A rapid upward shift in temperature, or heat shock, inhibits normal protein synthesis and induces the new synthesis of proteins called heat shock proteins (HSP). Corn seedlings synthesize a set of HSP in response to short exposure to high temperature (Baszczynski et al., Can. J. Biochem. 60:569, 1982). Most studies on heat shock in corn as well as other higher plants have utilized short (1-4 hr) heat shock incubations and detection of HSP by fluorography of newly synthesized proteins. HSPs accumulate to 10% of total protein detectable in Drosophila embryos after 10 hr heat shock incubation (Moran et al., Phil. Trans. Roy. Soc. Lond. B. 283:391, 1978). We have investigated the effect of long term heat shock on synthesis of HSP in corn. Oh43 seeds were germinated for 4-5 days and subjected to heat shock of 41C in an incubator under high moisture conditions for varying lengths of time (2, 4, 6, 8, 10, 12, 18, 24 hr). Proteins were extracted according to Baszczynski et al. and separated by SDS-PAGE. After 10-12 hr of heat shock treatment (27C to 41C) we found significant accumulation of higher molecular weight HSP (85K to 70K proteins). By 24 hours the HSPs corresponded to 10-15% of total protein detectable by Coomassie blue staining. Our results directly contradict the published results of Cooper and Ho (Plant Physiol. 71:215, 1983). These investigators report that HSPs do not accumulate in excised corn roots in response to various intervals of heat shock. There may be an important difference between excised roots and intact seedlings in terms of accumulation of proteins. Newly synthesized proteins were also prepared by subjecting seedlings to various intervals of heat shock and subsequently labelling for 2 hr with 35S-methionine. Proteins were extracted and separated as described above, and fluorographs compared for detection of newly synthesized proteins. New HSP synthesis occurs up to 18 hr, with no detectable protein synthesis at 24 hr. This indicates that proteins accumulated at 24 hr are previously synthesized and stable.
Thermotolerance is the proposed function of HSPs. Pretreatment of soybean seedlings at a high incubation temperature (40C) for a short period of time (10 to 20 min) provides protection from an otherwise lethal exposure to 45C (Lin et al., Plant Physiol. 74:152, 1984). We have investigated the role of HSP in the induction of thermotolerance in corn by subjecting seedlings to various incubation time and temperature regimes. A 2 hr exposure of 45C is lethal to 5 day Oh43 seedlings. However, if seedlings are incubated at 41C for periods of not less than 2 hr and not more than 6 hr (heat shock) and then exposed to the lethal temperature for 2 hr, the seedlings survive to grow. Growth is severely inhibited but seedlings will eventually grow to maturity. Longer heat shock times (greater than 6 hr) do not protect the seedlings from lethal temperature. Seedlings incubated at the original temperature after long heat shock incubations (6 to 24 hr) survive to maturity, but growth is inhibited. Measures of growth (seedling length in mm, fresh weight in g, dry weight in g) are all decreased by 40-60% at long heat shock incubations compared to controls. Our conclusion is that accumulation of HSPs in corn seedlings after long term heat shock (10-24 hr) does not correlate with the induction of thermotolerance. This does not preclude a causative role for HSPs in the phenomena but points to a more complex model for induction of proteins, accumulation of HSPs and thermotolerance, at least in corn.
Bruce H. Rockwell, Patricia Lehman and Christine M. Nebiolo
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