University of Milan
The heat-shock response of developing kernels (7, 14, 21 days after pollination) was investigated in two genotypes. In order to optimize a procedure for the analysis, different protocols were used for both lines:
a ) A first set of kernels was excised from the cob and singularly incubated at 42 C over a drop of distilled water containing 20 uCi of 35S Methionine.
b ) A second set of kernels from the same cob was injected without excision with an equal amount of labelled amino acid into the endosperm, and the whole cob put at 42 C.
After 3 hrs of treatment kernels from both sets were dissected into scutellum+embryo, endosperm and pericarp. The tissues were extracted and analyzed separately by 1 D SDS PAGE.
Incorporation of the label into proteins was good and more or less the same in the case of both procedures in all tissues from 7 DAP kernels. However, in 14 and 21 DAP embryo and endosperm from excised kernels, incorporation, although sufficient for the analysis, was significantly lower than in those directly injected. This discrepancy was not found in the pericarp.
The hsp pattern showed marked differences depending on the procedure adopted. Excised heat-shocked kernels synthesize the typical set of HSPs in all tissues considered, even though at a very reduced level in the endosperm as compared with scutellum and pericarp. No significant differences in the HSPs synthesized were detected between genotypes or developmental stages. In injected kernels, however, scutellar (+embryo) tissues show a very similar pattern of protein synthesis at 25 and 42 C. Surprisingly, in the early stages of development (7 DAP) this common pattern is typically that of non-shocked tissues, while by 14 DAP it appears to switch to that of heat-shocked tissues. At 3 weeks (21 DAP) the typical heat-shock response is induced by high temperature. Pericarp tissues exhibit the same heat-shock response as those from excised kernels.
The first approach (excised kernels) appears more reliable and will
be adopted for further studies. However, the unusual behaviour of injected
kernels is under investigation.
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