Environmental stress treatments, such as heavy metal and heat shock treatments, lead to altered gene expression in plants. Different metal-ions have been found to induce some, or all proteins induced by heat shock (heat shock proteins, or hsps) in different plants. The best-characterized environmental response, at the molecular level, is the heat shock response (Vierling, Annu Rev Plant Physiol Plant Mol Biol 42, 1991). Heat shock response regulation has been found at the transcription, RNA processing and translation levels (Lindquist, Ann Rev Biochem 55, 1986). In maize, a group of 18 kDa hsps referred to as the small hsps or shsps are induced when seedlings are exposed to methomyl (Rees et al., Plant Physiol 90, 1989) and metal-ions (Rees, Ph.D thesis. London, Ontario: UWO, 1989).
To detect and localize the mRNA from the 18 kDa hsp genes in metal-ion insulted maize radicles, four-day-old seedlings grown at 27 C were immersed in the insult solution and incubated at 27 C for 3 h. Controls were immersed either in ddH2O and incubated at 27 C or incubated at 27 C for 3 h. The insult solutions were cadmium chloride (CdCl2, 10mM), zinc sulphate (ZnSO4, 100 mM), potassium chloride (KCl, 100 mM), sodium chloride (NaCl, 100 mM), and copper sulphate (CuSO4, 0.1 mM). The radicles of insulted seedlings were fixed (FAA), dehydrated (TBA series), embedded in wax, cut on a microtome, and mounted on slides. DIG-labeled 18 kDa hsp common ORF probe antisense RNA in situ hybridization was carried out as reported above in this volume. Quantitative estimates of hybridization intensity were made from black and white images of longitudinal sections viewed through a microscope via a video monitor and digitized using Northern Exposure Analysis Software (ver. 2.5) (Image Expert, Mississauga, Ontario). Black and white images (255 grey levels) were captured, and average relative video intensities of areas of the overall, the epidermis region, the cortex region, and the stele region of the proximal meristematic region of the root-tip were prepared separately. Duncan's Multiple Range (DMR) tests were performed with the average relative video intensity of overall, epidermis, cortex, and stele region of the meristematic region of the root-tip and are presented in Table 1. Means connected by a bar are not significantly different (p=0.05).< p> Note in the overall area (Table 1a) and cortex regions (Table 1c) of the meristematic regions of root-tips, means of relative video intensity of cadmium, zinc, and potassium insults are not significantly different, but are significantly different from those of copper and sodium insults, control, and water treatments. In the epidermis region (Table 1b), means of relative video intensity of cadmium and zinc are significantly different from those of other insults, water and control treatments. These results indicate that cadmium insult is restricted to the epidermis and cortex regions and zinc insult can be mediated through the epidermis to the stele in the meristematic region of maize root-tips.
Table 1. DMR tests of mean relative video intensity* of overall area (a), epidermis region (b), cortex region (c), and stele region (d) of the meristematic region of the metal-ion insulted maize root-tips.
*Lower mean relative video intensity is attributable to greater concentration of the DIG-labelled antisense RNA in situ hybridization.
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