We have previously reported on the localization and timing of the accumulation of small heat shock gene transcripts in active meristems and immature vascular bundles of seedling radicle and plumule during heat shock induction (Greyson et al., 1996. Developmental Genetics 18:244-253) as well as the localization of developmentally modulated and heat induced accumulation in spikelets (MNL 71:87, 1997). We now report preliminary results of quantitative RNA-Dot experiments comparing the temperature and timing of heat induction in spikelets and somatic tissues.

Relative induction temperatures were examined in spikelets from the central (microspores in uninucleate stages) and lateral (microsporocytes in prophase and division stages) branches of a tassel of Ohio43, as compared with the somatic tissue of the growing leaves taken from around the same tassel. Samples were incubated for 1 hour each at 30, 33, 35, 37, 39, or 42C in a Robbins Incubator and then snap-frozen in liquid nitrogen for later RNA isolation. RNA Dot-blots were prepared with equal amounts of RNA from these samples, from control samples frozen at harvest , and from post-control samples held at ambient temperature until all incubations were concluded. Duplicate blots were probed with Mhsp18-9-2, a subclone containing the ORF of clone Mhsp 18-9 (map designation uwo11), which is a common probe for mRNAs from all members of the maize shsp gene family, and subclone and Mhsp18-3-3, which is a gene-specific 3'-UTR region for the shsp family member with map designation uwo10. Additional blots were probed for RNAs representing two distinct hsp families: a gene-specific 3'-UTR fragment for hsp82 and an Arabadopsis probe for the highly-conserved hsp100 family. The results with all these probes were qualitatively identical. There is little or no hsp RNA accumulation from any of the three families in leaf samples incubated below 37C, which squares with what we reported earlier for seedlings. For spikelets, however, accumulation is at a peak in the 35C samples, and is already down somewhat in the 37C samples. Figure 1 shows results for the Mhsp18-3-3 probe which illustrate the common pattern. The first row shows the signals seen on RNA from lateral spikelets, the second central spikelets, and the third leaves taken from around the tassel.

Figure 1.

We have also performed a preliminary study of the timing of shsp RNA accumulation in spikelets by freezing an initial sample of spikelets, then incubating an intact plant at heat shock temperature and freezing additional spikelet samples at intervals. In this case, we have so far probed only with the shsp18ORF and 18-3-3 probes. In both cases, the results show a very rapid response in spikelets, with substantial RNA accumulation by 30 minutes, a peak at 1 hour, and a considerable drop-off by 2 hours of heat shock. This contrasts with our earlier observations on radicles (Greyson et al, 1996), where accumulation did not peak until 2 hours. These results are illustrated by an exposure of the shspORF probing as shown in Figure 2, which displays R-Dots at full concentration (5 micrograms) on the first row and a one-fifth dilution on the second.

Figure 2.

Taken together, these results strongly suggest that in addition to producing some heat shock RNAs as part of their normal sequence of development, spikelets are also distinctive in responding to heat shock more rapidly and at lower temperatures than somatic tissues. The possibility for a potential role of hsps in maize male fertility thus remains a continuing focus for our ongoing research.
 
 

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