The College of Wooster
The University of Western Ontario

Localization of shsp mRNA synthesis in maize spikelets
--Bouchard, RA; Greyson, RI; Walden, DB

As we have shown recently using in situ techniques, heat-induced accumulation of RNA detected by an ORF probe specific for members of the gene family of the small heat-shock protein (shsp) genes of maize is localized to specific cell and tissue-types in the radical and plumule of seedlings (Greyson et al., Devel Genet 18:244-253, 1996). We are now extending these studies to developing spikelets containing anthers whose microspores are in the meiotic prophase or early uninucleate stages of development, since we have previously demonstrated both developmental and heat shock induction of shsp transcript accumulation in such anthers using RNA-dot hybridization (Bouchard et al., Maydica 38:135-144, 1993).

In these initial experiments, field-grown plants of inbred Ohio 43 were used. The tassels of plants at the appropriate stage were heat shocked by maintaining them between 41 C and 44 C in the field for two hours, using a system of plastic jackets containing heated water; a control tassel was harvested from an untreated sibling of the same stage in the same accession. Upon harvest, tassels were fixed immediately in FAA using vacuum infiltration and shaken overnight, then stored in 70% ethanol at -20 C. Spikelets were dehydrated, embedded, and processed for in situ hybridization as in the seedling study.

Based on our initial observations, vigorous and localized shsp transcript accumulation in cross-sections of heat shocked spikelets is seen in both early prophase and late prophase/early uninucleate anthers. The strongest signal is in the dense cytoplasm of the tapetum, but transcript accumulation also appears to occur in the developing microsporocytes, and also to some degree in the immature vascular bundles of the anthers. Interestingly, when the lower-level developmental accumulation that exists in untreated material is examined using spikelets from the control plant, it appears to be localized to the same regions.

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