Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate

To elucidate determinants of thermostability and folding pathways of the intrinsically stable proteins from extremophilic organisms, we are studying β-glucosidase from Pyrococcus furiosus. Using fluorescence and circular dichroism spectroscopy, we have characterized the thermostability of β-glucosidase at 90°C, the lowest temperature where full unfolding is achieved with urea. The chemical denaturation profile reveals that this homotetrameric protein unfolds at 90°C with an overall ΔG° of ∼ 20 kcal mol⁻¹. The high temperatures needed to chemically denature P. furiosus β-glucosidase and the large ΔG° of unfolding at high temperatures shows this to be one of the most stable proteins yet characterized. Unfolding proceeds via a three-state pathway that includes a stable intermediate species. Stability of the native and intermediate forms is concentration dependent, and we have identified a dimeric assembly intermediate using high temperature native gel electrophoresis. Based on this data, we have developed a model for the denaturation of β-glucosidase in which the tetramer dissociates to partially folded dimers, followed by the coupled dissociation and denaturation of the dimers to unfolded monomers. The extremely high stability is thus derived from a combination of oligomeric interactions and subunit folding.     

Change in kinetic regime of protein aggregation with temperature increase. Thermal aggregation of rabbit muscle creatine kinase

Creatine kinase thermal aggregation kinetics has been studied in 30 mM Hepes-NaOH buffer, pH 8.0, at two temperatures: 50.6 and 60°C. Aggregation kinetics was analyzed by measuring the growth of apparent absorption (A) at 400 nm. It was found that the limiting value of apparent absorption (A _lim) is proportional to protein concentration at both temperatures. The first order rate constant (k _I) does not depend on protein concentration in the range 0.05–0.2 mg/ml at temperature 50.6°C, but at temperature 60°C it increases with the growth of protein concentration in the range 0.1–0.4 mg/ml. Kinetic curves, shown in coordinates {A/A _lim; t}, in experiments at 50.6°C fuse to a common curve, which coincides with the theoretical curve of creatine kinase denaturation calculated using the denaturation rate constant determined from differential scanning calorimetry. At temperature 60°C, half-transformation time t _1/2 = ln2/k _I decreases when protein concentration grows. We conclude that when temperature increased from 50.6 to 60°C, change in the kinetic regime of thermal creatine kinase aggregation took place: at 50.6°C aggregation rate is limited by the stage of protein molecule denaturation, but at 60°C it is limited by the stage of protein aggregate growth, which proceeds as a reaction of pseudo-first order. Small heat shock protein Hsp 16.3 Mycobacterium tuberculosis suppresses the creatine kinase aggregation. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 3, pp. 408–416.     

Hybrid complexes of photosynthetic reaction centers and quantum dots in various matrices: resistance to UV irradiation and heating

The effects of ultraviolet (UV) irradiation (up to 0.6 J/cm²) and heating (65 °C, 20 min) on the absorption spectra and electron transfer in dehydrated film samples of photosynthetic reaction centers (RCs) from purple bacterium Rhodobacter (Rb.) sphaeroides, as well as in hybrid structures consisting of RCs and quantum dots (QDs), have been studied. The samples were placed in organic matrices containing the stabilizers of protein structure—polyvinyl alcohol (PVA) and trehalose. UV irradiation led to partially irreversible oxidation of some RCs, as well as to transformation of some fraction of the bacteriochlorophyll (BChl) molecules into bacteriopheophytin (BPheo) molecules. In addition, UV irradiation causes degradation of some BChl molecules that is accompanied by formation of 3-acetyl-chlorophyll a molecules. Finally, UV irradiation destroys the RCs carotenoid molecules. The incorporation of RCs into organic matrices reduced pheophytinization. Trehalose was especially efficient in reducing the damage to the carotenoid and BChl molecules caused by UV irradiation. Hybrid films containing RC?+?QD were more stable to pheophytinization upon UV irradiation. However, the presence of QDs in films did not affect the processes of carotenoid destruction. The efficiency of the electronic excitation energy transfer from QD to P865 also did not change under UV irradiation. Heating led to dramatic destruction of the RCs structure and bacteriochlorins acquired the properties of unbound molecules. Trehalose provided strong protection against destruction of the RCs and hybrid (RC?+?QD) complexes.

     

Biochemical characterization of a novel thermostable xyloglucanase from an alkalothermophilic Thermomonospora sp.

Xyloglucanase from an extracellular culture filtrate of alkalothermophilic Thermomonospora sp. was purified to homogeneity with a molecular weight of 144 kDa as determined by SDS-PAGE and exhibited specificity towards xyloglucan with apparent K _m of 1.67 mg/ml. The enzyme was active at a broad range of pH (5–8) and temperatures (40–80°C). The optimum pH and temperature were 7 and 70°C, respectively. The enzyme retained 100% activity at 50°C for 60 h with half-lives of 14 h, 6 h and 7 min at 60, 70 and 80°C, respectively. The kinetics of thermal denaturation revealed that the inactivation at 80°C is due to unfolding of the enzyme as evidenced by the distinct red shift in the wavelength maximum of the fluorescence profile. Xyloglucanase activity was positively modulated in the presence of Zn²⁺, K⁺, cysteine, β-mercaptoethanol and polyols. Thermostability was enhanced in the presence of additives (polyols and glycine) at 80°C. A hydrolysis of 55% for galactoxyloglucan (GXG) from tamarind kernel powder (TKP) was obtained in 12 h at 60°C and 6 h at 70°C using thermostable xyloglucanases, favouring a reduction in process time and enzyme dosage. The enzyme was stable in the presence of commercial detergents (Ariel), indicating its potential as an additive to laundry detergents.     

Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus

We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and 37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS 6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks. Received 20 January 1999/ Accepted in revised form 30 April 1999     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of ΔC p of protein unfolding

Cold and heat denaturation of the double mutant Arg 3→Glu/Leu 66→Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D ¹H NMR spectroscopy in the temperature range from −12°C in supercooled water up to +70°C. The fraction of unfolded protein, f _u, was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be −11°C and 39°C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, ΔC _p of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.     

Structural characteristic,pH and thermal stabilities of apo and holo forms of caprine and bovine lactoferrins

Apo and holo forms of lactoferrin (LF) from caprine and bovine species have been characterized and compared with regard to the structural stability determined by thermal denaturation temperature values (T _m), at pH 2.0–8.0. The bovine lactoferrin (bLF) showed highest thermal stability with a T _m of 90 ± 1°C at pH 7.0 whereas caprine lactoferrin (cLF) showed a lower T _m value 68 ± 1°C. The holo form was much more stable than the apo form for the bLF as compared to cLF. When pH was gradually reduced to 3.0, the T _m values of both holo bLF and holo cLF were reduced showing T _m values of 49 ± 1 and 40 ± 1°C, respectively. Both apo and holo forms of cLF and bLF were found to be most stable at pH 7.0. A significant loss in the iron content of both holo and apo forms of the cLF and bLF was observed when pH was decreased from 7.0 to 2.0. At the same time a gradual unfolding of the apo and holo forms of both cLF and bLF was shown by maximum exposure of hydrophobic regions at pH 3.0. This was supported with a loss in α-helix structure together with an increase in the content of unordered (aperiodic) structure, while β structure seemed unchanged at all pH values. Since LF is used today as fortifier in many products, like infant formulas and exerts many biological functions in human, the structural changes, iron binding and release affected by pH and thermal denaturation temperature are important factors to be clarified for more than the bovine species.     

Allosteric serine hydroxymethyltransferase from monkey liver: Temperature induced conformational transitions

The homogeneous serine hydroxymethyltransferase from monkey liver was optimally activate at 60°C and the Arrhenius plot for the enzyme was nonlinear with a break at 15°C. The monkey liver enzyme showed high thermal stability of 62°C, as monitored by circular dichroism at 222 nm, absorbance at 280 nm and enzyme activity. The enzyme exhibited a sharp co-operative thermal transition in the range of 50°–70°(T _m= 65°C), as monitored by circular dichroism. L-Serine protected the enzyme against both thermal inactivation and thermal disruption of the secondary structure. The homotropic interactions of tetrahydrofolate with the enzyme was abolished at high temperatures (at 70°C, the Hill coefficient value was 1.0). A plot ofh values vs. assay temperature of tetrahydrofolate saturation experiments, showed the presence of an intermediate conformer with anh value of 1.7 in the temperature range of 45°–60°C. Inclusion of a heat denaturation step in the scheme employed for the purification of serine hydroxymethyltransferase resulted in the loss of cooperative interactions with tetrahydrofolate. The temperature effects on the serine hydroxylmethyltransferase, reported for the first time, lead to a better understanding of the heat induced alterations in conformation and activity for this oligomeric protein.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Isolation and characterization of OmpF-like porin from <Emphasis Type="Italic">Yersinia ruckeri</Emphasis>

The polypeptide profile of the porin protein fraction of Yersinia ruckeri, a Gram-negative bacterium causing yersiniosis in fish, has been shown to depend on cultivation temperature. OmpF-like porins are expressed mainly in the outer membrane (OM) of the “cold” variant (4°C) of the microorganism and OmpC-like proteins are expressed in the OM of the “warm” variant (37°C). Both types of porins are present in the OM of Y. ruckeri at room temperature. The OmpF-like porin of the “cold” variant was isolated and characterized. The molecular weight and primary structure of the protein were determined. The methods of optical spectroscopy (circular dichroism and intrinsic protein fluorescence) have shown that the protein has a spatial structure typical of β-structured porins from the OM of Gram-negative bacteria. The functional activity of isolated protein was characterized by the bilayer lipid membrane (BLM) technique. The most probable level of channel conductivity was 320 ± 60 pS, corresponding to the channel conductivity of OmpF porins of the genus Yersinia. The distinctive feature of OmpF porin from Y. ruckeri is high thermostability of its functionally active conformation: the protein forms stable pores in the BLM even after heating to 85°C.     

Pigment exchange of Photosystem II reaction center by chlorophyll d

Pigment exchanges among photosystem reaction centers (RCs) are useful for the identification and functional analysis of chromophores in photosynthetic organisms. Pigment replacement within the spinach Photosystem II RC was performed with Chl d derived from the oxygenic alga Acaryochloris marina, using a protocol similar to that reported previously [Gall et al. (1998) FEBS Lett 434: 88–92] based on the incubation of reaction centers with an excess of other pigments. In this study, we analyzed Chl d-modified monomeric RC which was separated from Chl d-modified dimeric RC by size-exclusion chromatography. Based on the assumption of a constant ratio of two Pheo a molecules per RC, the number of Chl a molecules in Chl d-modified monomeric RCs was found to decrease from six to four. The absorption spectrum of the Chl d-modified monomeric RC at room temperature showed a large peak at 699.5 nm originating from Chl d and a small peak at 672.5 nm orignating from Chl a. Photoaccumulation of the Pheo a⁻ in Chl d-modified monomeric RC, in the presence of sodium dithionate and methyl viologen, did not differ significantly from that in control RC, showing that the Chl d-modified monomeric RC retains its charge separation activity and photochemically active Pheo a.     

Reconstitution of photosynthetic reaction centers and core antenna-reaction center complexes in liposomes and their thermal stability

Photosynthetic reaction centers (RCs) and their core light-harvesting complexes (LH1-RCs), purified from a thermophile, Thermochromatium (T.) tepidum, and a mesophile, Allochromatium (A.) vinosum, were reconstituted into liposomes. The RC and the LH1-RC in the reconstituted liposomes were found intact from the absorption spectra at about 4 and 40 degrees C respectively. The thermal stability of the RCs of T. tepidum in the liposome was dependent on whether they were surrounded directly by lipids or by the core light-harvesting complexes. The results show that the RC of T. tepidum gains its thermostability through interactions with the LH1. These results are consistent with the result that the thermal stability of the LH1 in T. tepidum is similar in both the reconstituted LH1-RC liposome and ICM. This is clearly different from the mesophilic bacterium, A. vinosum. The thermal stability of RC was also affected by its subunit constitution: the RC containing a cytochrome subunit was more thermostable than the cytochrome-detached RC. This suggests that the cytochrome subunit might play a role in protecting the special pair pigments from denaturation. The thermal denaturation showed a second-order reaction dependence on time. The interaction of the pigments with proteins and/or lipids might be the cause of the second-order reaction profile.     

Biophysical analysis of phaseolin denaturation induced by urea,guanidinium chloride,pH, and temperature

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.     

The effect of some osmolytes on the activity and stability of mushroom tyrosinase

The thermodynamical stability and remained activity of mushroom tyrosinase (MT) fromAgaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40°C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obeye the first order law. Inactivation rate constant (k_inact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of k_inact(0.7×10⁻⁴s⁻¹) in comparison with their absence (2.5×10⁻⁴s⁻¹). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodyanamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.     

Thermal Denaturation of Bacterial and Bovine Dihydrofolate Reductases and their Complexes with NADPH,Trimethoprim and Methotrexate

Abstract

Scanning microcalorimetry was used for the study of thermal denaturation of E.coli and bovine liver dihydrofolate reductases (cDHFR and bDHFR, respectively) and their complexes with NADPH, trimethoprim (TMP) and methotrexate (MTX) at pH 6.8. It was shown that the denaturation temperature of bDHFR is 7.2°C less than that of cDHFR and that ionic strength is equally important for the thermostability and cooperativity of the denaturation process of the two proteins. Binding of antifolate compounds significantly stabilizes DHFR against heat denaturation. The stabilizing effect and the transition cooperativity depend on the nature of the inhibitor, the presence of NADPH and the origin of the enzyme. The dependence of calorimetric denaturation enthalpy (calculated per gram of protein) on denaturation temperature for DHFRs, their complexes with NADPH and binary/ternary complexes with TMP/MTX fits to the same straight line with the slope of 0.66 J/K g. This relatively high value indicates an essential role of hydrophobic contacts in the stabilization of DHFR structure. The change of denaturation temperatures in binary complexes with MTX/TMP (in comparison with the free enzymes) is as much as 14.2°C/8.5°C and 13.3°C/3.2°C for cDHFR and bDHFR, respectively. The same change in ternary complexes with MTX/TMP is much more pronounced and equals to 21.9°C/16.8°C and 29.0°C/16.4°C. The vast difference of binary and ternary complexes thermostability demonstrates the important role of cofactor in the stabilization of enzyme. Moving from binary to ternary systems causes a significant increase in denaturation temperatures, even when corresponding association constants do not change (cDHFR binary/ternary complexes with MTX) or increases very slightly (bDHFR binary/ternary complexes with TMP). In all other cases the increase of denaturation temperature     

Effect of Thermal Treatment on the Physicochemical Properties of Emulsion Stabilized by Gelatin from Black Tilapia (Oreochromis mossambicus) Skin

This study investigated the influence of thermal treatment (30 °C to 110 °C, 30 min) on the physicochemical and rheological properties of an emulsion stabilized by black tilapia (Oreochromis mossambicus) skin at pH 4. The protein pattern of tilapia gelatin did not have any significant difference after the gelatin was heated within a temperature range of 30 °C to 70 °C. However, at 90 °C and 110 °C, denaturation occurred where α-, β- and γ-chains of the gelatin were degraded, leading to a concomitant increase in low molecular peptides. The emulsion stability was investigated through a particle size analyzer, zeta potential, microscopic observation and creaming index. The gelatin emulsion was physically stable at 30 °C to 70 °C with a mean droplet size of less than 13 μm. When the heating temperature was increased to 90 °C and 110 °C, the emulsion showed a pronounced increase in droplet size due to coalescence. The gelatin emulsion heated at 90 °C and 110 °C also displayed instability against creaming after storage at room temperature for 7 days. As the heating temperature increased, the gelatin emulsion exhibited a decrease in apparent viscosity and the flow behavior changed from shear thinning to Newtonian. The rheological data also showed that the storage modulus (G′) of emulsion became more frequency dependent as the heating temperature increased, indicating weak droplet interactions. The tilapia gelatin emulsion was physically unstable when subjected to thermal treatment above 70 °C. The data reported in this study provides useful insight into the formulation of acidic food emulsions that require thermal treatment.

     

Effects of decomposition and storage conditions on the δ13C and δ15N isotope values of killer whale (Orcinus orca) skin and blubber tissues

Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ¹⁵N and δ¹³C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ¹⁵N value within 3 d at 20°C. Storage at ?20°C was as effective as ?80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze‐dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO‐salt solution, frozen or at room temperature, did not significantly change the δ¹⁵N and δ¹³C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected.     

Effects of temperature, light, storage conditions, sowing time, and burial depth on the seed germination of Cardiocrinum cordatum var. glehnii (Liliaceae)

In this study, we conducted experiments to accumulate practical information on the propagation and establishment of a population of Cardiocrinum cordatum var. glehnii by seed sowing. C. cordatum var. glehnii seeds require approximately 19 months from seed dispersal to cotyledon emergence in the field. However, the period from seed dispersal to radicle emergence was shortened to approximately 7–8 months by the temperature transition of 25/15°C (60 days) → 15/5°C (30 days) → 0°C (120 days) → 15/5°C (i.e., 15/5°C represents alternating temperature treatment wherein the seeds were placed at 15°C for 12 h during the day and then at 5°C for 12 h during the night). More than 90% of the seeds, which were stored dry at 5°C for 12 months and sown in pots in the field, showed cotyledon emergence, whereas in seeds stored dry at 25°C, dry at room temperature, and non-dry at room temperature, cotyledon emergence was decreased by less than 1%. More than 88% of the seeds that were stored dry at 5°C and sown in the field in October 2002 immediately after collecting, November, and from April to July 2003 showed cotyledon emergence in spring 2004. However, seeds sown in August, September, and October 2003 showed cotyledon emergences of 57.6%, 0%, and 0% in spring 2004, respectively. Seeds collected in October 2002 and sown until July 2003 in the field received adequate high temperature in summer, moderate temperature in autumn, and cold temperature in winter; therefore, the percentage of cotyledon emergence was high in spring 2004. On the other hand, seeds sown in August 2003 or later could not receive enough high temperature; thus, cotyledons emerged from only a few seeds.