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In addition to heat shock (HS), a proline analog, azetidine-2-carboxylic acid (Aze), also induces heat shock protein (HSP) synthesis. Here, we describe our further characterization of the induced effects by Aze treatment in soybean seedlings. Northern blot analyses revealed transcription of the soybean HS genes hsp70 and hsc70 as well as hspl7.5 activated after 6 h of Aze incubation, as reported previously. However, Aze-induced activation of HSP genes was not attenuated after transferring seedlings pretreated with Aze to shaking buffer, whereas the activation was decreased when seedlings were treated with Aze and then proline. Besides triggering an HS-like response, Aze treatment triggered a continuous increase of cellular leakage in soybean seedlings at room temperature. Nonlethal HS pretreatment protected seedlings from cellular leakage induced by Aze and allowed them to survive subsequent lethal HS treatment. As well, 10 mM proline prevented electrolyte leakage after 6 h of 10 mM Aze treatment and increased the survival of seedlings under lethal treatment. These results indicate that Aze treatment causes a permeability change in the plasma membrane by leading to cellular leakage before accumulation of HSPs. In vitro chaperone activity of 10 mM Aze-induced sHSP-CIs (class I small HSPs) with the model substrate citrate synthase was less efficient than that from 2 h of 40 °C treatment.

Accumulation of class I small heat shock proteins (sHSPs) is induced by the proline analog, azetidine-2-carboxylic acid (Aze) in soybean seedlings to a level similar to that induced by exposure to 40 degrees C. However, only the treatment with 10 mM Aze for 6 h and subsequently with 10 mM proline for 24 h protected the seedlings from damage during subsequent exposure to 45 degrees C as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. A chaperone activity assay showed that the purified class I sHSPs induced by Aze were functional in vitro and protected proteins from thermal denaturation. Amino acid composition analysis indicated that Aze was not incorporated into de novo synthesized class I sHSPs. Accumulation of class I sHSPs in the soluble post-ribosomal supernatant fraction was found to be important for acquisition of thermotolerance. We suggest that both the accumulation of class I sHSPs and their presence in the soluble fraction are important for establishment of thermotolerance.

Etiolated leaves of 28 degrees C-dark-grown mung bean (Vigna radiata L. cv. 2937) seedlings fail to turn green after being shifted to a light and cold environment. At the visible phenotypic level, incapability of leaf greening is the only failure event for the de-etiolation of mung bean seedlings at low temperature. Ultrastructural studies revealed that chloroplast development was completely suppressed by chilling treatment. A cDNA library originating from 28 degrees C-light-grown seedling leaves was constructed for screening cold-suppressed (cos) genes. Thirteen full-length cDNA clones were obtained, with 12 clones encoding chloroplast proteins, which, according to their known physiological functions, were important for chloroplast development and photosynthesis. Another cos cDNA encodes CYP90A2, which is a cytochrome P450 protein involved in the biosynthesis of brassinosteroid hormones. All cos genes are light-regulated at normal temperature. The influence of chilling stress on cos expression was examined in 10 degrees C-light- and 10 degrees C-dark-grown etiolated seedlings, and in 10 degrees C-light-grown green plants. The data show that cos expression in these three treatments is severely suppressed. This suppression is controlled at the transcriptional level, as demonstrated by nuclear runoff experiments, and is reversible because cos mRNAs accumulate again after the cold-treated plants have been transferred to 28 degrees C.

The cytosolic class I small heat shock proteins (sHSP-CI) represent the most abundant sHSP in plants. Here, we report the characterization and the expression profile of nine members of the sHSP-CI gene family in rice (Oryza sativa Tainung No.67), of which Oshsp16.9A, Oshsp16.9B, Oshsp16.9C, Oshsp16.9D and Oshsp17.9B are clustered on chromosome 1, and Oshsp17.3, Oshsp17.7, Oshsp17.9A and Oshsp18.0 are clustered on chromosome 3. Oshsp17.3 and Oshsp18.0 are linked by a 356-bp putative bi-directional promoter. Individual gene products were identified from the protein subunits of a heat shock complex (HSC) and from in vitro transcription/ translation products by two-dimensional gel electrophoreses (2-DE). All sHSP-CI genes except Oshsp17.9B were induced strongly after a 2-h heat shock treatment. The genes on chromosome 3 were induced rapidly at 32 and 41 degrees C, whereas those on chromosome 1 were induced slowly by similar conditions. Seven of these genes, except Oshsp16.9D and Oshsp17.9B, were induced by arsenite (As), but only genes on chromosome 3 were strongly induced by azetidine-2-carboxylic acid (Aze, a proline analog) and cadmium (Cd). A similar expression profile of all sHSP-CI genes at a lower level was evoked by ethanol, H2O2 and CuCl2 treatments. Transient expression assays of the promoter activity by linking to GUS reporter gene also supported the in vivo selective expression of the sHSP-CI genes by Aze treatment indicating the differential induction of rice sHSP-CI genes is most likely regulated at the transcriptional level. Only Oshsp16.9A abundantly accumulated in mature dry seed also suggested additionally prominent roles played by this HSP in development.

Rice (Oryza sativa) class I low-molecular mass (LMM) heat shock protein (HSP), Oshsp16.9, has been shown to be able to confer thermotolerance in Escherichia coli. To define the regions for this intriguing property, deletion mutants of this hsp have been constructed and overexpressed in E. coliXL1-blue cells after isopropyl β-d-thioglactopyranoside induction. The deletion of amino acid residues 30 through 36 (PATSDND) in the N-terminal domain or 73 through 78 (EEGNVL) in the consensus II domain of Oshsp16.9 led to the loss of chaperone activities and also rendered the E. coli incapable of surviving at 47.5°C. To further investigate the function of these two domains, we determined the light scattering changes of Oshsp16.9 mutant proteins at 320 nm under heat treatment either by themselves or in the presence of a thermosensitive enzyme, citrate synthase. It was observed that regions of amino acid residues 30 through 36 and 73 through 78 were responsible for stability of Oshsp16.9 and its interactions with other unfolded protein substrates, such as citrate synthase. Studies of two-point mutants of Oshsp16.9, GST-N74E73K and GST-N74E74K, indicate that amino acid residues 73 and 74 are an important part of the substrate-binding site of Oshsp16.9. Non-denaturing gel analysis of purified Oshsp16.9 revealed that oligomerization of Oshsp16.9 was necessary but not sufficient for its chaperone activity.

A rice class I low-molecular-mass heat shock protein (LMM HSP) Oshsp 16.9 was overexpressed in Escherichia coli. Oligomerized complexes of Oshsp16.9 were harvested and electron microscopic observations of purified complexes revealed globular structures of 10-20 nm in diameter (with majority of 15-18 nm) and calculated to comprise approx. 12 monomers per complex. In comparison, complexes from native rice class I LMM HSPs were observed as larger ellipsoid- or globular-like random aggregated hetero-oligomers. To characterize the biochemical functions of the hydrophobic N-terminal region of Oshsp16.9, a truncation in the N-terminal region was constructed and introduced into E. coli. Results showed that the N-terminal truncated Oshsp16.9 mutant was capable of forming complexes similar to the full-length Oshsp16.9; however, the deletion protein failed to confer in vitro protein thermostability under elevated temperatures. Protein assays from in vivo treatments at higher temperatures exhibited that non-specific interactions of E. coli cellular proteins only occurred with full-length Oshsp16.9 complexes but not with the mutant complex. In vitro immunoprecipitation of cellular proteins from E. coli overexpressing full-length Oshsp16.9 showed that interactions between plant LMM HSP and E. coli cellular proteins are temperature-dependent. Taken together, the hydrophobic N-terminal region of rice class I LMM HSP is critical in the ability of the protein to interact/bind with its potential substrates.

A gene encoding the rice 16.9-kDa class I low-molecular-mass (LMM) heat-shock protein (HSP), Oshsp16.9, was introduced into Escherichia coli using the pGEX-2T expression vector to analyze the possible function of this LMM HSP under heat stress. It is known that E. coli does not normally produce class I LMM HSPs. We compared the survivability of E. coli XL1-Blue cells transformed with a recombinant plasmid containing a glutathione S -transferase (GST)–Oshsp16.9 fusion protein (pGST-FL cells) with the control E. coli cells transformed with the pGEX-2T vector (pGST cells) under heat-shock (HS) after isopropyl β- d -thiogalactopyranoside induction. The pGST-FL cells demonstrated thermotolerance at 47.5°C, a treatment that was lethal to the pGST cells. When the cell lysates from these two E. coli transformants were heated at 55°C, the amount of protein denatured in the pGST-FL cells was 50% less than that of the pGST cells. Similar results as pGST-FL cells were obtained in pGST-N78 cells (cells produced a fusion protein with only the N-terminal 78 aa in the Oshsp16.9 portion) but not in pGST-C108 cells (cells produced a fusion protein with C-terminal 108 aa in the Oshsp16.9 portion). The acquired thermotolerant pGST-FL cells synthesized three types of HSPs, including the 76-, 73-, and 64-kDa proteins according to their abundance at a lethal temperature of 47.5°C. This finding indicates that a plant class I LMM HSP, when effectively expressed in transformed prokaryotic cells that do not normally synthesize this class of LMM HSPs, may directly or indirectly increase thermotolerance.

Sporamin accounts for about 60% to 80% of total soluble protein in sweet potato tubers, and the predicted protein sequence of sporamin shares significant amino acid sequence identity with some Kunitz-type trypsin inhibitors. We constructed three recombinant plasmids with cDNAs that encode preprosporamin, prosporamin, and sporamin, and these three were expressed in Escherichia coli cells as fusion proteins. All three forms of sporamin expressed in E. coli were shown to have strong inhibitory activity to trypsin in vitro, suggesting that post-translational modifications are not essential for trypsin inhibitory activity. Northern blot analysis showed that sporamin transcripts could be systemically induced in leaf tissue of sweet potato by wounding. Therefore, sporamin may have a defense role as a protease inhibitor, in addition to its role as a storage protein.

When all living organisms are exposed to elevated temperatures, they exhibit induced synthesis of a set of proteins known as HSPs.Y Using rice heat-shock cDNA pTSS.1 (5) as a probe, we isolated two 16.9-kD HSP genes from a rice genomic library (Table I). The rice genomic library was constructed in a XgtlO vector using EcoRI-digested rice genomic DNA fragments (size between 2.0 and 4.0 kb) as inserts that were eluted from agarose gel pieces by a NA 45 DEAE membrane (Schleicher and Schuell). The heat-shock cDNA pTS1 hybridized to 2.5and 3.4-kb EcoRI-digested rice genomic DNA fragments in Southern hybridization. The DNA sequences of the two rice genomic HSP genes, Oshspl6.9A and Oshspl6.9B, were determined and analyzed as shown in Figure 1. Oshspl6.9A contained the sequence of cDNA pTS1. Comparison of Oshspl6.9A and Oshspl6.9B genes reveals that they share 98.8% homology in terms of the nucleotide sequence in the coding regions and 99.3% homology at the level of the deduced amino acid sequence. Multiple copies of the HSE-like sequence, similar to the Drosophila heat-shock consensus sequence (4), can be identified upstream from the putative TATA box, TATAAATA. The initiation site of the transcription of Oshspl6.9A and Oshspl6.9B, which was identified by the primer extension method (1), is located 131 bases upstream from the coding region and 26 bases downstream from the TATA-like region. The deduced polypeptide sequence of the Oshspl6.9A gene exhibits 73 and 84% homology to Gmhspl7.5E from soybean (2) and c5-8 from wheat (3), respectively. Table I. Characteristics of Two 16.9-kD Heat-Shock Genes from Oryza sativa

A monospecific polyclonal antibody was used to study the tissue-type specificity and intracellular localization of class I low-molecular-weight (LMW) heat-shock proteins (HSPs) in soybean (Glycine max) under different heat-shock regimes. In etiolated soybean seedlings, the root meristematic regions contained the highest levels of LMW HSP. No tissue-type-specific expression of class I LMW HSP was detected using the tissue-printing method. In immunolocalization studies of seedlings treated with HS (40[deg]C for 2 h) the class I LMW HSPs were found in the aggregated granular structures, which were distributed randomly in the cytoplasm and in the nucleus. When the heat shock was released, the granular structures disappeared and the class I LMW HSPs became distributed homogeneously in the cytoplasm. When the seedlings were then given a more severe heat shock following the initial 40[deg]C -> 28[deg]C treatment, a large proportion of the class I LMW HSPs that originally localized in the cytoplasm were translocated into the nucleus and nucleolus. Class I LMW HSPs may assist in the resolubilization of proteins denatured or aggregated by heat and may also participate in the restoration of organellar function after heat shock.