Plants (Berl.

) 126, 25--35 (1975) 9 by Springer-Verlag 1975

The Metabolism of Applied Gibberellic Acid in Pharbitis nil Choisy: Tentative Identification of Its Sole Metabolite as Gibberellic Acid Glucoside and Some of Its Properties *
G. W . M. B a r e n d s e a n d G. J. M. de K l e r k Botany Department, University of Nijmegen, Toernooiveld, The Netherlands Received 4 April; accepted 30 May 1975
Summary. Radioactive gibberellic acid ([14CJGA3) applied to seedlings of Pharbitis nil strain Violet is converted to one single metabolite (R-[nC]GAa), which has been tentatively identified as GA3-glucoside. As with authentic GA3-glucoside, R-[zdC]GA3 can be hydrolysed to some extent with cellulase and fl-glucuronidase, but hardly at all with fl-glueosidase. Acid hydrolysis, which is more effective than enzymatic hydrolysis, yielded GA3 as well as a biological inactive compound. The latter represents a degradation product of GA3 due to the sensitivity of GA3 to acidic conditions. The R-[zdC]GA3, like authentic GAa-glucoside, possesses little or no biological activity. R-[zaC]GAa applied to developing seeds is partly hydrolysed during imbibition of the mature seed but is, however, reconverted to R-[14C]GA3 during subsequent germination. Applied R-[14C]GA8 is strongly accumulated in the cotyledons of Pharbitis seedlings, to a greater extent than [zdC]GA3. However, unlike [14C]GA3it is not accumulated in the apical regions of the hypocotyl. Furthermore no competition was observed between R-[zdC]GA3 and [z~C]GAa, which suggests that they do not compete for the same sites.

Introduction
I t has been shown p r e v i o u s l y (Barendse, 1973) t h a t a p p l i e d [14CJGAa accumulates in t h e growing p l a n t parts, e.g. t h e apical region of t h e h y p o c o t y l a n d t h e cotyledons of Pharbitis nil seedlings. Moreover, i t was d e m o n s t r a t e d t h a t t h e a p p l i e d [14CJGAs is v e r y r a p i d l y c o n v e r t e d t o one single compound, a GA3-conj u g a t e w i t h low biological a c t i v i t y . T h e r a t e of conversion was higher in lightg r o w n seedlings c o m p a r e d with d a r k - g r o w n ones. I t was i n d i c a t e d t h a t t h e lighti n h i b i t i o n of g r o w t h in Pharbitis seedlings could be p a r t l y due t o t h e r a p i d conversion, i.e. i n a c t i v a t i o n , of t h e h o r m o n e b y light. This p a p e r deals w i t h t h e t e n t a t i v e i d e n t i f i c a t i o n of t h e GAa-metabolite a n d describes some e x p e r i m e n t s a i m e d a t e l u c i d a t i n g t h e possible physiological significance of this m e t a b o l i t e .

Materials and Methods

Plant Material. Seeds of Pharbitis nil Choisy, strain "Violet" or "Kidachi", were treated

with concentrated sulfuric acid for 45 min, extensively rinsed thoroughly with water and subsequently soaked for 24 h. The imbibed seeds were sown in vermiculite and germinated in the dark at 27~ Four-day-old seedlings, hypocotyl length approximately 7 era, from which the roots were removed were placed in aqueous solutions of [14C]GA8 or its metabolite R* Abbreviations: GA8 = Gibberellic acid, R-GA 3 = l~Ietabolite of GA8.

26

G.W. ~ . Barendse and G. J. 1~. de Klerk

[~C]GA3, usually 5 ml of solution per 10 plants at a cone. of 10-6 M in the light (approx. 4000 lux at plant level). After treatment the cotyledons were removed and the hypocotyls cut into 5 mm sections. The cotyledons as well as the hypoootyl sections, usually 5 sections of the corresponding regions, were pooled, weighed and subsequently added to Bray's scintillation fluid and left to stand at least 48 h before being counted. Radioactive Gibberellic Acid. [laC]GA3 (8 methylene-[14C], )specific activity 6.1 mCi/mmol was purchased from the Radiochemical Centre, Amersham, England. Extraction Procedures. The plant material was homogenized in methanol with a Sorvall omnimixer, filtered, evaporated to dryness and subsequently stored at --20~ for future analysis. The storage time was kept to a minimum and never exceeded one month. The residues of some experiments were taken up in methanol and directly chromategraphed on thin layers of silica gel G, developed in benzene/n-butanol/acetic acid (70: 25:5, v/v) or in chloroform/ethyl acetate/acetic acid (60:40:5, v/v). l~-[14C]GA~ was prepared by purifying the extracts as follows: the residue was taken up in ample water and partitioned several times with ethyl acetate until the latter remained clear, the ethyl acetate phase was discarded. The pH of the water phase was raised to p i t 8 and subsequently partitioned with n-butanol. The aqueous phase was discarded and the combined butanol fraction was evaporated to dryness. Subsequently the residue was taken up in a mixture of methanol-acetone (1 : 1, v/v) and chromatographed on thin layers which were developed in chloroform/methanol/acetic acid/water (40:15: 3:2, v/v). The radioactive zone between Rf 0.4-0.6 was eluted 4 times with methanol and yielded a reasonably pure fraction of R-[I~C]GAz. Hydrolysis. Acid hydrolysis was carried out in culture tubes containing 1 ml of diluted HC1 varying in normality and 1000 epm of R-[14C]GAs. The tubes were incubated in a waterbath for 1 or 2 hrs at different temperatures. After hydrolysis the tubes were quickly evaporated to dryness, immediately taken up in methanol and chromatographed on thin layers. Enzymatic hydrolysis took place by adding the different enzymes in several concentrations to 1 ml 0.1 M KH2PO ~ buffer with the required p i t and 1500 cpm of R-[14C]GA3. After incubation for 24 hrs at 37~ approximately 2 ml acetone was added. The tubes were subsequently centrifuged for 5 min at 2200 g (pellet discarded, contained very little radioactivity) and the supernatant evaporated to dryness. The residue was taken up in methanol and chromatographed on thin layer. The thin layers were developed in the above mentioned benzene/butanol/acetic acid solvent. Scintillation counting was carried out by scraping ten equal zones of the chromatograms directly into the vials containing 10 ml Bray's solution. Column Chromatography. A DEAE-Sephadex A-25 column, bed volume 18 2.5 cm, was stepwise eluted with 80% methanol plus increasing concentrations of aqueous acetic acid, according to the procedure obtained from Dr. G. Sembdner (Halle, DDR), e.g. 50 ml methanol -k 30 ml of 0.25%, 0.25 N, 0.50 lq, 0.75 N, 1.00 N and 3.00 N acetic acid respectively. Bioassay. Dwarf corn (dh) was sown in vermiculite and germinated at 27~ After 7 days seedlings were selected and transferred to boxes containing half-stregth Hoagland solution by placing them with the roots through holes in the box covers. Four plants per treatment received 0.1 ml of aqueous extract or standard cone of GAs per plant. The elongation of the first and second leaf sheath was measured after seven days. The biological activity was expressed as percentage of increase or growth over the control (see also Kende and Lang, 1964).

Results Chromatographic Identi]ication el R-GA 3. Since it was suspected t h a t t h e GA 3

m e t a b o l i t e R - G A 3 r e p r e s e n t e d GA3-glucoside, it was c h r o m a t o g r a p h e d on t h i n layers d e v e l o p e d in several s o l v e n t systems w i t h a u t h e n t i c 3-0-fl-D-glucopyranos y l - G A 3 (which was k i n d l y supplied b y Prof. N. T a k a h a s h i , J a p a n ) as reference. T h e results are g i v e n in T a b l e 1. I n all four s o l v e n t sy st em s only one r a d i o a c t i v e zone was o b s e r v e d of w h ic h t h e R f v a l u e of t h e m a x i m u m a c t i v i t y is g i v e n in T a b l e 1. Th es e v a l u e s correspond w i t h t h e R f values of t h e e o e h r o m a t o g r a p h e d a u t h e n t i c GAa-glucoside. I n a personal discussion w i t h Dr. G. S e m b d n e r (Halle,

Metabolism of Applied GA
cpm

27

0000 Gh3-g[ucosyt ester 5000 I-H+++H-H I

I&C-GA3 Oh3_gtucaside

~000

3000

RJ~C.-GA 3

2000

1000

~I~" I 80
i

I 160
1

I 2L~O
I

I I 320
1

L__I I. . . .

I /+00
[

l /+80rnl
1

0,25~176

0,25N

0.50 N

0.75N

1.00N

3,00N Aceticacid

Fig. 1. Column chromatography of [140]GA3,R-[14C]GA3, GA~-glucosylester, and GA3-glucoside. The column contained DEAE-Sephadex A-25 and was stepwise eluted with 80 % methanol plus increasing concentrations of acetic acid. GAa-glucosyl ester and GAa-glucoside were detected on thin layer, developed in benzene/n-butanol/acetic acid (70:25:5, v/v), by spraying with an ethanolic solution of sulfuric acid. The [14C]GA3and l~-[14C]GAawere determined by liquid scintillation counting of the reduced eluants Table 1. Thin-layer chromatography of R-[14C]GA3 in four different solvent systems Solvent system (1) Benzene/n-butanol/acetic acid (2) n-Butanol/acetic acid/water (3) Chloroform/ethyl acetate/acetic acid (4) Chloroform/methanol/acetic acid/water v/v 70 : 25 : 5 60 : 15 : 30 60: 40 : 5 40:15:3:2 Rf max R-[zdC]GA3 0.2-0.3 0.7-0.8 0.0-0.1 0.4-0.5 Rf GAsgtucoside 0.23-0.31 0.68-0.80 0.00-0.08 0.47-0.53

DDR) it was suggested that the present R-[14C]GA3 preparation could also represent GA3-glucosyl ester instead of GAa-glucoside and he proposed a columnchromatographic procedure to distinguish between these two possibilities. When a DEAE-Sephadex A-25 column is stepwise eluted with 80% methanol plus increasing cone. of aqueous acetic acid, GAa-glucosyl ester will be eluted before GAa and GAs-glueoside after GAz. Column chromatography has been carried out on several consecutive occasions with GAs, R-[14C]GA~, authentic GAa-glueosyl ester (kindly supplied by Dr. G. Sembdner) and authentic GAa-glucoside. The results are summarized in Fig. 1. The results show that most of the R-[I~C]GAa is eluted after the [14C]GAa. However, rechromatography on thin layer of the eluted R-[14C]GAa fraction cochromatographing with the [14CJGAa revealed that a major part of this fraction consisted of hydrolysed R-[14C]GAa, probably due to the increasing acidity of the eluant, which explains the broad range of eluted radioactivity from R-[14C]GA3 as well as of authentic GAa-glucoside. I t is clear that the R-[14C]GAa does not represent GAa-glucosyl ester as had been suggested.

28

G.W.M. Barendse and G. J. 1Yr.de KIerk

Hydrolysis o/R-[14C]GAa. The R f values of R-[14C]GA3 on thin layer concur with those of the earlier reported so-called bound gibberellins in Pharbitis (Barendse, 1971, and Barendse and Lung, 1972). These bound gibberellins could be conv e t t e d to biologically active fractions b y means of mild acid hydrolysis or m u c h less effectively with the enzymes fl-glucosidase, cellulase or ficin. I n view of these data, it was decided to carry out acid as well as enzymatic hydrolyses under v a r y i n g conditions. A compilation of the m a i n results is presented in Table 2.
Table 2. Acid and enzymatic hydrolysis of R-[14C]GAa Acid hydrolysis: 1000 cpm R-[14C]GA3/ml Control, 2 hrs, 60~ C Control, 2 hrs, 70~ C Control, 2 hrs, 85~ C 1.0 N HC1, 2 hrs, 60~ C 0.25 N tICI, 2 hrs, 60~ C 0.25 N HC], 2 hrs, 70~ C 0.25 1~ HC1, 2 hrs, 85~ C 1.0 N KOH, 1 hr, 60~ C Enzymatic hydrolysis: 1500 cpm 1~-[14C]GAa/ml Control, pH 4.5, 37~ C Cellulase, pH 4.5, 37~ C fl-glucosidase, pH 5.0, 37~ C fl-glucuronidase, pH 5.1, 37~ C 93.6 27.0 92.6 28.4 6.4 73.0 7.4 71.6 Percentage of to~aI epm R- [14C]GAa 95.3 85.6 80.3 32.1 68.3 53.0 33.3 98.0 [14C]GA3 1.7 9.3 8.3 7.6 10.7 17.8 13.5 0.5 inactivated [llC] G A 3 3.0 5.1 11.4 60.3 21.0 29.2 53.1 1.5

T h e acid hydrolysis experiments were always straightforward and yielded reproducible results. The enzymatic hydrolysis d a t a varied considerably from experiment to experiment although the trends remained the same. I t is k n o w n and has also been indicated previously (Barendso, 1973) t h a t GA a is sensitive to acidic conditions a n d m a y be converted to a biologically inactive compound, p r o b a b l y gibberic acid (Cross et al., 1958), which has a higher l~f value t h a n GA a. Thus after hydrolysis of R-[14C]GA3 three radioactive zones were obtained on the thin-layer chromatograms, developed in solvent system 1 (Table 1), e.g. at ~ f 0.0-0.2, at l~f 0.6-0.7 and at l~f 0.9-1.0 representing I~-[laC]GAs, [14C]GAa and inactivated [~dCJGAa respectively. The results in Table 3 show that acid hydrolysis results in inactivation of a p a r t of the released [14C]GAa. A complete acid-hydrolysis was never obtained in the present experiments, while under alkaline conditions no hydrolysis occurred. The latter indicates a/~-glucosidic linkage. E n z y m a t i c hydrolysis was obtained quite effectively with cellulase and fl-glucuronidase (from Helix pomatia), while -glucosidase appeared to be ineffective. These hydrolysis results agree to some extent with those reported by u et al. (1969, 1970) and those obtained b y lq. Takahashi (personal communication). T h e y found t h a t GAa-glucoside resists enzymatic hydrolysis, particularly fl-gluco-

Metabolism of Applied GA

29

sidase was ineffective while some hydrolysis with cellulase could be obtained. However, Sembdner and eoworkers (personal communication) were even unable to obtain significant hydrolysis with cellulase whereas, in agreement with the present results, they had also problems with acid hydrolysis of GAa-glueoside. Biological activity. The activity of both R-[14C]GA8 and partially hydrolysed R-[14C]GA3 was tested with the dwarf maize (d5) assay. The results are shown in Table 3. Since with extraction of the R-[14C]GA8 from plant material also endogenous GAa-glucosides are extracted means that the specific activity of the R-GA s preparation is not known. Therefore, extracts were made of seedlings, which are known to contain relatively high amounts of biologically active fractions, and of seedpods (from which immature seeds had been removed) which have very little biological activity. This is confirmed b y the results of Table 3, which show t h a t hydrolysis of seedling extracts results in a higher activity than hydrolysis of seedpod extracts. Non-hydrolysed R-[14C]GA3 from seedlings show only some biological activity at high concentrations. This activity is possibly due to hydrolysis in the maize plants. Thus R-[laC]GA3 itself has little or no biological activity which is again in agreement with the findings of Tamura et al. (1968) and of Sembdner et al. (1972) for GA3-glucoside. Table 3. Biological activity of R-[14C]GAa in the (dS) dwarf maize assay Treatment Control 10-~ M [1~C]GAa, 1300 cpm/plant 10-6 M GA3, 0.1 ml/plant 10-~ M GAa, 0.1 ml/plant Seed extract: 150 cpm R- [14C]GA3/plant 1500 cpm R-[14C]GAs/plant 15000 cpm R-[laC] GAs/plant Enzymatic hydrolysis with cellulase: Control 10-6 M GA3, 0.1 ml/plant Seedling extract; hydrolysis 48 % : 130 cpm/plant 1300 epm/plant Seedpod extract; hydrolysis 56% : 250 cpm/plant 2500 cpm/plant 1st -k 2nd sheath (mm) 20.3 29.7 29.0 47.1 22.4 19.1 34.1 22.0 30.6 32.3 45.1 23.4 32.4 % of control 100 147 143 233 111 95 169 100 139 147 205 106 147

To investigate the possibility t h a t the R-[14C]GA3 is partly hydrolysed by the dwarf maize and in this manner exerts some biological activity, the treated plants were re-extracted at the end of the assay. Thin-layer chromatograms of extracts of both R-[I~C]GAa- and [14C]GA3-treated plants are given in Fig. 2. The R-[laC]GA3 extract shows a small peak of radioactivity cochromatographing with GA 8 which suggests t h a t some hydrolysis (approx. 4%) has taken

30

G.W.M. Barendse and G. J. M. de Klerk

dprn
1000

tOA3 =:
apptied

900
800

--~-~C-GA
--11+CGh3

3F

r-7

700 000 000 ~+00 300 200 100 0

I L-- I

0.1 0.2 0.30.L+ 05 0.0 0.7 0.8 0.9 1.0 I~f

-i

Fig. 2. Thin-layer chromatograms of extracts from dwarf (d5) maize after treatment with either [z4C]GAzor R-[i40]GA3
hypocotyi[ength increment Irnm)

12 -

VIOLET ......~=~
:,F"

.+

KIDACHI

11 - +.-.-.* 2500 cpm R-Y:C-GA3/ptant ./" 10 - o---o 12500 cpm . . . . 9 - , , - - * controt 0 76 /

....

13000 cpm lt=C-GA3]ml,=10-6H 0A3

- +-..-.+13000 cpm
- ~--.

R-l~c-6h3/m[
~...~

control

s.f j f S ~ j ' ~

/://"
2 1 0 ~.-." ~/.j,,o, " [~ft I 0 B I 2h 32 I

/../ // /"

/
/

/'
.]~
j"

/.
I /+8 [ 72 hr
8 2t, 32 G8 72 104 hr

Fig. 3. Growth curves of Pharbitis seedlings: (a) strain Violet treated with R-[I~C]GA3applied via the cotyledons, and (b) the dwarf strain Kidaehi in which an aqueous solution of R-[z4CJGAs was supplied to de-rooted seedlings

place. A rough calculation reveals that even a one percent hydrolysis would account for the biological activity observed with the 15000 cpm R-[I40]GA3 (see Table 3), since, for example the hydrolysed seedling extract of 130 cpm/plant, approximately equivalent with 65 cpm released GA3, results in a significant biological activity. These results indicate that I~-[z4C]GAa itself has no or only little biological activity. I n contrast, the opposite process also takes place, since the applied [itC]GAa is partly converted to R-[14C]GA3, approximately 17 % after 7 days which is slow compared to the conversion in Pharbitis. The 1~-[140]GAa preparation has been repeatedly applied in different coneentrations to Pharbitis seedlings of both the normal strain "Violet" and the dwarf strain " K i d a c h i " . Application took place either on the cotyledons or b y standing de-rooted seedlings on aqueous solutions of R-[z4C]GA3 . Particularly in

Metabolism of Applied GA Table 4=.Hydrolysis of R-[14C]GA3by seedlings of Pharbitis nil Fraction Solution at application Solution after 8 hours Roots Hypoeotyls Cotyledons Recovered (epm) 3050 1542 729 1048 1650 R-[I~C]GA3 (%) 99.1 93.4 91.7 98.1 99.9 [14C]GA3 (%) 0.9 5.6 8.3 1.9 0.1

31

the latter case damage often occurred in the seedlings at high concentrations due to impurities in the extract which also resulted in stunted growth. I n Fig. 3 an example is given whereby 2500 cpm and 12500 cpm of R-[14C]GA3 was supplied to the cotyledons of the strain "Violet" and a concentration of 13000 cpm [14C]GA3/ml (10 -8 M GAs), and 13000 cpm of R-[14C]GA~/ml was supplied in aqueous solution to de-rooted "Kidachi" seedlings. These and other experiments showed that high concentrations of R-[14C]GA3 are inhibitory or even damaging while low concentrations are generally without effect. The results presented in Fig. 3 suggest some growth promotion 48 h after application. These findings were, however, not reproducible. Nevertheless, the possibility has been investigated t h a t some of the applied R-[14C]GA~ was hydrolysed b y the Pharbitis seedlings by re-extracting treated seedlings after 8 hrs supply with R-[14C]GA3 (13000 cpm/ml). Since Sembdner (1972) reported t h a t roots m a y be the site where GA-glucosides are hydrolysed, roots as well as hypocotyls and cotyledons were extracted separately. After thin-layer chromatography the relative amounts R-[14C]GA3 and [14C]GA3 were determined. The results are summarized in Table 4. There appears to be no significant hydrolysis in the cotyledons and hypocotyls. However, roots as well as the surrounding solution suggest some hydrolysis which would support the findings of Sembdner (t972). I t can, however, not be said whether roots themselves or microorganisms on or in the roots are responsible for the hydrolysis. Metabolism o/R-[14C]GA8 in Developing and Germinating Seeds. The possible role of bound gibberellins (see Barendse et al., 1968) during germination of Phar. bitis has been reinvestigated with our R-[14C]GA8 preparation. Approximately 750 cpm/seed of R-[14C]GA3 was injected into the developing seeds 21 days after anthesis. Samples of seeds were harvested at 1.5 and 12 days after injection, and at m a t u r i t y and analysed for possible metabolism of the applied R-[14C]GA3. Mature seeds were imbibed at 27~ and subsequently germinated for 2 days in vermiculite at the same temperature. The extracts were chromatographed on thin layers developed in benzene/n-butanol/acetic acid (70:25:5, v/v) and the relative amounts of R-[14C]GAa and released [14C]GA 3 were determined. The results are compiled in Table 5. There is some hydrolysis in the developing seeds. However, this process has apparently been reversed towards m a t u r i t y of the seeds, since the latter do not contain significant amounts of [14C]GA3. There also appears some hydrolysis during imbibition, but this process is again reversed during subsequent germina-

32

G. W. ~. Barendse and G. J. M. de Klerk Table 5. Metabolism of R-[laC]GAa in developing and germinating seeds Stage at extraction 1 day after injection 5 days after injection 12 days after injection Mature seeds Imbibed seeds, 24 hrs 2 days after germination: cotyledons roots plus hypocotyls No. of seeds 13 15 12 8 10 12 Recovered (cpm) 1201 1802 1378 758 1791 1336 1141 195 R-[14C]GA3 [14C]GAa ( %) ( %) 97.6 95.7 94.2 99.5 90.6 98.2 98.9 94.2 2.4 4.3 5.8 0.5 9.4 1.8 1.1 5.8

tion. There is also some indication of hydrolysis in the roots (hypoeotyls were still very short at this stage) however, the number of counts is too small to be conclusive. The hydrolysis during imbibition is in accordance with previous results with applied [3H]GA1 (Barendse et al., 1968).

Distribution o] R-p4C ]GAs and [14C]GA~ in Seedlings. The distribution pattern of [14C]GA3 has been reported previously (Barendse, 1974). I t was found t h a t applied [14C]GA3 accumulated in the growing zones, e.g. the apical region of the hypocotyl and the cotyledons. I n the following experiment the uptake of both R-[14C]GA3 and [14C]GA~ was compared at three different concentrations in the cotyledons, the apical 1st, 2nd and 3rd cm sections of the hypoeotyl of Pharbitis seedlings. The radioactivity was determined after 8 hrs of supply. The results are presented in Fig. 4. I t appears t h a t R-[ltC]GA3 is strongly accumulated in the cotyledons, even more so t h a n [~4C]GA3. I n contrast with the [14C]GA3 it appears that R-[ltC]GA3 does not accumulate in the apical region of the hypocotyl, suggesting a different. behaviour of R-[14C]GA3 . Fig. 5 gives the distribution pattern of R-[ltC]GA3 in hypocotyls of seedlings with and without cotyledons as well as of decapitated seedlings. As we have seen in Fig. 4, intact seedlings do not accumulate radioactivity in the apical region of the hypoeotyl. Removal of the cotyledons results in strong accumulation of the upper 5 m m of the hypocotyl. However, there is no accumulation in the apical region of decapitated seedlings. Competition o/R-p4C]GA3 with Cold GA3. I n order to investigate the possibility t h a t R-GA 3 competes for different sites t h a n GA3, an experiment was carried out in which R-[14C]GA3 (13000 cpm/ml) and [14C]GAs (13000 cpm/ml, 10-6 M GAs) were supplied to seedlings with or without an additional saturating supply of cold GA s (10-4 M) for 8 h. The results are compiled in Table 6. I t appears t h a t the accumulation of [I~C]GA3 in the cotyledons is hardly affected b y the addition of 1O-4 M cold GA3, the latter does, however, lower the uptake of [~4C]GAs b y the hypocotyls, which indicates saturation of GA~ in this tissue. The results with R-[14C]GA3 are somewhat less clear, probably due to the different distribution pattern of R-[14C]GAa compared with [14C]GAa. There even

Metabolism of Applied GA
d pm/gr 80.000 70000 60.000 50.000 /4.000 30.000 20.000 10.000 / 0 / ~ ; /// R-lZ~c-oA3 14C-GA3 / / ~ cotytedons /, cotytedons

33

///
//'" j , ~

/I I

////i//////I
/1 ~

hypocoty[:l

st

cm

//,/"
)z
/~"'/..S/J E E C L

~JJ-..
_ ~ i ' - ' : " ~

_j.

hypocotyL:2 nd cm hypocoty[: 3 rd cm

////
. ..'hypocotyt:2 cm
hypocotyt: 1 st cm ".. hypocotyt: 3 rd cm d p m/rot

/ /f-;/j---~_S j'"
18.000

36.000

5&.O00

Fig. 4. Uptake of [!40]GA3 and R-[14C]GA3 at 3 concentrations by the cotyledons and the apical 1st, 2nd and 3rd cm of the hypocotyls after 8 hrs supply
dpm 1,50 400 | I

R-I~c-6A3 distribution .......... with cotyledons -without coty[edons --dec2pilated

350 300 250 208

150 100 .......

50

'1

I----1
i ,

=-i

.-J

L 7--...=__~=q

5 10 15 20 25 30 35 ~0 z,5 50 rnmfrom apex

Fig. 5. The distribution of ]{-[I~C]GA~ in the hypocotyls of intact seedlings, of seedlings without cotyledons, and of seedlings decapitated 2 cm below the apex Table 6. Competition of [14CJGA3 and 1R-[14C]GAa with cold GA 3. Radioactivity after 8 h in dpm/g fresh weight Treatment Cotyledons tgypocotyl 1st cm [I~CJGAa E14CJGAa4-GAa R-[14CJGAa R-[I~CJGA~+GA3 a Percentage 3 Planta (Berl.) 7114 (100) a 6678 (94) 6429 (100) 8320(129) 3509 1412 519 408 (100) (40) (100) (78) 2nd cm 1607 540 246 305 (100) (34) (100) (124) 3rd cm 1011 435 253 165 (100) (43) (100) (65) Total 6127 2387 1018 878 (100) (39) (106) (80)

34

G.W.M. Barendse and G. J. M. de Klerk

appears some stimulation of R-[14C]GAa in the presence of cold GAa, whereas the overall picture in the hypocotyls reveals only a small reduction in R-[14C]GAa uptake. Discussion The fact that only one new radioactive zone appears after thin-layer chromatography, in several developing solvents, of extracts from Pharbitis seedlings treated with [14C]GAs indicates that GA s is converted to one single metabolite (R[14C]GAs). The chromatographic properties, on both thin layer and column, are in close agreement with those of authentic GAs-glucoside. Quite effective enzymatic hydrolysis of R-[14C]GAs has been obtained with cellulase and fl-glucuronidase (from Helix pomatia), while fi-glucosidase (from almonds) appeared to be ineffective. Sembdner (personal communication) was unable to obtain significant hydrolysis of GAs-glucoside with cellulase, while Yokota et al. (1969, 1970) also reported t h a t GAs-glucoside resists enzymatic hydrolysis, particularly hydrolysis with /~-glucosidase. Possibly the source of the enzyme is of importance for its effectiveness with regard to hydrolysis of GAs-glucoside. Furthermore the experiments with enzymatic hydrolysis were not straightforward. The l~-[14C]GAs preparation used possesses little or no biological activity which is in agreement with the findings of T a m u r a et al. (1968) and of Sembdner et al. (1972) for GA sglucoside. Together with the fact t h a t GAs-glucoside does occur endogenously in Pharbitis nil (Yokota eta/., 1969), there remains little doubt that the R-[14C] GA s represents GAs-glucoside, or at least a very closely related compound. Acid hydrolysis of 1~-[14C]GA s gives rise to three radioactive fractions e.g. nonhydrolysed R-[14C]GAs, [14CJGAs and a biologically inactive compound, possibly gibberic acid. The latter compound is due to the acidic conditions to which GA s is very sensitive. Thus during acid hydrolysis a part of the released GA s is inactivated as far as biological activity is concerned. These findings relate to a certain extent with a previous report (Barendse and Lang, 1972) in which acid hydrolysis with 0.4 N HC1 for 60 min at 60~ was used as a standard procedure for determining the amount of so-called bound gibberellins. A significant part of these bound gibberellins must have consisted of GAs-glucoside. First of all the acid hydrolysis must have been incomplete under the conditions used, while at the same time a part of the released GA s must have been inactivated. Thus the actual amounts of bound gibberellins measured in those experiments must have been much higher. However, since a particular acid hydrolysis yields straightforward and reproducible results, the observed relative differences remain valid and do not affect the general conclusions drawn. Since l~-[14C]GAs itself has no significant biological activity in Pharbitis seedlings, while the small activity observed in the dwarf maize assay could be attributed to endogenous hydrolysis, the conversion of [14C]GAa to R-[14C]GAs can be considered as an inactivation of the hormone. Having tentatively identified the R-[14C]GAa as GAs-glucoside, it was decided to reinvestigate the possible role of this metabolite during germination of Pharbitis. I n accordance with previous findings (Barendse et al., 1968) it was found t h a t some hydrolysis of 1~-[14C]GA 8 occurs during the imbibition of the seeds. However, this process is again reversed during the subsequent germination. Whether such

Metabolism of Applied GA

35

a hydrolysis is f u n c t i o n a l or merely a consequence of increased hydrolitic a c t i v i t y d u r i n g the i m b i b i t i o n r e m a i n s a n open question. Since moist rapid growth takes place between a p p r o x i m a t e l y three a n d six days after g e r m i n a t i o n . The d i s t r i b u t i o n of applied R-[14C]GA8 is different from t h a t of [14C]GA3. 1~-[14C]GA a is strongly a c c u m u l a t e d in the cotyledons, even more so t h a n [laC] GAa, while i n c o n t r a s t with [14C]GAs the R@4C]GA3 is n o t a c c u m u l a t e d b y the apical zone of the growing hypocotyl, unless the cotyledons are removed. I n addition, a competition experiment, whereby [14C]GAa as well as R-[~4C]GAa were applied together with s a t u r a t i n g concentrations of cold GA~, revealed t h a t these comp o u n d s do n o t compete for the same sites. To sum up the d a t a so far o b t a i n e d suggest t h a t R-[laC]GAa, i.e. GAa-glucoside, represents a biologically inactive endp r o d u c t of the p a t h w a y which leads to the biologically very active GA 3. The authors are much indebted to Dr. G. Sembdner for the generous gift of authentic GAa-glueosyl ester and for the very fl'uitfull discussions with Barendse. The authors also gratefully acknowledge the gift of authentic GA3-glueoside from Dr. N. Takahashi.

References
Barendse, G. W. 2~[.: Formation of bound gibberellins in Pharbitis nil. Planta (Berl.) 99, 290-301 (1971) Barendse, G. W. M. : Accumulation and metabolism of radioactive gibbercllic acid in seedlings of Pharbitis nil Chois. Proc. 8th Intern. Conf. Plant Growth Substances, Tokyo, 1973, p. 332-341. Hirokawa Publ. Cy., Inc. Tokyo 1974 Barendse, G. W. M., Kende, H., Lang, A. : Fate of radioactive gibberellin A1 in maturing and germinating seeds of peas and Japanese morning glory. Plant Physiol. 43, 815-822 (1968) Barendse, G. W. 1 . , Lang, A. : Comparison of endogenous gibberellins and of fate of applied radioactive gibberellin A1 in a normal and dwarf strain of Japanese morning glory. Plant Physiol. 49, 836-841 (1972) Cross, B.E., Grove, J . F . , MacMillan, J., Mulholland, T. P. C.: Gibberellic acid. Part VII. The structure of gibberellic acid. J. chem. Soc. (Lond.) 2520-2536 (1958) Kende, H., Lang, A. : Gibberellins and light inhibition of stem growth in peas. Plant Physiol. 89, 435M40 (1964) Sembdner, G., Weiland, J., Schneider, G., Schreiber, K., Focke, J. : Recent advances in the metabolism of gibberellins. In: Plant growth substances 1970. Proc. 7th Intern. Conf. Plant Growth Substances, Canberra, Australia. D. J. Carr (ed.), p. 143-150. Ber]imHeidelberg-New York: Springer 1972 Tamura, S., Takahashi, N., Murofushi, N., Yokota, T., Kato, J. : Isolation of new gibbercilins from higher plants and their biological activity. In: Biochemistry and physiology of plant growth substances. Proc. 6th Intern. Conf. Plant Growth Substances, Carleton University, Ottawa 1967. Wightm-m F. and Settcrfield, G. (eds.), p. 85-99. Ottawa, Canada: l~unge Press Ltd. 1968 Yokota, T., Murofushi, N., Takahashi, N. : Structure of a new gibberelIin glucoside in immature seeds of Pharbitis nil. Tetrahedron Letters No. 18, 1489-1492 (1970) Yokota, T., Takahashi, N., Murofushi, N., Tamura., S. : Structures of new gibberellinglucosides in immature seeds of Pharbitis nil. Tetrahedron Letters No. 21, 2081-2084 (1969)