metabolism of ga3

8/13/2019 metabolism of GA3

1/11

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

The Metabolism of Applied Gibberellic Acidin harbiti s nil Choisy:

Tentative Identification of Its Sole Metaboliteas 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 . d e K l e r kBotan y Depar tmen t , Universi ty of Ni jmegen, Toernooive ld, The Nether lands

Received 4 Ap ril ; accepted 30 M ay 1975S u m m a r y . Rad ioac tive gibberellic acid ([14CJGA3) app lied to seedlings of Pharbi t is n i lstrain Violet is converted to one single metaboli te (R-[nC]GAa), which has been tentativelyiden tified as GA3-glucoside. A s wit h auth enti c GA3-glucoside, R-[zdC]GA3 can be h ydr olys edto some ex tent with cellulase and fl-glucuronidase, but har dly at a l l with fl-glueosidase. Acidhydrolysis, which is more effective than enzym atic hydrolysis, yielded G A3 as well as a b io-logical inactive compound. The la t ter represen ts a degrada tion prod uct of GA3 due to the sen-si t iv i ty of GA3 to acidic condit ions.The R-[zdC]GA3, like a uth enti c GAa-glucoside, possesses litt le or n o biological act ivit y.R-[zaC]GAa applied to developing seeds is part l y hyd rolyse d during imbib it ion of the matu reseed bu t is, however, reconv erted to R-[14C]GA3 during subsequent germination. A ppliedR-[14C]GA8 is stro ngly accumu lated in t he cotyledons of Pharbi t is seedlings, to a greaterexte nt tha n [zdC]GA3. However, unlike [14C]GA3 it is not accum ulated in th e apical regionsof the hypocotyl . Furth erm ore no competit ion was observed between R-[zdC]GA3 and [z~C]GAa,which suggests tha t they do not compete for the same si tes .

ntroductionI t h a s b e e n s h o w n p r e v i o u s l y ( B a r e n d s e , 1 97 3) t h a t a p p l i e d [14CJG Aa a c c u m u -

l a t e s i n t h e g r o w i n g p l a n t p a r t s , e.g. t h e a p i c a l r e g i on o f t h e h y p o c o t y l a n d t h ec o t y l e d o n s o f P h a r b i t i s n i l s e ed l in g s . M o r e o v er , i t w as d e m o n s t r a t e d t h a t t h ea p p l i e d [14 CJG As i s v e r y r a p i d l y c o n v e r t e d t o o n e s i n g le c o m p o u n d , a G A 3 -c o n-j u g a t e w i t h l o w b i o l o g i c a l a c t i v i t y . T h e r a t e o f c o n v e r s i o n w a s h i g h e r i n l ig h t -g r o w n s e e dl i n gs c o m p a r e d w i t h d a r k - g r o w n o n e s. I t w a s i n d i c a t e d t h a t t h e l i g h t -i n h i b i t i o n o f g r o w t h i n P h a r b i t i s s e e d l i n g s c o u l d b e p a r t l y d u e t o t h e r a p i d c o n -v e r s i o n , i .e . i n a c t i v a t i o n , o f t h e h o r m o n e b y l i g h t. T h i s p a p e r d e a l s w i t h t h et 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 G A a - m e t a b o l i t e a n d d e s c ri b e s s o m e e x p e r i m e n t sa i m e d a t e l u c i d a t i n g t h e p o s s i b l e p h y s i o l o g i c a l s i g n i f ic a n c e o f t h i s m e t a b o l i t e .

Materials and MethodsPlant Mater ial . Seeds of Pharbi t is n i l Choisy , s t ra in "Viole t" or "Kid achi" , were t rea tedwith concentra ted su l fur ic ac id for 45 min , extensive ly r insed thoroughly wi th water and sub-sequently soak ed for 24 h. T he im bibed seeds were sown in vermiculi te and germ inated inthe d ark a t 27~ Four-da y-old seedl ings, hypocotyl length approxim ate ly 7 e ra, f rom whichthe roots w ere remo ved w ere placed in aqueous solutions of [14C]GA8 or i ts metabo li te R-

Abbreviations: GA8 = Gibberell ic acid, R-GA 3 = l~Ietabolite of G A8.


2/11

26 G.W. ~ . Barendse and G. J. 1~. de Klerk[~C]GA3, usual ly 5 ml of solution per 10 plants at a cone. of 10-6 M in the ligh t (approx.4000 lux at plant level). After treatment the cotyledons were removed and the hypocotylscut into 5 mm sections. The cotyledons as well as the hypoootyl sections, usually 5 sectionsof the corresponding regions, were pooled, weighed and subsequently added to Bray's scin-tillation fluid and left to stand at least 48 h before being counted.Radioact ive Gibberel lic Ac id. [laC]GA3 (8 methylene-[14C], )specific act iv ity 6.1 mCi/mmolwas purchased from the Radiochemical Centre, Amersham, England.Extract ion Procedures . The plant material was homogenized in methanol with a Sorvallomnimixer, filtered, evaporated to dryness and subsequently stored at --20~ for futureanalysis. 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 chromate-graphed 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 upin ample water and partitioned several times with ethyl acetate until the latter remainedclear, the ethyl acetate phase was discarded. The pH of the water phase was raised to pit 8and subsequently partitioned with n-butanol. The aqueous phase was discarded and thecombined butanol fraction was evaporated to dryness. Subsequently the residue was takenup in a mixture of methanol-acetone (1 : 1, v/v) and chromatographed on thin layers whichwere developed in chloroform/methanol/acetic acid/water (40:15: 3:2, v/v). The radioactivezone between Rf 0.4-0.6 was eluted 4 times wi th methanol and yielded a reasonably purefraction of R-[I~C]GAz.H y d r o l y s i s . Acid hydrolysis was carried out in culture tubes containing 1 ml of dilutedHC1 varying in normali ty and 1000 epm of R-[14C]GAs. The tubes were incubated in a water-bath for 1 or 2 hrs at different temperatures. After hydrolysis the tubes were quickly evapora-ted to dryness, immediately taken up in methanol and chromatographed on thin layers.Enzyma tic hydrolysis took place by adding the different enzymes in several concentrationsto 1 ml 0.1 M KH2PO ~ buffer with t he requi red pi t and 1500 cpm of R-[14C]GA3. Afte r in-cubation for 24 hrs at 37~ approximately 2 ml acetone was added. The tubes were sub-sequen tly centrifuged for 5 min a t 2200 g (pellet discarded, contained very lit tle radioacti-vity) and the supernatant evaporated to dryness. The residue was taken up in methanol andchromatographed on thin layer. The thin layers were developed in the above mentionedbenzene/butanol/acetic acid solvent. Scintillation counting was carried out by scraping tenequal zones of the chromatograms directly into the vials containing 10 ml Bray's solution.Column Chromatography . A DEAE-Sephadex A-25 column, bed vo lume 18 2.5 cm, wasstepwise 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 me-thanol -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.B i o a s s a y . Dwarf corn (dh) was sown in vermiculi te and germinated at 27~ After 7 daysseedlings were selected and transferred to boxes containing half-stregth Hoagland solutionby placing them with the roots through holes in the box covers. Four plants per treatmentreceived 0.1 ml of aqueous extract or standard cone of GAs per plant. The elongation of thefirst and second leaf sheath was measured after seven days. The biological activity was ex-pressed as percentage of increase or growth over the control (see also Kende and Lang, 1964).

e s u l t sC h r o m a t o g r a p h i c I d e n t i ] i c a t io n e l R G A 3. Since i t was suspected t hat the GA 3

me tabo l i t e R-GA3 represented GA3-glucoside, i t was ch roma togr aphed on thinlayers developed in several solvent systems with authentic 3-0-fl-D-glucopyrano-syl-GA3 (which was kindl y supplied b y Prof. N. Takahashi, Japa n) as reference.The results are given in Table 1. In a ll four solvent systems only one radio activezone was observed of which the Rf va lue of the maximum ac t iv i ty is g iven inTable 1 . These va lues correspond with the Rf va lues of the eoehromatographedaut hen ti c GAa-glucoside. In a personal discussion wit h Dr. G. Sem bdn er (Halle,


3/11

Metabolism of Applied GA 27c pm

0000

5000

~000

3000

2000

1000

0

Gh3 g[ucosy t es terI - H H - H I

I C - G A 3O h 3 _ g t u c a s i d e

R J ~ C . G A

L _ _ I~ I ~ I I . . . .I I I I I l8 0 1 6 0 2 L ~ O 3 2 0 / + 0 0 / + 8 0 rn li I [

0 , 2 ~ 1 7 6 0 , 2 5 0 . 5 0 0 . 7 5 1 . 0 0 3 , 0 0 A c e t ic c i dFig. 1. Column chromatography of [140]GA3,R-[14C]GA3, GA~-glucosylester, and GA3-gluco-side. The column contained DEAE-Sephadex A-25 and was stepwise eluted with 80 methanolplus increasing concentrations of acetic acid. GAa-glucosyl ester and GAa-glucoside weredetected on thin layer, developed in benzene/n-butanol/acetic acid (70:25:5, v/v), by sprayingwith an ethanolic solution of sulfuric acid. The [14C]GA3and l~-[14C]GAawere determined byliquid scintillation counting of the reduced eluantsTable 1. Thin-layer chromatography of R-[14C]GA3 in four different solvent systemsSolvent system v/v Rf max Rf GA -R-[zdC]GA3 gtucoside(1) Benzene/n-butanol/acetic acid(2) n-Butanol/acetic acid/water(3) Chloroform/ethyl acetate/acetic acid(4) Chloroform/methanol/acetic acid/water

70 : 25 : 5 0.2-0.3 0.23-0.3160 : 15 : 30 0.7-0.8 0.68-0 .8060: 40 : 5 0.0-0.1 0.00-0.0840:15:3:2 0.4-0.5 0.47-0 .5 3

DDR) it was suggested that the present R-[14C]GA3 preparation could also re-present GA3-glucosyl ester instead of GAa-glucoside and he proposed a column-chromatographic procedure to distinguish between these two possibilities. Whena DEA E-S eph adex A-25 column is stepwise eluted with 80 methanol plusincreasing cone. of aqueous acetic acid, GAa-glucosyl ester will be eluted beforeGAa and GAs-glueoside after GAz. Column chromatography has been carried outon several consecutive occasions with GAs, R-[14C]GA~, authentic GAa-glueosylester (kindly supplied by Dr. G. Sembdner) and authentic GAa-glucoside. Theresults are summarized in Fig. 1. The results show that most of the R-[I~C]GAais eluted after the [14C]GAa. However, rechr omatography on thin layer of theeluted R-[14C]GAa frac tion cochromatographing with the [14CJGAa revealed th ata major part of this fraction consisted of hydrolysed R-[14C]GAa, probably dueto the increasing acidity of the eluant, which explains the broad range of elutedradioactivity from R-[14C]GA3 as well as of authentic GAa-glucoside. It is clearth at the R-[14C]GAa does not represent GAa-glucosyl ester as ha d been suggested.


4/11


5/11

Metabolism of Applied GA 29s i d a s e w a s i n e f f e c t i v e w h i l e s o m e h y d r o l y s i s w i t h c e l l u l a s e c o u l d b e o b t a i n e d .H o w e v e r , S e m b d n e r a n d e o w o r k e r s (p e rs o n a l c o m m u n i c a t i o n ) w e re e v e n u n a b l et o o b t a i n s i g n i f i c a n t h y d r o l y s i s w i t h c e l l u l a s e w h e r e a s , i n a g r e e m e n t w i t h t h ep r e s e n t r e s u l ts , t h e y h a d a l so p r o b l e m s w i t h a c i d h y d r o l y s i s o f G A a -g lu e o si de .B i o l o g i c a l a c t i v i t y T h e a c t i v i t y o f b o t h R -[1 4C ]G A 8 a n d p a r t i a l l y h y d r o l y s e dR -[1 4C ]G A 3 w a s t e s t e d w i t h t h e d w a r f m a i z e (d 5 ) a s s a y . T h e r e s u l t s a r e s h o w ni n T a b l e 3 . S i n c e w i t h e x t r a c t i o n o f t h e R -[1 4C ]G A 8 f r o m p l a n t m a t e r i a l a l soe n d o g e n o u s G A a - g lu c o s id e s a r e e x t r a c t e d m e a n s t h a t t h e s p e c i fi c a c t i v i t y o f t h eR - G A s p r e p a r a t i o n i s n o t k n o w n . T h e r e f o r e , e x t r a c t s w e r e m a d e o f se e d li n g s,w h i c h a r e k n o w n t o c o n t a i n r e l a t i v e l y h i g h a m o u n t s o f b i o l o g i c a l l y a c t i v e f r a c -t io n s , a n d o f s e e d p o d s ( fr o m w h i c h i m m a t u r e s e ed s h a d b e e n r e m o v e d ) w h i chh a v e v e r y l i t tl e b i o l o g ic a l a c t i v i t y . T h i s i s c o n f i r m e d b y t h e r e s u l t s o f T a b l e 3 ,w h i c h s h o w t h a t h y d r o l y s i s o f s e e d li n g e x t r a c t s r e s u l t s i n a h i g h e r a c t i v i t y t h a nh y d r o l y s i s o f s e e d p o d e x t r a c t s . N o n - h y d r o l y s e d R - [ 1 4 C ] G A 3 f r o m s e e d l i n g s s h o wo n l y s o m e b io l o g ic a l a c t i v i t y a t h i g h c o n c e n t r a t i o n s . T h i s a c t i v i t y i s p o s s i b l y d u et o h y d r o l y s i s i n t h e m a i z e p l a n t s . T h u s R - [l aC ] G A 3 i ts e l f h a s l i t t l e o r n ob i o l o g i c a l a c t i v i t y w h i c h i s a g a i n i n a g r e e m e n t w i t h t h e f i n d i n g s o f T a m u r a e t a l( 1 9 6 8 ) a n d o f S e m b d n e r e t a l ( 1 9 7 2 ) f o r G A 3 - g l u c o s i d e .

Tab le 3. Biological act i vi ty of R-[14C] GAa in the (dS) dw arf ma ize assayTre atm ent 1s t -k 2nd sheath of control(mm)Control 20.3 10010 ~ M [1~C]G Aa, 1300 cpm /plan t 29.7 14710 6 M GA3, 0.1 m l/p lan t 29.0 14310 ~ M GA a, 0.1 m l/plan t 47.1 233Seed ext ra ct :150 c p m R [14C] GA 3/plant 22.4 1111500 cp m R-[14C] GA s/plant 19.1 9515000 cpm R-[laC] GA s/plant 34.1 169En zy m atic hydrolysis with cel lulase:Control 22.0 10010 6 M GA3, 0.1 m l/p lan t 30.6 139Seedling ex trac t ; hydrolysis 48 :130 cp m /pla nt 32.3 1471300 epm /plant 45.1 205Seedpod ex trac t ; hydrolysis 56 :250 cpm /plan t 23.4 1062500 cp m /pla nt 32.4 147

T o i n v e s t i g a t e t h e p o s s i b i l it y t h a t t h e R -[1 4C ]G A 3 is p a r t l y h y d r o l y s e d b y t h ed w a r f m a i z e a n d i n t h i s m a n n e r e x e r t s s o m e b io l og i ca l a c t i v it y , t h e t r e a t e d p l a n t sw e r e r e - e x t r a c t e d a t t h e e n d o f t h e a s s a y . T h i n - la y e r c h r o m a t o g r a m s o f e x t r a c t so f b o t h R - [ I~ C ] G A a - a n d [ 1 4 C ]G A 3 - t re a te d p l a n t s a r e g i v e n i n F i g . 2 .

T h e R - [l aC ] G A 3 e x t r a c t s h o w s a s m a l l p e a k o f r a d i o a c t i v i t y c o c h r o m a t o -g r a p h i n g w i t h G A 8 w h i c h s u g g e st s t h a t s o m e h y d r o l y s i s (a p p r o x . 4 ) h a s t a k e n


6/11


7/11

Metabolism of Applied GATa ble 4=. H yd ro lys is of R-[14C]GA3 b y seedlings of Pharbitis nil

31

F r a c ti o n R e co ve re d R - [ I ~ C ] G A 3 [1 4C ]G A 3(epm) ( ) ( )Solution at app licat ion 3050 99.1 0.9Solution afte r 8 hou rs 1542 93.4 5.6R oo ts 729 91.7 8.3H yp oeo tyls 1048 98.1 1.9Cotyledons 1650 99.9 0.1

t h e l a t t e r c a s e d a m a g e o f t e n o c c u r r e d i n t h e s e e d li n g s a t h ig h c o n c e n t r a t io n s d u et o i m p u r i t i e s i n t h e e x t r a c t w h i c h a ls o r e s u l t e d i n s t u n t e d g r o w t h .

I n F i g . 3 a n e x a m p l e is g i v e n w h e r e b y 2 5 0 0 c p m a n d 1 2 5 0 0 c p m o f R-[1 4C ]G A 3w a s s u p p l i e d t o t h e c o t y l e d o n s o f t h e s tr a i n V i o l e t a n d a c o n c e n t r a t i o n o f1 3 0 0 0 c p m [1 4 C] GA 3 /m l ( 1 0 - 8 M G A s ) , a n d 1 3 0 0 0 c p m o f R - [1 4 C ] G A ~ / m l w a s s u p -p l i e d i n a q u e o u s s o lu t i o n t o d e - r o o t e d K i d a c h i s e e d li n g s. T h e s e a n d o t h e r e x -p e r i m e n t s s h o w e d t h a t h i g h c o n c e n t r a t i o n s o f R-[1 4C ]G A 3 a r e i n h i b i t o r y o r e v e nd a m a g i n g w h i l e l o w c o n c e n t r a t i o n s a r e g e n e r a l l y w i t h o u t e f f e c t . T h e r e s u l t sp r e s e n t e d i n F i g . 3 s u g g e s t s o m e g r o w t h p r o m o t i o n 4 8 h a f t e r a p p l i c a t i o n . T h e s ef i n d in g s w e r e , h o w e v e r , n o t r e p r o d u c i b l e . N e v e r t h e l e s s , th e p o s s i b i l i ty h a s b e e ni n v e s t i g a t e d t h a t s o m e o f t h e a p p l i e d R -[1 4C ]G A ~ w a s h y d r o l y s e d b y t h e P h a r b i t i ss e e d l in g s b y r e - e x t r a c t i n g t r e a t e d s e e d l in g s a f t e r 8 h r s s u p p l y w i t h R -[1 4C ]G A 3( 1 30 0 0 c p m / m l ) . S i n c e S e m b d n e r ( 1 97 2) r e p o r t e d t h a t r o o t s m a y b e th e s i t ew h e r e G A - g lu c o s id e s a re h y d r o l y s e d , r o o t s a s w e l l a s h y p o c o t y l s a n d c o t y l e d o n sw e r e e x t r a c t e d s e p a r a t e ly . A f t e r t h i n - la y e r c h r o m a t o g r a p h y t h e r e l a t i v e a m o u n t sR -[1 4C ]G A 3 a n d [1 4C ]G A 3 w e r e d e t e r m i n e d . T h e r e s u l t s a r e s u m m a r i z e d i n T a b l e 4 .

T h e r e a p p e a r s t o b e n o s i g n i f i c a n t h y d r o l y s i s i n t h e c o t y l e d o n s a n d h y p o -c o t y l s. H o w e v e r , r o o t s a s w e l l a s t h e s u r r o u n d i n g s o l u ti o n s u g g e s t s o m e h y d r o l y s i sw h i c h w o u l d s u p p o r t t h e f i n d i n g s o f S e m b d n e r ( t 9 7 2 ) . I t c a n , h o w e v e r , n o t b es a i d w h e t h e r r o o t s t h e m s e l v e s o r m i c r o o r g a n i s m s o n o r i n t h e r o o t s a r e r e s p o n s ib l ef o r t h e h y d r o l y s i s .

M e t a b o l i s m o / R - [ 1 4 C ] G A 8 i n D e v e l o p i n g a n d G e r m i n a t i n g S e e d s . T h e p o s s i b l er o l e o f b o u n d g i b b e r e l l i n s ( s ee B a r e n d s e et al. 1 9 6 8 ) d u r i n g g e r m i n a t i o n o f P h a r .bi t is h a s b e e n r e i n v e s t i g a t e d w i t h o u r R-[1 4C ]G A 8 p r e p a r a t i o n . A p p r o x i m a t e l y7 5 0 c p m / s e e d o f R - [ 1 4 C ] G A 3 w a s i n j e c t e d i n t o t h e d e v e l o p i n g s e e d s 2 1 d a y s a f t e ra n t h e s i s . S a m p l e s o f s e e d s w e r e h a r v e s t e d a t 1 .5 a n d 1 2 d a y s a f t e r in j e c ti o n , a n da t m a t u r i t y a n d a n a l y s e d f o r p o s si b le m e t a b o l i s m o f th e a p p l i e d R -[1 4C ]G A 3.M a t u r e s e ed s w e r e i m b i b e d a t 2 7 ~ a n d s u b s e q u e n t l y g e r m i n a t e d f o r 2 d a y s i nv e r m i c u l i t e a t t h e s a m e t e m p e r a t u r e . T h e e x t r a c t s w e r e c h r o m a t o g r a p h e d o nt h i n la y e r s d e v e l o p e d i n b e n z e n e / n - b u t a n o l / a c e t i c a c i d ( 7 0 : 2 5 : 5 , v / v ) a n d t h er e l a t i v e a m o u n t s o f R -[ 14 C ]G A a a n d r e l e a s e d [1 4C ]G A 3 w e r e d e t e r m i n e d . T h e r e s u l tsa r e c o m p i l e d i n T a b l e 5 .

T h e r e i s s o m e h y d r o l y s i s i n t h e d e v e l o p i n g s e e d s . H o w e v e r , t h i s p r o c e s s h a sa p p a r e n t l y b e e n r e v e r s e d t o w a r d s m a t u r i t y o f t h e s e e ds , si nc e th e l a t t e r d o n o tc o n t a i n s i g n i fi c a n t a m o u n t s o f [ 14 C]G A 3. T h e r e a l s o a p p e a r s s o m e h y d r o l y s i sd u r i n g i m b i b i t i o n , b u t t h i s p r o c e s s i s a g a i n r e v e r s e d d u r i n g s u b s e q u e n t g e r m i n a -


8/11

32 G. W. ~ . Barendse and G. J. M. de Kle rkTab le 5. Metabolism of R-[laC]GAa in developing an d g ermin ating seeds

Stage at extr act ion No. of Rec overed R-[14C]GA3 [14C]GAaseeds (cpm) ( ) ( )1 da y afte r injection 13 1201 97.6 2.45 da ys aft er injection 15 1802 95.7 4.312 day s afte r injection 12 1378 94.2 5.8Ma ture seeds 8 758 99.5 0.5Im bi be d seeds, 24 hrs 10 1791 90.6 9.42 day s afte r germ inatio n: 12 1336 98.2 1.8coty ledo ns 1141 98.9 1.1roots plushyp oco tyls 195 94.2 5.8

t i o n . T h e r e i s a l s o s o m e in d i ca t i o n o f h y d r o ly s i s i n t h e r o o t s ( h y p o eo ty l s w er es t i l l v e r y s h o r t a t t h i s s t a g e ) h o w e v e r , t h e n u m b e r o f c o u n t s i s t o o s m a l l t o b eco n c lu s iv e . T h e h y d r o ly s i s d u r in g im b ib i t i o n i s i n acco r d an ce w i th p r ev io u s r e -s u l t s w i th ap p l i ed [ 3 H ] G A 1 ( Bar en d s e et al. 1968).

Distribution o] R- p4 C ]GAs and [14C GA~ in Seedlings. T h e d i s t r i b u t i o n p a t t e r nof [14C]GA3 has bee n r epo r ted p re v io us ly (Barendse , 1974) . I t was found t ha tapp l ied [14C]GA3 ac cu m ula ted in the g rowing zones , e .g . the ap ica l r eg ion o f theh y p o c o t y l a n d t h e c o t y l e d o n s . I n t h e f o l l o w i n g e x p e r i m e n t t h e u p t a k e o f b o t hR- [14C]GA3 an d [14C]GA~ was co m pa red a t th ree d i f f e ren t con cen t ra t ion s in th eco ty l ed o n s , t h e ap i ca l 1 s t , 2 n d a n d 3 r d cm s ec t i o n s o f t h e h y p o eo ty l o f Pharbitiss eedl i ng s . T h e r ad io ac t i v i t y w as d e t e r m in ed a f t e r 8 h r s o f s u p p ly . T h e r e s u l t s a r ep r e s en t ed i n F ig . 4 .

I t a p p e a r s t h a t R - [ lt C ]G A 3 i s s t r o n g l y a c c u m u l a t e d i n t h e c o t y le d o n s, e v e nm ore so tha n [~4C]GA3. In co n t ra s t w i th t he [14C]GA3 i t ap pe ar s t ha t R- [ l tC]GA3d o es n o t accu m u la t e i n t h e ap i ca l reg io n o f t h e h y p o co ty l , s u g g es t in g a d i f f e r e n t .b eh av io u r o f R -[ 14 C]G A 3 F ig . 5 g iv es t h e d i s t r i b u t i o n p a t t e r n o f R-[ l tC ] G A 3 inh y p o c o t y l s o f s e e dl in g s w i t h a n d w i t h o u t c o t y l e d o n s a s w e l l a s o f d e c a p i t a t e dseedl ings .A s w e h av e s een in F ig . 4 , i n t a c t s eed li n g s d o n o t accu m u la t e r ad io ac t i v i t y i nt h e a p i c a l re g i o n o f t h e h y p o e o t y l . R e m o v a l o f t h e c o t y l e d on s r e s u lt s i n s t r o n ga c c u m u l a t i o n o f t h e u p p e r 5 m m o f t h e h y p o c o t y l . H o w e v e r , t h e r e i s n o a c c u m u l a -t i o n i n t h e ap i ca l r eg io n o f d ecap i t a t ed s eed l i n g s .

Competition o /R-p 4C ]G A3 with Cold GA3. I n o r d e r t o i n v es t i g a t e t h e p o s s ib i l i t yth a t R - G A 3 co m p e te s f o r d i f f e r en t s i te s t h an G A3, an ex p e r im e n t w as ca r r i ed o u tin whic h R-[14C]GA3 (13000 cpm /m l) an d [14C]GAs (13000 cpm /ml , 10-6 M GAs)w er e s u p p l i ed t o s eed l i n g s w i th o r w i th o u t an ad d i t i o n a l s a tu r a t i n g s u p p ly o fco ld G A s (10-4 M) fo r 8 h . T he r esu l t s a re com pi led in T ab l e 6 .

I t a p p e a r s t h a t t h e a c c u m u l a t i o n o f [ I ~ C ] G A3 in t h e co ty l ed o n s i s h a r d l yaf fec te d by th e add i t ion o f 1O 4 M co ld GA3, the la t t e r does, howe ver , lowe r theu p t ak e o f [~4C]G As b y t h e h y p o co ty l s , w h ich i n d i ca t e s s a tu r a t i o n o f GA ~ in t h i st i s sue . The r esu l t s w i th R- [14C]GA3 are som ew hat l ess c lear , p r ob ab ly due to thed i f f eren t d i s t r ibu t ion pa t te rn o f R- [14C]GAa co mp are d wi th [14C]GAa. The re ev en


9/11

M e t a b o l i sm o f A p p l i e d G A 3 3

d p m / g r80.0007000060.00050.000/4.00030.000

20.00010.000

/ , co ty tedonsR-lZ~c-oA3 /14C-GA3 / ~ co tyt ed on s

i II I s t/ 1 ~ hyp oco ty [ : l cm

/ / / / / ' _ j . hypocotyL:2 nd cm/ / , / ~ J J - . . hypo coty[: 3 rd cmz / / / /

/ / ~ ' / . . S / J j . . . 'hypocotyt:2 cm/ /f-;/j---~_S ' _ ~ i ' - ' : - hypo cotyt: 1 st cm/ ~ ; E E C L , ~ ~ .. hypoco tyt: 3 rd cm

0 18.000 36.000 5 .O00 d p m /rotF i g . 4 . U p t a k e o f [ 4 0] G A3 a n d R - [1 4C ]G A 3 a t 3 c o n c e n t r a t i o n s b y t h e c o t y l e d o n s a n d t h ea p i c a l 1 s t, 2 n d a n d 3 r d c m o f t h e h y p o c o t y l s a f t e r 8 h r s s u p p l y

d p m1 5 0 R - I ~ c - 6 A 3 d i s t r i b u t io n. .. .. .. .. . w i t h c o t y l e d o n s- I - - w i t h o u t c o t y [ e d o n s4 | - - - d e c 2 p i l a t e d1350 -300250208150 I - - - - 1100 ....... i5 = i . J L 7 . . .=__~=q

5 10 15 20 25 30 35 ~0 z,5 50rnm from apexF i g . 5 . T h e d i s t r i b u t i o n o f ]{ -[ I~ C ]G A ~ i n t h e h y p o c o t y l s o f i n t a c t s e e d l i n g s , o f s e e d l i n g sw i t h o u t c o t y le d o n s , a n d o f s e ed l in g s d e c a p i t a t e d 2 c m b e l o w t h e a p e x

T a b l e 6 . C o m p e t i t i o n o f [ 1 4C JG A 3 a n d 1 R -[1 4C ]G A a w i t h c o l d G A 3. R a d i o a c t i v i t y a f t e r 8 hi n d p m / g f r e s h w e i g h tT r e a t m e n t C o t y l e d o n s t g y p o c o t y l

1 st c m 2 n d c m 3 r d c m T o t a l[ I~CJGAa 71 14 (100) a 35 09 (100) 160 7 (100) 1011 (100) 612 7 (100)E14CJGAa4-GAa 66 78 (94) 141 2 (40) 540 (34) 435 (43) 23 87 (39)R-[14CJGAa 64 29 (100) 519 (100) 246 (100) 253 (100) 101 8 (106)R- [ I~ CJ G A ~+ G A 3 83 20 (12 9) 408 (78 ) 305 (124) 165 (65 ) 878 (80)a P e r c e n t a g e3 Planta (Berl.)


10/11

34 G .W .M . Barendse and G. J . M . de Kle rka p p e a r s s o m e s t i m u l a t i o n o f R - [1 4 C ]G A a i n t h e p r e s e n c e o f c o l d G Aa , w h e r e a s t h eo v e r a l l p i c tu r e i n t h e h y p o c o t y l s r e v e a l s o n l y a s m a l l r e d u c t i o n i n R -[1 4 C] G A au p t a k e .

i s c u s s i o nT h e f a c t t h a t o n l y o n e n e w r a d i o a c t i v e z o n e a p p e a r s a f t e r t h in - l a y e r c h r o m a t o -

g r a p h y , in s e v e r a l d e v e l o p i n g s o l v e n t s , o f e x t r a c t s f r o m P h a r b i t i s s e e d l i n g s t r e a t -e d w i t h [ 14 C]G A s i n d i c a t e s t h a t G A s i s c o n v e r t e d to o n e s in g l e m e t a b o l i t e R -[ 1 4 C ] G A s ) . T h e c h r o m a t o g r a p h i c p r o p e r t i e s , o n b o t h t h i n l a y e r a n d c o l u m n , a r ei n c lo s e a g r e e m e n t w i t h t h o s e o f a u t h e n t i c G A s - g lu c o si d e. Q u i t e e f f e c ti v e e n z y -m a t i c h y d r o l y s i s o f R - [1 4 C ]G A s h a s b e e n o b t a i n e d w i t h c e l l u l a se a n d f l -g l u c u r o n i -d a s e f r o m H e l i x p o m a t i a , w h i l e f i- g lu c o s id a s e f r o m a l m o n d s ) a p p e a r e d t o b ei n e f fe c t iv e . S e m b d n e r p e r s o n a l c o m m u n i c a t i o n ) w a s u n a b l e t o o b t a i n s i g n i fi c a n th y d r o l y s i s o f G A s - g l u c o s id e w i t h c e l lu l a s e, w h i l e Y o k o t a et al . 1969, 1970) a l sor e p o r t e d t h a t G A s - g l u co s id e r e s is t s e n z y m a t i c h y d r o l y s is , p a r t i c u l a r l y h y d r o l y s i sw i t h / ~ -g lu c o si da se . P o s s i b l y t h e s o u r c e o f t h e e n z y m e is o f i m p o r t a n c e f o r i tse f f e c ti v e n e s s w i t h r e g a r d t o h y d r o l y s i s o f G A s -g l u co s id e . F u r t h e r m o r e t h e e x p e r i-m e n t s w i t h e n z y m a t i c h y d r o l y s i s w e r e n o t s t r a i g h t f o r w a r d . T h e l ~ - [ 1 4 C ] G A s p r e -p a r a t i o n u s e d p o s s e s s e s l i t t l e o r n o b i o l o g i c a l a c t i v i t y w h i c h i s i n a g r e e m e n tw i t h t h e f i n di n g s o f T a m u r a et al . 1 9 6 8 ) a n d o f S e m b d n e r et al . 1972) fo r GA s-g l u c o s i d e . T o g e t h e r w i t h t h e f a c t t h a t G A s - g l u c o s i d e d o e s o c c u r e n d o g e n o u s l yin P h a r b i t i s n i l Y o k o t a e t a / . , 1 9 69 ), t h e r e r e m a i n s l i t t l e d o u b t t h a t t h e R -[1 4C ] G A sr e p r e s e n t s G A s - g lu c o s id e , o r a t l e a s t a v e r y c l o s e l y r e l a t e d c o m p o u n d .A c id hy dr o l ys i s o f 1~-[14C]G A s g i v e s r i se t o t h r e e r a d i o a c t i v e f r a c t i o n s e .g . n o n -h y d r o l y s e d R - [1 4 C ]G A s , [1 4C JG A s a n d a b i o l o g i c a l l y i n a c t i v e c o m p o u n d , p o s s i b l yg i b b e r i c a c i d . T h e l a t t e r c o m p o u n d i s d u e t o t h e a c i d i c c o n d i t i o n s t o w h i c h G A si s v e r y s e n s it iv e . T h u s d u r i n g a c i d h y d r o l y s i s a p a r t o f t h e r e l e a s e d G A s i s in-a c t i v a t e d a s f a r a s b io l o g ic a l a c t i v i t y i s co n c e r n e d . T h e s e f in d i n g s r e l a t e t o a c e r t a i ne x t e n t w i t h a p r e v i o u s r e p o r t B a r e n d s e a n d L a n g , 1 9 72 ) i n w h i c h a c i d h y d r o l y s i sw i t h 0 . 4 N H C 1 f o r 6 0 m i n a t 6 0 ~ w a s u s e d as a s t a n d a r d p r o c e d u r e fo r d e t e r m i n -i n g t h e a m o u n t o f s o - c a ll e d b o u n d g i b b e re l li n s . A s i g n if i c a n t p a r t o f th e s e b o u n dg i b b e r e ll in s m u s t h a v e c o n s i s te d o f G A s - g lu c o si d e. F i r s t o f a l l t h e a c i d h y d r o l y s i sm u s t h a v e b e e n i n c o m p l e t e u n d e r t h e c o n d it io n s u s e d , w h il e a t th e s a m e t i m e ap a r t o f th e r e l e a se d G A s m u s t h a v e b e e n in a c t i v a t e d . T h u s t h e a c t u a l a m o u n t s o fb o u n d g i b b e r e l l i n s m e a s u r e d i n t h o s e e x p e r i m e n t s m u s t h a v e b e e n m u c h h i g h e r .H o w e v e r , s i n c e a p a r t i c u l a r a c i d h y d r o l y s i s y i e l d s s t r a i g h t f o r w a r d a n d r e p r o -d u c i b l e r e su l ts , t h e o b s e r v e d r e l a t iv e d i f f e re n c e s r e m a i n v a l i d a n d d o n o t a f f e c tt h e g e n e r a l c o n c l u s i o n s d r a w n .

S i n c e l~ -[ 14 C ]G A s i t s e l f h a s n o s i g n i f i c a n t b i o l o g i c a l a c t i v i t y i n P h a r b i t i ss e ed l in g s , w h i le t h e s m a l l a c t i v i t y o b s e r v e d in t h e d w a r f m a i z e a s s a y c o u l d b ea t t r i b u t e d t o e n d o g e n o u s h y d r o l y s i s , t h e c o n v e r s i o n o f [1 4C ]G A a t o R - [1 4 C ]G A sc a n b e c o n s i d e r e d a s a n i n a c t i v a t i o n o f t h e h o r m o n e .

H a v i n g t e n t a t i v e l y i d e n t if i e d t h e R -[ 14 C ]G A a a s G A s - g lu c o s id e , it w a s d e c i d e dt o r e i n v e s t i g a t e t h e p o s s ib l e r o le o f t h i s m e t a b o l i t e d u r i n g g e r m i n a t i o n o f P h a r b i t i s .I n a c c o r d a n c e w i t h p r e v io u s f in d i n g s B a r e n d s e et al . , 1 9 6 8 ) i t w a s f o u n d t h a tsome hydro lys i s o f 1~ - [14C]G A 8 o c c u r s d u r i n g t h e i m b i b i t i o n o f t h e s e ed s . H o w e v e r ,t h is p r o c e ss is a g a in r e v e r s e d d u r i n g t h e s u b s e q u e n t g e r m i n a t io n . W h e t h e r s u c h


11/11

Metabolism of Applied GA 35a hydrolysis is funct ional or merely a consequence of increased hydroli t ic ac tivit yduring the imb ibit ion remains a n open question. Since moist rapid growth takesplace between approximately three and six days after germination.

The di str ib ut io n of appl ied R-[14C]GA8 is differ ent fro m t ha t of [14C]GA3.1~-[14C] GAa is strong ly ac cum ula ted in the cotyledons, e ven more so tha n [laC] GAa,while in contrast with [14C]GAs the R@4C]GA3 is not accumulated by the apicalzone of the growing hypocotyl , unless the cotyledons are removed. In addit ion,a co mpe tit ion ex per ime nt, whe reb y [14C]GAa as well as R-[~4C]GAa were a ppli edtogether with satu rati ng concent rations of cold GA~, revealed tha t these com-pounds do not compete for the same si tes . To sum up the data so far obtainedsuggest tha t R-[laC]GAa, i e GAa-glucoside represent s a biologically inac tive end-product of the p ath way which leads to the biologically very active GA3.

The authors are much indebted to Dr. G. Sembdner for the generous gift of authenticGAa-glueosyl ester and for the very fl uitfull discussions with Barendse. The authors alsogratefully acknowledge the gift of authentic GA3-glueoside from Dr. N. Takahashi.

e f e r e n c e sBarendse, G. W. 2~[.: Formation of bound gibberellins in P h a r b i t i s n i l Planta (Berl.) 99,290-301 (1971)Barendse, G. W. M. : Accumulation and metabolism of radioactive gibbercllic acid in seedlings

o f P h a r b i t i s n i l Chois. Proc. 8th Intern. Conf. Plant Growth Substances, Tokyo, 1973,p. 332-341. Hirokawa Publ. Cy., Inc. Tokyo 1974Barendse, G. W. M., Kende , H., Lang, A. : Fate of radioactive gibberellin A 1 in matur ing andgerminating 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 appliedradioactive gibberellin A1 in a normal and dwarf strain of Japanese morning glory. PlantPhysiol. 49, 836-841 (1972)Cross, B.E ., Grove, J .F . , MacMillan, J., Mulholland, T. P. C.: Gibberellic acid. Pa rt VII.The s tructure of gibberellic acid. J. chem. Soc. (Lond.) 2520-2536 (1958)Kende, H. , Lang, A. : Gibberellins and light inhibi tion of stem growth in peas. P lant Physiol.89, 435M40 (1964)Sembdner, G., Weiland, J., Schneider, G., Schreiber, K., Focke, J. : Recent advances in themetabolism of gibberellins. In: Pla nt growth substances 1970. Proc. 7th Intern . Conf.Plant Growth Substances, Canberra, Australia. D. J. Carr (ed.), p. 143-150. Ber]imHeidel-berg-New York: Springer 1972Tamura, S., Takahashi, N., Murofushi, N., Yokota, T., Kato, J. : Isolation of new gibbercilinsfrom higher plants and their biological activity. In: Biochemistry and physiology ofpla nt growth substances. Proc. 6th Intern . Conf. Pla nt Growth Substances, Carleton Uni-versi ty, Ottawa 1967. Wightm-m F. and Settcrfield, G. (eds.), p. 85-99. Ottawa, Canada:l~unge Press Ltd. 1968Yokota, T., Murofushi, N., Takahashi, N. : Structure of a new gibberelIin glucoside in im-mature seeds of P h a r b i t i s n i l Tetrahedron Letters No. 18, 1489-1492 (1970)Yokota, T., Takahashi, N., Murofushi, N., Tamura ., S. : Structures of new gibberell inglucosidesin immature seeds of P h a r b i t i s n i l Tetrahedron Letters No. 21, 2081-2084 (1969)

metabolism of ga3

Documents