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

Two major polypeptides of the 15-to 18-kilodalton class of soybean (Glycine max) heat shock proteins (HSPs)

Examination of an ammonium sulfate-enriched fraction (70-1 00% saturation) of heat-shock proteins (HSPs) by nondenaturing polyacrylamide gel electrophoresis revealed the presence of a high molecular m a s 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 L M W HSPs that could be detected by staining, radiolabeling, and western blotting. A similar LMW-HSP complex was observed in mung bean (Vigna radiafa L.; 295 kD), in pea (Pisum safivum 1.; 270 kD), and in rice (Oryza sativa 1.; 310 kD). l h e complex was stable under high salt conditions (250 mM KCI), and the integrity was not affected by 1 % Nonidet P-40 and 3 & n L RNase treatment. l h e size of the isolated HSP complex in vitro was conserved to 55°C; however, starting at 37.5"C, it changed to higher molecular forms in the presence of soluble proteins. l h e isolated HSP complex was able to protect up to 75% of the soluble proteins from heat denaturation in vitro.The induction of HSP synthesis in response to thermal stress occurs in a11 organisms that have been examined, ranging from bacteria to humans (Schlesinger et al., 1982;Vierling, 1991). Whereas the physiological functions of HSPs have not been clearly established, these proteins are well correlated with the acquisition of thermotolerance in a time-and temperature-dependent manner. Under HS conditions the expression of the LMW HSPs in soybean (Glycine max) is increased, yielding final concentrations of up to 1% of the total cellular proteins (Hsieh et al., 1992). Based on this correlation, it has been hypothesized that accumulation of HSPs is a n essential component of a process that prevents heat damage (Lin et al., 1984;Key et al., 1985;Kimple et al., 1990). Some HSPs become selectively localized in cellular organelles in a temperature-dependent fashion and relocate to the cytoplasm after a 28°C incubation period (Lin et al., 1984). Isolated mitochondria with associated HSPs are functional in oxidative phosphorylation under thermal stress, suggesting that association of HSPs with organelles provide protection from heat damage (Chou et al., 1989 (Jinn et al., 1993). In soybean, depletion of the 15-to 18-kD HSPs in the HSP-enriched fraction resulted in the loss of the thermostabilizing ability, and this ability was again restored when these HSPs were added. The protection provided by these HSP-enriched fractions is effective mainly for membrane-associated proteins (Jinn et al., 1993).The LMW HSPs of plants and a11 other eukaryotes have a conserved carboxyl-terminal region homologous to the a-crystallin of the eye lens at the amino acid sequence leve1 (Ingolia and Craig, 1982; Augusteyn and Koretz, 1987;Lindquist and Craig, 1988). The LMW HSPs in vertebrates (Arrigo and Welch, 1987;Merck et al., 1993), Drosopkila (Arrigo and Pauli, 1988), ye...

Mitochondria isolated from 2-day-old etiolated soybean (Glycine max) seedlings which had been subjected to various heat shock treatments, i.e. (A) 280C (2 h), (B) 380C (2 h), (C) 380C (2 h)-42.50C (0.5 h), and (D) 38°C (2 h)-42.50C (0.5 h)-280C (4 h), were monitored for 02 uptake using an oxygen electrode. Mitochondria isolated after all four heat shock treatments were active in 02 consumption at 280C in response to succinate and ADP (derived P/O ratios were 1.6, 1.7, 1.3, and 1.3, respectively.) The mitochondria from all four treatments were also active in 02 uptake at 42.50C. However, only mitochondria isolated after treatment (C) were tightly coupling at 42.50C (derived ADP/O ratio was about 1.4). Combined with our earlier findings on the subcellular localization of heat shock proteins, our present data demonstrate that association of heat shock proteins with mitochondria by treatment (C) enables them to phosphorylate at 42.50C (i.e. they become thermotolerant). Isolated mitochondria from treatment (C) and treatment (A) were compared by electron microscopy. They appeared to be very similar and no significant ultrastructural differences were noted.When soybean seedlings are shifted from a normal growth temperature of 28°C to 40°C HS,3 there is a dramatic change in protein synthesis and a new set of proteins known as HSPs are produced (1, 6, 7).There is a strong positive correlation between the accumulation of HSPs and the acquisition of thermotolerance (9-12, 14, 15, 19). The acquisition of thermotolerance appears to depend not only upon the synthesis of HSPs but also on their cellular localization (10, 24). In soybean seedlings, several HSPs become selectively localized in or associated with nuclei, mitochondria, ribosomes, and plasma membranes, as demonstrated by sucrose gradient centrifugation (10, 1 1). Specifically, the complex group of 15 to 18 kD HSPs was localized in these fractions during HS of soybean seedlings. The 70 kD HSP also exhibited similar localization, although to a lesser extent. In addition, some 22 and 24 kD HSPs were selectively localized in the mitochondrial fraction (10). This selective localization of HSPs was shown to be temperature dependent (10).The nature ofthe association ofthe HSPs with the organelle fraction of soybean is not known. We have tested whether the 'Supported by National Science Council of ROC under grant NSC76-0201-B002-1 1 to C. Y. Lin.

Two new prenylflavanones, tanariflavanones A (1), and B (2), and one known compound, (-)-nymphaeol-C (3), were isolated from the fallen leaves of Macaranga tanarius. Compounds 1 and 2 showed inhibition of radical growth of lettuce seedlings at 200 ppm. Their structures were elucidated primarily by NMR, circular dichroism, and mass spectroscopic methods.