra; Professor N. Takahashi, University of Tokyo, for a sample of 2fl-OHGAg; and Imperial Chemical Industries Ltd. and the Agricultural Research Council for financial support to V.M.F.

References Baldev, B., Lang, A., Agatep, A. O. : Gibberellin production in pea seeds developing in excised pods: effects of growth retardant AM0-1618. Science 147, 155-157 (1965) Barendse, G.W.M., Kende, It., Lang, A. : Fate of radioactive gibberellin A s in maturing and germinating seeds of peas and Japanese morning glory. Plant Physiol. 43, 815-822 (1968) Bearder, J. 1~., MacMillan, J. : Fungal products. Part IX. Gibberellins A18, Aa6, A~, A~I and A~2 from Gibberella ]ufikuroi. J. chem. Soc. Perkin 1 2824-2830 (1973) Frydman, V.M., MacMillan, J. : Identification of gibberellins A20 and A29 in seed of Pisum sativum cv. Progress No. 9 by combined gas chromatography-mass spectrometry. Planta (Berl.) 115, 11-15 (1973) Frydman, V.M., MacMillan, J.: Gibberellins in developing seed of Pisum 8ativum cv. Progress No. 9. Presented at the International Symposium on Plant Growth Regulators, Torun, Poland (1974) Frydman, V.M., Gaskin, P., MacMillan, J.: Qualitative and quantitative analyses of fibberellins throughout seed maturation in Pisum sativum cv. Progress No. 9. Planta (Berl.) 118, 123-132 (1974)

Metgbolism of Gibberellins in Seeds

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Graebe, J. : Biosynthesis of gibberellins in a ceil-free system. Presented at the International Symposium on Plant Growth Regulators, Halle, G.D.R. (1974) Graebe, J., Hedden, P., Gaskin, P., MacMillan, J. : The biosynthesis of g Cls-gibbercllin from mevglonic acid in g cell-free system from g higher plant. Plgntg (Berl.) 120, 307-309 (1974) Jones, E. R. H. : The microbiologicgl hydroxylation of steroids and related compounds. Pure and Applied Chemistry 33, 39-52 (1973) Lang, A.: Gibberellins: structure gnd metgbolism. Ann. Rev. Plant Physiol. 21, 537 570 (1970) MacMillan, J.: Diterpenes--the gibberellins. In: Aspects of terpenoid chemistry gnd biochemistry, p. 153-180, Goodwin, T.W. ed. London: Academic Press Inc. 1971 MgcMillgn, J., Wels, C.M.: Detgiled analyses of mctgbolites from mevalonic lgctone in Gibberella ]u]ikuroi. Phytochcmistry 13, 1413-1417 (1974) Musgrgve, A., Kende, tt. : Rgdioactive gibberellin A5 and its metabolism in dwgrf pegs. Plgnt Physiol. 45, 56-61 (1970) Nitsch, J. P. : Growth gnd development in vitro of excised ovaries. Amer. J. Bot. 38, 566-577 (1951) Rgilton, I.D.: Studies on gibberellins in shoots of light-grown peas II. The metgbolism of tritigted gibberellin A9 gnd gibberellin A20 by light- and dgrk-grown shoots of dwgrf P i s u m sativum vgr. Meteor. Plant Sci. Letters 3, 207 212 (1974g) Railton, I.D.: Effects of N6-benzyladcnine on the rgtc of turnover of [3H]GA26 by shoots of dwarf P i s u m sativum. Plantg (Berl.) 120, 197-200 (1974b) Railton, I.D., Durley, R.C., Pharis, R.P. : ~etgbolism of tritigted gibberellin A9 by shoots of dgrk-grown dwgrf peg, cv. Meteor. Plgnt Physiol. ~4, 6-12 (1974g) Railton, I.D., Murofushi, N., Durley, R.C., Pharis, R.P.: Interconversion of gibberellin A20 to gibberellin A29 by etiolgted seedlings and germingting seeds of dwgrf P i s u m sativum. Phytochemistry 13, 793-796 (1974b) Sembdner, G., Weilgnd, J., Aurich, 0., Schreiber, K.: Isolgtion, structure and metabolism of a gibberellin glucoside. In: Plgnt growth regulators. Soc. Chem. Ind. Monogr. No. 31, 70-86 (1968). Yamgne, H., Murofushi, N., Takghashi, N. : Metgbolism of gibberellins in mgturing gnd germinating bean seeds. Phytochemistry, in press (1975) Yokotg, T., Murofushi, N., Takahgshi, N., T~mura, S.: Gibbcrellin in immature seeds of Pharbitis nil. Part 3. Isolation gnd structure of gibberellin glucosides. Agr. Biol. Chem. 35, 583-595 (1971)