As reported last year (MGCNL 59:77, 1985), selected agrichemicals may elicit a variety of responses in five day old maize plumules and/or radicles, as measured by 35S-methionine uptake and protein synthesis. We have extended this study to examine other possible stress inducing factors.
Maize 5 day old seedlings were treated with one of four heavy metals (ZnSO4, PbNO3, CUSO4, CdCl2) or salt (NaCl). Each heavy metal or salt treatment ranged in concentration from 2x10-8M to 5x10-1M. Control seedlings were placed in double-distilled water. Plumules or radicles were treated for three hrs. Proteins were extracted, precipitated with TCA, and incorporated radioactivity was measured using a liquid scintillation counter (C. L. Baszczynski et al., Can. J. Biochem. 60:569-579, 1982). A dose response was seen as both radicles and plumules showed increasing incorporation of 35S-methionine into protein with decreasing concentrations of the factor. Copper sulphate and cadmium chloride showed the greatest inhibitory effect on 35S-methionine incorporation. Least inhibition was seen with sodium chloride.
Two dimensional IEF-SDS PAGE and fluorography were carried out at the highest non-toxic concentration of treatment using standard techniques (C. L. Baszczynski et al., op. cit.) to examine the impact of the treatments on protein synthesis. Preliminary analysis of the fluorograms showed some differences between treated plumules or radicles and controls. Radicles and plumules treated with 2x10-3M cadmium chloride and radicles treated with 1x10-1M zinc sulphate showed synthesis of apparent heat shock or "stress" proteins (hsps) of molecular weight 18, 23, 73, 76, 84, and 89 kD. Radicles treated with 1x10-1M PbNO3 showed an increase in synthesis of higher molecular weight hsps and possibly hsps of 18 and 23 kD. Treatment of plumules with 1x10-1M zinc sulphate or 2x10-3M copper sulphate did not induce a stress response. Treatment of radicles and plumules with varying concentrations of sodium chloride did not alter the protein pattern as compared to controls.
Since the pH of the heavy metal solutions used as treatments ranged from 3.4 to 6.1, it was possible that pH alone could be responsible for the change in the polypeptide patterns observed on fluorograms of 2D gels. To examine this possibility further, the plumule/radicle system was treated with an assortment of organic and inorganic buffers ranging in pH from 3.0 to 8.8. As compared to the dd H2O controls, the incorporation of 35S-methionine into protein was inhibited to some extent by all buffer solutions, although the inhibition was not as great as that seen with the heavy metals. No significant differences in incorporation were observed between buffers across the range of pH's for both plumules and radicles. Examination of fluorograms showed no major differences in polypeptide patterns among any buffer treatments and controls (dd H2O), for radicles and plumules.
These results suggest that differences in the polypeptide pattern as visualized by 2D IEF-SDS PAGE and fluorography that were seen between treatments and controls were due to the treatments alone, and not due to changes in the pH of the solutions within the ranges tested. Thus high salt concentration or inorganic and organic buffers of varying pH's do not seem to significantly alter polypeptide patterns with respect to controls. However, from preliminary analysis it appears that treatment of radicles or plumules with some heavy metals will elicit varying degrees of a heat shock or "stress" response.
A. Gullons, A. Mackenzie and D. B. Walden
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