Heat Shock Induced Lipid Changes and Solute Leakage in Germinating Seeds of Pigeonpea

Heat shock (HS) reduced total lipid and phospholipid contents and their synthesis in germinating seeds of pigeonpea [Cajanus cajan (L.) Millspaugh]. Lipid peroxidation was also enhanced with increasing temperature and HS duration. HS influenced lipid metabolism to a higher extent at 45°C than at 40°C. This altered lipid metabolism and lipid peroxidation was associated with the loss of various solutes from the germinating seeds, and modification of growth and development. Pretreatment of germinating seeds at 40°C for 1 h or at 45°C for 10 min followed by incubation at 28°C for 3 h prior to 45°C for 2 h ameliorated solute leakage due to reduced lipid peroxidation and improvement in lipid content and membrane function.     

Ethanol treatment triggers a heat shock-like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.     

induction of heat shock response and acquisition of thermotolerance in callus cultures of Gerbera jamesonii

Summary The heat shock (HS) response in callus cultures of the ornamental plant Gerbera jamesonii H. Bolus var. hybrida was analyzed. A HS at 35° C or 40° C for 4 h induced (a) the synthesis of several heat shock proteins (HSPs), especially in the small molecular weight range and some spots corresponding to HSP70 components, and (b) an increase in the steady state levels of some specific mRNAs. At the nonstressing temperature (26° C), a sustainable level of translation for HSP70 was indeed carried out, as confirmed by immunological analysis with a monoclonal antibody against cotton HSP70. The steady state levels of mRNAs measured before and after a HS by Northern hybridization showed an increase with the heterologous probes HSP17.4, HSP17.6, and HSP21, whereas the probes HSC70 and HSP70 did not show any difference between the levels of control and HS-mRNAs. A pretreatment at 35° C, which induced a set of HSPs in the callus cultures, decreased the cell damage upon exposure to a temperature of 45° C as determined either with a regrowth test or by the tetrazolium reduction assay. Typically, as with the whole plants, callus of Gerbera jamesonii possessed the ability to respond to HS both by inducing HSPs and by developing an acquired thermotolerance.     

Prosopis chilensis is a plant highly tolerant to heat shock

At temperatures between 25 and 35°C, 100% of Prosopis chilensis seeds germinated within 24 h. At higher temperatures, the germination rate was reduced; at 50°C, seeds did not germinate. After germination at 25°C, the optimal temperature for seedling growth was 35°C and the seedlings did not grow at a temperature of 50°C. However, when germination was at 35°C, the optimal temperature for seedling growth was 40°C and some seedlings grew at 50°C, suggesting that thermotolerance was induced during seed germination at 35°C. Further thermotolerance can be induced in seedlings germinated at 35°C, by exposing them to 40°C for 2h. Under these conditions, seedlings exhibited increased growth rate at 45 and 50°C. Fluorography of SDS-polyacrylamide gel electrophoresis of the proteins synthesized and accumulated during 2 h at temperatures of 35, 40, 45 and 50°C in the presence of [³⁵S]methionine revealed the expression of 11 proteins not detectable at 35°C. Most of the proteins present at 35°C also increased in expression. The temperature for maximal expression of these proteins was 45°C.     

Heat shock induced proteins in plant cells

Tobacco (Nicotiana tabacum) and soybean (Glycine max) tissue culture cells were exposed to a heat shock and protein synthesis studied by SDS-polyacrylamide gel electrophoresis after labeling with radioactive amino acids. A new pattern of protein synthesis is observed in heat-shocked cells compared to that in control cells. About 12 protein bands, some newly appearing, others synthesized in greatly increased quantities in heat-shock cells, are seen. Several of the heat-shock proteins (HSPs) in both tobacco and soybean are similar in size. One of the HSPs in soybean (76K) shares peptide homology with its presumptive 25°C counterpart, indicating that the synthesis of at least some HSPs may not be due to activation of new genes. The optimum temperature for maximal induction of most HSPs is 39–40°C. Total protein synthesis decreases as heat-shock temperature is increased and is barely detectable at 45°C. The heat-shock response is maintained for a relatively short time in tobacco cells. After 3 hr at 39°C, a decrease is seen in the synthesis of the HSPs, and after 4 hr practically no HSPs are synthesized. After exposure to 39°C for 1 hr, followed by a return of tobacco cells to 26°C, recovery to the control pattern of synthesis requires greater than 6 hours. These results indicate that cells of flowering plants exhibit a heat-shock response similar to that observed in animal cells.     

<Emphasis Type="Italic">Paenibacillus</Emphasis> strain MP-1: a new source of mutanase

A mutan-degrading bacterium, closely related to Paenibacillus curdlanolyticus, was isolated from soil. It produced 0.4 U mutanase ml⁻¹ in 2 days in shake-flask cultures when bacterial mutan was the sole carbon source. Mutanase activity was optimal at pH 6.2 and 45°C over 1 h and was stable between pH 5.8 and 12 at 4°C for 24 h and up to 40°C for 1 h. Mutan produced by Streptococcus mutans was rapidly hydrolyzed by this enzyme. The hydrolysis of mutan (1 g l⁻¹) resulted in 17% saccharification over 2 h and, at the same time, glucan was entirely solubilized.     

Contents of soluble carbohydrates in yellow lupin seeds maturated at various temperatures

The objective of this paper was to compare the levels of soluble sugars in seeds of yellow lupin cv. Juno matured at different temperatures. The temperature regimes applied were 1). 26 °C for 24 h (high temperature), 2). 24 °C for 12 h and 19 °C for the next 12 h (optimum temperature regime), 3). 26 °C for 16 h and 4 °C for the next 8 h (high-low temperatures). Six soluble carbohydrates (d-galactose, myo-inositol, sucrose, raffinose, stachyose and verbascose) were quantified. Seeds maturing at constant temperature 26 °C accumulated more raffinose (by 100 %) than seeds maturing at optimum temperature regime. Seeds maturing at high temperature accumulated less stachyose and verbascose than those maturing at optimum temperature conditions, the differences being 45 and 24 %, respectively. In seeds maturing at high-low temperature the level of raffinose decreased while the level of stachyose and verbascose increased, compared to those maturing at optimum conditions. The contents of sucrose, d-galactose and myo-inositol in seeds maturing at optimum temperatures was lower than in seeds maturing at both high and high-low temperature regimes. It was shown, that temperature conditions — constant high temperature, or physiologically optimal thermal oscillations (24 °/19 °C) or high-low temperature regime — differently affect the contents of six soluble carbohydrates in maturing seeds of yellow lupin.     

Exposure of Fischerella [Mastigocladus] to high and low temperature extremes: strain evaluation for a thermal mitigation process

In conjunction with a proposed algal cultivation scheme utilizing thermal effluent, twelve Fischerella strains were tested for tolerance to temperatures above and below their growth range. Exposure to 65 °C or 70 °C for 30 min caused bleaching and death of most or all cells. Effects of 60 °C exposure for periods of up to 2 h ranged from undetectable to severe for the various strains. Chlorophyll a content typically decreased 21–22% immediately following 60 °C or 65 °C (1 h) exposure. However, the 60 °C-shocked cultures regained normal Chl a content after 24 h at 45 °C, whereas Chl a in 65 °C-shocked cultures immediately lost visible autofluorescence and was later degraded. Exposure to 15 °C virtually stopped growth of all strains during a 48 h exposure period. Most strains grew as rapidly as 45 °C controls when restored to 45 °C, while a few strains recovered more slowly. Comparison with dark-incubated controls indicated that photooxidative damage did not occur during cold shock. Certain strains exhibited relatively rapid recovery from both heat and cold exposure, thus meeting the temperature tolerance criteria for the proposed algal cultivation process.     

Induction of secondary dormancy in Amaranthus caudatus seeds

Nondormant A. caudatus seeds germinated in the darkat temperatures between 20 and 35° but not at 45 °C.Incubation at this temperature for at least 10 h inhibited seedgermination over the temperature range 20 to 35 °C,temperatures previously suitable for germination. Thus incubation at 45°C induced secondary dormancy. Mechanical or chemicalscarification or exposure to pure oxygen caused complete or almost completegermination of dormant seeds although more slowly in comparison to nondormantseeds. Secondary dormant scarified seeds required a lower concentration of ABAthan nondormant seeds to inhibit germination. The high temperature, whichinduced dormancy, 45 °C, caused the seed coat to be partiallyresponsible for secondary dormancy. Involvement of ABA (synthesis orsensitivity) in the induction and/or maintenance of this dormancy should beconsidered.     

Viability of rape (Brassica napus L.) seeds following selection on the basis of newly-emerged radicles then subsequent drying and storage

Germinating rape seeds selected on the basis of newly-emerged radicles (1 ± 0.5 mm) were dried to an equilibrium moisture content (c. 11%) in air at 20°C and 80% relative humidity without loss of viability. Storage life of these low-moisture-content germinating (LMCG) seeds at 15°C was limited to 7 days before viability was significantly reduced. However, viability of LMCG seeds was maintained for 84 days in storage at -20°C. Longer periods in store reduced viability, but 96% of seeds still remained viable after 336 days at - 20°C. Increasing periods of storage at -20°C reduced the subsequent seed longevity at 15°C, indicating a reduction in vigour during storage. Storage under reduced pressure or in a nitrogen atmosphere had little significant effect on seed longevity. Reduction of moisture content below 11% using vacuum drying at a range of temperatures reduced seed vigour.     

Decrease in sunflower (Helianthus annuus) seed viability caused by high temperature as related to energy metabolism, membrane damage and lipid composition

The aim of the present work was to investigate whether loss of germination ability and viability of sunflower (Helianthus annuus L.) seeds during incubation at a high temperature (45°C) was related to changes in energy metabolism, loss of membrane integrity, and/or changes in lipid composition. Pre‐treatment of seeds at 45°C progressively reduced subsequent germination at the optimal temperature (25°C). Seeds did not germinate at 45°C and almost all of them were dead after 72 h of soaking at this high temperature. This loss of seed viability was associated with a large increase in leakage of K⁺ and total electrolytes into the incubation medium, and with production of malondialdehyde in the embryonic axis and cotyledons, suggesting a loss of membrane integrity probably due to lipid peroxidation. ATP and ADP levels increased sharply during the first hours of imbibition at 45°C, remained high for about 24 h and then decreased. As a consequence, the energy charge followed a similar pattern. If the treatment at 45°C did not exceed 48 h, seeds recovered an apparently normal energy metabolism after transfer to 25°C, even though they lost their ability to germinate at this temperature. Therefore, energy metabolism at the whole embryo level cannot be considered as an indicator of germination ability. Incubation of seeds at 45°C resulted in an increase in triacylglycerols and diacylglycerols without a significant change in their fatty acid composition. It also induced a slight increase in phospholipid content with an increase in C_16:0, C_18:0 and C_18:1, but with no change in C_18:2. In phospholipids, the C_18:2/C_18:1 and (C_18:1 + C_18:2)/ (C_16:0 + C_18:0) ratios thus declined during treatment at 45°C. The results obtained suggest that deterioration of sunflower seeds during incubation at a high temperature is mainly related to membrane damage and alteration of energy metabolism, and that accumulation of malondialdehyde, which is an index of lipid peroxidation, does not correspond to a decrease in total lipids and phospholipids nor to a significant change in fatty acid composition, except in PL in which the C_18:2/C_18:1 and (C_18:1 + C_18:2)/ (C_16:0 + C_18:0) ratios slightly declined.     

DNA synthesis during the early stage of germination in the barley embryo meristems and its inhibition by N-methyl-N-nitrosourea

3 peaks of DNA synthesis were observed in the barley embryo of seeds, germinating for 49 h in running tap water at 25°C. The first peak, found after 22h, was formed by S-cells in the roots and in the 1st leaf meristem. The second peak (after 34–37h) and third peak (after 46–49 h) represents the S-cells in the roots, apex and 1st, 2nd and 3rd leaf meristems. Application of N-methyl-N-nitrosourea for 3 h at the onset of germination inhibited the rate of DNA synthesis and postponed the peaks of DNA synthesis in individual meristems of the embryo.     

Characteristics of the amylase of Arthrobacter psychrolactophilus

Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations was found to be a nonlinear function of time at 30 and 40 °C. Concentrates of supernatant fractions incubated without substrate exhibited poor stability at 30, 40, or 50 °C, with 87% inactivation after 21 h at 30 °C, 45% inactivation after 40 min at 40 °C and 90% inactivation after 10 min at 50 °C. Proteases known to be present in crude preparations had a temperature optimum of 50 °C, but accounted for a small fraction of thermal instability. Inactivation at 30, 40, or 50 °C was not slowed by adding 20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases. Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 °C, and the amylase required Ca⁺² for activity. The optimal pH for activity was 5.0–9.0 on soluble starch (30 °C), and the amylase exhibited a K _m with 4-nitrophenyl-α-D-maltoheptaoside-4,6-O-ethylidene of 120 μM at 22 °C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 °C at about the same rate as an equal number of units of barley α-amylase, but lost most of its activity after only a few hours.     

Solute Leakage in Soybean Seedlings under Various Heat Shock Regimes

The leakage of solute from intact seedlings during incubationunder various heat shock (HS) regimes was studied. ContinuousHS at 40?C did not induce leakage of amino acids, soluble sugarsand electrolytes into the incubation medium, when compared withcontrol incubation at 28?C. Continuous HS at 45?C (lethal treatment)caused leakage to increase continuously and linearly duringa 5-h treatment period. However, brief HS at 47.5?C, (lethaltreatment), unlike continuous HS at 45?C, induced leakage ata slower rate which reached a plateau within 2 to 3 h at 28?C.Preincubation for 2 h at 40?C completely prevented the leakagecaused by the brief HS at 47.5?C, but not that caused by continuous45?C HS. The amount of leakage during 2 h of incubation at 45?Cwas reduced to half by 30 min preincubation at 40?C and wasreduced to a minimal level by 1-h preincubation. Greater reductionof leakage at 45?C HS was observed when an additional 4 h ofincubation at 28?C immediately followed the 40?C preincubation.These results and previous findings (Lin et al. 1984) indicatethat the synthesis and accumulation of HS proteins (HSPs) areimportant for preventing HS-induced leakage from the cells.One of the HSPs, 15 kD in size appeared to be associated withthe plasma membrane. (Received February 12, 1985; Accepted August 30, 1985)     

Acquisition of Thermotolerance in Soybean Seedlings : Synthesis and Accumulation of Heat Shock Proteins and their Cellular Localization

When soybean Glycine max var Wayne seedlings are shifted from a normal growth temperature of 28°C up to 40°C (heat shock or HS), there is a dramatic change in protein synthesis. A new set of proteins known as heat shock proteins (HSPs) is produced and normal protein synthesis is greatly reduced. A brief 10-minute exposure to 45°C followed by incubation at 28°C also results in the synthesis of HSPs. Prolonged incubation (e.g. 1-2 hours) at 45°C results in greatly impaired protein synthesis and seedling death. However, a pretreatment at 40°C or a brief (10-minute) pulse treatment at 45°C followed by a 28°C incubation provide protection (thermal tolerance) to a subsequent exposure at 45°C. Maximum thermoprotection is achieved by a 2-hour 40°C pretreatment or after 2 hours at 28°C with a prior 10-minute 45°C exposure. Arsenite treatment (50 micromolar for 3 hours) also induces the synthesis of HSP-like proteins, and also provides thermoprotection to a 45°C HS; thus, there is a strong positive correlation between the accumulation of HSPs and the acquisition of thermal tolerance under a range of conditions.

During 40°C HS, some HSPs become localized and stably associated with purified organelle fractions (e.g. nuclei, mitochondria, and ribosomes) while others do not. A chase at 28°C results in the gradual loss over a 4-hour period of the HSPs from the organelle fractions, but the HSPs remain selectively localized during a 40°C chase period. If the seedlings are subjected to a second HS after a 28°C chase, the HSPs rapidly (complete within 15 minute) relocalize in the organelle fractions. The relative amount of the HSPs which relocalize during a second HS increases with higher temperatures from 40°C to 45°C. Proteins induced by arsenite treatment are not selectively localized with organelle fractions at 28°C but become organelle-associated during a subsequent HS at 40°C.

     

Expression of heat shock proteins in adult honey bee (Apis mellifera L.) workers under hot-arid subtropical ecosystems

Heat stress elicits the expression of heat shock proteins (HSPs) in honey bee subspecies. These highly conserved proteins have significant role in protecting cells from thermal-induced stresses. Honey bees in subtropical regions face extremely dry and hot environment. The expression of HSPs in the nurses and foragers of indigenous (Apis mellifera jemenitica) and imported European (Apis mellifera ligustica and Apis mellifera carnica) honey bee subspecies after heat shock treatment were compared using SDS-PAGE. Hsp70 and Hsp82 were equally expressed in the nurses of all tested bee subspecies when exposed to 40 °C and 45 °C for 4 h. The forager bees exhibited differential expression of HSPs after heat stress. No HSPs was expressed in the foragers of A. m. jemenitica, and Hsp70 was expressed only in the foragers of A. m. ligustica and A. m. carnica at 40 °C. A prominent diversity in HSPs expression was also exhibited in the foragers at 45 °C with one HSP (Hsp70) in A. m. jemenitica, two HSPs (Hsp40 and Hsp70) in A. m. carnica, and three HSPs (Hsp40, Hsp60 and Hsp70) in A. m. ligustica. No HSPs was expressed in the control nurse and forager bees at any of the tested temperatures. These findings illustrated the differences in HSP expression among nurse and forager bees. It is obvious that the native foragers are more heat tolerant with least HSPs expression than exotic bee races. Further investigations will help to understand the potential role of HSPs in the adaptability, survival, and performance of bee subspecies in harsh climate of the subtropical regions.     

Germination traits and seed-bank dynamics of a biennial plant,Oenothera glazioviana Micheli

A study was conducted on the germination traits and seed-bank dynamics ofOenothera glazioviana (=O. erythrosepala), which sets seed in August in sand-dune systems in Japan. More than 90% of freshly matured seeds germinated over a wide range of temperature in light, but less than 10% did so in continuous darkness. Stratification (chilling under moist conditions) was ineffective in diminishing the light-requirement for germination. When fresh seeds were imbibed for 24 h including a 12-h light period, followed by 7-day air-drying, 94% of them became germinable in the dark at 25°C, but remained dormant at less than 15°C. of seeds collected in March from capsules of dead plants, 58% germinated in the dark at 25°C. After four cycles of alternatc 1-day wetting followed by 2-day drying or 1.5-day wetting followed by 1.5-day drying under a 12-h photoperiod, the fraction of viable seeds declined from 76% to 40% and 22%, respectively, due to germination during the wet periods. Seed-bag experiments were conducted in the field, using seeds given and not given a light-stimulus. Forty percent of the light-stimulated seeds germinated in the soil, whereas the seeds without a light-stimulus remained dormant throughout the experiment. When seeds were placed on the soil surface or at a depth of 0.5-1 cm, the proportion of germinable seeds declined during late spring and autumn, but not during winter and early spring. The seed-bank size of a natural population just prior to current seed dispersal was 2–3% of the seed production in the previous year, suggesting a high turnover rate of the seed-bank.     

A butenolide, isolated from smoke, can overcome the detrimental effects of extreme temperatures during tomato seed germination

The butenolide, 3-methyl-2H-furo[2, 3-c]pyran-2-one, is an highly active compound isolated from plant-derived smoke. This compound is known to stimulate seed germination in a wide range of plants akin to smoke or aqueous extracts of smoke. The present study attempted to elucidate the role of the butenolide in overcoming detrimental effects of low and high temperatures on tomato seed germination and seedling growth. The germination percentage followed a parabolic curve for temperatures ranging from 10 to 40°C, with 25°C being the optimum for all treatments. Control seeds showed radicle emergence at two extreme temperatures (10 and 40°C) and seedlings failed to develop further, even upon prolonged incubation. By comparison the butenolide-treated seeds grew into phenotypically normal seedlings at these non-optimum temperatures. The smoke–water-treated seeds had an intermediate response as only a fraction of germinated seed developed into normal seedlings. Seedling vigour indices as well as seedling weight were significantly higher (p ≤ 0.05) for butenolide-treated seeds at all temperatures. Furthermore, seedlings developed in the presence of the butenolide had about a 1:1 correspondence between root and shoot length. Butenolide-treated seeds grew better than the control seeds in the temperature shift experiments. A gradual decline in the vigour index values was recorded with an increased duration of incubation at the extreme temperatures. Results of the present study are very important from an horticultural point of view as they indicate the potential use of the butenolide compound in restoring normal seed germination and seedling establishment in tomato below and above optimum temperatures.     

Effect of Temperatures on Dormancy and Germination in Three Species in the Lamiaceae Occurring in Northern Wetlands

In the temperate region temperature is the main factor influencing the germination period of plant species. The purpose of this study was to examine effects of constant and fluctuating temperatures on dormancy and germination under laboratory and field conditions in the three wetland species Lycopus europaeus, Mentha aquatica and Stachys palustris. The results should give indications if the temperature-dependent regulation of dormancy and germination is phylogenetically constrained. Tests for germination requirements showed a minimum temperature for germination of 9 °C in Mentha and 12 °C in Lycopus and Stachys, and a maximum temperature of 33 °C for Lycopus and 36 °C for Mentha and Stachys. Fluctuating temperatures promoted germination in all three species but the amplitude required for high germination (>50%) differed: it was 8 °C in Mentha, 10 °C in Stachys and 14 °C in Lycopus (mean temperature 22 °C). The effect of temperatures on the level of dormancy was examined in the laboratory by imbibing seeds at temperatures between 3 °C and 18 °C for periods between 2 and 28 weeks, as well as by a 30-month burial period, followed by germination tests at various temperatures, in light and darkness. In the laboratory only low temperatures (≤12 °C) relieved primary dormancy in seeds of Lycopus, while in Mentha and Stachys also higher temperatures lead to an increase of germination. Dormancy was only induced in Lycopus seeds after prolonged imbibition at 12 °C in the laboratory. Buried seeds of all species exhibited annual dormancy cycles with lower germination in summer and higher germination from autumn to spring. Exhumed seeds, however, showed considerable differences in periods of germination success. Dormancy was relieved when ambient temperatures were below 12 °C. Ambient temperatures that caused an induction of dormancy varied depending on species and test condition, but even low temperatures (8 °C) were effective. At high test temperatures (25 °C) in light, exhumed seeds of all three species showed high germination throughout the year. The three species showed various differences in the effects of temperatures on dormancy and germination. Similarities in dormancy and germination found among the species are in common with other spring-germinating species occurring in wetlands, so it seems that the temperature dependent regulation of dormancy and germination are related to habitat and not to phylogenetic relatedness.