Planta

Planta 142, 37-40 (1978)

9 by Springer-Verlag 1978

Total Protein Release from Barley Aleurone Layers as a Bioassay for Gibberellins S. Mapelli 1 a n d A . M . R a n i e r i 2 C.N.R., Laboratorio Biosintesi Vegetali, and 2 Istituto Chimica Agraria, Universit~tdi Milano, Via Celoria 2, 1-20133 Milano, Italy

Abstract. The effect of gibberellic acid o n the secretion of proteins f r o m barley ( H o r d e u m vulgare L.) a l e u r o n e layers has been investigated for its suitability as a gibberellin bioassay. C o n c e n t r a t i o n s from 10 4 gg/ml to 100 i_tg/ml of GA3 resulted in the release of p r o p o r tionally increasing a m o u n t s of total protein. The release of proteins is n o t affected by indoleacetic acid a n d kinetin. This m e t h o d has been applied a n d comp a r e d with the a-amylase assay for the e s t i m a t i o n of gibberellin in extracts of t o m a t o fruits a n d maize seedlings.

Key words: A l e u r o n e -

Gibberellin -

Hordeum

c o n c e n t r a t i o n . The possibility that the d e t e r m i n a t i o n of the total p r o t e i n released could be used as a bioassay for gibberellin was p o s t u l a t e d by A u d u s (1972). The present work deals with the feasibility of this m e t h o d , a n d the possible i n t e r a c t i o n s between GA3, indoleacetic acid (IAA), a n d k i n e t i n (K). The gibberellin c o n t e n t of t o m a t o fruits and maize seedlings was also d e t e r m i n e d by this m e t h o d a n d the results c o m p a r e d with those o b t a i n e d with the c~-amylase assay.

-

P r o t e i n release.

Introduction It was s h o w n previously that GA3 a n d other gibberellins induce the release of r e d u c i n g sugars in barley e n d o s p e r m (Paleg, 1960a, b ; Y o m o , 1960). The release of these r e d u c i n g sugars is p r i m a r i l y the result of the action of de n o v o synthesized a n d secreted c~-amylase by a l e u r o n e layers ( Y o m o , 1960; Paleg, 1960b; Varner, 1964; Chrispeels a n d Varner, 1967); both synthesis a n d secretion are d e p e n d e n t o n the presence of gibberellins. I n barley a l e u r o n e layers, GA3 also p r o m o t e s the release of other hydrolitic enzymes (Yomo, 1961; Briggs, 1963; H a r m e y a n d M u r r a y , 1968 ; Chrispeels a n d Varner, 1967 ; Taiz a n d Jones, 1970) a n d of a greater a m o u n t of reserve p r o t e i n (Melcher a n d Varner, 1971). This knowledge has led to bioassays of gibberellins in which either the a m o u n t of r e d u c i n g sugars (Nicholls a n d Paleg, 1963) or the activity of a-amylase (Jones a n d Varner, 1967) is p r o p o r t i o n a l to the l o g a r i t h m of the h o r m o n e Abbreviations." GA3 = gibberellic acid; IAA = indoleactic acid; K = kinetin

Materials and Methods Barley seeds, Hordeum vulgare cv. Himalaya, crop 1971 (Botanical Club, Pulmann, Wa., USA) were prepared essentially according to Jones and Varner (1967). After three days of imbibition ten half-seeds were incubated in a 25 ml Erlenmayer flask with 2 ml of a solution containing 2 mM acetate buffer, pH 4.8, 10 mM CaC12, chloramphenicol, and the sample to be tested. Incubations were carried out at 25~ C on a continuous shaker at 50 oscillations/ min. After incubation, the medium was decanted, the flask and the half-seeds washed with a total addition of 3 ml of acetate buffer. GA 3 was tested in a concentration range that varied between 10 5 ~tg/ml and 100 gg/ml. IAA at 0.1 and 1 gg/ml, K at 0.03 and 0.3 Itg/ml and, the 16 possible combinations between GA 3 at 10 3 and 10 2 gg/ml, were also tested. e-Amylase activity was determined with 0.04% amylose solution as substrate. One a-amylase unit was defined as the amount of enzyme causing the decrease of 1 A (measured at 620 nm) per ml of the tested solution in 1 min. The protein concentration was determined spectrophotometrically, either directly or after proper dilution with acetate buffer, by the method of Murphy and Kies (1960), with a Jasco UV 5 spectrophotometer, using bovine serum albumin as a standard. Tomato flowers (Lycopersicon esculentum Mill. cv. Ventura) were tagged at anthesis and ovaries or berries were harvested from anthesis to maturation. Fruits of each harvest were weighted, and only a uniform population, within a suitable weight range, was selected. The samples were lyophilized and stored at -20 ~ C (Mapelli et al., 1976). The gibberellin was extracted by the method of Abdel-Ramhan et al. (1975). The extracts were partitioned by paper chromatography on Whatman No. 1 paper, using isopropa-

0032-0935/78/0142/0037/$01.00

38

S. Mapelli and A.M. Ranieri: Protein Release from Aieurone

nol: ammonia:water (10:1:1, vol/vol) as the solvent mixture. The chromatogram was divided into ten fractions, and growth substances were eluted with methanol. After reducing to dryness gibberellins were redissolved in acetate buffer for the biossay. Maize seedlings (Zea rnays L.) were obtained by growing isolated embryos on Redei medium (1955) with or without added GA 3 at 10 lag/ml. The seedlings were harvested after about one week of growth, when they had developed three leaves, and the gibberellins were extracted and partitioned as described above for tomato fruits. Each experiment was repeated at least three times, and each sample replicated twice. The data reported in the figures are averages of three experiments.

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Results

F i g u r e 1 s h o w s t h e t o t a l p r o t e i n r e l e a s e d a n d c~-amylase a c t i v i t y s e c r e t e d f r o m b a r l e y h a l f - s e e d s as a f u n c t i o n o f G A 3 c o n c e n t r a t i o n . It is e v i d e n t t h a t the inc r e a s e o f t o t a l p r o t e i n has a p a t t e r n s i m i l a r to t h a t o f ~ - a m y l a s e b u t o v e r a w i d e r r a n g e ; f r o m 1 0 - ~ lag/ml to 100 lag/ml o f G A 3 . T h e release o f p r o t e i n s b e g i n s a f t e r a few h o u r s o f i n c u b a t i o n . i F i g u r e 2 s h o w s t h a t a f t e r 10 h o f i n c u b a t i o n d i f f e r e n c e s b e t w e e n c o n t r o l a n d G A 3 - t r e a t e d s a m p l e s b e c o m e e v i d e n t at a G A 3 c o n c e n t r a t i o n h i g h e r t h a n 10- 3 g g / m l , a n d a f t e r 24 h t h e y b e c o m e e v i d e n t at a G A 3 c o n c e n t r a t i o n h i g h e r t h a n 10 - 4 ~tg/ml. I n c r e a s i n g t h e i n c u b a t i o n t i m e d o e s n o t l o w e r t h e t h r e s h o l d G A 3 c o n c e n t r a t i o n , a n d the c u r v e f o r 10 - 4 I.tg/ml G A 3 a n d c o n t r o l are the s a m e for all t i m e s tested. A t 100 g g / m l G A 3 , t h e release o f p r o t e i n r e a c h e s a m a x i m u m level a f t e r a b o u t 40 h a n d at 10 - t ~tg/ml G A 3 , a f t e r 72 h o f i n c u b a t i o n . I A A a n d K a l o n e o r in c o m b i n a t i o n h a v e n o effect

Fig. 2. Kinetics of protein release from barley half-seeds treated with various concentrations of GA3: A, 100gg/ml; o, 1 ~tg/ml; m, 0.1 gg/ml; x, 10 2 ~tg/ml; [], 10 -3 ~tg/ml; 9 10 _4 ~tg/ml and control

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Fig. 3. Effect of IAA and K on the stimulation of protein release by GA 3. A: A, control; cz, 0.03 ~tg/ml K; m, 0.3 gg/mlK; 9 0.1 pg/ml IAA; *, 1 gg/ml IAA; B: A, 10 3 lag/m1 GA3; [], ", 9 9 as in A plus 10 3gg/ml GA3; C: A, 10-2gg/ml GA3; [], m, o, 9 as in A plus 10 -2 gg/ml GA~. D: A, cor~troI; ~, 0.03 ixg/ ml K+0.1 gg/ml IAA; o, 0.03 gg/ml K + l gg/ml IAA; m, 0.3 ~tg/ ml K+0.1gg/ml IAA; e, 0.3gg/ml K + l g m / m l IAA; E: A, 10 3~tg/ml GA3; D, - , 0 , 9 as in D plus 10 -3gg/ml GA3; F: , , 10 -2gg/ml GA3; :z, m, 9 9 as in D plus 10 -2gg/ml GAa

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Fig. 1. Total protein and or-amylase released from barley half-seeds treated with various concentrations of GA3. ", total protein; A, cz-amylase activity

o n p r o t e i n release, a n d d o n o t m o d i f y the a c t i o n o f G A 3 (Fig. 3). T h e g i b b e r e l l i n c o n t e n t in t o m a t o b e r r i e s at different stages o f d e v e l o p m e n t , d e t e r m i n e d as c~-amylase a c t i v i t y o r as t o t a l p r o t e i n r e l e a s e d , a r e r e p o r t e d in F i g u r e 4. N o s i g n i f i c a n t q u a l i t a t i v e o r q u a n t i t a t i v e

S. Mapelli and A.M. Ranieri: Protein Release from Aleurone -4.

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Discussion

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Fig. 4. Gibberellin content in tomato fruit extracts, cromatographed on W h a t , a n No. 1 paper, determined either by total protein estimation (right) or by the a-amylase assay (left). The number in each histogram indicates the days after anthesis of berry harvest

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