The College of Wooster and Ohio Agricultural Research & Development Center
The University of Western Ontario
Seedling growth, heat shock (HS), and heavy metal-ion insults were performed as reported in the notes cited above, while RNA extraction, filter-binding, preparation of labeled DNA probes, hybridization conditions, scanning, and quantification were as described in Bouchard et al., 1993. Maydica 38: 135-144). Seedlings subjected to heat shock at 40C were shifted to 25C at the end of the 2 hour period and held on damp filter paper until harvest. Seedlings incubated in CdCl2 solution were rinsed in sterile distilled water for 15 minutes with gentle agitation, then shifted to damp filter paper at 25C until harvest. The RNA Dots were probed with the insert fragment from plasmid pscMHSP18-9-2, a sub-clone containing the ORF of clone pMHSP18-9 (map designation uwo11), which is a common probe for mRNAs from all members of the maize shsp gene family. The results, relative to the signals observed after each treatment in radicles harvested immediately after the period of stress set to 100%, are shown in the accompanying graph.
These results reveal an interesting difference in the persistence of relative RNA levels after the two types of stress treatment. Once heat shock ends, shsp18 mRNA levels decline quickly, and are already close to baseline after only two hours. By contrast, the overall levels of shsp18 mRNA remain essentially unchanged for many hours after cadmium insult.
It is possible that substantial amounts of cadmium remain in the tissues of seedlings even after the 15-minute rinse that follows treatment with the metal. However, the perdurance of shsp18 mRNA following cadmium insult would still contrast with what is seen with prolonged heat stress. As reported in Greyson et al. (1996, Developmental Genetics 18: 244-253), under continuous heat shock shsp18 mRNAs essentially decline to baseline levels after eight hours. It is thus likely that the control mechanisms modulating shsp-gene response to metal stress are at least partially distinct from those governing their response to heat stress.
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