Plants start to up-regulate the transcription and/or translation of the heat-shock genes and down-regulate most other genes when shifted to temperatures five or more degrees above the optimal growing temperature (Vierling, Ann Rev Plant Physiol Plant Mol Biol 42, 1991). Maize seedlings respond to a brief heat-shock exposure with the synthesis of a variety of hsps, particularly a group of 18 kDa hsps referred to as the small hsps (shsps) and their mRNAs (Baszczynski et al., Can J Biochem 60, 1982; Baszczynski et al., Can J Biochem Cell Biol 61, 1983). Three members of the maize 18 kDa gene family have been characterized and sequenced (Atkinson et al., Genome 31, 1989; Goping et al., Plant Mol Biol 16, 1991), and they bear high identity (over 90%) in their open reading frames (ORFs). One common ORF fragment (18-9-2) can recognize all three genes (Atkinson et al., Dev Genet 14, 1993). Maize hsp induction in response to different growing temperatures, and thermal shifts within and beyond the normal growing temperature, have been studied. The major factors that may affect maize heat-shock response include: 1) the initial growing or "preshift" temperature; 2) the thermal shift increment ; and 3) the duration of the thermal shift (Baszczynski et al., in Changes in Eucaryotic Gene Expression in Response to Environment Stress, Academic Press, New York, 1985). In maize plumules, the 18 kDa hsps are not induced when maize seedlings are shifted from 15 C, 20 C, or 25 C to 25 C, 30 C, or 35 C respectively, but they are synthesized when seedlings are shifted from 30 C to 40 C, and from one of the preshift temperatures (15 C, 20 C, 25 C, or 30 C) to 42 C (Baszczynski et al., 1985).

Antisense RNA in situ hybridization can detect mRNAs of low abundance present only in a few cells and permits localization of specific mRNA transcripts at the cell level. The 18 kDa hsp mRNA localizes to the meristematic regions of root- and shoot-tips, the vasculature, and the young leaves (Greyson et al., Dev Genet 18, 1996).

To determine the threshold temperature for induction of 18 kDa hsp mRNA, four-day-old maize seedlings grown at either 22 C or 27 C were shifted to one of several temperatures (33 C, 35 C, 38 C, 40 C or 43 C), and incubated at that shift temperature for 2 h. Controls were kept at the preshift temperatures. The radicles of "non-thermal shifted" and the "thermal-shifted" seedlings were fixed (FAA), dehydrated (TBA series), embedded in wax, cut on microtome, and mounted on slides. DIG-labeled 18 kDa hsp common ORF (18-9-2) probe antisense RNA in situ hybridization with the Hybaid Omnislide System was carried out as described elsewhere in this contribution. 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 which would include the quiescent zone and the proximal meristem of the root-tip were prepared (Figure 1).

Hybridization signal was observed when the shift temperature was at and above 35 C from either of the preshift temperatures. At 35 C, hybridization was found only in cells near the cap initials and in the immature stele region of the meristem. These results suggest that the threshhold temperature for heat shock induction of 18 kDa hsp mRNA in maize radicles is 35±1 C, and cells near the cap initials and in the immature stele region of the meristem respond to heat shock first .

Fig. 1. Relative intensity of DIG-labelled antisense RNA in situ hybridization of radicles which underwent a thermal shift from one preshift temperature (22 C, black dots or 27 C, open dots) to one of several shift temperatures. Means of five observations are plotted. error bar = standard deviation.

*Lower average relative video intensity is attributable to greater concentration of the DIG-labelled antisense RNA in situ hybridization.

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