Immunogold localization of the 18-kDa hsp in Zea mays L. radicles
--Denison, SJ; Smith, RJ; Walden, DB

Heat-shock proteins (hsps), induced by temperature shifts and other stresses, are among the most highly conserved proteins. In plant species, the low-molecular weight heat-shock proteins (LMW hsps) are among the most abundant and are highly conserved, in contrast to mammals in which the LMW hsps make up only a small fraction of total hsp content.

Immuno-gold cytochemistry and transmission electron microscopy were used to examine the distribution, subcellular and cellular localization of the 18-kDa hsp in inbred Oh43 heat-shocked and non-heat-shocked radicles. A recombinant protein, from our UWO 10 cDNA clone, produced in the pTrcHisB expression vector, was used to raise polyclonal antibodies (Greyson et al., Dev. Genet. 18:244-253, 1996). These antibodies, specific to the 18-kDa hsp proteins, were used to probe ultra-thin silver-gold sections of radicles. Colloidal gold particles (15 nm) conjugated to goat-anti-rabbit IgG secondary antibodies were employed to detect the presence of antibody-hsp-18 complexes. From these studies, we report five observations:

1) Research has suggested that the Secale cereale L.) leaves (Hon et al., Plant Physiol. 109:879-889, 1995).

2) Observations reveal greater density of labelling in 'thick' cell walls of heat-shocked radicles. We observed that the 'thin' cell walls have gold label at the plasmalemma membrane, whereas in the thicker walls label is observed within the wall. Different biochemical composition of thicker walls may be the cause of this differential labelling.

3) hsp 18-antibody complexes are abundant in cytoplasmically dense cell types relative to less cytoplasmically dense cells (Figure 1). The cytoplasmically dense cell types are those cells typically engaged in rapid cell division and therefore meristematic. It can be seen further that granular bodies, described as heat-shock granules (HSGs) by Nover et al. (Mol. Cell Biol. 3:1648-1655, 1983) have the greatest labelling density within these cells. Heat-shock granules contain RNA synthesized immediately preceding the heat-shock, as well as distinct subsets of HSPs. It is suggested, probably in connection with cytoskeletal elements, that HSGs serve as mRNA storage sites (Nover et al., 1983).

4) Gold labelling to membrane structures (see the companion article below - Denison et al., MNL 71, 1997), provide additional support for the role of the small hsps as 'molecular chaperones'.

5)Companion studies in our lab, using non-radioactive in-situ anti-sense RNA hybridization, to determine the localization of the mRNA for the 18-kDa gene, reveal similar distribution patterns for the 18-kDa hsp mRNA relative to the hsp 18 (Greyson et al., 1996).

In conclusion, localization of the hsp 18-antibody complexes to the intercellular spaces, membraneous whorl structures, thick cell walls, and cytoplasmically dense cells of maize radicles suggest multiple roles for members of the 18-kDa hsp gene family, ranging from the osmotic pressure or osmolyte transport regulation to serving as a component in the storage of mRNAs in the meristematic regions of the root.

Figure 1. Electron micrograph of a heat-shocked radicle, probed with primary antibody and gold labelled, demonstrating the preferential gold labelling of the cytoplasmically dense cell types of the root meristem (x30,000). Note the absence of gold label in the cytoplasmically less dense neighbouring cells (NC). Cell wall (CW), heat shock granules (HSG).


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