Ronald T. Nagao

A heat shock protein localized to chloroplasts is a member of a eukaryotic superfamily of heat shock proteins.

We have isolated cDNA clones from soybean and pea that specify nuclear‐encoded heat shock proteins (HSPs) which localize to chloroplasts. The mRNAs for these HSPs are undetectable at control temperatures, but increase approximately 150‐fold during a 2‐h heat shock. Hybridization‐selection followed by in vitro translation demonstrates that these HSPs are synthesized as precursor proteins which are processed by the removal of 5‐6.5 kd during import into isolated chloroplasts. The nucleotide sequence of the cDNAs shows the derived amino acid sequences of the mature pea and soybean proteins are 79% identical. While the predicted transit peptide encoded by the pea cDNA has some characteristics typical of transit sequences, including high Ser content, multiple basic residues and no acidic residues, it lacks two domains proposed to be important for import and maturation of other chloroplast proteins. The carboxy‐terminal region of the chloroplast HSP has significant homology to cytoplasmic HSPs from soybean and other eukaryotes. We hypothesize that the chloroplast HSP shares a common structural and functional domain with low mol. wt HSPs which localize to other parts of the cell, and may have evolved from a nuclear gene.

Molecular and cellular biology of the heat-shock response

Publisher Summary This chapter discusses the three major aspects of the heat-shock (HS) response including a comparative analysis of HS gene families and their expression, regulation of HS gene transcription, and functions of the heat-shock proteins (HSPs). This major protein family is also referred to as HSP70, reflecting the molecular weight of the major Drosophila HSP. This heat-inducible protein is conserved throughout evolution, and close related proteins are identified in a wide range of organisms including prokaryotes. In all eukaryotic organisms that are carefully examined, the HSP70 genes consist of a family of close related genes. The complexity and number of genes comprising the HSP70 family differ among species. Despite the number of organisms that are investigated, it is difficult to make generalizations about the composition of groups of HSPs from various species. Biochemical and genetic studies indicate that some HSPs or their cognates are present in organisms at normal growth temperatures and perform essential roles in normal cell function. The identification of some of these functions is valuable in providing clues to the function of HSPs at high temperatures.

Regulatable endogenous production of cytokinins up to toxic levels in transgenic plants and plant tissues

The effects of expressing a chimeric gene consisting of a soybean heat shock gene promoter and a sequence that encodes an enzyme catalyzing the synthesis of a potent phytohormone, the cytokinin iPMP, have been analyzed in transgenic tobacco plants. The production of cytokinin endogenously produced several effects previously undocumented. The differentiation of shoots independent of exogenous cytokinin from heat-treated transgenic plant leaf explants demonstrates that long-term heat treatments do not interfere with complex developmental processes. This extends the potential usefulness of heat shock gene promoters to conditionally express genes during windows of development that span several weeks.

Isolation and characterization of three families of auxin down-regulated cDNA clones

Five cDNA clones (ADR6, ADR11-1, ADR11-2, ADR12-1 and ADR12-2), representing three families of auxin down-regulated (ADR) genes were isolated and characterized. These were isolated by screening a λZap cDNA library with the partial cDNA clones p6, p11 and p12, isolated earlier (Baulcombe and Key, J Biol Chem 255: 8907–8913, 1980). Hybrid-select translation of ADR6, ADR11-2 and ADR12-2 clones produced polypeptides of 33 kDa 22.5 kDa and a 6 and 7 kDa respectively, when analyzed by SDS-PAGE. ADR6 and ADR12-2 gave one and two spots, respectively, on an IEF-SDS 2D gel. ADR11-2 probably encodes a basic protein as it was only resolved on non-equilibrium pH gradient gel electrophoresis (NEPHGE). Genomic Southern blot analysis of ADR6, ADR11 and ADR12 suggests that each represents a small multigene family. The RNA levels corresponding to ADR6, ADR11 and ADR12 decrease in response to applied auxin by 100-, 15- and 10-fold, respectively (Baulcombe and Key, 1980). Runoff transcription, done in the presence and absence of auxin, showed that the rate of transcription of the genes corresponding to ADR6, ADR11-2 and ADR12-2 was reduced in the presence of auxin, but the decrease was small relative to the decrease in the cytoplasmic levels of these mRNAs, in response to auxin. A comparative analysis of the influence of auxin on in vitro transcription and steady state RNA levels corresponding to these ADR cDNAs suggests that the decrease in rate of transcription due to auxin is not enough to account for the auxin-induced decrease in the steady state levels. Northern analysis showed developmental and organ/tissue-specific response of these ADR genes. Furthermore, the expression of the genes corresponding to ADR6 and ADR12-1 appears to be upregulated by light, whereas the gene corresponding to ADR11 appears to be down-regulated by light.

Identification of protein-binding DNA sequences in an auxin-regulated gene of soybean

The promoter region of a soybean auxin-responsive gene, GmAux28, was analyzed to identify protein-binding DNA sequences that may be involved in regulation of expression. Using DNase I footprinting and gel mobility shift assays, multiple regions of interaction, including eight major protein-binding sites, were observed in the GmAux28 gene. Two sequence motifs, TGACGACA and TCCACGTGTC, related to as-1/Hex and G-box elements, respectively, found in several plant promoters, were identified. Four distinct A/T-rich domains were identified; such A/T-rich domains appear to modulate, but not to specify, the expression of many genes. Two new sequence motifs, delta-1 (D1) and delta-4 (D4) were also identified. D1 and D4 share a very similar core sequence, TAGTxxCTGT and TAGTxCTGT, respectively. In gel mobility shift analyses, D1 and D4 elements exhibit a complex interaction of binding proteins. The GmAux22 promoter also contains D1-related elements which compete with the GmAux28 elements. Sequence comparisons have identified D1/D4-like sequences in several other auxin-responsive genes suggesting the possible importance of D1/D4 and the respective binding proteins in the regulation of expression of these genes.

Molecular characterization of cDNAs encoding low-molecular-weight heat shock proteins of soybean.

Three cDNA clones (GmHSP23.9, GmHSP22.3, and GmHSP22.5) representing three different members of the low-molecular-weight (LMW) heat shock protein (HSP) gene superfamily were isolated and characterized. A fourth cDNA clone, pFS2033, was partially characterized previously as a full-length genomic clone GmHSP22.0. The deduced amino acid sequences of all four cDNA clones have the conserved carboxyl-terminal LMW HSP domain. Sequence and hydropathy analyses of GmHSP22, GmHSP22.3, and GmHSP22.5, representing HSPs in the 20 to 24 kDa range, indicate they contain amino-terminal signal peptides. The mRNAs from GmHSP22, GmHSP22.3, and GmHSP22.5 were preferentially associated in vivo with endoplasmic reticulum (ER)-bound polysomes. GmHSP22 and GmHSP22.5 encode strikingly similar proteins; they are 78% identical and 90% conserved at the amino acid sequence level, and both possess the C-terminal tetrapeptide KQEL which is similar to the consensus ER retention motif KDEL; the encoded polypeptides can be clearly resolved from each other by two-dimensional gel analysis of their hybrid-arrest translation products. GmHSP22.3 is less closely related to GmHSP22 (48% identical and 70% conserved) and GmHSP22.5 (47% identical and 65% conserved). The fourth cDNA clone, GmHSP23.9, encodes a HSP of ca. 24kDa with an amino terminus that has characteristics of some mitochondrial transit sequences, and in contrast to GmHSP22, GmHSP22.3, and GmHSP22.5, the corresponding mRNA is preferentially associated in vivo with free polysomes. It is proposed that the LMW HSP gene superfamily be expanded to at least six classes to include a mitochondrial class and an additional endomembrane class of LMW HSPs.

The separation of RNA polymerases I and II achieved by fractionation of plant chromatin

Abstract Chromatin-bound RNA polymerase I and II from soybean hypocotyl can be separated by differential centrifugation. While all detectable RNA polymerase I is pelleted in association with chromatin, nearly all of the RNA polymerase II activity is not pelleted even at high speeds. Substitution of pH 6 for pH 8 isolation medium results in several-fold greater recovery of chromatin-bound RNA polymerase II.

Induction and Regulation of Heat-Shock Gene Expression by an Amino Acid Analog in Soybean Seedlings.

The effect of the proline analog azetidine-2-carboxylic acid (Aze) on the induction and the regulation of heat-shock (HS) mRNA accumulation and heat-shock protein (HSP) synthesis in soybean (Glycine max) seedlings was studied. Treatment with Aze elicited an HS-like response at the normal growth temperature, 28[deg]C, with seven of nine HS cDNA clones tested. Two cDNA clones, Gm-Hsp22.5 and pFS2033, share 78% identity; however, transcripts hybridizing to GmHsp22.5 but not pFS2033 accumulated with Aze treatment at 28[deg]C. Substantial incorporation of radioactive amino acid into high molecular weight HSPs but not low molecular weight HSPs was observed in vivo during Aze treatment at 28[deg]C. Low molecular weight HSPs were detected using antibodies raised against an abundant member of low molecular weight class I HSPs, indicating that low molecular weight HSPs were synthesized at normal growth temperatures during Aze treatment despite a lack of substantial in vivo radioactive amino acid incorporation. In summary, Aze treatment induced accumulation of most but not all HS mRNAs and HSPs in soybean seedlings; the observations presented here suggest differential regulation among various HS genes at the transcriptional and posttranscriptional levels.

Construction and application of a modified "gene machine": a circular concentrating preparative gel electrophoresis device employing discontinuous elution.

Abstract A modified version of a preparative circular gel electrophoresis apparatus, first described by Edwin Southern (Medical Research Council, University of Edinburgh, Edinburgh, Scotland), has been constructed. The apparatus fractionates a large volume of sample into concentric bands which migrate toward a small circular collection chamber. Samples exiting the gel into the collection chamber are concentrated against a dialysis membrane which encloses the inner electrode and are pumped from this center chamber into a fraction collector at fixed time intervals. The apparatus has been employed to fractionate samples of DNA (10 mg) by electrophoresis through either agarose or acrylamide gels. Two examples of nucleic acids which have been successfully fractionated are given: restriction endonuclease cleavage fragments of total soybean DNA, and a heterogeneous mixture of covalently closed circular plasmid DNA from Bacillus megaterium. Franctionated DNA is suitable for molecular cloning directly from acrylamide and, after one additional treatment, from agarose. The run time for DNA treated with restriction endonuclease is from 24 to 48 h. Purification of 60- to 200-fold is common for a DNA restriction fragment from a total genome.

The Low Molecular Weight Heat Shock Proteins of Soybean Seedlings

The heat shock (HS) response has been studied extensively for about 30 years since the discovery of the phenomenon in Drosophila in the early 1960s; studies in plants have been restricted generally to less than 15 years. Heat shock is the subject of numerous reviews in recent years (e.g. Craig, 1985; Kimpel and Key, 1985a; Key et al., 1985a; 1985b; Lindquist, 1986; Lindquist and Craig, 1988; Nagao et al., 1986; Key et al., 1987a; 1987b; Nagao and Key, 1989; Nagao et al., 1990; Neumann et al., 1989; Vierling, 1991; Gurley and Key, 1991). Since various aspects of the HS response in a number of plant systems are covered in this volume, the comments of this contribution will focus on our work on the low molecular weight (LMW) heat shock proteins (hsps) of soybean seedlings, much of which is detailed in some of the reviews cited above, with emphasis on expression of the HS genes encoding hsps, and some properties of those proteins, in the 15 to 27 kD range. This relates to a rather unique feature of HS in plants in that most plants synthesize a large number of LMW hsps, and some of these are generally the most abundant hsps expressed in plants. Most other eukaryotes, in contrast, synthesize a limited number of LMW hsps, and these are generally much less abundant than the 70 kD and 83/90 kD hsps, with hsp70 proteins often representing the majority of hsp synthesis in many organisms. Plants synthesize a complement of hsps in the 60 kD, 70 kD, 83/90 kD, and 104/110 kD MW range which appear to be homologs of the corresponding hsps of other eukaryotes.