Heat shock proteins in mitochondria

Hybrid vigor occurs when the F1 is superior in growth, productivity, resistance to disease, or some other measurable attribute. It has been shown that hybrid corn seedlings manifest hybrid vigor in plumule length and dry weight/germinated seed, as well as seedling protoplast poly A+ RNA content and transcriptional response to the phytohormone gibberellic acid (Nebiolo, 1983, Plant Science Letters 28:195, 1982/83). Several investigators have implicated mitochondria in the generation of hybrid vigor (Sarkissian, Genetics 57:843, 1967). In corn, the inheritance of mitochondrial DNA is strictly maternal (Pring, Genetics 89:121, 1978). However, the maternally derived mitochondria may be affected by a hybrid environment. Mitochondria are the primary target to heat shock stimuli (Leenders, Sub-Cell Biochem. 3:119, 1974). A non-dialyzable, heat-labile activity has been isolated from heat shocked mitochondria which, when injected into non-heat shocked salivary gland cells, induces heat shock puffs (Sin, Nature 258:159, 1975). Isolated mitochondria can be subjected to a cell-free translation system to detect proteins synthesized from mitochondrially-expressed mRNA (Forde, PNAS 75:3841, 1978).

Isolated mitochondria of two inbred (W23 and OH43) and two heterotic hybrid lines (W23 X OH43 and OH43 X W23) of maize (generously supplied by David Walden, Western Ontario University, London, Ontario) incorporate 35S-methionine into acid-insoluble material at significant rates for 90 min at 27 C. Heterosis was observed in plumule length of imbibed pedigree seed stock of the four lines for up to 96 hr. Purified mitochondria from 4-day seedlings were obtained by differential centrifugation and banding through a discontinuous sucrose gradient according to Forde. Respiratory activity was determined by measurement of O2 consumption by a Clarke electrode of mitochondria incubated in an osmotically stable solution containing ADP/succinate as energy source. 35S-methionine incorporation by mitochondria incubated according to Forde was sensitive to 10 ug/ml chloramphenicol and insensitive to 200 ug/ml cyclohexamide. Genotype-specific differences were observed in rate and total amount of incorporation at 90 min, with mitochondria of W23 and W23 X OH43 incorporating 20-30 times the amount of label incorporated by OH43 and OH43 X W23. No effect of hybridity was observed. Pelleted mitochondria were subjected to 15% SDS-PAGE electrophoresis after 30 min of incubation in label mix containing 19 amino acids, 35S-methionine and ADP/succinate. Eighteen to 20 proteins were detectable by fluorography. No qualitative or quantitative differences among lines were observed. Hybrid vigor is not manifested in mitochondrial protein synthesis.

Utilizing the cell-free mitochondrial translation system developed as outlined above, we characterized the maize mitochondrial response to heat shock. Actively respiring purified mitochondria (200-250 ug protein) were incubated for 10 min at 27 C in label mix without 35S-methionine. After this short equilibration, mitochondria were incubated in water baths at either 27 C or 37 C for 10 min, followed by addition of 5 uci 35S-methionine, and were returned to the water baths. The 40 min incubation at 37 C, after a 10 min incubation at 27 C, constitutes a heat shock. Heat shock occurs rapidly; transcripts of heat shock genes are observable within 3-5 min. Sin (op. cit.) incubated salivary gland mitochondria for 30 min prior to injection of supernatant and induction of heat shock puff in non heat-shocked tissues. A significant increase in rate of acid-insoluble 35S-methionine incorporation at 37 C compared to 27 C was observed in this study in all lines.

Mitochondria incubated at both 37 C and 27 C were pelleted (10,000 rpm, 5 min) and dissolved in SDS sample buffer, and an aliquot was taken for measurement of radioactivity and subjected to SDS-PAGE electrophoresis and fluorography. Proteins in the supernatants of pelleted mitochondria were extracted and electrophoresed. Our results include: 1) a significant difference between genotypes in 35S-methionine incorporation at 30 min, similar to acid-insoluble incorporation data discussed above; 2) a significant increase in 35S-methionine incorporation at 37 C compared to 27 C for all lines; 3) a significant amount of 35S-methionine detectable in supernatants of mitochondria incubated at 37 C, not detectable at 27 C; 4) detection of a strongly labelled protein, estimated molecular weight 52 Kdaltons, in fluorographs of electrophoresed mitochondria of all lines incubated at 37 C, not detectable at 27 C; 5) detection of a 52 Kdalton protein in mitochondrial supernatants incubated at 37 C, not detectable at 27 C. This protein is estimated to account for 70-80% of the labelled protein in the supernatant at 37 C, while accounting for only 10-20% of total labelled protein in the intact mitochondria. Synthesis of this protein induced at 37 C is sensitive to chloramphenicol and insensitive to cyclohexamide. This protein may be analogous to a short-lived heat shock protein (50 Kdaltons) observed by Meyer (Plant Phys. 72:26, 1983) in tobacco mesophyll protoplasts. Further investigation may strengthen the implication that this protein is a mitochondrial effector of heat shock gene expression. No qualitative effect of hybridity on proteins synthesized in response to heat shock by mitochondria is evident. Further characterization of this protein, including sensitive quantitation, may provide more information pertaining to the possible differential response of hybrids and inbreds to this stress phenomenon.

Christine M. Nebiolo and Elizabeth M. White
 
 


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