Cryoprotective and osmotic responses to cold acclimation and freezing in freeze-tolerant and freeze-intolerant earthworms

In this paper we present the results of physiological responses to winter acclimation and tissue freezing in a freeze-tolerant Siberian earthworm, Eisenia nordenskioeldi, and two freeze-intolerant, temperate earthworm species, Lumbricus rubellus and Aporrectodea caliginosa. By analysing the physiological responses to freezing of both types we sought to identify some key factors promoting freeze tolerance in earthworms. Winter acclimation was followed by a significant increase in osmolality of body fluids in E. nordenskioeldi, from 197 mosmol kg⁻¹ in 10 °C-acclimated animals to 365 mosmol kg⁻¹ in animals acclimated to 0 °C. Cold acclimation did not cause any change in body fluid osmolality in the two freeze-intolerant species. As a response to ice formation in the body, the freeze-intolerant species produced copious amounts of slime and expulsion of coelomic fluids, and thereby lost 10–30% of their total water content. Contrary to this, the freeze-tolerant species did not lose water upon freezing. At temperatures down to −6.5 °C, the ice content in the freeze-tolerant E. nordenskioeldi was significantly lower than in L. rubellus. At lower temperatures there were no differences in ice content between the two species. Cold acclimated, but unfrozen, specimens of all three species had low levels of ammonia, urea, lactate, glycerol and glucose. As a response to ice formation, glucose levels significantly increased within the first 24 h of freezing. This was most pronounced in E. nordenskioeldi where a 153-fold increase of glucose was seen (94 mmol · l⁻¹). In L. rubellus and A. caliginosa a 19-fold and 17-fold increase in glucose was seen. This is the first study on physiological mechanisms promoting freeze tolerance in E. nordenskioeldi, or any other oligochaete. Our results suggest that the cryoprotective system of this species more closely resembles that of freeze-tolerant anurans, which synthesize cryoprotectants only after tissues begin to freeze, than that of cold-hardy invertebrates which exhibit a preparatory accumulation of cryoprotectants during seasonal exposure to low temperature. Accepted: 10 February 1999     

A novel yeast gene, RHK1 , is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits β-1,3-glucan synthesis

The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall β-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast. A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin. These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups. A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated. DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids. Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S. cerevisiae cells with complete killer resistance. A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of β-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin. Received: 27 June 1996 / Accepted: 14 October 1996     

Inhibition of yeast Δ-pyrroline-5-carboxylate dehydrogenase by common amino acids and the regulation of proline catabolism

A yeast glutamate auxotroph (glt_{1 − 1}), blocked in the tricarboxylic acid cycle at aconitase, is shown to possess catabolic pathways to glutamate from proline, arginine and glutamine, and grows on any of these amino acids in a minimal medium. This mutant does not, however, grow on these amino acids in a medium containing the full complement of common amino acids minus glutamate. The mechanism of this growth failure involves partial inhibition of the catabolic routes to glutamate by more than half the common amino acids. In the case of proline catabolism, this inhibition is localized principally at the enzyme Δ¹-pyrroline-5-carboxylate: NAD(P)⁺ oxidoreductase by in vitro studies. Similar results with this enzyme prepared both from yeast and from beef kidney mitochondria suggest that the inhibition observed may be the basis of a regulatory mechanism of general significance.     

Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications

Proline is an important amino acid in terms of its biological functions and biotechnological applications. In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. However, it has been shown that proline levels are not increased under various stress conditions in the yeast Saccharomyces cerevisiae cells. Proline is believed to serve multiple functions in vitro such as protein and membrane stabilization, lowering the T _m of DNA, and scavenging of reactive oxygen species, but the mechanisms of these functions in vivo are poorly understood. Yeast cells biosynthesize proline from glutamate in the cytoplasm via the same pathway found in bacteria and plants and also convert excess proline to glutamate in the mitochondria. Based on the fact that proline has stress-protective activity, S. cerevisiae cells that accumulate proline were constructed by disrupting the PUT1 gene involved in the degradation pathway and by expressing the mutant PRO1 gene encoding the feedback inhibition-less sensitive γ-glutamate kinase to enhance the biosynthetic activity. The engineered yeast strains successfully showed enhanced tolerance to many stresses, including freezing, desiccation, oxidation, and ethanol. However, the appropriate cellular level and localization of proline play pivotal roles in the stress-protective effect. These results indicate that the increased stress protection is observed in yeast cells under the artificial condition of proline accumulation. Proline is expected to contribute to yeast-based industries by improving the production of frozen dough and alcoholic beverages or breakthroughs in bioethanol production.     

Microclimate and variations in haemolymph composition in the freezing-tolerant New Zealand alpine weta Hemideina maori Hutton (Orthoptera: Stenopelmatidae)

The microclimate in the habitat of the New Zealand alpine weta Hemideina maori is very variable with winter temperatures down to −6 °C under the rocks where the insects are found. Subfreezing temperatures may in winter prevail for up to 17 days but diurnal cycles of freezing and thawing are common, as is also the case in summer. Rates of temperature change can be very high and up to −7.20 °C/h. During winter, humidity was high for extended periods ranging from 70% to 100% relative humidity (RH). In the summer, humidity ranged from 30% RH during the day to 100% RH at night. The supercooling point of the haemolymph was approximately −8 °C year round, caused by a heat labile substance. The supercooling point of the haemolymph of an insect of the same genus, Hemideina femorata not regularly exposed to subfreezing temperatures, was ca. −16.5 °C. Thermal hysteresis was not detected in the haemolymph of H. maori. Haemolymph osmolality varied from 380 mOsm (summer) to 700 mOsm (winter). Body water content was ca. 75% all year round. Total concentrations of sodium, potassium and chloride in haemolymph varied from 170 mM (winter) to 250 mM (summer). The total concentration of free amino acids varied from 58 mM (summer) to 263 mM (winter). This variation was mostly due to proline which varied from ca. 15 mM (summer) to ca. 100 mM (winter). The freeze-tolerant weta H. maori is exposed to a highly variable and cold environment all year round and several properties of its haemolymph composition can be attributed to these climatic conditions, e.g. the presence of ice-nucleating agents and an increase in the concentration of proline during cold hardening in the autumn. Accepted: 22 February 1999     

The concentration of cryoprotective lectins in mistletoe (Viscum album L.) leaves is correlated with leaf frost hardiness

The leaves of mistletoe (Viscum album L.) contain three galactose- and N-acetylgalactosamine-specific isolectin groups (ML I, II, III). The groups ML I and ML III showed strong cryoprotective activity during freezing and thawing of isolated spinach (Spinacia oleracea L.) thylakoid membranes, while ML II showed no such activity. The cryoprotective efficiency of the proteins was correlated with their relative hydrophobicity, as determined by a fluorescence titration assay. We found that the frost hardiness of mistletoe leaves was seasonally regulated under natural conditions. While leaves harvested in winter were not damaged by freezing to −20 °C, leaves harvested in July had already suffered 70% electrolyte leakage after freezing to −5 °C. Likewise, the amount of ML I and ML III varied during the year, with the highest amounts of these cryoprotective lectins in winter and early spring and the lowest amounts during the summer months. There was no comparable change in the amount of ML II. These data suggest that some lectins may play a role in the stabilization of cellular membranes under environmental stress conditions. Received: 18 December 1996 / Accepted: 29 March 1997     

Volume-regulatory Amino Acid Release from the Protozoan Parasite Crithidia luciliae

The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue α-aminoisobutyrate. α-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of α-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated α-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for α-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated α-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mm. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells. Received: 13 May 1996/Revised: 9 July 1996     

Changes in protein profile and amino acids in <Emphasis Type="Italic">Cladophora vagabunda</Emphasis> (Chlorophyceae) in response to salinity stress

The aim of the present study was to detect organic substances functioning as osmoticants that are used by the intertidal alga, Cladophora vagabunda (L.) Hoek (Chlorophyceae), to adapt to a wide range of salinity. The major constituents of the amino acid pool were aspartate, glutamate, glycine, valine, lysine, histidine, arginine, and proline. There were concomitant increases in the acidic amino acids: aspartate and the glutamate and the basic amino acids: lysine, histidine and arginine in response to salinity stress. The appearance of proline at hypersalinity alone showed that it acts as an osmoticant. As salinity increased, there was a progressive shift in the electrophoretic pattern of protein bands. New peptide bands appeared under hyposalinity (10‰) and hypersalinity (65‰) stress conditions in addition to the usual bands which appeared in the control (35‰). Glycine betaine, which has been considered a novel organic osmolyte in a number of organisms, has also been observed in C. vagabunda in response to salinity stress. The synthesis of the compatible solute glycine betaine and the amino acid proline with increasing salinity illustrates the contention that marine algae establish an osmotic equilibrium primarily by the synthesis of organic compounds. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase is known as the rate-limiting enzyme in early sterol biosynthesis in eukaryotic cells. To eliminate this regulation in the yeast Saccharomyces cerevisiae, a truncated HMG1 gene, producing a form of the enzyme that lacks the membrane-binding region (i.e. amino acids 1–552), was constructed and overexpressed in this yeast. The transformed strains accumulated large amounts of the sterol precursor squalene, while the levels of ergosterol and a number of other sterol compounds were only slightly elevated. These findings suggest that HMG-CoA reductase is not the only rate-limiting step in sterol synthesis and its overexpression cannot significantly influence this pathway beyond the sterol precursor squalene. Received: 9 June 1997 / Received revision: 1 September 1997 / Accepted: 19 September 1997     

Influence of culture conditions on production and freeze-drying tolerance of Paecilomyces fumosoroseus blastospores

With the goal of developing a defined medium for the production of desiccation-tolerant blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus, we evaluated the impact of various media components such as amino acids, carbohydrates, trace metals and vitamins on hyphal growth and sporulation of P. fumosoroseus cultures and on the freeze-drying tolerance of blastospores produced under these conditions. A comparison of 13 amino acids as sole nitrogen sources showed that glutamate, aspartate, glycine and arginine supported biomass accumulations (12–16 mg ml⁻¹) and blastospore yields (6–11 × 10⁸ blastospores ml⁻¹) comparable to our standard production medium which contains casamino acids as the nitrogen source. Using glutamate as the sole nitrogen source, tests with various carbohydrates showed that P. fumosoroseus grew best on glucose (18.8 mg biomass ml⁻¹) but produced similar blastospore concentrations (7.3–11.0 × 10⁸) when grown with glucose, glycerol, fructose or sucrose. P. fumosoroseus cultures grown in media with sodium citrate or galactose as the sole carbohydrate produced lower blastospore concentrations but more-desiccation-tolerant spores. Zinc was the only trace metal tested that was required for optimal growth and sporulation. In a defined medium with glutamate as the nitrogen source, vitamins were unnecessary for P. fumosoroseus growth or sporulation. When blastospores were freeze-dried in the absence of a suspension medium, residual glucose (>2.5% w/v) was required for enhanced spore survival. Thus, a defined medium containing basal salts, glucose, glutamate and zinc can be used to produce optimal concentrations of desiccation-tolerant blastospores of P. fumosoroseus. Received 27 October 1998/ Accepted in revised form 06 May 1999     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

Effect of xylitol and trehalose on dry resistance of yeasts

The effects of dehydration/rehydration on two strains of Saccharomyces cerevisiae: S600, a metabolically engineered xylose-utilising strain, and H158, the non-xylose-utilising host strain; and on the naturally xylose-utilising yeast Pachysolen tannophilus CBS 4044, were compared after glucose and xylose utilisation respectively. The yeast strains differed in their ability to excrete and accumulate intracellular xylitol. A high intracellular xylitol content before and after dehydration coincided with a higher viability after a dehydration/rehydration cycle. The intracellular trehalose content increased during dehydration in all three yeast strains, but this did not correspond to enhanced cell viability after dehydration/rehydration. The results are discussed in relation to the ability of xylitol and trehalose to structure water. Received: 9 July 1996 / Received revision: 29 October 1996 / Accepted: 2 November 1996     

Inhibition of yeast Δ1-pyrroline-5-carboxylate dehydrogenase by common amino acids and the regulation of proline catabolism

A yeast glutamate auxotroph (glt_{1 ? 1}), blocked in the tricarboxylic acid cycle at aconitase, is shown to possess catabolic pathways to glutamate from proline, arginine and glutamine, and grows on any of these amino acids in a minimal medium. This mutant does not, however, grow on these amino acids in a medium containing the full complement of common amino acids minus glutamate. The mechanism of this growth failure involves partial inhibition of the catabolic routes to glutamate by more than half the common amino acids. In the case of proline catabolism, this inhibition is localized principally at the enzyme Δ¹-pyrroline-5-carboxylate: NAD(P)⁺ oxidoreductase by in vitro studies. Similar results with this enzyme prepared both from yeast and from beef kidney mitochondria suggest that the inhibition observed may be the basis of a regulatory mechanism of general significance.     

Effect of strength training session on plasma amino acid concentration following oral ingestion of arginine or taurine in men

This study examined the acute effects of a one-hour hypertrophic strength training session (STS) on plasma amino acid concentration following oral ingestion of arginine or taurine in nine physically active men participating in a double-blind and randomised experiment. The subjects took placebo, arginine or taurine capsules (50 mg/kg) in either rest (REST) or STS condition. Blood samples were taken before and at 30, 60, 90, and 120 min after the beginning of the treatment and assayed for plasma amino acids with HPLC. There was a significant interaction effect with STS and sample time for both arginine and taurine in the raw data (p < 0.05). The modelled polynomial data for the arginine treatment showed that the peak concentration of arginine occurred at 69 min at rest and at 104 min in STS, and for the taurine treatment, the peak concentration of taurine occurred at 89 min at rest and at 112 min in STS. In conclusion, one hour of hypertrophic STS slows the increase in the peak concentration of plasma arginine and taurine after oral ingestion of the respective amino acids.     

Desensitization of Feedback Inhibition of the Saccharomyces cerevisiae γ-Glutamyl Kinase Enhances Proline Accumulation and Freezing Tolerance

In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. The yeast Saccharomyces cerevisiae induces trehalose or glycerol synthesis but does not increase intracellular proline levels during various stresses. Using a proline-accumulating mutant, we previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. This mutant was recently shown to carry an allele of PRO1 which encodes the Asp154Asn mutant γ-glutamyl kinase (GK), the first enzyme of the proline biosynthetic pathway. Here, enzymatic analysis of recombinant proteins revealed that the GK activity of S. cerevisiae is subject to feedback inhibition by proline. The Asp154Asn mutant was less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation. To improve the enzymatic properties of GK, PCR random mutagenesis in PRO1 was employed. The mutagenized plasmid library was introduced into an S. cerevisiae non-proline-utilizing strain, and proline-overproducing mutants were selected on minimal medium containing the toxic proline analogue azetidine-2-carboxylic acid. We successfully isolated several mutant GKs that, due to extreme desensitization to inhibition, enhanced the ability to synthesize proline better than the Asp154Asn mutant. The amino acid changes were localized at the region between positions 142 and 154, probably on the molecular surface, suggesting that this region is involved in allosteric regulation. Furthermore, we found that yeast cells expressing Ile150Thr and Asn142Asp/Ile166Val mutant GKs were more tolerant to freezing stress than cells expressing the Asp154Asn mutant.     

Phenylalanine- and tyrosine-auxotrophic mutants of Saccharomyces cerevisiae impaired in transamination

This paper reports the first isolation of Saccharomyces cerevisiae mutants lacking aromatic aminotransferase I activity (aro8), and of aro8 aro9 double mutants which are auxotrophic for both phenylalanine and tyrosine, because the second mutation, aro9, affects aromatic aminotransferase II. Neither of the single mutants displays any nutritional requirement on minimal ammonia medium. In vitro, aromatic aminotransferase I is active not only with the aromatic amino acids, but also with methionine, α-aminoadipate, and leucine when phenylpyruvate is the amino acceptor, and in the reverse reactions with their oxo-acid analogues and phenylalanine as the amino donor. Its contribution amounts to half of the glutamate:2-oxoadipate activity detected in cell-free extracts and the enzyme might be identical to one of the two known α-aminoadipate aminotransferases. Aromatic aminotransferase I has properties of a general aminotransferase which, like several aminotransferases of Escherichia coli, may be able to play a role in several otherwise unrelated metabolic pathways. Aromatic aminotransferase II also has a broader substrate specificity than initially described. In particular, it is responsible for all the measured kynurenine aminotransferase activity. Mutants lacking this activity grow very slowly on kynurenine medium. Received: 21 October 1996 / Accepted: 23 September 1997     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress. Received: 17 April 1996 / Accepted: 21 October 1996