Characterization of a cDNA encoding an 80kDa HSP and its expression in somatic and male gametophytic tissue
--J. Roger H. Frappier, Robert A. Bouchard, David B. Walden and Burr G. Atkinson

When an organism is exposed to an environmental stress, such as hyperthermia, the organism responds in a very predictable manner. The most striking aspect of the cellular response is the synthesis of a select set of proteins, the so-called heat-shock proteins (HSPs). Until recently, these highly-conserved HSPs were considered to solely protect the cell/organism from the damaging effects of an external stressor. However, an increasing number of reports have demonstrated that the expression of these HSPs is not restricted to a stress response, but their expression also appears to be developmentally regulated (reviewed in Lindquist and Craig, Ann. Rev. Genetics 22:631-637, 1988; Nagao and Key in Culture and Somatic Cell Genetics of Plants, Vol. 6 pp. 297-328, 1989). We have previously reported on the stage-specific expression of the low molecular weight HSPs (Atkinson et al., Dev. Genetics, in press) during microsporogenesis and male gametophyte development. We report here, the isolation and characterization of a partial 80kDa sequence from maize, designated scMHSP80-6-4, and on its expression in somatic and male gametophytic tissue.

A comparison of the 1887 base pairs in scMHSP80-6-4 with the Genbank database revealed that this sequence was in fact related to known HSP 80 genes. This sequence showed 80.6% identity with a rice HSP82A gene (Van Greusegen et al., unpublished) and 74.4% identity with the Arabidopsis thaliana HSP81-1 (Takahaski et al., unpublished). Since scMHSP80-6-4 is a cDNA clone, while the rice and Arabidopsis sequences are genomic clones, the homology was, as expected, restricted to exon 3 of HSP82A and exon 4 of HSP81-1. Further inspection revealed that scMHSP80-6-4 is lacking approximately 500 nucleotides downstream from the putative ATG start codon. A 587 nucleotide XhoI/PstI fragment from scMHSP-6-4 was subcloned into pBluescript II SK- (designated as scMHSP80-6-5) and used in further studies of the maize HSP 80 gene(s).

Northern hybridization analyses with scMHSP80-6-5 of total RNA isolated from radicles and plumules of control (25 C) and heat-shocked (42.5 C) 5-day-old etiolated 0h43 maize seedlings, revealed the presence of a 2.6kb heat-inducible transcript. Furthermore, dot-blot hybridization analyses revealed that transcripts from the HSP 80 gene(s) are present at elevated levels (as compared to the level in control plumule RNA) throughout microspore development with peak accumulation in anthers containing early-to-midprophase I microsporocytes. The detectable amount of HSP 80 RNA is reduced in mature pollen. The developmental regulation of HSP 80 has previously been reported in diverse organisms such as yeast (Kurtz et al., Science 231:1154-1157, 1986) and mammalian male germ cells (S-J. Lee, S-J, Mol. Cell Biol. 10:3239-3242, 1990). It has been shown that the 80kDa gene family is divided into heat-inducible members (HSP 80), as well as developmentally regulated members (heat-shock cognate or HSC 80; Koning et al., Plant Physiol. 100:801-811, 1992).

In order to further characterize the expression of specific 80kDa family members in maize, screening of a genomic library was undertaken. A number of clones have been isolated and are presently under scrutiny. Furthermore, Southern blot hybridization analysis with scMHSP80-6-5 of different maize inbreds was performed in order to determine the number of family members present in this monocot. It is hoped that gene-specific probes for the 80kDa family may be found in order to clearly assess developmental expression during microsporogenesis. 

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors

Return to the MNL 67 On-Line Index
Return to the Maize Newsletter Index
Return to the Maize Genome Database Page