Yih-Ming Chen

Functional activity of sporamin from sweet potato (Ipomoea batatas Lam.) : a tuber storage protein with trypsin inhibitory activity

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.

Characterization of the genomic structures and selective expression profiles of nine class I small heat shock protein genes clustered on two chromosomes in rice (Oryza sativa L.)

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 °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 invivo 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.

Characterization and Physiological Function of Class I Low-Molecular-Mass, Heat-Shock Protein Complex in Soybean

Examination of an ammonium sulfate-enriched fraction (70–100% saturation) of heat-shock proteins (HSPs) by nondenaturing polyacrylamide gel electrophoresis revealed the presence of a high molecular mass complex (280 kD) in soybean (Glycine max) seedlings. This complex cross-reacted with antibodies raised against soybean class I low-molecular-mass (LMW) HSPs. Dissociation of the complex by denaturing polyacrylamide gel electrophoresis showed the complex to contain at least 15 polypeptides of the 15-to 18-kD class I LMW HSPs that could be detected by staining, radiolabeling, and western blotting. A similar LMW-HSP complex was observed in mung bean (Vigna radiata L.; 295 kD), in pea (Pisum sativum L.; 270 kD), and in rice (Oryza sativa L.; 310 kD). The complex was stable under high salt conditions (250 mM KCI), and the integrity was not affected by 1% Nonidet P-40 and 3 [mu]g/ML RNase treatment. The size of the isolated HSP complex in vitro was conserved to 55[deg]C; however, starting at 37.5[deg]C, it changed to higher molecular forms in the presence of soluble proteins. The isolated HSP complex was able to protect up to 75% of the soluble proteins from heat denaturation in vitro.

Nucleotide sequence of a sporamin gene in sweet potato.

Sporamin is tissue specific and may account for more than 80% of the soluble protein found in tuberous root of sweet potato (Ipomoea batatas Lam.) (Maeshima et al., 1985). SDS-PAGE analysis indicates that sporamin may be resolved into two bands with molecular masses in the range of 25 kD. These two protein bands were characterized and classified into two subfamilies, based on the homology of their nucleotide sequences (Murakami et al., 1986; Hattori et al., 1989). Within the subfamilies, the homology may be greater than 90%, whereas the cDNAs between the two subfamilies share only 80% homology. Recent studies of two sporamin genes, gSPO-A1 and gSP0-B1, which belong to the two distinct subfamilies A and B, respectively, have shown that they are intronless genes (Hattori and Nakamura, 1988). Comparison of the 5’ flanking region also revea led t h e presence of two conserved sequence blocks, namely Suc box 2 and box 3 (Hattori and Nakamura, 1988; Tsukaya et al., 1991). In this paper, we present a nove1 sporamin gene, gSPOR5-31, which was identified to be an isogene of the gSPO-A1 within subfamily A. A hEMBL3 genomic library comprising 6 X 105 plaqueforming units was constructed with partially Sau3A-digested genomic DNA of tuberous roots. The library was screened by plaque hybridization using the cDNA probe of SP-B (an antisense strand of sporamin cDNA; K.-W. Yeh, unpublished results). One positive signal was obtained during the primary screening. It was purified to a single plaque, from which the DNA was extracted and characterized by restriction endonuclease and Southern blot analysis (Table I). The results showed that the clone (gSPOR5-31) contained an insert DNA of approximately 7 kb. When the insert DNA was digested by BamHI, resolved by agarose gel electrophoresis, and hybridized with the SP-B cDNA probe, only a 2.2-kb fragment clearly showed a strong signal. Therefore, it was subsequently subcloned into pGEM3Z (Promega) at the BamHI site for further studies. The nucleotide sequence of the 2.2-kb BamHI fragment was determined by nested deletion and primer walking. It included an open reading frame of 660 nucleotides corresponding to 220 amino acid residues. The genomic clone of gSPOR5-31 was similar to those of the gSPO-A1 and gSPO-B1 containing no intron. These observations suggest that sporamin genes are intronless. Analysis of the coding

Functional Regions of Rice Heat Shock Protein, Oshsp16.9, Required for Conferring Thermotolerance in Escherichia coli

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.

Molecular characterization of Oryza sativa 16.9 kDa heat shock protein.

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.

Cloning and characterization of a cDNA encoding an 18.0-kDa class-I low-molecular-weight heat-shock protein from rice

A novel cDNA clone, Oshp18.0 cDNA, encoding a rice (Oryza sativa L. cv. Tainong 67) 18.0-kDa heat-shock protein (HSP), was isolated from a cDNA library of heat-shocked rice seedlings by use of the rice HSP cDNA, Oshsp17.3 cDNA, as a probe. The sequence showed that Oshsp18.0 cDNA contains a 749-bp insert encoding an ORF of 160 amino acids, with a predicted molecular mass of 18.0 kDa and a pI of 7.3. Sequence comparison reveals that Oshsp18.0 cDNA is highly homologous to other low-molecular-weight (LMW) HSP cDNAs. Also, the results of hybrid-selected in vitro translation clearly establish that Oshsp18.0 cDNA is the rice 18.0-kDa LMW HSP-encoding cDNA clone. The recombinant Oshsp18.0 fusion protein produced in Escherichia coli was of the size predicted, and was recognized by the class-I rice 16.9-kDa HSP antiserum. The results suggest that Oshsp18.0 cDNA is an 18.0-kDa class-I LMW HSP- encoding cDNA clone from rice.

Class I low molecular weight heat shock proteins in plants: immunological study and thermoprotection against heat denaturation of soluble proteins

Antibodies prepared against two major polypeptides of the 15- to 18-kD class of soybean heat shock proteins (HSPs) individually reacted with its antigen and cross-reacted with 12 other 15- to 18-Kd HSPs. We also found that this antibody preparation cross-reacted with the same class low molecular weight (LMW) HSPs of mung bean, rice and other seven plant species tested based on western blot analysis. The 70 to 100% ammonium sulfate (AS) fraction from heat shocked seedlings of mung bean and rice, as in soybean, contained a high percentage of all the HSPs. The proteins in this fraction were resistant to heat denaturation, as judged by their unpelletability after heat treatment. Moreover, this fraction showed a significant ability to protect the soluble proteins from heat denaturation. The HSPsenriched fractions prepared from mung bean and rice heat shocked seedlings were able to thermostabilize the homologous soluble proteins. Additionally, the HSPs-enriched fractions were exchangeable among these three plant species for thermostabilization.