Corn seedling mitochondria respond to temperature elevation and 50 uM arsenite treatment by the enhanced synthesis in organello of a 52 kD protein. We have defined this protein as a mitochondrial heat shock protein (HSP) (Nebiolo and White, Plant Phys. 79:1129, 1985). We are continuing to investigate the potential role of this protein in the mitochondria, as well as in the cellular heat shock response in light of various lines of evidence implicating the mitochondria as the primary target to heat and chemical stress (Nebiolo and Walden, J. Cell Biol. 79:258a, 1986).

Erythromycin (100 -150 uM) inhibits plastid in organello protein synthesis, while having no significant inhibitory effect on the mitochondrial translation apparatus (Tassi et al., Plant Sci. Lett. 29:215, 1983; Newton and Walbot, PNAS 82:68, 1985). To ensure that our purified etiolated corn seedling mitochondria prepared according to Forde and Leaver (PNAS 77:418, 1980) were not significantly contaminated by plastids, we treated an aliquot of each sample with 100 uM erythromycin. In organello translation products were labelled as previously described (Nebiolo, op. cit.) and subjected to SDS-PAGE/fluorography. Fluorographic profiles of erythromycin-treated and control samples were identical for both control incubation temperature (27C) and heat shock temperature (37C), as well as for mitochondria chemically stressed by 50 uM arsenite treatment. We have concluded that our preparations are not significantly contaminated by plastids and that the 52 kD mitochondrial HSP is not affected by erythromycin treatment. Other controls were run in previous experiments (e.g., measuring amount of bacterial contamination in purified mitochondrial preparations).

We are interested in the potential role of mitochondrial protein synthesis, specifically synthesis of the 52 kD HSP in response to temperature and chemical stress, in seedling growth and seedling thermotolerance. We have grown 3d (27C) corn seedlings (Oh43) for varying lengths of time (24, 48, and 72 hr) in sterile solutions of 100 uM erythromycin, 200 uM erythromycin, 200 uM chloramphenicol (inhibitor of organelle protein synthesis), 10 ug/ml cycloheximide (inhibitor of eukaryotic protein synthesis), and sterile distilled water as a control. Rates of growth, measured as fresh weight/hour of 3d seedlings in the various solutions were all decreased relative to the control. The lowest rate was manifested by seedlings incubated in cycloheximide, the next lowest by 200 and 100 uM erythromycin and the next by chloramphenicol. Similar results were obtained when using 4 and 5d seedlings. Therefore, erythromycin inhibits growth of corn seedlings to an extent greater than chloramphenicol but not as severely as cycloheximide.

To test the effect of erythromycin on the acquisition of thermotolerance (ability to survive an otherwise lethal temperature) we incubated 5d seedlings in the various sterile solutions of erythromycin, chloramphenicol and cycloheximide and at various temperature regimes (27C, control; 45C for 2 hr, lethal treatment; 37C for 2 hr followed by 2 hr at 45C, heat shock and lethal treatment). We found that seedlings in all drugs acquired thermotolerance and growth curves after heat shock were similar to those obtained for controls.

We are investigating the expression of this protein by the mitochondrial genome and its role in the mitochondria during temperature/chemical stress. We have separated membrane and non-membrane fractions of control and heat shocked mitochondria by the method of Boutry et al. (J. Biol. Chem. 258:8524, 1984). By subjecting mitochondrial proteins to SDS-PAGE/fluorography we have localized the 52 kD protein exclusively to the membrane in heat shocked mitochondria. Its role may be to protect membrane components of the respiratory complex from stress. We are currently designing experiments to isolate from the mitochondrial genome the gene coding for the 52 kD protein and to localize the protein in tissue sections using polyclonal antibodies raised against purified protein.

Christine M. Nebiolo and David B. Walden
 
 

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