Planta
Planta 151, 75-80 (1981)
9 Springer-Verlag 1981
Effects of High Temperature on Protein Synthesis During Germination of Maize (Zea mays L.) G r a h a m J. P. Riley Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth SY23 3EB, Dyfed, U.K.
Abstract. The poor germination of maize seeds at high temperatures ( > 37 ~ is related to the low rate of protein synthesis by the embryo. The apparatus of translation was not heat-labile when embryos were incubated for 2 h at 41 ~ and cell free extracts from seeds imbibed for 16 h at this temperature were able to translate exogenous m R N A , indicating that ribosomes and other subcellular components were present and functional. Analysis of polysome profiles from embryos imbibing at high temperature indicated that the low rate of protein synthesis was due to the nonavailability of active m R N A . Key words: Embryos (protein synthesis) - Germination (seeds) - Protein synthesis and temperature - Temperature and protein synthesis - Z e a . Introduction
Germination of many maize cultivars is severely inhibited above about 37 ~ This is related to the low rate of protein synthesis in the embryos of seeds imbibing at high temperatures; the rate of incorporation of [14C]leucine by the embryos of seeds imbibing at 41 ~ was less than one third of the rate in seeds imbibing at 28 ~ (Riley 1981). There are several possible reasons for the low rate of protein synthesis in seeds imbibing at high temperature : (i) the energy state of the embryos could be too low to support anabolic reactions; (ii) the supply of amino acids could be limiting; (iii) the rate of R N A synthesis could be lower at high temperature; Abbreviations." ATP=adenosine-5'-triphosphate; EDTA =ethylen-
ediaminetetra-acetic acid; GTP = guanosine-5'-triphosphate ; HEPES = N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; Poly (U) = polyuridylic acid; PPO =2,5-diphenyloxazole; SDS = sodium lauryl sulphate; TMV = tobacco mosaic virus
(iv) the rate of R N A turnover could be higher at high temperature; (v) the processing of R N A molecules or their release from the nucleus could be inhibited; (vi) the assembly of ribosomes or polysomes, or their attachment to membranes could be disrupted; (vii) there could be a deficiency of some of the enzymes and other factors involved in polypeptide synthesis; (viii) some of these enzymes etc could be heatlabile. Possibilities (i) and (ii) may be excluded on the basis of previous results, which showed that energy metabolism proceeds normally at temperatures which severely depress the rate of protein synthesis, and that this is not increased by incubating embryos with an excess of amino acids. Furthermore, the activity of ribonuclease is significantly lower in seeds imbibing at 41 ~ (Riley 1981), indicating that (iv) is also unlikely. The experiments reported below were undertaken in order to test some of the other possibilities. The activity of cell-free extracts from seeds imbibing at 41 ~ was assayed in order to test the ability of isolated ribosomes, enzymes and other factors to synthesize proteins when supplied with exogenous m R N A , and the polysome profiles of imbibing seeds were analysed to determine whether active m R N A was present in the cytoplasm.
Material and Methods Materials'. Maize seeds cv. Marls Jade were purchased from Hurst
Gunson Cooper Taber, Colchester, Essex, U.K. They were imbibed on filter paper moistened with 1 mmol 1 i streptomycin sulphate or sterile water. Tobacco mosaic virus was prepared from the leaves of infected Nicotiana tabacum plants by precipitation with n-butanol (Tomlinson et al. 1959), and RNA was extracted with SDS and phenol buffered with 5 mmol 1-1 EDTA, pH 7.3 (Roberts et al. 1973a;
0032-0935/81/0151/0075/$01.20
76
G.J.P. Riley: Temperature and Protein Synthesis in Germination
Brawerman 1974). It was dialysed against distilled water for 6 h and stored in liquid N 2. All glassware was oven-sterllised before use, and solutions were autoclaved or filtered, and stored frozen.
Protein Synthesis. The rate of protein synthesis by whole embryos was determined by incubating them with U-[l*C]leucine as described elsewhere (Riley 1980). Polypeptide synthesis in vitro was measured in cell-free extracts from seeds which had imbibed for I6 h. Isolated axes were frozen in liquid N2, crushed, and homogenised in an equal volume (w/v) extraction buffer (20 mmol 1-2 HEPES, 0.1 mol 1-1 KC1; 1 mmol 1-1 MgAc2; 2mmol 1 1 CaC12; 7 mmol 1-1 dithiothreitol, adjusted to pH 7.6 with KOH). The homogenate was centrifuged (I 7,000 g, 12 min), and the supernatant was loaded onto a column of Sephadex G-25 equilibrated with 20 mmol 1-1 HEPES-KOH, pH 7.6; 0.1 mol 1 1 KCI; 3 mmol 1 1 MgAc2 and 1 mmol 1 - 1 dithiothreitol. Fractions were collected and the most turbid were pooled and recentrifuged (17,000g, 20 rain). The supernatant was used immediately after preparation, although it retained most of its activity when stored in liquid N2. Polypeptide synthesis was measured in a final volume of 50 gl, containing (final concentrations) 14 mmol 1-1 HEPES-KOH, pHT.9; 3 0 m m o l i - 1 K A c ; 2 m m o l l - l M g A % ; ( l m m o l 1 - 1 MgA% when TMV-RNA was used); 1 mmol 1-~ ATP; 0.1 mmol 1 i GTP; 8 mmol 1-1 phosphocreatine; 40 mg 1-1 creatine phosphokinase; 0.25 mmol 1-1 spermine; 20 lamol 1-1 of each amino acid except phenylalanine or leucine; and 0.74.109 Bq 1 1 L-[2, 4, 6-3H]phenylalanine (0.77 gmol 1 1) or L-[4, 5-3H]leucine (0.16 ~tmol 1-1). Extract (15gl) and RNA (10/.tg poly(U) or 18/.tg TMV-RNA) were added, and the samples were incubated at 28 ~ Incorporation was determined by spotting 20 gl aliquots onto glass fibre discs, and the hot trichloroacetic acid-insoluble radioactivity was determined by liquid scintillation spectrometry in toluene: Triton: PPO scintillant after washing the filters according to the procedure of Marts and Novelli (1961). Potassium and magnesium concentrations were optimised separately for each message. The rate of reaction was insensitive to pH between 7.4 and 8.2. Polyacry&mideGel Electrophoresis,Cell-free extracts were incubated with TMV-RNA for 30rain, and an equal volume of I25mmol 1-1 tris-HCl, pH6.8, containing 4% (w/v) SDS and 10% (w/v) glycerol, was added. The protein solution was denatured at 100 ~ (5 min), and 0.05 vol. 2-mercaptoethanol was added after it had cooled. The discontinuous gel system employed was based on that of Laemmli (1970). The running get was a 0.7 mm thick sIab of 12.5% polyacrylamide (acrylamide: NN'-methylenebisacrylamide ratio 29.2:0.8), containing 375mmoll l tris-HC1, pH 8.9, and 0.1% (w/v) SDS. The stacking gel was 2 cm long and consisted of 5% polyacrylamide, 150 mmol 1-1 tris-HC1, pH 6.7 and 0.025% (w/v) SDS. The electrode buffer contained 25 mmoll l tris, 190 mmoll ~ glycine, and 0.1% (w/v) SDS. Denatured protein solution (20-30 gl) was introduced into each sample welt, and electrophoresis was carried out at 15 mA per gel for 1 h, followed by 25 mA until the front reached the bottom of the running gel. Radioactive polypeptides were detected by the quantitative fluorographic procedure of Laskey and Mills (1975). Analysis of Polysomes. Embryos (0.75 g) were frozen in liquid
N 2,
crushed, and homogenised in 6 ml 0.1 moI 1-1 tris-HC1, pH 8.0, containing 0.3 mol 1-1 KCI; 50 mmoI 1-1 MgCI2; l0 mmol 1 1 dithiothreitol; and 0.25 tool 1 1 sucrose (Sigma, Grade I; added immediately before use). The homogenate was filtered through Miracloth and centrifuged at 20,000 g (20 min). Supernatant (5 ml) was layered over 2 ml 50% (w/v) sucrose in gradient buffer
(40mmoll ltris_HC1, pH8.0; 20mmo11-1 KC1; 10mmoll-X MgC12) and centrifuged at 155,000 gay (90 min). The pellet, containing polysomes and monosomes, was resuspended in 0.5 ml 5% (w/v) sucrose in gradient buffer and layered over i2 ml of a linear sucrose gradient (12.5-50% w/v) above 2 ml of a 60% (w/v) sucrose cushion. This was centrifuged at 130,000 gmax (2 h), and the gradient was pumped through a LKB Uvicord, measuring transmittance at 258 nm. Polysome yield was best when the embryos were homogenised at high pH and high ionic strength (Davies et al. I972), but K + concentrations above 0.4 tool 1-1 appeared to cause dissociation of monosomes. Incubation of the polysome pellet with pancreatic ribonuclease A (2 mg 1 1; 10 rain, 30 ~ destroyed large polysomes and caused a corresponding increase in di- and tri-somes, showing that the profiles represent polysomes and not ribosomal aggregates. Prolonged centrifugation during the preparative stage did not alter the monosome: polysome ratio.
Results
Effect of Temperature on Protein Synthesis. E m b r y o s w e r e i s o l a t e d f r o m seeds i m b i b e d for 16 h at 28 ~ a n d t h e n i n c u b a t e d for 1 h at v a r i o u s t e m p e r a t u r e s . M a x i m u m i n c o r p o r a t i o n o f [ l * C ] l e u c i n e o c c u r r e d at 36 ~ a n d the r a t e o f p r o t e i n s y n t h e s i s at 40 ~ was o v e r l l / z t i m e s the r a t e at 28 ~ (Fig. 1). T h e r a t e o f i n c o r p o r a t i o n was l i n e a r f o r 90 rain at b o t h 28 a n d 41 ~
Protein Synthesis in vitro. C e l l - f l e e e x t r a c t s o f e m b r y o n i c a x e s i m b i b i n g at 28 ~ i n c o r p o r a t e d [ 3 H ] a m i n o acids i n t o p o l y p e p t i d e s w h e n t h e y w e r e i n c u b a t e d with either poly(U) (which codes for the synthesis of polyphenylalanine) or TMV-RNA. Incorporation was n o t s t i m u l a t e d by b a c t e r i o p h a g e M S 2 R N A , a m e s s a g e w h i c h is also i n a c t i v e in w h e a t g e r m cell-free e x t r a c t s ( K l e i n et al. 1972). TMV-RNA-stimulated incorporation continued f o r at least 40 rain (Fig. 2), a l t h o u g h there was s o m e i n d i c a t i o n o f an initial lag ( M a r c u s 1970). I n c o r p o r a t i o n was also l i n e a r l y r e l a t e d to the a m o u n t o f e x t r a c t added. The translation of TMV-RNA was essentially i n i t i a t i o n d e p e n d e n t , since it was i n h i b i t e d by a u r i n t r i c a r b o x y l i c a c i d ( M a r c u s et al. 1970; T a b l e 1). A p r o p o r t i o n o f the e n d o g e n o u s t e m p l a t e a c t i v i t y was also i n h i b i t e d , s u g g e s t i n g t h a t this a c t i v i t y is n o t d u e exclusively to the ' r u n - o f f ' o f p r e f o r m e d p o l y s o m e s . I n c o r p o r a t i o n was r e d u c e d in t h e p r e s e n c e o f c y c l o h e x i m i d e b u t was r e l a t i v e l y i n s e n s i t i v e to c h l o r a m p h e n i col, s h o w i n g t h a t t r a n s l a t i o n o f T M V - R N A o c c u r r e d o n e u k a r y o t i c r i b o s o m e s a n d was n o t d u e to b a c t e r i a l c o n t a m i n a t i o n ( T a b l e 1). T h e e n d o g e n o u s activity o f e x t r a c t s f r o m axes imbibing at 41 ~ was l o w e r , b u t w h e n t h e y w e r e s u p plied w i t h e x o g e n o u s m e s s e n g e r s t h e r a t e o f p o l y p e p t i d e s y n t h e s i s was s i m i l a r to t h a t at 28 ~ (Fig. 3). The products ofTMV RNA-stimulated incorporation w e r e i d e n t i c a l in e x t r a c t s o f axes i m b i b i n g at b o t h
77
G.J.P. Riley: Temperature and Protein Synthesis in Germination T
E 0.3
14000 /
O..
(D
//
12000-
/
E
9
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0.2-
"~ 10000 O
go
////
O
8000
go
"13
P ~6
0.1
u 6000
g
E
k~
X g t.. gO ~o ~6 -= ~-
20
30 40 Temperature
[~
z~O00
2000
50
//
Fig. 1. Effect of temperature on protein synthesis. Embryos were isolated from seeds imbibed at 28 ~ (16 h), and then incubated for 1 h in [14C]leucine. Incorporations are expressed as nmol leucine mg- x protein
/
~ 12000-
/
O
/
/
. . . . . . . . . . . . . . . . . . q3- ........... ....... D
10
o 8000-
/
o
9"o
~,
/
/
/
Control +20 gmol 1 ~ cycloheximide + 20 gmol 1 - 1 chloramphenicol + 100 gmol 1 1 aurintricarboxylic acid
/
6000-
o 4000.
i
/
/
4'0
/ /I
/
9
9
C
/
/
3'0 [mini
Table 1. Effect of inhibitors on activity of cell-free extract of maize axes
9
~,mo0o-
20
Time Fig. 3. Time course of [3H] amino acid incorporation by a cell-free extract of embryos imbibed at 41 ~ (16 h). Details as for Fig. 2
9/ /
Endogenous activity (% of control)
TMV R N A Stimulated activity (% of control)
100 19 67 53
100 5 117 14
///
20000
/
9
lb
Incorporation was measured after 30 rain at 28 ~
2'0
3'0 [mi'n]
/~0
Time Fig. 2. Time course of [3H]amino acid incorporation by a cell-free extract of embryos imbibed at 28 ~ (16 h). Extracts were incubated with [3H]phenylalanine and poly(U) (o), or [3H]leucine with ( - ) or without (El) TMV-RNA. All points are the averages of 2 experiments, each in duplicate, and are expressed as cpm E26~0. Figures for T M V - R N A and poly(U)-stimulated incorporation are minus endogenous template activity. Poly(U)-stimulated incorporations are expressed as cpm E26 ~ - 10-1
cell-free extract, but they were able to demonstrate the presence of a specific TMV coat peptide, suggesting that TMV-RNA is faithfully translated by extracts of cereal embryos. The failure of these extracts to synthesise coat protein when supplied with TMVRNA is presumably related to the observation that only a small part of the chain is translated in vivo (Lodish 1976).
Polysome Profiles. Polysomes were isolated from 28 and 41 ~ (Fig. 4). Polypeptides with molecular weights up to at least 65,000 were synthesized, and major bands were present with mobilities corresponding to molecular weights of 32 and 36,000. There was no indication of a band corresponding to TMV coat protein (molecular weight=IT,400). Roberts etal. (1973a) also found a mixture of products, including two major bands at 30 to 35,000 molecular weight, when TMV-RNA was translated by a wheat germ
seeds imbibing at 28 and 41 ~ and analysed by density gradient centrifugation. The proportion of large polysomes increased throughout the first 2 days of germination at 28 ~ (Fig. 5), with a similar time course to that described for imbibing wheat seeds (Tateyama et al. 1978). Spiegel and Marcus (1975) found that large polysomes predominated after only 40 min imbibition of wheat embryos, but this is probably due to the faster rates of H20 and Oz uptake by isolated embryos.
78
G.J.P. Riley: Temperature and Protein Synthesis in Germination
Top
Bottom
E ul
/,,1~ 16h
28~ 24h
41~ 24 h
28~ 48 h
Fig. 5. Polysome profiles of embryos from seeds imbibing at 28 and 41 ~ Each profile was analysed in duplicate
Fig. 4A and B. Fluorographs of the polypeptides coded for by TMV-RNA in cell-free extracts of seeds imbibing at 28 (A) and 41 ~ (B). The molecular weight markers were bovine serum albumin, ovalbumin, lactate dehydrogenase subunits and equine cytochrome c
At 41 ~ however, there were very few polysomes throughout imbibition (Fig. 5). This difference is unlikely to be due to degradation by ribonuclease during extraction, since endolytic ribonuclease activity (measured by the method of Davies and Larkins 1974) was similar in extracts of embryos imbibing at both 28 and 41 ~
Discussion
Studies on animal cells and microorganisms (reviewed by Bernstam 1978) have indicated that, in general, translation is the step of protein synthesis which is most sensitive to external influences, including high temperatures. Transferring plasmodia of the slime mould Physarum polycephalum to 40 ~ for 10 min inhibited protein synthesis and caused polysome disaggregation (Schiebel et al. 1969). This was not due
to ribonuclease action, since when the cells were returned to favourable temperatures the polysomes reformed, even in the presence of inhibitors of RNA synthesis (Brewer 1972). A similar disaggregation of polysomes has been observed when ripening pears are transferred to 40 ~ (Romani and French 1977). Aminoacyl-tRNA synthetases and other factors involved in the initiation and elongation of polypeptides may also be temperature sensitive in animals and microorganisms (Bernstam 1978). Some of these factors may be temperature-labile in plant cells too, since the polypeptide elongation factors of cereal embryos are sensitive to the conditions under which the seeds are stored (Roberts et al. 1973b). In bacterial cells RNA synthesis exhibits much higher thermostability than DNA or protein synthesis, but rRNA synthesis is inhibited at 42-44 ~ in several mammalian cell lines (Bernstam 1978). Inhibition of RNA synthesis has also been observed when imbibing maize seeds are exposed to 46 ~ for 5 h (Fransolet et al. 1979), while Feierabend and Schrader-Reichhardt (1976) reported that the formation of active chloroplast ribosomes was prevented when winter rye was grown at 32 ~
G.J.P. Riley: Temperature and Protein Synthesis in Germination
During a short (1 h) incubation the optimum temperature for protein synthesis by isolated maize embryos was 36 ~ and the rate was considerably higher at 41 than at 28 ~ Since incorporation continued for at least 2 h at 41 ~ the low rate of protein synthesis in seeds imbibed at this temperature is unlikely to be due to heat-inactivation of any components of the protein-synthesising apparatus. The rate of polypeptide synthesis in vitro was comparable in extracts of seeds imbibing at 28 and 41 ~ when exogenous mRNA was supplied. The lower endogenous template activity in the 41 ~ extract reflects the lower rate of protein synthesis in vivo in seeds imbibing at this temperature, and the higher poly(U)-stimulated activity in this extract is probably due to the larger number of free ribosomes. Translation of poly(U) requires two elongation factors, responsible for the binding of aminoacyl-tRNA to the ribosomes and the translocation of peptidyl-tRNA from the ribosomal acceptor site to the donor site, while the translation of TMV-RNA additionally requires certain initiation factors (Seal et al. 1972; Ciferri 1975). A ribosome dissociation factor is also present in cereal embryos (Russell and Spremulli 1979). All these factors must be functional in the embryonic axes of seeds imbibing at both 28 and 41 ~ since TMV-RNA is translated as specific polypeptides by a process which is sensitive to aurintricarboxylic acid. Dry cereal embryos contain all the components necessary for translation, since extracts are able to synthesise proteins in vitro when supplied with mRNA. These components must be stable during imbibition at high temperature, or they must be renewed at 41 ~ because the rate of polypeptide synthesis was similar in extracts of seeds which had imbibed at either temperature. The increasing number of large polysomes in embryos imbibing at 28 ~ reflects the rising rate of protein synthesis in the seeds. Since protein synthesis continues for at least 2 h at 41 ~ the low number of polysomes in seeds imbibed at this temperature cannot be due to heat-induced polysome breakdown. Furthermore the rate of protein synthesis in vitro is stimulated by the addition of mRNA, so it seems likely that many of the ribosomes present in the monosome peak are potentially active and that the reason for the low rate of protein synthesis in seeds imbibing at 41 ~ is the absence of active mRNA. The large number ofmonosomes in embryos imbibing at both temperatures, together with the stimulation of protein synthesis in vitro by exogenous mRNA, suggests that mRNA activity is the factor which regulates the rate of protein synthesis in germinating maize embryos. Although dry cereal embryos probably contain
79
some long-lived mRNA which is translated during imbibition (Payne 1976; Cuming and Lane 1979), this turns over quite quickly and newly synthesised messengers are required to support further protein synthesis (Caers et al. 1979; Cheung et al. 1979). Synthesis of ribosomal and messenger RNA commences as soon as cereal embryos begin to rehydrate (Sen et al. 1975; Spiegel et al. 1975; Payne 1977), and synthesis of mRNA precursors has also been observed at an early stage of the imbibition of maize seeds (van de Walle and Deltour 1974; van de Walle et al. 1976). Although Fransolet et al. (1979) have observed inhibition of rRNA synthesis in maize seeds heatshocked at 46 ~ the experiments reported here suggest that the major reason for the failure of maize seeds to germinate at continuous high temperatures is the inhibition of the synthesis or processing of mRNA. I wish to thank Dr. J.L. Stoddart, head of the Department of Plant Biochemistry, and Dr. H. Thomas for their advice, and Professor J.P. Cooper, F.R.S., Director of the Welsh Plant Breeding Station, for his interest in this project. The assistance of Denise Mills is once again gratefully acknowledged. This research was financed by a grant from the U.K. Overseas Development Administration.
References Bernstam, V.A. (1978) Heat effects on protein biosynthesis. Ann. Rev. Plant Physiol. 29, 25-46 Brawerman, G (1974) The isolation of messenger RNA from mammalian cells. In: Methods in enzymology, vol. 30, pp. 605-612, Colowick, S.P., Kaplan, N.O., eds., Academic Press, New York Brewer, E.N. (1972) Polysome profiles, amino acid incorporation in vitro and polysome reaggregation following disaggregation by heat shock through the mitotic cycle in Physarum polycephalure. Biochim. Biophys. Acta 277, 639-645 Caers, L.I., Peumans, W.J., Carlier, A.R. (1979) Preformed and newly synthesized messenger RNA in germinating wheat embryos. Planta 144, 491-496 Cheung, C.P., Wu, J., Suhadolnik, R.J. (1979) Dependence of protein synthesis on RNA synthesis during the early hours of germination of wheat embryos. Nature 277~ 66 67 Ciferri, O. (1975) Mechanism of protein synthesis in higher plants. In: The chemistry and biochemistry of plant proteins, pp. 113135, Harborne, J.B., van Sumere, C.F., eds., Academic Press, New York Cuming, A.C., Lane, B.G. (1979) Protein synthesis in imbibing wheat embryos. Eur. J. Biochem. 99, 217-224 Davies, E., Larkins, B.A. (1974) Polysome degradation as a sensitive assay for endolytic messenger ribonuclease activity. Anal. Biochem. 61, 155-164 Davies, E., Larkins, B.A., Knight, R.H. (1972) Polyribosomes from peas. An improved method for their isolation in the absence of ribonuclease inhibitors. Plant Physiol. 50, 581-584 Feierabend, J., Schrader-Reichhardt, U. (1976) Biochemical differentiation of plastids and other organelles in rye leaves with a high-temperature-induced deficiency of plastid ribosomes. Planta 129, 133-145
80 Fransolet, S., Deltour, R., Bronchart, R., van de Walle, C. (1979) Changes in ultrastructure and transcription induced by elevated temperature in Zea mays embryonic root cells. Planta 146, 7 18 Klein, W.H., Nolan, C., Lazar, J.M., Clark, J.M. (1972) Translation of satellite tobacco necrosis virus ribonucleic acid. I. Characterization of in vitro procaryotic and eucaryotic translation products. Biochemistry 11, 2009-2014 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-684 Laskey, R.A., Mills, A.D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341 Lodish, H.F. (1976) Translational control of protein synthesis. Ann. Rev. Biochem. 45, 39-72 Mans, R.J., Novelli, G.D. (1961) Measurement of the incorporation of radioactive amino acids into protein by a filter-paper disk method. Arch. Biochem. Biophys. 94, 48-53 Marcus, A. (1970) Tobacco mosaic virus ribonucleic acid-dependent amino acid incorporation in a wheat embryo system in vitro. Analysis of the rate-limiting reaction. J. Biol. Chem. 245, 955-961 Marcus, A., Bewley, J.D., Weeks, D.P. (1970) Aurintricarboxylic acid and initiation factors of wheat embryo. Science 167, 1735 1736 Payne, P.I. (1976) The long-lived messenger ribonucleic acid of flowering-plant seeds. Biol. Rev. 51, 329-363 Payne, P.I. (1977) Synthesis of poly (A)-rich RNA in embryos of rye during imbibition and early germination. Phytochemistry 16, 431-434 Riley, G.J.P. (1981) Effects of high temperature on the germination of maize (Zea mays L.). Planta 151, 68 74 Roberts, B.E., Mathews, M.B., Bruton, C.J. (1973a) Tobacco mosaic virus RNA directs the synthesis of a coat protein peptide in a cell-free system from wheat. J. Mol. Biol. 80, 733-742 Roberts, B.E., Payne, P.I.0 Osborne, D.J. (1973b) Protein synthesis and the viability of rye grains. Biochem. J. 131, 275-286
G.J.P. Riley: Temperature and Protein Synthesis in Germination Romani, R., French, K. (1977) Temperature-dependent changes in the polysomal population of senescent (ripening) pear fruit. Plant Physiol. 60, 930-932 Russell, D.W., Spremulli, L.L. (1979) Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ. J. Biol. Chem. 254, 8796-8800 Schiebel, W., Chayka, T.G., DeVries, A., Rusch, H.P. (1969) Decrease of protein synthesis and breakdown of polyribosomes by elevated temperature in Physarum polycephalum. Biochem. Biophys. Res. Commun. 35, 338 345 Seal, S.N., Bewley, J.D., Marcus, A. (1972) Protein chain initiation in wheat embryo. Resolution and function of the soluble factors. J. Biol. Chem. 247, 2592-2597 Sen, S., Payne, P.I.~ Osborne, D.J. (1975) Early ribonucleic acid synthesis during the germination of rye (Secale cereale) embryos and the relationship to early protein synthesis. Biochem. J. 148, 381-387 Spiegel, S., Marcus, A. (1975) Polyribosome formation in early wheat embryo germination independent of either transcription or polyadenylation. Nature 256, 228-230 Spiegel, S., Obendorf, R.L., Marcus, A. (1975) Transcription of ribosomal and messenger RNAs in early wheat germination. Plant Physiol. 56, 502-509 Tateyama, M., Ishikawa, H.A.. Ishikawa, K. (1978) Nucleic acid contents and polyribosome formation in wheat embryos during germination and vernalization. Plant Cell Physiol. 19, 411-418 Tomlinson, J.A., Shepherd, R.J., Walker, J.C. (1959) Purification, properties and serology of cucumber mosaic virus. Phytopathology 49, 293-299 van de Walle, C., Deltour, R. (1974) Presence of heterodisperse nuclear RNA in a plant: Zea rnays. FEBS Lett. 49, 87 91 van de Walle, C., Bernier, G., Deltour, R., Bronchart, R. (1976) Sequence of reactivation of ribonucleic acid synthesis during early germination of the maize embryo. Plant Physiol. 57, 632639
Received 3 July; accepted 3 September 1980
Planta 151, 75-80 (1981)
9 Springer-Verlag 1981
Effects of High Temperature on Protein Synthesis During Germination of Maize (Zea mays L.) G r a h a m J. P. Riley Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth SY23 3EB, Dyfed, U.K.
Abstract. The poor germination of maize seeds at high temperatures ( > 37 ~ is related to the low rate of protein synthesis by the embryo. The apparatus of translation was not heat-labile when embryos were incubated for 2 h at 41 ~ and cell free extracts from seeds imbibed for 16 h at this temperature were able to translate exogenous m R N A , indicating that ribosomes and other subcellular components were present and functional. Analysis of polysome profiles from embryos imbibing at high temperature indicated that the low rate of protein synthesis was due to the nonavailability of active m R N A . Key words: Embryos (protein synthesis) - Germination (seeds) - Protein synthesis and temperature - Temperature and protein synthesis - Z e a . Introduction
Germination of many maize cultivars is severely inhibited above about 37 ~ This is related to the low rate of protein synthesis in the embryos of seeds imbibing at high temperatures; the rate of incorporation of [14C]leucine by the embryos of seeds imbibing at 41 ~ was less than one third of the rate in seeds imbibing at 28 ~ (Riley 1981). There are several possible reasons for the low rate of protein synthesis in seeds imbibing at high temperature : (i) the energy state of the embryos could be too low to support anabolic reactions; (ii) the supply of amino acids could be limiting; (iii) the rate of R N A synthesis could be lower at high temperature; Abbreviations." ATP=adenosine-5'-triphosphate; EDTA =ethylen-
ediaminetetra-acetic acid; GTP = guanosine-5'-triphosphate ; HEPES = N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid; Poly (U) = polyuridylic acid; PPO =2,5-diphenyloxazole; SDS = sodium lauryl sulphate; TMV = tobacco mosaic virus
(iv) the rate of R N A turnover could be higher at high temperature; (v) the processing of R N A molecules or their release from the nucleus could be inhibited; (vi) the assembly of ribosomes or polysomes, or their attachment to membranes could be disrupted; (vii) there could be a deficiency of some of the enzymes and other factors involved in polypeptide synthesis; (viii) some of these enzymes etc could be heatlabile. Possibilities (i) and (ii) may be excluded on the basis of previous results, which showed that energy metabolism proceeds normally at temperatures which severely depress the rate of protein synthesis, and that this is not increased by incubating embryos with an excess of amino acids. Furthermore, the activity of ribonuclease is significantly lower in seeds imbibing at 41 ~ (Riley 1981), indicating that (iv) is also unlikely. The experiments reported below were undertaken in order to test some of the other possibilities. The activity of cell-free extracts from seeds imbibing at 41 ~ was assayed in order to test the ability of isolated ribosomes, enzymes and other factors to synthesize proteins when supplied with exogenous m R N A , and the polysome profiles of imbibing seeds were analysed to determine whether active m R N A was present in the cytoplasm.
Material and Methods Materials'. Maize seeds cv. Marls Jade were purchased from Hurst
Gunson Cooper Taber, Colchester, Essex, U.K. They were imbibed on filter paper moistened with 1 mmol 1 i streptomycin sulphate or sterile water. Tobacco mosaic virus was prepared from the leaves of infected Nicotiana tabacum plants by precipitation with n-butanol (Tomlinson et al. 1959), and RNA was extracted with SDS and phenol buffered with 5 mmol 1-1 EDTA, pH 7.3 (Roberts et al. 1973a;
0032-0935/81/0151/0075/$01.20
76
G.J.P. Riley: Temperature and Protein Synthesis in Germination
Brawerman 1974). It was dialysed against distilled water for 6 h and stored in liquid N 2. All glassware was oven-sterllised before use, and solutions were autoclaved or filtered, and stored frozen.
Protein Synthesis. The rate of protein synthesis by whole embryos was determined by incubating them with U-[l*C]leucine as described elsewhere (Riley 1980). Polypeptide synthesis in vitro was measured in cell-free extracts from seeds which had imbibed for I6 h. Isolated axes were frozen in liquid N2, crushed, and homogenised in an equal volume (w/v) extraction buffer (20 mmol 1-2 HEPES, 0.1 mol 1-1 KC1; 1 mmol 1-1 MgAc2; 2mmol 1 1 CaC12; 7 mmol 1-1 dithiothreitol, adjusted to pH 7.6 with KOH). The homogenate was centrifuged (I 7,000 g, 12 min), and the supernatant was loaded onto a column of Sephadex G-25 equilibrated with 20 mmol 1-1 HEPES-KOH, pH 7.6; 0.1 mol 1 1 KCI; 3 mmol 1 1 MgAc2 and 1 mmol 1 - 1 dithiothreitol. Fractions were collected and the most turbid were pooled and recentrifuged (17,000g, 20 rain). The supernatant was used immediately after preparation, although it retained most of its activity when stored in liquid N2. Polypeptide synthesis was measured in a final volume of 50 gl, containing (final concentrations) 14 mmol 1-1 HEPES-KOH, pHT.9; 3 0 m m o l i - 1 K A c ; 2 m m o l l - l M g A % ; ( l m m o l 1 - 1 MgA% when TMV-RNA was used); 1 mmol 1-~ ATP; 0.1 mmol 1 i GTP; 8 mmol 1-1 phosphocreatine; 40 mg 1-1 creatine phosphokinase; 0.25 mmol 1-1 spermine; 20 lamol 1-1 of each amino acid except phenylalanine or leucine; and 0.74.109 Bq 1 1 L-[2, 4, 6-3H]phenylalanine (0.77 gmol 1 1) or L-[4, 5-3H]leucine (0.16 ~tmol 1-1). Extract (15gl) and RNA (10/.tg poly(U) or 18/.tg TMV-RNA) were added, and the samples were incubated at 28 ~ Incorporation was determined by spotting 20 gl aliquots onto glass fibre discs, and the hot trichloroacetic acid-insoluble radioactivity was determined by liquid scintillation spectrometry in toluene: Triton: PPO scintillant after washing the filters according to the procedure of Marts and Novelli (1961). Potassium and magnesium concentrations were optimised separately for each message. The rate of reaction was insensitive to pH between 7.4 and 8.2. Polyacry&mideGel Electrophoresis,Cell-free extracts were incubated with TMV-RNA for 30rain, and an equal volume of I25mmol 1-1 tris-HCl, pH6.8, containing 4% (w/v) SDS and 10% (w/v) glycerol, was added. The protein solution was denatured at 100 ~ (5 min), and 0.05 vol. 2-mercaptoethanol was added after it had cooled. The discontinuous gel system employed was based on that of Laemmli (1970). The running get was a 0.7 mm thick sIab of 12.5% polyacrylamide (acrylamide: NN'-methylenebisacrylamide ratio 29.2:0.8), containing 375mmoll l tris-HC1, pH 8.9, and 0.1% (w/v) SDS. The stacking gel was 2 cm long and consisted of 5% polyacrylamide, 150 mmol 1-1 tris-HC1, pH 6.7 and 0.025% (w/v) SDS. The electrode buffer contained 25 mmoll l tris, 190 mmoll ~ glycine, and 0.1% (w/v) SDS. Denatured protein solution (20-30 gl) was introduced into each sample welt, and electrophoresis was carried out at 15 mA per gel for 1 h, followed by 25 mA until the front reached the bottom of the running gel. Radioactive polypeptides were detected by the quantitative fluorographic procedure of Laskey and Mills (1975). Analysis of Polysomes. Embryos (0.75 g) were frozen in liquid
N 2,
crushed, and homogenised in 6 ml 0.1 moI 1-1 tris-HC1, pH 8.0, containing 0.3 mol 1-1 KCI; 50 mmoI 1-1 MgCI2; l0 mmol 1 1 dithiothreitol; and 0.25 tool 1 1 sucrose (Sigma, Grade I; added immediately before use). The homogenate was filtered through Miracloth and centrifuged at 20,000 g (20 min). Supernatant (5 ml) was layered over 2 ml 50% (w/v) sucrose in gradient buffer
(40mmoll ltris_HC1, pH8.0; 20mmo11-1 KC1; 10mmoll-X MgC12) and centrifuged at 155,000 gay (90 min). The pellet, containing polysomes and monosomes, was resuspended in 0.5 ml 5% (w/v) sucrose in gradient buffer and layered over i2 ml of a linear sucrose gradient (12.5-50% w/v) above 2 ml of a 60% (w/v) sucrose cushion. This was centrifuged at 130,000 gmax (2 h), and the gradient was pumped through a LKB Uvicord, measuring transmittance at 258 nm. Polysome yield was best when the embryos were homogenised at high pH and high ionic strength (Davies et al. I972), but K + concentrations above 0.4 tool 1-1 appeared to cause dissociation of monosomes. Incubation of the polysome pellet with pancreatic ribonuclease A (2 mg 1 1; 10 rain, 30 ~ destroyed large polysomes and caused a corresponding increase in di- and tri-somes, showing that the profiles represent polysomes and not ribosomal aggregates. Prolonged centrifugation during the preparative stage did not alter the monosome: polysome ratio.
Results
Effect of Temperature on Protein Synthesis. E m b r y o s w e r e i s o l a t e d f r o m seeds i m b i b e d for 16 h at 28 ~ a n d t h e n i n c u b a t e d for 1 h at v a r i o u s t e m p e r a t u r e s . M a x i m u m i n c o r p o r a t i o n o f [ l * C ] l e u c i n e o c c u r r e d at 36 ~ a n d the r a t e o f p r o t e i n s y n t h e s i s at 40 ~ was o v e r l l / z t i m e s the r a t e at 28 ~ (Fig. 1). T h e r a t e o f i n c o r p o r a t i o n was l i n e a r f o r 90 rain at b o t h 28 a n d 41 ~
Protein Synthesis in vitro. C e l l - f l e e e x t r a c t s o f e m b r y o n i c a x e s i m b i b i n g at 28 ~ i n c o r p o r a t e d [ 3 H ] a m i n o acids i n t o p o l y p e p t i d e s w h e n t h e y w e r e i n c u b a t e d with either poly(U) (which codes for the synthesis of polyphenylalanine) or TMV-RNA. Incorporation was n o t s t i m u l a t e d by b a c t e r i o p h a g e M S 2 R N A , a m e s s a g e w h i c h is also i n a c t i v e in w h e a t g e r m cell-free e x t r a c t s ( K l e i n et al. 1972). TMV-RNA-stimulated incorporation continued f o r at least 40 rain (Fig. 2), a l t h o u g h there was s o m e i n d i c a t i o n o f an initial lag ( M a r c u s 1970). I n c o r p o r a t i o n was also l i n e a r l y r e l a t e d to the a m o u n t o f e x t r a c t added. The translation of TMV-RNA was essentially i n i t i a t i o n d e p e n d e n t , since it was i n h i b i t e d by a u r i n t r i c a r b o x y l i c a c i d ( M a r c u s et al. 1970; T a b l e 1). A p r o p o r t i o n o f the e n d o g e n o u s t e m p l a t e a c t i v i t y was also i n h i b i t e d , s u g g e s t i n g t h a t this a c t i v i t y is n o t d u e exclusively to the ' r u n - o f f ' o f p r e f o r m e d p o l y s o m e s . I n c o r p o r a t i o n was r e d u c e d in t h e p r e s e n c e o f c y c l o h e x i m i d e b u t was r e l a t i v e l y i n s e n s i t i v e to c h l o r a m p h e n i col, s h o w i n g t h a t t r a n s l a t i o n o f T M V - R N A o c c u r r e d o n e u k a r y o t i c r i b o s o m e s a n d was n o t d u e to b a c t e r i a l c o n t a m i n a t i o n ( T a b l e 1). T h e e n d o g e n o u s activity o f e x t r a c t s f r o m axes imbibing at 41 ~ was l o w e r , b u t w h e n t h e y w e r e s u p plied w i t h e x o g e n o u s m e s s e n g e r s t h e r a t e o f p o l y p e p t i d e s y n t h e s i s was s i m i l a r to t h a t at 28 ~ (Fig. 3). The products ofTMV RNA-stimulated incorporation w e r e i d e n t i c a l in e x t r a c t s o f axes i m b i b i n g at b o t h
77
G.J.P. Riley: Temperature and Protein Synthesis in Germination T
E 0.3
14000 /
O..
(D
//
12000-
/
E
9
/
/
//
0.2-
"~ 10000 O
go
////
O
8000
go
"13
P ~6
0.1
u 6000
g
E
k~
X g t.. gO ~o ~6 -= ~-
20
30 40 Temperature
[~
z~O00
2000
50
//
Fig. 1. Effect of temperature on protein synthesis. Embryos were isolated from seeds imbibed at 28 ~ (16 h), and then incubated for 1 h in [14C]leucine. Incorporations are expressed as nmol leucine mg- x protein
/
~ 12000-
/
O
/
/
. . . . . . . . . . . . . . . . . . q3- ........... ....... D
10
o 8000-
/
o
9"o
~,
/
/
/
Control +20 gmol 1 ~ cycloheximide + 20 gmol 1 - 1 chloramphenicol + 100 gmol 1 1 aurintricarboxylic acid
/
6000-
o 4000.
i
/
/
4'0
/ /I
/
9
9
C
/
/
3'0 [mini
Table 1. Effect of inhibitors on activity of cell-free extract of maize axes
9
~,mo0o-
20
Time Fig. 3. Time course of [3H] amino acid incorporation by a cell-free extract of embryos imbibed at 41 ~ (16 h). Details as for Fig. 2
9/ /
Endogenous activity (% of control)
TMV R N A Stimulated activity (% of control)
100 19 67 53
100 5 117 14
///
20000
/
9
lb
Incorporation was measured after 30 rain at 28 ~
2'0
3'0 [mi'n]
/~0
Time Fig. 2. Time course of [3H]amino acid incorporation by a cell-free extract of embryos imbibed at 28 ~ (16 h). Extracts were incubated with [3H]phenylalanine and poly(U) (o), or [3H]leucine with ( - ) or without (El) TMV-RNA. All points are the averages of 2 experiments, each in duplicate, and are expressed as cpm E26~0. Figures for T M V - R N A and poly(U)-stimulated incorporation are minus endogenous template activity. Poly(U)-stimulated incorporations are expressed as cpm E26 ~ - 10-1
cell-free extract, but they were able to demonstrate the presence of a specific TMV coat peptide, suggesting that TMV-RNA is faithfully translated by extracts of cereal embryos. The failure of these extracts to synthesise coat protein when supplied with TMVRNA is presumably related to the observation that only a small part of the chain is translated in vivo (Lodish 1976).
Polysome Profiles. Polysomes were isolated from 28 and 41 ~ (Fig. 4). Polypeptides with molecular weights up to at least 65,000 were synthesized, and major bands were present with mobilities corresponding to molecular weights of 32 and 36,000. There was no indication of a band corresponding to TMV coat protein (molecular weight=IT,400). Roberts etal. (1973a) also found a mixture of products, including two major bands at 30 to 35,000 molecular weight, when TMV-RNA was translated by a wheat germ
seeds imbibing at 28 and 41 ~ and analysed by density gradient centrifugation. The proportion of large polysomes increased throughout the first 2 days of germination at 28 ~ (Fig. 5), with a similar time course to that described for imbibing wheat seeds (Tateyama et al. 1978). Spiegel and Marcus (1975) found that large polysomes predominated after only 40 min imbibition of wheat embryos, but this is probably due to the faster rates of H20 and Oz uptake by isolated embryos.
78
G.J.P. Riley: Temperature and Protein Synthesis in Germination
Top
Bottom
E ul
/,,1~ 16h
28~ 24h
41~ 24 h
28~ 48 h
Fig. 5. Polysome profiles of embryos from seeds imbibing at 28 and 41 ~ Each profile was analysed in duplicate
Fig. 4A and B. Fluorographs of the polypeptides coded for by TMV-RNA in cell-free extracts of seeds imbibing at 28 (A) and 41 ~ (B). The molecular weight markers were bovine serum albumin, ovalbumin, lactate dehydrogenase subunits and equine cytochrome c
At 41 ~ however, there were very few polysomes throughout imbibition (Fig. 5). This difference is unlikely to be due to degradation by ribonuclease during extraction, since endolytic ribonuclease activity (measured by the method of Davies and Larkins 1974) was similar in extracts of embryos imbibing at both 28 and 41 ~
Discussion
Studies on animal cells and microorganisms (reviewed by Bernstam 1978) have indicated that, in general, translation is the step of protein synthesis which is most sensitive to external influences, including high temperatures. Transferring plasmodia of the slime mould Physarum polycephalum to 40 ~ for 10 min inhibited protein synthesis and caused polysome disaggregation (Schiebel et al. 1969). This was not due
to ribonuclease action, since when the cells were returned to favourable temperatures the polysomes reformed, even in the presence of inhibitors of RNA synthesis (Brewer 1972). A similar disaggregation of polysomes has been observed when ripening pears are transferred to 40 ~ (Romani and French 1977). Aminoacyl-tRNA synthetases and other factors involved in the initiation and elongation of polypeptides may also be temperature sensitive in animals and microorganisms (Bernstam 1978). Some of these factors may be temperature-labile in plant cells too, since the polypeptide elongation factors of cereal embryos are sensitive to the conditions under which the seeds are stored (Roberts et al. 1973b). In bacterial cells RNA synthesis exhibits much higher thermostability than DNA or protein synthesis, but rRNA synthesis is inhibited at 42-44 ~ in several mammalian cell lines (Bernstam 1978). Inhibition of RNA synthesis has also been observed when imbibing maize seeds are exposed to 46 ~ for 5 h (Fransolet et al. 1979), while Feierabend and Schrader-Reichhardt (1976) reported that the formation of active chloroplast ribosomes was prevented when winter rye was grown at 32 ~
G.J.P. Riley: Temperature and Protein Synthesis in Germination
During a short (1 h) incubation the optimum temperature for protein synthesis by isolated maize embryos was 36 ~ and the rate was considerably higher at 41 than at 28 ~ Since incorporation continued for at least 2 h at 41 ~ the low rate of protein synthesis in seeds imbibed at this temperature is unlikely to be due to heat-inactivation of any components of the protein-synthesising apparatus. The rate of polypeptide synthesis in vitro was comparable in extracts of seeds imbibing at 28 and 41 ~ when exogenous mRNA was supplied. The lower endogenous template activity in the 41 ~ extract reflects the lower rate of protein synthesis in vivo in seeds imbibing at this temperature, and the higher poly(U)-stimulated activity in this extract is probably due to the larger number of free ribosomes. Translation of poly(U) requires two elongation factors, responsible for the binding of aminoacyl-tRNA to the ribosomes and the translocation of peptidyl-tRNA from the ribosomal acceptor site to the donor site, while the translation of TMV-RNA additionally requires certain initiation factors (Seal et al. 1972; Ciferri 1975). A ribosome dissociation factor is also present in cereal embryos (Russell and Spremulli 1979). All these factors must be functional in the embryonic axes of seeds imbibing at both 28 and 41 ~ since TMV-RNA is translated as specific polypeptides by a process which is sensitive to aurintricarboxylic acid. Dry cereal embryos contain all the components necessary for translation, since extracts are able to synthesise proteins in vitro when supplied with mRNA. These components must be stable during imbibition at high temperature, or they must be renewed at 41 ~ because the rate of polypeptide synthesis was similar in extracts of seeds which had imbibed at either temperature. The increasing number of large polysomes in embryos imbibing at 28 ~ reflects the rising rate of protein synthesis in the seeds. Since protein synthesis continues for at least 2 h at 41 ~ the low number of polysomes in seeds imbibed at this temperature cannot be due to heat-induced polysome breakdown. Furthermore the rate of protein synthesis in vitro is stimulated by the addition of mRNA, so it seems likely that many of the ribosomes present in the monosome peak are potentially active and that the reason for the low rate of protein synthesis in seeds imbibing at 41 ~ is the absence of active mRNA. The large number ofmonosomes in embryos imbibing at both temperatures, together with the stimulation of protein synthesis in vitro by exogenous mRNA, suggests that mRNA activity is the factor which regulates the rate of protein synthesis in germinating maize embryos. Although dry cereal embryos probably contain
79
some long-lived mRNA which is translated during imbibition (Payne 1976; Cuming and Lane 1979), this turns over quite quickly and newly synthesised messengers are required to support further protein synthesis (Caers et al. 1979; Cheung et al. 1979). Synthesis of ribosomal and messenger RNA commences as soon as cereal embryos begin to rehydrate (Sen et al. 1975; Spiegel et al. 1975; Payne 1977), and synthesis of mRNA precursors has also been observed at an early stage of the imbibition of maize seeds (van de Walle and Deltour 1974; van de Walle et al. 1976). Although Fransolet et al. (1979) have observed inhibition of rRNA synthesis in maize seeds heatshocked at 46 ~ the experiments reported here suggest that the major reason for the failure of maize seeds to germinate at continuous high temperatures is the inhibition of the synthesis or processing of mRNA. I wish to thank Dr. J.L. Stoddart, head of the Department of Plant Biochemistry, and Dr. H. Thomas for their advice, and Professor J.P. Cooper, F.R.S., Director of the Welsh Plant Breeding Station, for his interest in this project. The assistance of Denise Mills is once again gratefully acknowledged. This research was financed by a grant from the U.K. Overseas Development Administration.
References Bernstam, V.A. (1978) Heat effects on protein biosynthesis. Ann. Rev. Plant Physiol. 29, 25-46 Brawerman, G (1974) The isolation of messenger RNA from mammalian cells. In: Methods in enzymology, vol. 30, pp. 605-612, Colowick, S.P., Kaplan, N.O., eds., Academic Press, New York Brewer, E.N. (1972) Polysome profiles, amino acid incorporation in vitro and polysome reaggregation following disaggregation by heat shock through the mitotic cycle in Physarum polycephalure. Biochim. Biophys. Acta 277, 639-645 Caers, L.I., Peumans, W.J., Carlier, A.R. (1979) Preformed and newly synthesized messenger RNA in germinating wheat embryos. Planta 144, 491-496 Cheung, C.P., Wu, J., Suhadolnik, R.J. (1979) Dependence of protein synthesis on RNA synthesis during the early hours of germination of wheat embryos. Nature 277~ 66 67 Ciferri, O. (1975) Mechanism of protein synthesis in higher plants. In: The chemistry and biochemistry of plant proteins, pp. 113135, Harborne, J.B., van Sumere, C.F., eds., Academic Press, New York Cuming, A.C., Lane, B.G. (1979) Protein synthesis in imbibing wheat embryos. Eur. J. Biochem. 99, 217-224 Davies, E., Larkins, B.A. (1974) Polysome degradation as a sensitive assay for endolytic messenger ribonuclease activity. Anal. Biochem. 61, 155-164 Davies, E., Larkins, B.A., Knight, R.H. (1972) Polyribosomes from peas. An improved method for their isolation in the absence of ribonuclease inhibitors. Plant Physiol. 50, 581-584 Feierabend, J., Schrader-Reichhardt, U. (1976) Biochemical differentiation of plastids and other organelles in rye leaves with a high-temperature-induced deficiency of plastid ribosomes. Planta 129, 133-145
80 Fransolet, S., Deltour, R., Bronchart, R., van de Walle, C. (1979) Changes in ultrastructure and transcription induced by elevated temperature in Zea mays embryonic root cells. Planta 146, 7 18 Klein, W.H., Nolan, C., Lazar, J.M., Clark, J.M. (1972) Translation of satellite tobacco necrosis virus ribonucleic acid. I. Characterization of in vitro procaryotic and eucaryotic translation products. Biochemistry 11, 2009-2014 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-684 Laskey, R.A., Mills, A.D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341 Lodish, H.F. (1976) Translational control of protein synthesis. Ann. Rev. Biochem. 45, 39-72 Mans, R.J., Novelli, G.D. (1961) Measurement of the incorporation of radioactive amino acids into protein by a filter-paper disk method. Arch. Biochem. Biophys. 94, 48-53 Marcus, A. (1970) Tobacco mosaic virus ribonucleic acid-dependent amino acid incorporation in a wheat embryo system in vitro. Analysis of the rate-limiting reaction. J. Biol. Chem. 245, 955-961 Marcus, A., Bewley, J.D., Weeks, D.P. (1970) Aurintricarboxylic acid and initiation factors of wheat embryo. Science 167, 1735 1736 Payne, P.I. (1976) The long-lived messenger ribonucleic acid of flowering-plant seeds. Biol. Rev. 51, 329-363 Payne, P.I. (1977) Synthesis of poly (A)-rich RNA in embryos of rye during imbibition and early germination. Phytochemistry 16, 431-434 Riley, G.J.P. (1981) Effects of high temperature on the germination of maize (Zea mays L.). Planta 151, 68 74 Roberts, B.E., Mathews, M.B., Bruton, C.J. (1973a) Tobacco mosaic virus RNA directs the synthesis of a coat protein peptide in a cell-free system from wheat. J. Mol. Biol. 80, 733-742 Roberts, B.E., Payne, P.I.0 Osborne, D.J. (1973b) Protein synthesis and the viability of rye grains. Biochem. J. 131, 275-286
G.J.P. Riley: Temperature and Protein Synthesis in Germination Romani, R., French, K. (1977) Temperature-dependent changes in the polysomal population of senescent (ripening) pear fruit. Plant Physiol. 60, 930-932 Russell, D.W., Spremulli, L.L. (1979) Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ. J. Biol. Chem. 254, 8796-8800 Schiebel, W., Chayka, T.G., DeVries, A., Rusch, H.P. (1969) Decrease of protein synthesis and breakdown of polyribosomes by elevated temperature in Physarum polycephalum. Biochem. Biophys. Res. Commun. 35, 338 345 Seal, S.N., Bewley, J.D., Marcus, A. (1972) Protein chain initiation in wheat embryo. Resolution and function of the soluble factors. J. Biol. Chem. 247, 2592-2597 Sen, S., Payne, P.I.~ Osborne, D.J. (1975) Early ribonucleic acid synthesis during the germination of rye (Secale cereale) embryos and the relationship to early protein synthesis. Biochem. J. 148, 381-387 Spiegel, S., Marcus, A. (1975) Polyribosome formation in early wheat embryo germination independent of either transcription or polyadenylation. Nature 256, 228-230 Spiegel, S., Obendorf, R.L., Marcus, A. (1975) Transcription of ribosomal and messenger RNAs in early wheat germination. Plant Physiol. 56, 502-509 Tateyama, M., Ishikawa, H.A.. Ishikawa, K. (1978) Nucleic acid contents and polyribosome formation in wheat embryos during germination and vernalization. Plant Cell Physiol. 19, 411-418 Tomlinson, J.A., Shepherd, R.J., Walker, J.C. (1959) Purification, properties and serology of cucumber mosaic virus. Phytopathology 49, 293-299 van de Walle, C., Deltour, R. (1974) Presence of heterodisperse nuclear RNA in a plant: Zea rnays. FEBS Lett. 49, 87 91 van de Walle, C., Bernier, G., Deltour, R., Bronchart, R. (1976) Sequence of reactivation of ribonucleic acid synthesis during early germination of the maize embryo. Plant Physiol. 57, 632639
Received 3 July; accepted 3 September 1980
