Effect of long periods of low temperature exposure on protein synthesis activity in wheat seedlings

Plant Science 149 (1999) 59 – 62 www.elsevier.com/locate/plantsci

Effect of long periods of low temperature exposure on protein synthesis activity in wheat seedlings Demeter La´sztity a, Ilona Ra´cz a,*, Emil Pa´ldi b b

a Institute of Plant Physiology, Eo¨t6o¨s Lora´nd Uni6ersity, Budapest, H-1445, POB 330, Hungary Agricultural Research Institute of the Hungarian Academy of Sciences, Marton6a´sa´r, H-2462, POB 19, Hungary

Received 9 February 1999; received in revised form 19 July 1999; accepted 20 July 1999

Abstract Long periods of low temperature exposure induce complex changes in the metabolism of nucleic acids and protein molecules in plants: i.e. new proteins; new tRNA isoacceptors and new mRNAs appear with altered minor nucleotide contents, implying that the components of the protein synthesising system change during cold treatment. To study the effect of changes in the RNA pool on the intensity of protein synthesis, different homologous and heterologous cell-free protein synthesising systems were constructed with polysome fractions and tRNAs isolated from non-treated wheat seedlings and from seedlings cold treated for a long period. The homologous cell-free protein synthesising systems contained polysome fractions from non-treated samples of the wheat cultivar Martonva´sa´ri 15 and from samples treated for 1, 5 or 7 weeks together with their own tRNA. Heterologous systems were constructed from the tRNA fractions of cold-treated seedlings with S23 fractions of non-treated ones and vice versa. Cell-free protein synthesis was carried out at 4 and 30°C. The results demonstrate that independently of the length of the cold period the intensity of protein synthesis in homologous cold-treated systems at 4°C was as high as the intensity of homologous non-treated systems at 30°C. Combinations of cold-treated S23 fractions with cold-treated tRNAs were about 30% more effective than cold-treated S23 fractions with non-treated tRNAs at 4°C, while combinations of cold-treated tRNAs with non-treated S23 fractions resulted in only a slight decrease in activity at 30°C. It can thus be concluded that long-term cold exposure leads to changes in the protein synthesising system, resulting in optimal synthesising capacity under the altered conditions. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cell-free protein synthesis; Wheat; Cold treatment

1. Introduction During the cold acclimation period, complex changes take place in the metabolism of plants. New proteins form due to the effect of low temperature, and these help to adapt the plant to the altered environment [1–3]. The quality and quantity of RNAs are also known to differ under altered physiological conditions [4–6]. As the result of long periods of low temperature, both the tRNA and rRNA pool exhibit characteristic Abbre6iations: S23 fraction, polysome fraction; tRNA, transfer ribonucleic acid; mRNA, messenger ribonucleic acid. * Corresponding author. Tel.: +36-1-2670820/2098; fax: +36-12660240. E-mail address: [email protected] (I. Ra´cz)

changes in addition to the well-known alterations in the mRNA composition. The isoacceptor spectrum of the tRNAs changes, as does the modified nucleotide content of the rRNAs; cold-treated wheat seedlings possess 5.8 S rRNA and 18 +26 S rRNA with altered minor nucleotide composition, i.e. new minor nucleotides appear while others are missing as compared to the non-treated samples [7–9]. The changes in nucleic acid and protein contents imply an alteration in the function and activity of the protein synthesising system resulting in cold acclimation. Since both tRNAs and rRNAs are involved in the process of protein synthesis, the present work aimed at investigating how the altered tRNA and rRNA pools influence the activity of the cell-free

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protein synthesising system. To study the effect of changes in the RNA pool, homologous and heterologous cell-free protein synthesising systems were constructed with polysome fractions and tRNAs isolated from cold treated and non-treated seedlings. Homologous cell-free systems contained polysome fractions from non-treated or coldtreated samples of the wheat cultivar Martonva´sa´ri 15, together with their own tRNA. Heterologous systems were constructed from tRNA fractions of cold-treated seedlings with polysome fractions of non-treated ones and vice versa.

2. Materials and methods

2.1. Plant material and cold treatment Sterilised seeds of the wheat cultivar Martonva´sa´ri 15 were germinated for 72 h on Knop medium containing 1% agar at 23°C and then transferred to 4°C for cold treatment. After 1–7 weeks of cold treatment, total tRNA and S23 fractions were prepared from each sample for the cell-free protein synthesising systems. Seedlings of the same age without cold treatment were regarded as the nontreated control.

2.2. Cell-free protein synthesising system Total RNA fractions were prepared according to the phenol method described by Kirby [10]. The cell-free protein synthesising systems were extracted using the method of Seal [11]. In every case the S23 fractions were used while still fresh.

Every 100 ml of reaction mixture contained 10 mM of each of the 19 unlabelled amino acids, 10 mM 14 C-phenylalanine (2.5 mCi), 40 mg tRNA, 25 ml S23, 20 mM HEPES KOH (pH 7.6), 1 mM ATP, 20 mM GTP, 8 mM creatine phosphate, 4 mg creatine phosphokinase, 2.5 mM dithiothreitol, 1.6 mM Mg(OAc)2, 80 mM spermine, 45 mM KCl and 20 mM KOAc. Incubation lasted for 1 h at 30 or 4°C. The reaction was stopped using 200 ml 16% TCA containing 50 mM phenylalanine and 0.1 mg/ml BSA in 1 mM EDTA, after which 2 ml 5% TCA was added. After standing in ice for 10 min, the mixture was heated to 90°C for 15 min, then kept for a further 10 min on ice before being filtered through a glass fibre filter (Whatman GF/ C). The discs were washed twice with 5% TCA, after which they were measured in 3 ml toluenebased scintillator using a liquid scintillation spectrometer. Data represent mean values of three independent experiments.

3. Results and discussion Metabolic changes during cold treatment imply changes in the protein synthesising system of the plants. In the present experiments the cell-free protein synthesis of non-treated wheat seedlings and of seedlings cold treated for 1, 5 and 7 weeks was studied at low (4°C) and high (30°C) temperatures in order to improve our understanding of these alterations. The results demonstrate that, independently of the cold period, the intensity of protein synthesis in homologous cold-treated systems at 4°C was as high as that of homologous non-treated systems at 30°C, and that homologous

Table 1 Activity of cell-free protein synthesising system in wheat cultivar MV-15 cold treated for 1, 5, and 7 weeksa Cell-free system

1. Homologous systems/with nontreated control S23 and tRNA/ 2. Homologous systems/with coldtreated S23 and cold-treated tRNA/ 3. Heterologous systems/with nontreated S23 and cold-treated tRNA/ 4. Heterologous systems/with coldtreated S23 and non-treated tRNA/ a

72 h control (cpm)

1 week (cpm)

5 weeks (cpm)

7 weeks (cpm)

30°C

4°C

30°C

4°C

30°C

4°C

30°C

4°C

71 850

40 920*

70 980

41 100*

71 560

40 840*

71 720

41 220b

71 680

72 100

70 970

71 430

71 750

72 060

64 980

65 200

65 110

64 840

64 900

65 030

49 930

48 750

48 900

51 200

47 850

50 300

Values are the mean of three independent experiments. Denotes significant difference from the treatment at 30°C at P50.05. Data were analysed by Scheffe’s one-way ANOVA using STATA version 1.5 (Computing Resources, Los Angeles, CA). b

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systems incorporated amino acids with greater efficiency than heterologous ones (Table 1). This phenomenon can probably be explained as the joint effect of a number of biochemical and molecular biological changes occurring at low temperature. Protein synthesis can be regulated at the translational level by the availability of certain tRNA isoacceptors, whose anticodon is able to read a codon present in the mRNAs which are to be translated in a given tissue at a given stage of differentiation. Earlier studies furnished experimental evidence in favour of this hypothesis in experiments with animal tissues [12,13]. At low temperature there are changes in the modification of ribosomal RNAs [7], including that of RNA regions involved in the function of the peptidyl transferase centre [14] influencing the conformational properties of RNAs [15] and the rate at which peptide bonds form. The Y-box proteins possibly synthesised at low temperature may also influence the rate of translation and promote protein synthesis under similar conditions [3,16]. The increase in polyamine content as the result of cold treatment stabilises the protein synthesising system [17], which again promotes the synthesis of proteins at low temperature. The present results are in good agreement with these findings. According to our data, cold-treated seedlings incorporate amino acids with higher efficiency than nontreated ones at low temperature. No significant differences could be detected at high temperature. Heterologous systems were also constructed in order to identify the component of cell-free systems responsible for this activity at low temperature. Combinations of cold-treated polysome fractions and non-treated tRNAs at 4°C were about 30% less effective than homologous systems, while combinations of non-treated polysome fractions and cold-treated tRNAs resulted in only a slight decrease in activity. These results are in good agreement with the results of LeMeur [18], who demonstrated that exogenously added tRNA fraction had the greatest effect on the intensity of cell-free protein synthesis. In an earlier article it was proved that the undermodification of the tRNA fraction has a greater influence on the activity of cell-free protein synthesising systems than the undermodification of the polysome fraction [19]. In the present work it was concluded that changes in the tRNA fraction have a greater effect on the activity than changes in the polysome fraction.

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Acknowledgements The authors thank J. To´th for her assistance. This work was supported by grants from the Hungarian National Scientific Research Foundation (OTKA No.I/2 1112 and I/3 140).

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