Molecular and cellular biology of the heat-shock response.
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Animals↗
Biomedical subjects
J L Key
Publications and source records attributed to J L Key.
Regulation of the heat shock response in soybean seedlings.
The transcriptional response of soybean (Glycine max) seedlings during heat shock (HS) was investigated under two different treatment regimes. During prolonged heat treatment at 40 degrees C, active transcription of the HS genes (as measured by "runoff" transcription assays) occurs only during the first few hours. Nonetheless, mRNAs for these genes are present at relatively high abundance even after 9 hours of exposure to 40 degrees C. Because HS mRNAs have a fairly short half-life (less than 3 hours) at 28 degrees C, these results indicate that HS mRNAs are inherently more stable at 40 degrees C. During a second type of heat treatment regime-short pulses of high (45 degrees C) heat followed by 1 to 2 hours at 28 degrees C-transcription of HS genes is comparable to that achieved at 40 degrees C for the first few hours, even though the tissue is maintained at non-HS temperatures. The transcriptional responses to these two different heat treatments indicate that regulatory controls for the transcription of the HS genes must involve more than a simple sensing of ambient temperature, since transcription of these genes can be turned off at 40 degrees C (in the case of prolonged exposure) and can continue at 28 degrees C (following a short, severe heat treatment). Additional results demonstrate that the response of soybean seedlings to a particular HS depends on their prior exposure to heat; seedlings given a preheat treatment (that is known to induce thermotolerance) respond more moderately to a short heat pulse at 45 degrees C. Overall, this research indicates that plants have mechanisms for both monitoring the severity of changes in temperature and for measuring the magnitude and duration of the stress. Such information is then used to regulate the plant's response to heat both transcriptionally and posttranscriptionally.
Journal Article↗
Sequence and Expression of a HSP83 from Arabidopsis thaliana.
A full-length cDNA encoding a heat shock protein (hsp) belonging to the 83 to 90 kilodalton hsp family of Arabidopsis thaliana has been isolated and sequenced. Truncated cDNA clones were isolated by nucleic acid hybridization to a truncated soybean HSP83 cDNA probe and a fragment generated from a Drosophila HSP83 gene. A single strand DNA vector/primer based extension procedure was employed to obtain the full-length cDNA. The level of transcripts homologous to this cDNA (AtHS83) is low in 2-week-old Arabidopsis plants but is rapidly enhanced by elevated temperatures. DNA sequence comparison between this cDNA and hsp83-90 sequences from human, yeast and Drosophila reveal amino acid identities of 63 to 69%, typical identities for interspecies comparisons between hsp83 to 90 kilodalton proteins. Genomic DNA blot analysis performed with probes derived from AtHS83 indicate the presence of a HSP83 gene family estimated to be comprised of at least three genes.
Journal Article↗
Developmentally regulated expression of soybean proline-rich cell wall protein genes.
Previously, we reported the characterization of a developmentally regulated proline-rich cell wall protein (SbPRP1) gene of soybean; the encoded protein is represented by a consensus amino acid repeat structure of Pro-Pro-Val-Tyr-Lys [Hong, J.C., Nagao, R.T., and Key, J.L. (1987). J. Biol. Chem. 262, 8367-8376]. Two other closely related members of this family of proline-rich protein (PRP) genes (SbPRP2 and SbPRP3), which differ from the extensin family of cell wall proteins, have been characterized (J.C. Hong, R.T. Nagao, and J.L. Key, unpublished results). Here we report studies on the regulation of expression of this gene family during soybean development by analyzing various plant organs, including leaves, stems, and roots of etiolated seedlings and light-grown plants, as well as young and mature pods, seed coats, and cotyledons. These organs were tested at different stages of development (young and fully mature tissue). Although a high level of sequence homology is observed at the nucleotide and amino acid sequence level among these three PRP genes/proteins, there are marked differences in the patterns of expression of each gene in different plant organs and organ regions. SbPRP1 is highly expressed and is the predominant form of PRP transcript in the mature hypocotyl, root, and immature seed coat. SbPRP2 is the major form of PRP in the apical hypocotyl and young suspension culture cells. SbPRP3 is the major form of PRP gene expressed in aerial parts; it is highly expressed in leaves, although no expression is detected in the roots.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Regulatory domains of the Gmhsp17.5-E heat shock promoter of soybean.
Promoter domains required for in vivo transcriptional expression of soybean heat shock gene Gmhsp17.5-E were identified by insertion-deletion mutagenesis with transgenic expression monitored in Agrobacterium tumefaciens-incited tumors of sunflower. Removal of the TATA-distal domain from position -1175 to position -259 had little effect on overall activity. The four regions contributing to promoter activity identified by this study all map within 244 base pairs from the start of transcription. The most distal cis-acting element of major significance was located from -244 to -179 and contains a conserved TATA-dyad motif centered at -220. Sequences from -179 to -40 comprise the TATA-proximal domain and include an AT-rich region and two sites containing heat shock consensus elements (HSEs). Deletion of the HSE centered at -93 (site 2) severely reduced transcriptional activity. Heat-inducible expression was also eliminated by internal deletion of either the TATA motif or the overlapping HSEs at site 1, indicating that each of these regions is also a major determinant of promoter activity.
Base Sequence↗
Sequence and characterization of two auxin-regulated genes from soybean.
The auxin-regulated expression of two poly(A)+ mRNAs in soybean hypocotyl was demonstrated by cloning of the cDNAs and Northern blot hybridization analyses (Walker, J.C., and Key, J.L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7185-7189). The corresponding genes, designated Aux28 and Aux22, have been isolated, and the cDNAs and genes have been sequenced. The Aux28 and Aux22 genes are present at one to two copies per haploid genome, contain four and two introns, and encode hydrophilic proteins of 26.8 and 21.5 kDa, respectively. Although the cDNAs were isolated independently and do not cross-hybridize under stringent hybridization conditions, the protein coding sequences of the two cDNAs have several colinear regions of high homology at the nucleic acid (77-80%) and the amino acid (80-100%) levels; together these regions constitute approximately a third of the protein coding sequences of the cDNAs. These data, together with genomic Southern blot hybridization analysis and hybrid-select translations of mRNAs homologous to the cDNAs, show that these genes belong to two related multigene families. We have identified two sequences, TGATAAAAG and GGCAGCATGCA, that occur at similar distances upstream of the transcription start site in each gene, and the spacing between these two elements is essentially identical in the two genes. The possible significance of these sequences is under evaluation.
Amino Acid Sequence↗
Comparative analysis of polyadenylated RNA complexity in soybean hypocotyl tissue and cultured suspension cells.
Growth parameters of suspension culture cells of soybean (Glycine max L.) were compared between cells grown in medium with (+) auxin and without (-) auxin. Growth rates were greater for (+) auxin cells. Cells transferred to (-) auxin medium primarily expanded in size while (+) auxin cells initially divided and then expanded. Two methods were used to estimate polyadenylated RNA sequence complexity. Kinetic analysis gave a sum of component complexity values of 36,000 and 64,000 diverse poly(A) RNA sequences of about 1,400 nucleotides in (+) and (-) auxin grown cells, respectively. The most striking difference between these cell populations was the increase in the poly(A) RNA sequence complexity in cells grown in medium without auxin. RNA complexities were also determined by the saturation of ;single' copy DNA by poly(A) RNAs from (+) and (-) auxin suspension cells. These saturation studies estimated the total complexity of (+) and (-) auxin suspension cells as 41,000 and 57,000 diverse sequences, respectively. Suspension cells in auxin-depleted medium produced about 20,000 more diverse sequences than (+) auxin cells. Comparisons of poly(A) complexities were also made among auxin-treated and untreated hypocotyl cells from the intact plant relative to suspension culture cells. Mixed populations of poly(A) RNA from these tissues and cells allowed the determination of shared sequences among them. When all combinations of poly(A) RNA were mixed, the percentage of ;single' copy DNA that saturated was equivalent to diverse sequence complexity estimates of about 60,000. When mixed poly(A) RNA from suspension cells from (+) and (-) auxin medium were compared, they shared about 40,000 sequences and (-) auxin cells contained an additional 20,000. Both (+) and (-) tissue culture cells shared a subset of about 20,000 sequences with cells from (+) and (-) auxin treated hypocotyl. A third subset of about 20,000 sequences was shared by (-) auxin suspension cells and hypocotyl treated with or without auxin, a subset most of which were not shared by (+) auxin suspension cells. Kinetic and saturation data estimates of poly(A) RNA complexity compared favorably and indicated that exogenous auxin treatment can dramatically alter the complexity of all classes of poly(A) RNAs in cultured cells.
Journal Article↗
Induction and Accumulation of Heat Shock-Specific Poly(A) RNAs and Proteins in Soybean Seedlings during Arsenite and Cadmium Treatments.
Northern blot hybridization analyzes revealed that poly(A(+)) RNAs homologous to eight heat shock (HS)-specific cDNA clones were induced by arsenite (As) or Cd treatments. The mRNAs accumulated slower, and maximum accumulations were consistently lower than HS-induced levels. Prolonged treatment with low concentrations (50-100 micromolar) of As for 6 hours, or Cd for 12 hours, resulted in decreased accumulations of HS-specific mRNAs. This response resembled the ;autoregulation' observed during continuous 40 degrees C HS. However, no autoregulation was evident when soybean seedlings were exposed to high concentrations of As (250 micromolar) or Cd (1 millimolar) for 12 hours. The cDNA probe pCE54 detected a second higher molecular weight poly(A(+)) RNA following As or Cd treatments which accumulated concomitantly with the lower molecular weight HS-specific poly(A(+)) RNA. The patterns of low molecular weight HS polypeptides from in vitro translations induced by HS, As, and Cd, and analyzed by one-dimensional and two-dimensional SDS-PAGE, were similar but temporal differences were apparent. In addition to HS proteins, many control proteins were also detected in both in vitro and in vivo labeling patterns from As and, to a lesser extent, Cd treatments. The chemical agents used in this study apparently induced the accumulation and translation of HS messages in vivo but not in the selective manner as observed during HS treatment.
Journal Article↗
Cytoplasmic distribution of heat shock proteins in soybean.
Previous analyses of the distribution of heat shock (hs) proteins in soybean (Glycine max L. Merr., var Wayne) have demonstrated that a fraction of the low molecular weight hs protein associates with ribosomes during hs. To more specifically characterize the nature of this association, isokinetic centrifugation of ribosomes through sucrose gradients was used to separate monosomes from polysomes. The present analysis demonstrated that hs proteins were bound to polysomes but not monosomes. Treatment of polysomes with puromycin, K(+), and Mg(2+), which caused dissociation of ribosomes into 40S and 60S subunits, also caused dissociation of the hs proteins. Using the procedure of Nover et al. (1983, Mol. Cell Biol, 3: 1628-1655), a hs granule fraction was also isolated. As in tomato cells, hs granules from soybean seedlings contained the low molecular weight hs proteins as a primary component and a number of other non-hs proteins of relative molecular mass 30 to 40 kilodaltons and 70 to 90 kilodaltons. On metrizamide gradients they exhibited a buoyant density of 1.20 to 1.21 grams per cubic centimeter, typical of ribonucleoprotein particles. Heat shock granules were characterized as unique cytoplasmic particles based on protein composition and buoyant density. Isopycnic centrifugation of ribosome preparations demonstrated that they contained hs granules, but the hs proteins bound to polysomes were not released by KCI/EDTA treatment. Thus, the polysome-bound hs proteins and the granule-bound hs proteins appear to represent two distinct populations of hs proteins in the cytoplasm. Heat shock granules were not distinguishable from ribosomes at the level of resolution used in transmission electron microscopy.
Journal Article↗
Characterization of Gmhsp26-A, a stress gene encoding a divergent heat shock protein of soybean: heavy-metal-induced inhibition of intron processing.
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. Schöffl, 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). S1 nuclease mapping of transcripts and a comparison of the cDNA sequence with the genomic sequence indicated the presence of a soybean seedlings with either CdCl2 or CuSO4. 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.
Amino Acid Sequence↗
Molecular cloning of a pea H1 histone cDNA.
A pea (Pisum sativum, var. Little Marvel) H1 histone cDNA has been isolated from a lambda gt11 expression vector library. This cDNA has been sequenced and shown to represent the entire protein-coding region of the mRNA. The deduced protein sequence is 265 amino acids long (28018 Da) and contains 70 lysines and 3 arginines. The structure of the encoded protein is comparable to animal lysine-rich histones. The central region, which has an amino acid composition similar to that found in the globular domains of animal lysine-rich histones, is flanked by an amino-terminal region rich in lysine, glutamic acid and proline and by a carboxyl-terminal region rich in lysine, alanine, valine and proline. Despite the structural similarities, the protein has little sequence homology with animal lysine-rich histones. This H1 protein is unusual because 12 of the first 40 amino acids are glutamic acid.
Amino Acid Sequence↗
Characterization and sequence analysis of a developmentally regulated putative cell wall protein gene isolated from soybean.
A cDNA clone, pTU04, which hybridizes to two different sizes of mRNA on Northern blots was isolated from soybean suspension culture cell poly(A) RNA. Northern analysis reveals that meristematic tissue produces a 1050-nucleotide mRNA while quiescent mature cells produce primarily a 1220-nucleotide mRNA homologous to pTU04. The cDNA and its corresponding genomic clone have been partially characterized. The nucleotide sequence of the gene predicts a proline-rich protein, designated SbPRP1, which contains a signal peptide sequence and 43 repeats of a sequence consisting primarily of Pro-Pro-Val-Tyr-Lys (CCA-CCA-GTT-TAC-AAG). From nuclease S1 and hybrid-select translation analyses, the cDNA clone pTU04 appears to represent the mRNA for the mature tissue 1220-nucleotide RNA observed on Northern blots. Although there is no direct proof that the encoded protein is a cell wall protein, it has the properties similar to previously isolated cell wall proteins: 1) it is very basic with a high content of Pro, Tyr, and Lys; 2) it has similar hydropathic properties; and 3) its repeating unit shares sequence homology with that of more highly characterized cell wall proteins, generally termed extensin (Chen, J., and Varner, J. E. (1985) EMBO J. 4, 2145-2151; Smith, J. J., Muldoon, E. P., Willard, J. J., and Lamport, D. T. A. (1986) Phytochemistry 25, 1021-1030.
Amino Acid Sequence↗
Synthesis of the low molecular weight heat shock proteins in plants.
Heat shock of living tissue induces the synthesis of a unique group of proteins, the heat shock proteins. In plants, the major group of heat shock proteins has a molecular mass of 15 to 25 kilodaltons. Accumulation of these proteins to stainable levels has been reported in only a few species. To examine accumulation of the low molecular weight heat shock proteins in a broader range of species, two-dimensional electrophoresis was used to resolve total protein from the following species: soybean (Glycine max L. Merr., var Wayne), pea (Pisum sativum L., var Early Alaska), sunflower (Helianthus annuus L.), wheat (Triticum aestivum L.), rice (Oryza sativa L., cv IR-36), maize (Zea mays L.), pearl millet (Pennisetum americanum L. Leeke, line 23DB), and Panicum miliaceum L. When identified by both silver staining and incorporation of radiolabel, a diverse array of low molecular weight heat shock proteins was synthesized in each of these species. These proteins accumulated to significant levels after three hours of heat shock but exhibited considerable heterogeneity in isoelectric point, molecular weight, stainability, and radiolabel incorporation. Although most appeared to be synthesized only during heat shock, some were detectable at low levels in control tissue. Compared to the monocots, a higher proportion of low molecular weight heat shock proteins was detectable in control tissues from dicots.
Journal Article↗
Auxin-regulated gene expression.
During the 1960s a wide range of studies provided an information base that led to the suggestion that auxin-regulated cell processes--especially cell elongation--may be mediated by auxin-regulated gene expression. Indirect evidence from our work, based on the influence of inhibitors of RNA synthesis (e.g. actinomycin D) and of protein synthesis (e.g. cycloheximide) on auxin-induced cell elongation, coupled with correlations of the influence of auxin on RNA synthesis and cell elongation, provided the basis for this suggestion. With the availability of techniques for DNA-DNA and DNA-RNA hybridization, mRNA isolation-translation, in vitro 2D gel analysis of the translation products, and ultimately the cloning by recombinant DNA technologies of genomic DNA and copy DNAs (cDNAs) made to poly(A)+ mRNAs, we and others have provided direct evidence for the influence of auxin on the expression of a few genes (i.e. poly(A)+ RNA levels). Our laboratory has provided evidence for auxin's both down-regulating and up-regulating the level of a few poly(A)+ mRNAs out of a population of about 4 X 10(4) sequences that are not significantly affected by auxin. In our studies on auxin-regulated cell elongation, two cDNA clones (pJCW1 and pJCW2) were isolated which corresponded to poly(A)+ mRNAs that responded during growth transitions in a way consistent with a potential role of their protein products in cell elongation. These mRNAs are most abundant in the elongating zone of the soybean hypocotyl. Upon excision and incubation in the absence of auxin, these mRNAs deplete in concert with a decreasing rate of cell elongation. Addition of auxin to the medium results in both increased levels of these mRNAs and enhanced rates of cell elongation. These mRNAs do not deplete if auxin is added to the medium at the onset of excised incubation, and cell elongation rates remain high. We have isolated and sequenced genomic clones that are homologous to these cDNAs. Of the two genes sequenced, both genes are members of small multigene families. There are regions of high amino acid homology even though the nucleotide sequences are sufficiently different in these regions for cross-hybridization of the clones not to be observed. More recently others, especially Guilfoyle's laboratory, have shown that auxin selectively and rapidly influences the level of certain mRNAs and proteins. We have worked on other gene systems such as ribosomal proteins and possible cell wall proteins that are responsive to auxin; again the nature of regulation of expression of these genes is not known.(ABSTRACT TRUNCATED AT 400 WORDS)
DNA↗
Isolation of nuclear encoded plastid ribosomal protein cDNAs.
A pea leaf cDNA library was constructed in the expression vector lambda gt11 and screened with antisera raised against proteins extracted from 30S and 50S ribosomal subunits and 70S ribosomes prepared from isolated pea chloroplasts. Six recombinant phage were identified that encoded fusion proteins containing plastid ribosomal protein antigenic determinants. Phage-induced cell lysate proteins, containing the fusion proteins, were bound to nitrocellulose membranes and used as affinity matrices to prepare monospecific antibodies. These antibodies were then used to identify by Western blotting which plastid ribosomal protein shared antigenic determinants with the fusion proteins. cDNA inserts from the antigen-producing phage were used to hybrid-select complementary mRNAs. The cell-free translation products of these mRNAs were added to a pea chloroplast in vitro transport system and imported proteins analyzed by two-dimensional gel electrophoresis. The imported proteins comigrated with the plastid ribosomal proteins that were identified as being antigenically related to the fusion proteins produced by the corresponding recombinant phage. The imported proteins were 3,500-5,500 daltons smaller than their precursors.
Cell Nucleus↗
Specific heat shock proteins are transported into chloroplasts.
We demonstrate that in three plant species-soybean, pea, and corn-certain nuclear-encoded heat shock proteins are transported into chloroplasts. In vitro translation products of poly(A)-RNA from control or heat-shocked plants were incubated with isolated intact pea chloroplasts and differences in the profile of imported proteins were analyzed. In all three species, abundant polypeptides between 21 and 27 kDa are present in the heat shock sample and absent in the controls. These polypeptides are protected from trypsin and chymotrypsin digestion after their import into chloroplasts and are recovered primarily with the soluble chloroplast protein fraction. Chloroplasts isolated from pea or corn leaves labeled in vivo at heat shock temperatures, but not at normal growth temperatures, contain the same polypeptides observed in vitro. Synthesis of the heat shock polypeptides can be inhibited in vivo by cycloheximide but not by chloramphenicol, further indicating they are products of cytoplasmic protein synthesis. The in vitro transport experiments demonstrate that synthesis of the chloroplast-localized heat shock proteins results from heat-induced accumulation of the corresponding poly(A)-RNAs. The same mRNAs are also produced in response to heat shock by a nonphotosynthetic tissue, the etiolated soybean hypocotyl.
Journal Article↗
Upstream sequences required for efficient expression of a soybean heat shock gene.
A soybean gene (Gmhsp17.5-E) encoding a small heat shock protein was introduced into primary sunflower tumors via T-DNA-mediated transformation. RNA blot hybridizations and S1-nuclease hybrid protection studies indicated that the heat shock gene containing 3.25 kilobases of 5'-flanking sequences was strongly transcribed in a thermoinducible (40 degrees C) manner. Transcriptional induction also occurred to a lesser extent upon treatment of whole tumors with sodium arsenite and CdCl2. Basal (26 degrees C) transcription was not detected in soybean seedlings, but it was quite evident in transformed tumor tissue. A 5' deletion to -1,175 base pairs with respect to the CAP site had no effect on the levels of thermoinducible transcription, but it resulted in a large increase in basal transcription. Further removal of DNA sequences (including the TATA-distal heat shock consensus element) to -95 base pairs reduced thermoinducible transcription by 95% and also greatly decreased basal transcription. The termini of the Gmhsp17.5-E RNA in the tumor were generally the same as those present in soybean RNA, with the exception of several additional 3' termini.
Base Sequence↗
Coordinate expression of ribosomal protein mRNAs following auxin treatment of soybean hypocotyls.
Nine recombinant plasmids containing soybean (Glycine max. var. Wayne) ribosomal protein mRNA sequences were identified by hybrid selection-translation followed by gel electrophoresis. Individual plasmids were used to determine the amount of ribosomal protein mRNA in soybean hypocotyls at various times after application of the synthetic auxin (2,4-dichlorophenoxy)acetic acid. Results from these experiments indicate that the relative abundance of ribosomal protein mRNAs increases 3-8-fold within 24 hr of (2,4-dichlorophenoxy)acetic acid application. Earlier in vitro translation data (Gantt, J. S., and Key, J. L. (1983) Biochemistry 22, 4131-4139) also indicated that the ribosomal protein message levels increase following treatment with auxin. However, the two methods of quantifying mRNA lead to significantly different values for the amount of induction for three ribosomal protein mRNAs. The cause of these differences is not known. An increase in the level of the ribosomal protein mRNAs can be detected 1 h after treatment with (2,4-dichlorophenoxy)acetic acid. The similar kinetics of induction of these messages suggests that their levels are coordinately regulated.
Cloning, Molecular↗