II. 9. Regulation of protein synthesis by heat shock in barley (H. vulgare L.) (1)
N. Marmiroli*, M. Odoardi Stanca**, B. Giovanelli*, V. Terzi*, F. Tassi*, F.M. Restivo*, and C. Lorenzoni***.
(1) Research work supported by CNR, Italy. Special grant I.P.R.A.-Sub-project
1. Paper N. 102.
* Istituto di Genetica, Universita di Parma (Italy);
** Istituto sperimentale per la Cerealicoltura, Fiorenzuola d'Arda
(Pc) (Italy);
*** Istituto di Genetica vegetale, Universita del S. Cuore, Piacenze
(Italy).
In a wide range of organisms a rapid increase of the incubation temperature determines a dramatic reduction in the synthesis of most normal proteins (heat stroke proteins) and the appearance of novel proteins (heat shock proteins). The heat shock response (HS) has been extensively studied in Drosophila (1, 2, 3) in which the transition from the growth temperature of 25°C to 37°C turns on the synthesis of eight major groups of polypeptides in the molecular weight range of 22,000-80,000 daltons and shuts off most of the normal proteins (4). This pattern of protein synthesis reflects the new regulation induced by thermal stress upon the transcription of several genes, some of which have been recently cloned and partially sequenced (5, 6, 7). At the cytoplasmic level the normal mRNAs are stripped from the ribosomes and replaced by the heat shock mRNAs (8). A similar modification of the protein pattern has been observed in microorganisms such as E. coli (9) and yeast (10).
In higher plants similar changes in the pattern of protein synthesis were observed with the growth temperature risen up to 40°C. Synthesis of heat shock proteins has been identified upon thermal insult in soybean seedlings (Glycine max) (11, 12, 13, 14), in corn seedlings (Zea ways) (12), in tobacco (Nicotiana sylvestris) tissue culture cells (11). The heat shock proteins of soybean seedlings resemble those found in Drosophila with an additional set of 15 polypeptides in the molecular weight range of 15,000-18,000 daltons (14) and a great similarity has been found in the heat shock induced variations of protein synthesis between soybean and corn seedlings (12). The synthesis of the heat shock proteins in plants is due to the synthesis of novel mRNAs which do not appear to be preferentially translated as the heat shock mRNA in Drosophila on the contrary are. Almost ubiquitous some of the heat shock proteins show highly conserved functional domains which are also reflected to a high commonalty in the sequence of the relative DNAs.
We have analyzed the incorporation of 35S-methionine by intact barley seedlings (Hordeum vulgare L. cv "Onice") in the range of temperatures 25-42°C. After 3 h incubation at 42°C the total incorporation was less than 2% of that observed at 25°C. At temperatures of 37-40°C the seedlings incorporated with 40% and 10% efficiencies. Intact plants were therefore labelled at 25-30-34-37-40°C in phosphate buffer (ph 6.5) in the presence of 2% sucrose and after 3 h the labelling was chased and the different organs: roots, caryopses, and hypocotyls, were separated and the proteins extracted and precipitated with ammonium sulphate. The precipitates were dialysed and the proteins separated by SDS-PAG electrophoresis on ultrathin horizontal slab gels casted on adhesive polyester films. After running the gels were Coomassie-stained, prepared for fluorography, dried and exposed to X-ray film at -80°C.
The analysis of the various electrofluorograms showed that the radioactive material was incorporated into several proteins whose presence depended both upon the incubation temperature and upon the organs from which these proteins were derived. The electrofluorograms of SDS-PAGE gels of protein extracted from roots which were labelled at the high temperature, showed the appearance or the enhancement of intensity of several radioactive bands, which corresponded to heat shock proteins and the disappearance or decrease of intensity of other bands which correspond to heat stroke proteins. Heat shock proteins appeared at 30-34°C and several bands were also detectable at 40°C. The most relevant modifications were concerned with species of apparent molecular mass of 78-70, 69, 42, 41, 38 kilodaltons (kd). The heat stroke proteins disappeared mostly at 37°C, such as the 91, 49 and 45 kd species, or at 34°C, such as 96 and 83 kd species. Heat shock proteins of molecular mass around 80, 74, 66, 59, 37 and 35 kd and heat stroke proteins of 90, 71, 56, 50 and 32 kd were also detected in the extracts obtained by separating the seed of labelled plants. In this case most of the heat stroke proteins disappeared at 40°C. The heat shock proteins detected in the extracts of labelled hypocotyle could be present at 40°C such as the 79-71-70 and 60 kd proteins or absent in the 40°C fluorograms such as the 92-87-77-58-40-35 and 16 kd proteins. The protein patterns shown by separated organs which were labelled either by floating or by dipping the sliced tissues in a buffered solution added with 35S-methionine.
The results obtained showed a variety of differences between heat shock response of these organs separated by the whole plants before exposure to high temperatures and the response shown by the same organ isolated from whole plants after exposure at high temperature. Moreover, the sliced tissues labelled with two different methods reported above showed the appearance of different heat shock and heat stroke proteins.
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