Plants synthesize several families of low molecular weight (LMW) heat shock proteins (HSPs) in response to elevated temperatures. We have characterized two cDNAs, HSP18.1 and HSP17.9, that encode members of the class I family of LMW HSPs from pea (Pisum sativum). In addition, we investigated the expression of these HSPs at the mRNA and protein levels during heat stress and recovery. HSP18.1 and HSP17.9 are 82.1% identical at the amino acid level and are 80.8 to 92.9% identical to class I LMW HSPs of other angiosperms. Heat stress experiments were performed using intact seedlings subjected to a gradual temperature increase and held at a maximum temperature of 30 to 42 degrees Celsius for 4 hours. HSP18.1 and HSP17.9 mRNA levels peaked at the beginning of the maximum temperature period and declined rapidly after the stress period. Antiserum against a HSP18.1 fusion protein recognized both HSP18.1 and HSP17.9 but not members of other families of LMW HSPs. The accumulation of HSPI8.1-immunodetected protein was proportional to the severity of the heat stress, and the protein had a half-life of 37.7 ± 8 hours. The long half-life of these proteins supports the hypothesis that they are involved in establishing thermotolerance.Plants respond to elevated temperatures by expressing several families of evolutionarily conserved HSPs2 (12,17,23,31). Unlike yeast and most animals, which produce only one to four LMW HSPs (17), plants synthesize many LMW HSPs, between 12 and 27 different polypeptides, depending on the plant species (E. Vierling, unpublished data) (9,18,20). There are at least four families of nuclear encoded LMW HSPs in higher plants (22,26,31), all of which are members of the eukaryotic LMW HSP gene superfamily (17,23,31 (29), maize (24), petunia (4), and A. thaliana (4). The relationship between these families has not been closely examined at the amino acid level.It has long been known that plants can develop the ability to withstand otherwise lethal HS temperatures, a phenomenon referred to as acquired thermotolerance (12,17,23). Thermotolerance can be induced by several regimens: a previous moderate HS, a gradual temperature increase, a short, severe HS followed by a recovery period, and pretreatment with arsenite (1,12,16

Three related gene families of low-molecular-weight (LMW) heat shock proteins (HSPs) have been characterized in plants. We

Higher plants synthesize small heat-shock proteins (smHSPs) from five related gene families. The class I and II families encode cytosolic smHSPs. We characterized the class II smHSPs of pea (Pisum safivum) and compared them with class I smHSPs. Antibodies against recombinant HSP17.7, a class II smHSP, recognized four heat-inducible 17-to 18-kD polypeptides and did not cross-react with class I smHSPs. On sucrose gradients the class II smHSPs sedimented primarily at 8 Svedberg units, indicating that they are components of large complexes similar in size to class I smHSP complexes. However, the class I and II complexes were readily distinguishable by nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Nondenaturing immune precipitations using anti-HSP17.7 or anti-HSP18.1 (a class I smHSP) antiserum provide further evidence that the class I and II smHSPs exist in HSP17.7 and HSP18.1 formed complexes of sizes similar to those formed in vivo. When these two smHSPs were mixed, denatured with urea, and then dialyzed, the distinct class I and II complexes again formed, each containing only HSP18.1 or HSP17.7. Thus, cytosolic smHSPs from two related gene families expressed simultaneously form distinct complexes in vivo, suggesting that they have subtly different functions. different complexes, composed primarily of smHSPs. Recombinant

Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 250C growth chamber cultivated, or from plants given 38°C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 250C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (420C, 90 minutes) were administered following 1.5, 16, and 24 hours of 250C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 250C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 250C was reached at 17. stresses are endured without leaves and a root system, which help moderate extremes of temperature and water status, respectively, in postemergent plants. The study of heat stress responses during early germination has proven interesting in regard to both of these problems. We found that wheat (Triticum aestivum L.) embryos are able to synthesize a full set of hsps3 from the earliest times of imbibition. Early imbibing embryos also synthesize several hsps which cannot be produced after 12 h of imbibition (1 1). Further, embryos imbibed less than 8 h are resistant to heat stresses that are lethal to embryos imbibed 12 or more h (1). These findings suggest that stress proteins and/or their mRNAs may be present in dry embryos. If present, these proteins or mRNAs might contribute both to the strong hs response and the high thermal tolerance found during early imbibition.In order to learn more about the developmental nature of several wheat hsps, as well as the high thermal tolerance of early imbibed wheat, we studied the protein and mRNA composition of quiescent and early imbibed embryos, as well as the way in which the embryos responded to heat stress during early imbibition. Further, we examined...

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