Damages at translational level in aged wheat embryos

Plant Science Letters, 12 (1978) 217--226

217

© Elsevier/North-Holland Scientific Publishers Ltd.

DAMAGES AT TRANSLATIONAL LEVEL IN AGED WHEAT EMBRYOS* A. DELL'AQUILA and P. DE LEO Laboratorio del Germoplasma del C.N.R., Via Amendola, 165/A, 70126 Bari (Italy) E. CALDIROL! and G. ZOCCHI Istituto di Chimica Agraria dell'Universita degli Studi di Milano, Via Celoria, 2, 20133 Milano, (Italy) (Received November 3rd, 1977) (Revision received February 11 th, 1978) (Accepted February l l t h , 1978)

SUMMARY

The functional properties of elongation factor 1 (EF1) in high viability and aged wheat embryos have been studied. The results show the presence of an inhibitory activity at the level of the translational machinery in aged germs: the significance of this activity on protein synthesis related to the physiological conditions of ageing seeds is discussed.

INTRODUCTION

In wheat embryos [ 1--3] as well as in several eukariotic sources [4,5] the elongation factor 1 (EF1) can be isolated in high molecular size forms (EF1H) having a heterogeneous peptide composition related to particular functions of EF1 in the translational machinery. Our previous work [6] reported that during ageing in wheat embryo some irregular modifications of EF1 occur along with a decrease in protein synthesis. The elution patterns of Sephadex Cr-150 of the GTP-binding activity present in the cytoplasmic fraction of embryos at low viability levels showed, in comparison to viable embryos, the gradual disappearance of the high tool wt proteins which are normally found in viable embryos, where they contain the maximum binding activity. Moreover, in germs from the low-viability seeds the elution profile of the gel filtration presents a large heterogeneity in GTP-binding activity and no distinct peaks were observed. After further purification by *Dedicated to Professor Gian Antonio Lanzani in memory of his scientific achievements. Abbreviations: EF1, elongation factor 1; DEAE-cellulose, diethylaminoethyl cellulose; S DS, sodium dodecylsulphate; V, viability.

218 hydroxyapatite chromatography this form showed a low activity in binding Phe-tRNA to the ribosomes and an inability to complement EF2 in the polyPhe synthesis. This led us to postulate the presence in these germs of an inhibitory activity on the polyphenylalanine synthesis, probably related to the disordered changes of the polypeptide composition of EFIH that occur during ageing. To this purpose we have investigated the presence, in aged wheat embryos, of an inhibitory activity modifying the process of protein synthesis and discussed the possible correlations with the modifications that occur during ageing at the level of the translational machinery. MA TER I ALS AND METHODS

Plant material Stocks of wheat seeds (Triticum durum cv Lamia) at 85% and 35% viability level were used: the low viability seeds were obtained by storage at 14% moisture content at 38°C, according to the method of Roberts and AbdaUa [7]. The embryos were prepared according to Johnston and Stern [8].

DEAE-cellulose chromatography The post-ribosomal supernatant was prepared as described elsewhere [6] from 150 g of germs. The 100 000 g supernatant (1,900 mg protein) was loaded onto a diethylaminoethyl cellulose (DEAE~ellulose) (DE 32 Watman) column (42 × 5 cm), conditioned with 30 mM potassium phosphate pH 7.2, 0.2 mM MgC12,7 mM 2-mercaptoethanol, 5% glycerine (buffer A} and eluted with a linear gradient between 30--300 mM potassium phosphate in buffer A. Fractions of 9 ml were collected. The protein profile was recorded at 280 nm. Fractions containing GTP-binding activity were pooled and precipitated with (NH4)2SO4 (80% saturated). After centrifugation at 10 000 rev./min at 4°C the pellets were dissolved in 50 mM potassium phosphate pH 7.2, 1 mM dithiothreitol (buffer B) and dyalized against the same buffer.

Hydroxyapatite chromatography The first peak (180 mg protein) was loaded onto a hydroxyapatite (Serva) column (20 X 1.4 cm) conditioned with buffer B and eluted with a linear gradient between 50--300 mM potassium phosphate in buffer B. Fractions of 2 ml were collected; the absorbance at 280 nm recorded and the GTP-binding activity assayed. The active peaks were pooled and precipitated with ammonium sulphate (80% saturated). After centrifugation, the pellets were dissolved in buffer B and dyalized against the same buffer.

SDS-gel electrophoresis 30 tzg of protein were used for the analysis of the peptide composition by 10% sodium dodecylsulphate (SDS)-polyacrylamide disc gel electrophoresis according to the method of Weber and Osborne [9]. Ribosomes prepared

219 from embryos from high viability wheat seeds and completely free from elongation factors were obtained as already described [10] ; electrophoretically homogeneous EF1R also from high viability wheat seed embryos was prepared from crude ribosomes as described [11] ;elongation factor EF2 was obtained according to the method of Twardowski and Legocki [12] ; [14C] Phe-tRNA was prepared from partially-purified tRNA Phe (high viability wheat seed embryo source) by the method of Vold and Sypherd [13]. The product contained 167 pmol of [14C] Phe-tRNA per A26o nm unit. [3H] GTP binding, [14C] Phe-tRNA binding to DEAE ribosomes and poly (U) directed poly-Phe synthesis assays were performed as reported elsewhere [11] ; [all] poly (U) binding activity was determined as already described [14]. Protein concentration

This was determined according to the method of Lowry et al. [15], using crystalline bovine serum albumin as a standard. RESULTS In wheat embryos, a preparation of EF1H contains both GTP-binding and poly(U)-binding activity which co~elute from hydroxyapatite and DEAE-cellulose chromatography as well as from gel-filtration on Sephadex G-200 [2,14]. We have studied the elution profiles of a DEAE-cellulose chromatography of the post-ribosomal supernatant from wheat embryos at 85% and 35% viability (V) levels. Fig. l a shows the elution profile of the GTP-binding activity from the cytoplasmic fraction of the 85% V germs. Three distinct peaks of EF1 activity were detected which correspond with the peaks of the UV absorbance profile: peak I (fractions 20--34), peak II (fractions 34--50) and peak III (fractions 50--76). The cytoplasmic fraction from the 35% V germs eluted from the cellulose column shows a different profile of the UV absorbance and of the ability of the fractions to form a binary complex EF1-GTP (Fig. lb). The first peak contains lower amounts of protein and of GTP-binding activity than the corresponding peak from high-viability germs. Furthermore, it is eviaent that the second peak from the low viability germs was eluted distinct from the first peak, whereas in the 85% V germs the corresponding fractions resulted less separated either in the protein and in the GTP-binding activity profiles. We examined the presence of EF1 functions in these fractions by testing the GTP and poly(U) binding activities, the ability to catalyze the binding of PhetRNA to the ribosomes and to synthesize polyphenylalanine. The fractions were pooled as indicated in Fig. 1, precipitated with (NH4)2 SO4 (80% saturated) and dyalized before assaying the mentioned EF1 activities. As reported in Table I and II significant differences were observed in fractions I: in the 85% V germs the GTP-binding activity (Table I) and the ability to catalyze the binding of Phe-tRNA to the ribosomes (Table II) were higher with respect to the aged embryos. Furthermore the fraction from aged embryos was unable to synthesize polyphenylalanine in contrast to the activity present in

220

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Fig. 1. DEAE-cellulose chromatography of the 100 000 g post-ribosomal supernatant from embryos o f wheat seeds at 85% ( a ) a n d 35% viability level (b), The [a H] GTP-binding activity was tested with 180 ul aliquots of each fraction according to the method described [ 11 ].

the corresponding fraction from high viability germs. Fractions II and III from high- and low-viability germs did n o t present significant differences with regard to the EF1 activities. We have furthermore studied the effect o f fraction I from both germ stocks on the poly(U) directed poly-Phe synthesis using increasing amounts o f protein, in a complete system containing saturating amounts of E F I R and EF2 purified from viable wheat embryos. As it can be seen in Table III, while no effect of peak I from the 85% V on the poly-Phe synthesis was observed, the corresponding peak from low-viability germs inhibits the polyphenylalanine synthesis and the inhibition increases at increasing concentration of protein.

22t TABLE I

[ sH ] GTP AND [ 3H ] POLY(U) BINDING ACTIVITIES OF THE FRACTIONS FROM DEAE-CELLULOSECHROMATOGRAPHY FROM EMBRYOS AT 85% AND 35% VIABILITY LEVEL. Embryo viability

D E A E 32 fraction (200 ug protein)

[3H ]G T P bound (counts/minute)

[3H ]Poly(U) bound (counts/minute)

85%

I II III

4010 882 837

781 582 296

35%

I II III

448 852 551

482 515 293

In order to study the effects o f this inhibitor which appears to be associated with EF1 functions, we further purified the first peak from the 35% V germs by h y d r o x y a p a t i t e chromatography. In Fig. 2 the profile o f the GTP binding activity t h a t elutes from the column is shown. 2 distinct peaks o f activity are separated: a large peak between fractions 20--40 (peak A) which correspond with the shoulder peak of the UV profile and a small peak between fractions 43--45 (peak B). The ability o f these fractions to bind either GTP or poly(U) is reported in Table IV. Both peaks show about the same ratio of GTP-binding versus poly(U)-binding activity. It should be noticed t h a t for both activities the values are higher in peak A than in peak B. Moreover while peak A was TABLE II [14C ]P H E - t R N A B I N D I N G T O T H E R I B O S O M E S A C T I V I T Y A N D P O L Y - P H E SYNTHESIS C A T A L Y Z E D B Y T H E F R A C T I O N S F R O M D E A E - C E L L U L O S E C H R O M A T O G R A P H Y F R O M E M B R Y O S A T 85% A N D 35% VIABILITY LEVEL. Embryo viability

Fraction (100 ~g protein)

85%

I II III

35%

[~4C ]Phe-tRNA bound to the ribosomes (pmol)

Poly-Phe formed (pmol)

I

1.8

1.3

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7.9 3.6

1.7 1.6

0.9 5.6 3.3

-1.8 1.9

222 T A B L E III E F F E C T OF D I F F E R E N T AMOUNTS OF PROTEIN FROM PEAK I OBTAINED F R O M D E A E - C E L L U L O S E C H R O M A T O G R A P H Y ON P O L Y ( U ) D I R E C T E D P O L Y PHE S Y N T H E S I S IN THE SYSTEM C O N T A I N I N G S A T U R A T I N G A M O U N T S O F E F 1 R (10 ug) AND E F 2 (10 ug) P U R I F I E D F R O M V I A B L E E M B R Y O S .

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Protein amount (ug)

85% V embryos

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Fig. 2 . H y d r o x y a p a t i t e c h r o m a t o g r a p h y o f peak I f r o m DEAE-cellulose c h r o m a t o g r a p h y o f t h e c y t o p l a s m i c f r a c t i o n o f e m b r y o s at low-viability level. [ 3H ] GTP p m o l p e r 180 ul a l i q u o t s o f each f r a c t i o n a c c o r d i n g t h e m e t h o d d e s c r i b e d [ 11 ].

223 TABLE IV [ 3H ] GTP AND [ 3H ] POLY(U) BINDING ACTIVITIES OF THE VARIOUS PEAKS FROM HYDROXYAPATITE CHROMATOGRAPHY OF DEAE 32 PEAK I FROM 35% V EMBRYOS.

Hydroxyapati~ peak (100 ~g protein)

[3H]GTPbound (counts/min)

[3H]poly(U) bound (counts/min)

A B

1124 264

1168 237

able to catalyze the binding of Phe-tRNA to the ribosomes and this activity increased at increasing concentration of protein peak B showed no activity (Table V). Both peaks inhibit poly-Phe synthesis even though the effect is much more pronounced in peak B which completely abolishes such activity. SDS gel electrophoresis showed some noteworthy differences in the peptide pattern of these fractions. The first peak presented minor bands of protein having mol. wts. in the range of the peptides composing functioning EF1H [5] and a major band having mol. wt. 33 000 (data n o t shown). This latter peptide was present in much larger amounts in the gel electrophoresis pattern of peak B (Fig. 3) at a concentration about 5--6-fold higher than the corresponding peptide of peak A.

TABLE V [ 14C ] PHE-tRNA BINDING ACTIVITY AND EFFECT ON POLY-PHE SYNTHESIS IN THE SYSTEM CONTAINING SATURATING AMOUNTS OF EF1 a (10 ug) AND EF2 (10 ug), PURIFIED FROM VIABLE GERMS, OF THE VARIOUS HYDROXYAPATITE PEAKS.

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Protein (ug)

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48 65 87

224

Fig. 3. SDS-polyacrylamide disc gel electrophoresis of peak B from hydroxyapatite chromatography (right). The mol. wt. of the peptide was determined by comparison with markers of known mol. wt. (left). The standards used were: katalase (K), mol. wt. 60 000; aldolase (A), tool. wt. 40 000;cytochrome c (C), tool. wt. 12 500.

DISCUSSION As r e p o r t e d here, t h e c o m p a r i s o n o f t h e e l u t i o n profiles a n d G T P - b i n d i n g activities f r o m D E A E - c e l l u l o s e c h r o m a t o g r a p h y o f t h e c y t o p l a s m i c f r a c t i o n o f 85% V a n d 35% V w h e a t e m b r y o s s h o w s s o m e significant differences, in t h e t w o s t o c k s o f seeds. In fact t h e investigation o f s o m e o f t h e f u n c t i o n a l p r o p e r -

225 ties of EF1 contained in the isolated chromatographic peaks indicates that the first peak from the aged germs consists of a molecular aggregate in which both EF1 activity and an inhibitory activity on poly-Phe synthesis are present. Further purification of this peak by hydroxyapatite chromatography led to the separation of two fractions which bind GTP. The first peak possessed EF1 activity which was almost undetectable in the second peak, in which a strong inhibitory effect on protein synthesis is present. Moreover the gel electrophoretic patterns of these peaks showed the presence of a large band of mol. wt. 33 000, clearest in the second peak, along with minor bands of peptides of high and low molecular sizes. The loss of the functionality of the protein-synthesizing system has been also observed in rye embryos [16,17] and in pea embryonic axes [18,19] together with several biochemical lesions occurring at both the transcriptional and the translational levels. These effects induced by ageing could be related to the breakdown of macromolecules and membranes along with the release of several hydrolitic enzymes which occurs in aged cells, as described in nonviable maize germs by Berjack and Villiers [20]. The damage induced by ageing in wheat embryos could be responsible for the appearance and the accumulation of peptides of low molecular size which inhibit, in a n o t yet clear way, the translational machinery. This hypothesis could be related to an incorrect working of EF1 in aged embryos, since in the high-viability germs the correct functioning of EF1H is associated with a regular dynamic of association and disassociation of its c o m p o n e n t peptides: A (mol. wt. 52 000), B (mol. wt. 47 000), and C (27 000) [3]. As reported in a previous work [14], peptide A is involved in the formation of the ternary complex: aminoacyl-tRNA-GTPEF1, whereas the polypeptide complex B, C might be involved in an inhibitory effect on poly-Phe synthesis. On the other hand it can be postulated that a specific hydrolytic activity, present in a preparation of extracts from the cytoplasm of aged wheat embryos, could affect several components of the protein synthesis system in vitro. This effect might be responsible for the disaggregation of the molecular complexes in which EF1 and EF2 exist in vivo with a mechanism like that described by Twardwoski et al. [21] in extracts of Arternia salina nauplii, which present a proteolytic activity disaggregating EF1H in peptides of lower molecular weight. Further studies on the processes that lead, in aged seeds, to the observed modifications in the efficiences of the translational machinery and on the mechanism by which the peptide we have purified inhibits protein synthesis are needed. Note added in proof While this manuscript was in preparation, Stewart et al. [BBA, 479 (1977) 31--38] reported on the presence of an inhibitor of protein synthesis in the high speed supernatant fraction from wheat germ. The step at which this inhibitor acts and its tool. wt. may indicate a similarity between it and our inhibitor.

226

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