Plant Cell Reports

Plant Cell Reports (1984) 3: 193-195

© Springer-Verlag t984

Amino acid pools and protein synthesis in germinating maize embryos Raul Aguilar and Estela Sanchez de Jiminez Plant Biochemistry Department, Facultad de Quimica, Universidad Nacionat Autonoma de Mexico, 04510, Mexico, DF Received February 17, 1984/Revised version received August 1, 1984 - Communicated by O. L, Gamborg

ABSTRACT Amino acid pools were analyzed in maize (Zea mays L.) axes germinated f o r O, 6 and 24 h. P r o t e i n s s y n t h e s i z e d at e a r l y (0-6 h) and l a t e (18-24 h) stages were c h a r a c t e r i z e d by gel e l e c t r o p h o r e s i s and f l u o r o g r a p h y a f t e r e i t h e r 14C-leucine or 1 4 C - l y s i n e p u l s e s . An i n c r e a s e of amino acid i n c o r p o r a t i o n after 18-24 h was nbserved, as w e l l as changes in the p r o t e i n p a t t e r n s of the corresponding f l u o r o g r a p h s . A n a l y s i s of the endogenous amino acid pools showed major changes in cont e n t s of p r o l i n e , a l a n i n e , i s o l e u c i n e , v a l i ne, l e u c i n e and l y s i n e . A s e l e c t i v e i n c r e a s e of l y s i n e i n c o r p o r a t i o n i n t o p r o t e i n s d u r i n g the l a t e stage was d e t e c t e d . INTRODUCTION Cereal embryos r e - e s t a b l i s h m e t a b o l i c activity f o l l o w i n g i m b i b i t i o n (Bewley and Black 1978). P r o t e i n s y n t h e s i s i s one of the major m o l e c u l a r events t a k i n g place in t h i s p e r i o d (Brooker et a l . 1977). Several authors have shown t h a t the p r o t e i n s s y n t h e s i z e d during g e r m i n a t i o n are e s s e n t i a l f o r f u r t h e r growth and p l a n t l e t development ( J e n d r i s a k 1980, Datta et a l . 1983). Among t h e s e , the r i b o s o mal p r o t e i n s have been i d e n t i f i e d as being s y n t h e s i z e d e a r l y d u r i n g wheat g e r m i n a t i o n (Lane and Tumaitis-Kennedy 1981), w h i l e h i s tones seem to be produced l a t e r in maize embryos (Van de Walle and D e l t o u r 1974). On the o t h e r hand, there is c o n f l i c t i n g i n f o r m a t i o n r e g a r d i n g the s i m i l a r i t y or d i f f e r e n c e s between e a r l y and l a t e p r o t e i n s produced during g e r m i n a t i o n (Brooker et a l . 1978, Caers et a l . 1979). These c o n t r a d i c t o r y observat i o n s might be the r e s u l t of the s p e c i f i c developmental programs f o r RNA s y n t h e s i s that seem to c h a r a c t e r i z e d i f f e r e n t kinds of seeds (Van de Walle et a l . 1983). T h e r e f o r e , data o b t a i n e d in one species might not apply to another. Furthermore, d i f f e r e n c e s in the endogenous amino acid pools might r e g u l a t e the r a t e of p r o t e i n s y n t h e s i s d u r i n g seed germin a t i o n (Marsh et a l . 1982). 1Under the Academic C o l a b o r a t i o n Program between the U n i v e r s i d a d Nacional Aut6noma de MExico and the Colegio de Postgraduados, Chapingo, MExico.

The aim of the present paper i s to i n q u i re about s p e c i f i c changes in p r o t e i n s y n t h e s i s t a k i n g place in maize embryos concomitant to the g e r m i n a t i o n process and t h e i r r e l a t i o n to t h e i r f r e e amino acid c o n t e n t s . Two stages of g e r m i n a t i o n were chosen f o r t h i s purpose: 0-6 h, when m e t a b o l i c a c t i v i t y i s b e g i n n i n g , and 18-24 h, before r a d i c l e p r o t u s i o n i s observed (23.5 h average) and c e l l d i v i s i o n i s initiated. MATERIALS AND METHODS A h y b r i d v a r i e t y of maize (Zea mays L . ) (H-30) donated by Dr. Joaqu~n O r t i z - C e r e c e r e s Colegio de Postgraduados, Chapingo, M~xico, was the source of the b i o l o g i c a l m a t e r i a l . Embryonic axes - w i t h o u t s c u t e l l u m - were excised from dry seeds and used in a l l the experiments. I n c u b a t i o n system. Batches of I00 to 150 mg of axes were incubated at 25°C in an incubat i o n medium (50 mM T r i s - H C l , pH 7 . 6 , 50 mM KCI, 10 mM MgCI2 and 2% s u c r o s e ) . A m i x t u r e made of 0.03 mM of each of the f o l l o w i n g amino acids was s u p p l i e d to the i n c u b a t i o n system, except f o r the experiments where the i n t e r n a l amino acid pools were determined: a l a nine, valine, proline, arginine, glycine, isoleucine, threonine, serine, tryptophan, cysteine, glutamate, aspartate, glutamine, asparagine, tyrosine, histidine, methionine, p h e n y l a l a n i n e , and e i t h e r l e u c i n e or l y s i n e depending on the 14C-amino acid s u p p l i e d . 20 ~Ci of e i t h e r 14C-leucine (314 mCi/mmol) or 1 4 C - l y s i n e (285 mCi/mmol) (New England) were a p p l i e d f o r a 6 h p e r i o d at e i t h e r 0 or 18 h of i n c u b a t i o n . A f t e r the i n c u b a t i o n p e r i o d , the t i s s u e s were washed, f r o z e n and homogenized in b u f f e r (50 mM T r i s pH 7 . 6 , 50 mM KCI, 10 mM MgCI 2, 5 mM ~ - m e r c a p t o e t h a n o l ) w i t h a m o r t a r and p e s t l e and c e n t r i f u g e d at 15,000 g f o r 30 min. The s u p e r n a t a n t was again c e n t r i f u g e d at 190,000 g f o r 80 min. P r o t e i n s were p r e c i p i t a t e d from the supernatant with trichloroacetic acid ( f i n a l c o n c e n t r a t i o n of 5%), c e n t r i f u g e d and r e s u s pended in w a t e r , n e u t r a l i z e d w i t h 0.5 M KOH and t h i s process was repeated t w i c e . P r o t e i n s were assayed by the Lowry method (Lowry et al. 1951) using bovine serum albumin (Sigma) as the s t a n d a r d . R a d i o a c t i v i t y was determined in 100 ~I of p r o t e i n samples w i t h 10 ml of

194 200

190 8O 09 U

20'

0

Asp Glu Pro

Vol Tyr Gly AIG Phe I l e

Leu Lys Arg

Met Thr ÷

Ser Figure 1. Composition of the f r e e amino acid pool at t h r e e stages of development. Batches of 100 axes were incubated at 25°C in b u f f e r 50 mM T r i s - H C l pH 7.6, f o r 0 ( ), 6 ( and 24 ( ) h, Samples were processed as e x p l a i n e d in M a t e r i a l and Methods. Amino acid pools were measured in a Beckman A u t o a n a l y z e r . scintillation m i x t u r e (Bray 1964) in a packard s c i n t i l l a t i o n counter. Gel e l e c t r o p h o r e s i s of r a d i o a c t i v e p r o t e i n s . Radioac~~no aci~'ns were a-nal y z e d by sodium dodecyl s u l f a t e (SDS) p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s (PAGE), using a s t a c k i n g gel of 5% and a r e s o l v i n g gel of 7-15% g r a d i e n t of p o l y a c r y l a m i d e (Laemmli 1970). Fluorographs were prepared by exposing Kodak X-O mat S-5 f i l m to the d r i e d gel at -70°C (Bonner and Laskey 1974), 20x103 counts/ min were a p p l i e d per channel. Amino acid a n a l y s i s . P r o t e i n s from the post~s-s-oma7 s u p e r n a t a n t were p r e c i p i t a t e d w i t h sulfosalicylic acid (35 mg/ml) and c e n t r i f u g ed at 3,500 rpm f o r 15 min. Amino acid a n a l y s i s was c a r r i e d out on the s u p e r n a t a n t using a Beckman model 120 B amino acid a n a l y z e r , o p h y s i o l o g i c a l mode f o l l o w i n g P i c k e r i n g and Soto (1978). RESULTS AND DISCUSSION The pool of amino acids from embryonic axes at O, 6 and 24 h of g e r m i n a t i o n were analyzed (Fig. 1).For most of the amino a c i d s , t h e r e is a s l i g h t i n c r e a s e i n the amino a c i d cont e n t from zero to 24 h of g e r m i n a t i o n ; l e u c i ne, v a l i n e , i s o l e u c i n e , a l a n i n e and l y s i n e , however, i n c r e a s e from 3.5 to 9.7 t i m e s . On the o t h e r hand, t h r e e of the amino acids de -~ crease d u r i n g t h i s p e r i o d : a s p a r t i c a c i d , g l u t a m i c acid and p r o l i n e ; the l a t e r decreases to about a t h i r d of i t s o r i g i n a l v a l ue. Leakage of amino acids was t e s t e d in the system. A f t e r 24 h of i n c u b a t i o n , p r o l i n e i s the main amino acid p r e s e n t in the surrounding medium. I t s c o n c e n t r a t i o n , however, does not r e p r e s e n t more than 20% of the o r i g i n a l amount found in the dry embryonic a x i s , Met h i o n i n e i s not detected in the dry a x i s , but d e t e c t a b l e amounts occur at 6 and 24 h. This amino acid however, might be underestimated

due to loss during the e x t r a c t i o n procedure. P r o l i n e , on the o t h e r hand, i s much more abundant in the dry embryo than any o t h e r amino a c i d . In order to d e t e c t major changes in the types of p r o t e i n s being s y n t h e s i z e d during t h i s p e r i o d , l e u c i n e and l y s i n e were chosen f o r the pulse l a b e l l i n g e x p e r i m e n t s . Both of them i n c r e a s e s i g n i f i c a n t l y in conc e n t r a t i o n from zero to 24 h of g e r m i n a t i o n , and l y s i n e i s c h a r a c t e r i s t i c of basic p r o t e ins l i k e H1, HzA and H3B p l a n t h i s t o n e s ( S p i t h i r 1982), whose s y n t h e s i s might be expected during t h i s p e r i o d . Pulse l a b e l l e d experiments w i t h e i t h e r 14C-leucine or 1 ~ C - l y s i n e were c a r r i e d out f o r 6 h and the p r o t e i n s from 0-6 h and 1824 h were TCA p r e c i p i t a t e d and counted. The amount of amino acid i n c o r p o r a t e d per m i l l i gram of p r o t e i n was determined a f t e r c o r r e c t i o n f o r amino acid pool s i z e . Results i n d i cated a l a r g e r i n c o r p o r a t i o n of both amino acids in the p r o t e i n s s y n t h e s i z e d from 18-24 h than from 0-6 h. Table 1. Amount of 14C-amino acid i n c o r p o r a t ed in p r o t e i n s . Embryonic axes (150 mg) were p u l s e - l a b e l l e d (20 ~Ci) at 0-6 or 18-24 h w i t h l ~ C - l e u c i n e or 14C-lysine. Pulse time (~) Relative Amino Acid 0-6 18-24 Increment

1~C-leucine l~4C-lysine Ratio Lysine/Leucine

nmoles of amino acid/mg p r o t e i n 2.44 45.80 6.51 202.47 2.66

4.41

18-24 0-6 18.80 31..10 1.65

The increments are not s i m i l a r , however, since the l y s i n e / l e u c i n e r a t i o s s i g n i f i c a n t l y i n c r e a s e in the 18-24 h p e r i o d . (Table 1). The l a b e l l e d p r o t e i n s were a l s o analyzed by gel e l e c t r o p h o r e s i s and f l u o r o g r a p h y .

195 to d i v i d e . Embryonic axes of V i c i a faba have shown a b u r s t in h i s t o n e p r o d u c t i o n at about 24 h of g e r m i n a t i o n , p r e v i o u s to the p e r i o d of DNA s y n t h e s i s , and again at c e l l d i v i s i o n (Funkuel et a l . 1978). In maize, the main DNA s y n t h e s i s seems to reach major r a t e s between 24 and 40 h a f t e r i m b i b i t i o n is i n i t i a t e d ( u n p u b l i s h e d r e s u l t s ) ; t h e r e f o r e , an i n c r e a s e in l y s i n e r i c h p r o t e i n s in the 18-24 h p e r i o d seems to be in accordance w i t h t h i s scheme. Furthermore the l a r g e i n c r e a s e in l y s i n e and a l a n i n e pools at t h i s time ( F i g u r e 1) is in accordance w i t h the c e l l r e q u i r e m e n t s f o r h i s t o n e p r o d u c t i o n , as i n d i c a t e d by the abundance of these two amino acids in some of the p l a n t h i s t o n e s ( S p i t h i r 1982). ACKNOWLEDGEMENTS The authors are g r a t e f u l to Dr. Mark Engleman, Colegio de Postgraduados de Chapingo, M#xico, f o r c r i t i c a l reading of the manusc r i p t and to Mrs. M a r i c e l a V i l l a r r e a l f o r her a s s i s t a n c e in t y p i n g i t .

Figure 2.

Fluorographs of the S D S - p o l y a c r y l a mide gel p r o f i l e s of the p r o t e i n s s y n t h e s i z e d between 0-6 or 18-24 h. Embryonic axes were l a b e l l e d w i t h e i t h e r 14C-leucine (A) or Lysine (B). P r o t e i n s were separate by PAGE-SDS and detected by f l u o r o g r a phy.

Major changes can be observed in the c o r responding f l u o r o g r a p h s ( F i g u r e 2). Several new bands are c l e a r l y apparent in the 18-24 h p e r i o d ( a r r o w s , Figure 2 ) , which are not seen in the 0-6 h p a t t e r n s . Some of them are more pronounced in the 18-24 h l ~ C - l y s i n e p a t t e r n (double arrows, Figure 2). These data indicate q u a n t i t a t i v e as w e l l as q u a l i t a t i v e changes fo the p r o t e i n s being s y n t h e s i z e d during both p e r i o d s . I t i s known t h a t p r o t e i n s synthesized at e a r l y stages of g e r m i n a t i o n are m a i n l y coded f o r preformed mRNAs (Brooker et a l . 1977), w h i l e at l a t e r p e r i o d s new mRNAs are t r a n s l a t ed in the c e l l s (Lane and Tumaitis-Kennedy 1981). In maize, the same p a t t e r n seems to occur, since the e a r l y p e r i o d (0-6 h) is not s e n s i t i v e to ~ - a m a n i t i n , w h i l e the l a t e r one (18-24 h) shows a s i g n i f i c a n t i n h i b i t o r y e f fect with this antibiotic (S~nchez de Jim~nez et a l . 1981). Among these l a t e r s y n t h e s i z e d p r o t e i n s , h i s t o r i e s might be a s i g n i f i c a n t f r a c t i o n , since the embryonic c e l l s are ready

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