Soybean seedlings were subjected to a wide range of physical (abiotic) or environmental stresses. Cloned cDNAs to heat shock (hs)-induced mRNAs were used to assess whether these diverse stresses induced the accumulation of poly(A)RNAs in common with those induced by hs. Northern blot hybridization analyses indicated that a wide range of stress agents lead to the accumulation of detectable levels of several of the hs-induced poly(A)RNAs; the relative concentration of those RNAs 'induced' by the wide range of stress agents (e.g. water stress, salt stress, anaerobiosis, high concentrations of hormones, etc.), was generally in the order of 100-fold lower than that induced by hs. There are two notable exceptions to that pattern of response to the stress agents. First, arsenite treatment resulted in accumulation of the 'hs poly(A)RNAs' to levels similar to those induced by hs. Cadmium also induced a somewhat normal spectrum of the 'hs poly(A)RNAs', but generally lower levels accumulated than in hs- and arsenite0treated tissues. Second, one set of poly(A)RNAs which are present at low and variable levels in control (non-stressed tissue) tissue, and which are increased some 5- to 10-fold by hs, increased in relative concentration in response to a wide range of the stress agents similarly to the response to hs. The physiological significance of the accumulation of this set of poly(A)RNAs (which translate into four electrophoretically different 27 kd proteins) is not known, but they certainly seem to serve as a monitor (or barometer) of physiological stress conditions. Cadmium treatment results in the accumulation of those same poly(A)RNAs and an additional band of higher molecular weight poly(A)RNA homologous to the same hs cDNA clone (clone pCE 54). Ethylene seems to have no obvious causal relationship to the hs response, even though hs-treated seedlings display some symptoms similar to those exhibited by ethylene-treated seedlings.

We determined the DNA sequence and mapped the corresponding transcripts of a genomic clone containing the Gmhsp26-A gene of soybean. This gene is homologous to the previously characterized cDNA clone pCE54 (E. Czarnecka, L. Edelman, F. Schoffl, and J. L. Key, Plant Mol. Biol. 3:45-58, 1984) and is expressed in response to a wide variety of physiological stresses including heat shock (HS). Si nuclease mapping of transcripts and a comparison of the cDNA sequence with the genomic sequence indicated the presence of a single intron of 388 base pairs. Intron removal from pre-mRNA was preferentially inhibited by treatment of soybean seedlings with either CdCl2 or CuS04. Analysis of the 5' termini of transcripts indicated the presence of one major and at least two minor start sites. In each case, initiation occurred 27 to 30 base pairs downstream from a TATA-like motif, and thus each initiation site appears to be promoted by the activity of a separate subpromoter. The three subpromoters are all associated with sequences showing low homology to the HS consensus element of Drosophila melanogaster HS genes and are differentially induced in response to various stresses. Within the carboxyl-terminal half of the protein, hydropathy analysis of the deduced amino acid sequence indicated a high degree of relatedness to the small HS proteins. A comparison of the primary amino acid sequence of hsp26-A with sequences of the small HS proteins suggested that this stress protein is highly diverged and may therefore be specialized for stress adaptation in soybean.The heat shock (HS) response is found in a wide range of organisms and is typically triggered when cells are exposed to hyperthermia or a variety of other stresses (1). In soybean the low-molecular-mass small heat shock proteins (hsps) represent the most abundant class of proteins expressed during HS (23) and include 30 to 50 proteins which range in molecular mass from 15 to 27 kilodaltons (kDa). The lowmolecular-mass family of hsps is more diverse than the high-molecular-mass hsps (68, 70, and 83 kDa) and can be subdivided into at least three subgroups on the basis of size and conservation of amino acid sequences (22,30,40,41).Analysis of hybridization of soybean seedling RNA to cloned HS-specific cDNAs indicates that most representatives of the low-molecular-mass hsps show various degrees of induction by numerous stress agents, but generally at levels much lower than with HS (8; L. Edelman, E. Czarnecka, and J.

The DNA sequence of a gene (Gmhspl7.5-E) encoding a small heat shock protein of soybean, Glycine max, has been determined. Nuclease Si mapping of the 5' terminus of the corresponding RNA indicates that the start site for transcription is located 82 bases upstream from the coding region and 24 bases downstream from a "TATA"-like region (-T-T-T-A-A-A-T-A-). The 5' flanking region of Gmhspl7.5-E contains two imperfect dyads that closely resemble regulatory elements present in the promoters of heat- (5) and genomic clones (unpublished work) indicate that in soybean most members of this class cross-hybridize to various degrees. In addition, some of these cDNAs also share homology with heat shock mRNAs from a variety of other plant species, such as pea, millet, corn, and sunflower (6), as well as tomato and tobacco (unpublished data). The 5' flanking sequences of Drosophila heat shock genes contain a 14-nucleotide dyad that is located upstream from the "TATA"-like region (7). This dyad is essential in the heat induction of transcription of the Drosophila hsp7O gene in monkey cells (7,8) and Xenopus oocytes (9, 10) and is necessary for optimal function of an hsp7O-Adh fused gene in transformed Drosaphila (11). The mechanism of heat activation of the heat shock promoter seems to be highly conserved among animals, since the cloned hsp7O gene of Drosophila is transcriptionally heat-inducible when introduced into a variety of heterologous systems.In this study we report the DNA sequence for one of the most actively expressed small HSP genes in soybean and map the 5' and 3' termini of the RNA product. The 5' flanking region is shown to contain sequences similar to those present in Drosophila heat shock genes. Analysis of the deduced amino acid sequence suggests that the small HSPs found in soybean are not unique to plants but represent a structural and functional class of stress proteins widely dispersed among eukaryotes. (13) by using the 32P-labeled insert of the heat shock-specific cDNA clone pFS2019 (5). The X clone characterized in this study was designated hsE2019. Restriction fragments (see Fig. 1) showing homology to the cDNA pFS2019 were inserted into their respective sites of pBR322 (14) and pUC8 (15) and cloned. MATERIALS AND METHODSTranscript Mapping by Nuclease S1 Hybrid Protection. Total RNA isolation and poly(A)+ RNA purification from control (2 hr at 280C) and heat-shocked (2 hr at 400C) intact soybean seedlings was performed as described (16). The 5' and 3' termini of soybean mRNA homologous to X clone hsE2019 were determined by nuclease S1 mapping with end-labeled DNA hybridization probes. The heat shock gene identified in this study was designated Gmhspl 7.5-E (Glycine max heat shock protein, 17.5 kDa). For 5'-end analysis, the BB1.55 fragment was end-labeled at the 5' termini (17) and redigested with EcoRI, and the resulting 250-base-pair (bp) BamHIEcoRI fragment (BEO.25) was isolated. This fragment was hybridized as described by Favaloro et al. (18) at various temperatures (42-50'C) to total ...

Soybeans, Glycine max, synthesize a family of low-molecular-weight heat shock (HS) proteins in response to HS. The DNA sequences of two genes encoding 17.5-and 17.6-kilodalton HS proteins were determined. Nuclease Si mapping of the corresponding mRNA indicated multiple start termini at the 5' end and multiple stop termini at the 3' end. These two genes were compared with two other soybean HS genes of similar size. A comparison among the 5' flanking regions encompassing the presumptive HS promoter of the soybean HS-protein genes demonstrated this region to be extremely homologous. Analysis of the DNA sequences in the 5' flanking regions of the soybean genes with the corresponding regions of Drosophila melanogaster HS-protein genes revealed striking similarity between plants and animals in the presumptive promoter structure of thermoinducible genes. Sequences related to the Drosophila HS consensus regulatory element were found 57 to 62 base pairs 5' to the start of transcription in addition to secondary HS consensus elements located further upstream. Comparative analysis of the deduced amino acid sequences of four soybean HS proteins illustrated that these proteins were greater than 90% homologous. Comparison of the amino acid sequence for soybean HS proteins with other organisms showed much lower homology (less than 20%). Hydropathy profiles for Drosophila, Xenopus, Caenorhabditis elegans, and G. max HS proteins showed a similarity of major hydrophilic and hydrophobic regions, which suggests conservation of functional domains for these proteins among widely dispersed organisms.All groups of organisms investigated undergo a response to high temperature referred to as heat shock (HS) (43). The HS response was first discovered in Drosophila melanogaster and has been studied in considerable detail in that organism (4,43). This response is characterized by control mechanisms which are operative at the levels of both transcription and translation and is generally characterized by the induction of synthesis of a new set of proteins (HS proteins), decreased synthesis of most normal proteins, and the acquisition of thermotolerance to a nonpermissive (or lethal) HS temperature by prior exposure to permissive elevated temperatures. The induction of HS proteins is dependent on the transcriptional activation of a unique set of genes at the elevated or HS temperature. In D. melanogaster, four HS proteins in the range of 22 to 27 kilodaltons (kDa) and three high-molecular-weight groups of 68, 70, and 84 kDa are induced (4, 50). The 70-kDa class of HS proteins, arising from three genetic loci, represents a major proportion of total HS-protein synthesis in D. melanogaster and several other animal systems. In soybean, the high-molecular-weight HS proteins range from 68 to 110 kDa, and the small HS proteins are grouped between 15 and 27 kDa (22,23,26,52).The high-molecular-weight HS proteins seem to be highly conserved across a broad spectrum of organisms (21), whereas the small HS proteins show much more diversity (12,13,23,46) in...

A 33 bp double-stranded oligonucleotide homologous to two AT-rich sequences located upstream (-907 to -889 and -843 to -826) to the start of transcription of heat shock gene Gmhsp17.5E of soybean stimulated transcription when placed 5' to a truncated (-140) maize Adh1 promoter. The chimeric promoter was assayed in vivo utilizing anaerobically stressed sunflower tumors transformed by a pTi-based vector of Agrobacterium tumefaciens. Nuclear proteins extracted from soybean plumules were shown to bind double-stranded oligonucleotides homologous to AT-rich sequences in the 5' flanking regions of soybean beta-conglycinin, lectin, leghemoglobin and heat shock genes. These proteins were also shown to bind AT-rich probes homologous to homeobox protein binding sites from the Antennapedia and engrailed/fushi tarazu genes of Drosophila. Binding activity specific for AT-rich sequences showed a wide distribution among various plant organs and species. Preliminary characterization indicated that two sets of nuclear proteins from soybean bind AT-rich DNA sequences: a diverse high-molecular-weight (ca. 46-69 kDa) group, and a low-molecular-weight (23 and 32 kDa) group of proteins. The latter meets the operational criteria for high-mobility group proteins (HMGs).