London, Ontario, Canada

University of Western Ontario

Silver staining root-tip sections in maize --Maillet, DS, Walden, DB From our ongoing investigation into the heat shock response in maize, Greyson et al. (1996 Developmental Genetics 18:244-253) reported that the cells in the root-tip which respond to heat shock by an increase in the expression of HSP 18 are located in the first one to three mm. We have observed (Maillet et al. 1999 MNL 73: 67-68) that heat shock can cause changes in the morphology of nucleoli in several cell types. To test the hypothesis that the cells that respond by altering their protein and mRNA expression patterns also exhibit changes in the morphology of their nucleoli, a silver staining method (Howell and Black 1980 Experientia 36:1014-1015) was adapted to stain root-tip sections. Live heat shocked (shifted from 27 C to 43 C for 3h) and control roots from 5 day old Ohio43 seedlings (8 to 10 cm long) were cut into sections approximately 60 um thick with a hand microtome. Sections at 0.5, 1.0, 2.0, and 4.0 mm from the tip including the root cap were fixed (3:1 ethanol to acetic acid) for 24 h. The sections were rinsed in water for 10 min and stained in a mixture of 50 ul of silver nitrate (0.5 g . ml) and colloidal developer (2 % [w/v] gelatin, 10 % [v/v] formic acid) in the dark for 60 min. The sections were rinsed several times in the developer until all of the silver precipitate was removed. The sections were mounted in developer on a microscope slide under a coverslip. Sections were scored for the number of cortex cells that had nucleoli which demonstrated an altered morphology (Table 1.). In Heat shocked material (0.5 mm from the root-tip) nearly all cells had nucleoli which were altered; sections farther from the root-tip had progressively fewer responding cells. Figure 1 presents a photograph of a section, (a) from a heat shocked root (section taken at 1 mm from the root-tip) and (b) a control section. Cells from heat shocked cells have several small stained nucleolar ovoids, controls have one or two spherical nucleoli.

Table 1. Heat shock (3 hr at 43 C)and control sections from Ohio 43 root-tips. n = number of roots, E = epidermis, C = cortex, ED = endodermis, P = pith. The changes in morphology seen in HS cells were not observed in any of the nuclei in the other experiments where the number of nuclei was counted.
 
HS
Respond
No Respond
  n E C ED P E C ED P
0.5 mm 6 200 266 15 102 0 2 0 3
1.0 mm 6 249 249 92 87 14 51 1 16
2.0 mm 5 130 140 20 22 20 110 9 7
4.0 mm 5 0 0 0 0 12 137 0 19
                   
C
Respond
No Respond
  n E C ED P E C ED P
0.5 mm 5 0 0 0 0 200 180 113 110

Figure 1.


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