Poly-β-Hydroxyalkanoate Exert a Protective Effect Against Carbon Starvation and Frozen Conditions in <Emphasis Type="Italic">Sphingopyxis chilensis</Emphasis>

Several bacterial species have developed physiological response to avoid the cellular damage when are exposed to carbon starvation or frozen stress. For example survival to inanition has been related to endogenous substrates consumptions. The aim of this study was to evaluate if poly-β-hydroxylkanoates (PHA) consumption enable Sphingopyxis chilensis S37 to survive under carbon starvation or frozen condition. Bacterial cells were grown in R₂A broth for 48 h, and suspended in mineral saline solutions, without carbon source. The cellular suspension was incubated for 48 or 120 h at 30°C, followed by a frozen period of 48 h at −20°C, and viable bacterial cells were evaluated by the microdrop method. The proportions of cells with PHA were also determined by flow cytometry using Nile Red dye. The results indicate that S. chilensis were able to survive under carbon starvation and frozen conditions. Simultaneously, a decrease in the number of cells containing PHA, and a decrease in the biovolume of the cells (c.a 2.5 times) were also observed under these conditions. The results suggest that consumptions of PHA contributed to the surviving of S. chilensis under frozen stress.     

Variation of total soluble seminal root proteins of tetraploid wild and cultivated wheat induced at cold acclimation and freezing

The relationship between total soluble seminal root proteins induced at cold acclimation and freezing tolerance in tetraploid wild wheat Aegilops L. (Ae. biuncialis, Ae. cylindrica) and cultivated wheat Triticum turgitum L. (Firat-93, Harran-95) was investigated. Cold acclimation was performed at 0 °C for 7 days. Freezing tolerance was determined with survived roots after freezing treatments at −5 and/or −7 °C for 3, 6, 12 and 24 h. At −5°C, all tetraploid genotypes showed over 60% tolerance for 3 h. This effect was also present in wild wheat for 6 h, but was decreased in cultivated wheat to 30–35% tolerance for 6 h. Only Ae. biuncialis was able to show 52% tolerance just for 3 h freezing period at −7 °C. However, all the genotypes were not survived at −7 °C, for 6, 12 and 24 h. Cold acclimation induced greater amounts of new soluble seminal root proteins in tolerant Ae. biuncialis (29–104 kDa, pI 5.4–7.4) than in sensitive Harran-95 (29–66 kDa, pI 6.1–8.3). Synthesis and accumulation of these proteins may be related to degree of freezing tolerance of these genotypes.     

Gut morphology,feeding rate and gut clearance in five species of caddis larvae

Feeding rate, the rate of movement of food through the gut and gut morphology of large larvae of five caddis species (Halesus radiatus, Hydropsyche instabilis, Polycentropus kingi, Rhyacophila dorsalis and Potamophylax cingulatus) were investigated in the laboratory. Following 72 hr starvation, P. cingulatus and H. radiatus larvae became satiated (refused prey offered directly to the mouthparts) after consuming 8–11 and 9–13 mayfly nymphs (Baetis rhodani, 3.5–4.6 mm) respectively. Hunger level affected prey consumption. In P. cingulatus, the number of prey consumed over 24 h (at 9.5–12 °C) increased with starvation periods from 0–72 h, but declined following longer starvation periods. Six clearly recognisable gut states (defined by the position of food material in various parts of the gut) can be identified at different times since commencement of a meal. As environmental temperature increased (from 8–12 °C to 15–17 °C), the rate of change of the gut state increased and the food retention time decreased in all species. Feeding periodicity (i.e. nocturnal/diurnal activity) in the field was estimated based on the evacuation rate and the gut state and environmental temperature at the known time of collection. Initiation of consumption of prey appeared to coincide with emptying of the foregut and proximal midgut, whereas actual feeding continued until complete satiation when some threshold fullness of the foregut had been reached. The length of time food was held in the foregut was positively correlated with increasing specialisation of the foregut (particularly elaboration of the proventriculus).     

Flight initiation byProstephanus truncatus in relation to time of day, temperature, relative humidity and starvation

Studies were carried out in the laboratory on the influences of time of day, temperature, relative humidity and starvation on flight initiation byProstephanus truncatus (Horn) (Coleoptera: Bostrichidae). Flight occurred throughout the 12 h photophase and at the beginning of the scotophase but peaked at 2–0 h before darkness. Temperature exerted a significant effect on flight. The frequency of flight take-off increased with temperature over the range 20–30°C but declined sharply at 35°C. Flight activity increased with starvation up to a maximum at 2 days after which it began to decline.     

Culturability and survival of an extreme thermophile isolated from deep-sea hydrothermal vents

The culturability of a strictly anaerobic, extremely thermophilic archaeon, Thermococcus peptonophilus (optimal growth temperature: 85° C), was studied during survival stages at various temperatures (98, 85, 70, and 4° C). Total cell number (determined by DAPI staining), active cells (rhodamine-stained cells), and culturable cells (using most-probable-number) were counted over time. The number of culturable cells decreased under each condition tested. The total number of cells significantly decreased only at temperatures close to the maximum for growth (98° C); at this temperature, the cells spontaneously lysed. Our results suggested that survival at 4° C in oxygenated waters might be a mechanism for the dispersion of extreme thermophiles in the ocean. In addition, we proved the existence of T. peptonophilus cells in several physiological states: culturable cells, active non-culturable cells, inactive non-culturable cells, and dead cells. Cell death was caused by cellular lysis. Received: 5 February 1996 / Accepted: 16 April 1996     

Isolation and characterization of a thermophilic bacillus strain,that degrades phenol and cresols as sole carbon source at 70 °C

A phenol-degrading thermophilic bacterium, designated Bacillus sp. A2, was isolated from a water and mud sample from a hot spring in Iceland. The aerobic isolate grew optimally on phenol at 65 °C. At 70 °C, 85% of the optimal growth rate was still observed. No growth was observed at 40 °C and 75 °C. Bacillus sp. A2 is a gram-positive spore-forming rod. According to 16S rDNA analysis Bacillus sp. A2 is closely related to Bacillus stearothermophilus, Bacillus kaustophilus and Bacillus thermoleovorans. Bacillus sp. A2 degraded phenol completely in concentrations up to 5 mM. In addition, all three isomers of cresol were utilized as sole carbon and energy sources. The degradation of phenols proceeds via the meta-cleavage pathway and the enzymes involved in its degradation are constitutively expressed. Received: 13 May 1996 / Received revision: 29 July 1996 / Accepted: 12 August 1996     

The fatty acid profile of vegetative Azotobacter vinelandii ATCC 12837: growth phase-dependence

Fatty acids of Azotobacter vinelandii ATCC 12837 were determined at various times during aerobic vegetative growth at 30°C to provide baseline data for studying the effects of chemical agents on the organism’s survival and fatty acid biosynthesis. Palmitate (16:0) was the highest at 36.7±4.3 mol% (mean±SD) after the first 5 h in fresh culture, decreasing slightly to 33.4±2.6 mol% at 49 h. The other fatty acids were therefore each normalized as a ratio of 16:0. At 5 h, as a ratio of 16:0, myristate (14:0) was 0.14±0.06, palmitoleate (16:1cΔ^9–10) 0.13±0.06, oleate (18:1cΔ^9–10) 0.21±0.12, cis-vaccenate (18:1cΔ^11–12) 0.30±0.17 and stearate (18:0) 0.68±0.02. As the growth phase advanced to 49 h, 14:0 and 16:1cΔ^9–10 increased, 18:1cΔ^9–10 decreased and cis-vaccenate reciprocally increased, whereas 18:0 decreased. These suggest that the saturated fatty acid biosynthesis pathway yielded 16:0 and 18:0 in the 5-h lag period. By desaturation, 18:0 formed the unsaturated fatty acid (UFA) 18:1cΔ^9–10. As the culture aged, the anaerobic UFA biosynthesis pathway formed 16:1cΔ^9–10, which was elongated to 18:1cΔ^11–12. These fatty acid alterations represent a homeoviscous adaptation, modulating the microbe’s membrane lipid viscosity for optimal cellular function.     

Mannitol-enhanced survival of Lactococcus lactis subjected to drying

Survival of Lactococcus lactis subjected to different drying conditions was investigated. Mannitol most remarkably enhanced the survival of dried cells to a level almost equalling that of viable cells [log₁₀ (cfu ml⁻¹) = 9.42] as was found prior to the drying process (log₁₀ = 9.6). In the absence of mannitol, a survival was reduced by a factor of 10⁴. Drying of cells at 20 °C led to higher survival rates than drying at 30 °C. Mannitol enhanced the survival rate at both temperatures, and at both 20 °C and 30 °C the highest reduction in survival occurred when cells were dried at a water activity of 0.76. In the presence of mannitol, differences in survival after drying at different water activities were less pronounced. Rehydration of cells dried in the presence of mannitol resulted in an extended lag phase of 4 h compared to fresh cells. No growth or acidification of the culture medium was observed for 12 h in the case of rehydrated cells dried in the absence of mannitol. It was hypothesized that a radical scavenging activity of mannitol could partly explain these observations. Received: 28 August 1998 / Accepted: 2 October 1998     

Temperature influences the performance and effectiveness of field and laboratory strains of the ichneumonid parasitoid, <Emphasis Type="Italic">Campoletis chlorideae</Emphasis>

To understand the influence of temperature on host–parasitoid interactions as a consequence of climatic change, we studied development, survival, and fecundity of field and laboratory strains of the Helicoverpa armigera larval endoparasitoid, Campoletis chlorideae at five different temperatures under laboratory conditions. Post-embryonic development period and degree-days required for completing the life cycle by both the strains decreased by 2.5 and 1.5 folds at 27°C compared to 18°C. Post embryonic development period showed a negative (r = −0.99, P < 0.001) and the development rate a positive (r = 0.99, P < 0.001) association with an increase in temperature. However, no parasitoid larvae survived in H. armigera larvae reared at 12 and 35°C after parasitization, suggesting that temperatures ≥35°C as a result of global warming will be lethal for development and survival of immature stages of C. chlorideae. Adult longevity was negatively associated (r = −0.91 to −0.96, P < 0.001) with temperatures between 12 and 35°C. The parasitoid adults stored at 12°C survived for longer period and exhibited higher fecundity than those kept at 27°C, but the efficiency of parasitism and adult emergence were quite low. Sex ratio of the progeny at 12°C was highly male-biased than the insects kept at 27°C. Laboratory strain of the parasitoid exhibited better survival, and the adults lived longer than the field strain at 18°C than at 27°C. Therefore, C. chlorideae adults stored at 18°C could be used for parasitism, while the immature stages should be reared at 27°C for mass production of the parasitoid for biological control of H. armigera.     

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.     

Impact of mild heat treatments on induction of thermotolerance in the biocontrol yeast Candida sake CPA-1 and viability after spray-drying

Aims: The objective of this study was to examine the induction of thermotolerance in the biocontrol agent Candida sake CPA‐1 cells by mild heat treatments to enhanced survival of formulations using spray‐drying. The possible role of heat‐shock proteins (HSPs) biosynthesis in induced thermotolerance and the role of sugars and sugar alcohols were also determined. Methods and Results: Studies were conducted on C. sake cells grown in molasses medium and exposed to mild temperatures of 30 and 33°C during mid‐ (16 h), late‐exponential (24 h), early‐ (30 h) and mid‐stationary (36 h) growth phases. The effect on viability was determined both before and after spray‐drying. Cycloheximide and chloramphenicol were used to examine the role of HSPs and HPLC was used to analyse the accumulation of sugar and sugar alcohols. The results indicate that both temperatures induced thermotolerance in cells of C. sake. Mild heat‐adapted cells at 33°C in the early‐ or mid‐stationary phases had survival values after spray‐drying significantly higher (P ≤ 0·05) than nonadapted cells. However, viabilities were not high enough to be considered for commercial use with values up to 17%. HSPs were not implicated in thermotolerance acquired by mild heat‐adapted cells as similar viabilities were obtained in the presence of protein inhibitors. Little change was observed in sugar and sugar alcohols with an increase in glucose and arabitol in some treatments. Conclusions: This study suggests that it is possible to induce thermotolerance in biocontrol yeasts such as C. sake. However, this does not improve survival of cells exposed to spray‐drying sufficiently to consider this a suitable formulation method for this biocontrol agent. HSPs, sugars and sugar polyols were not directly responsible for induced thermotolerance in yeast cells. Significance and Impact of the Study: This type of information can be effectively applied to improve the viability of cells in the process of formulation.     

Thermoanaerobacter mathranii sp. nov., an ethanol-producing, extremely thermophilic anaerobic bacterium from a hot spring in Iceland

The extremely thermophilic ethanol-producing strain A3 was isolated from a hot spring in Iceland. The cells were rod-shaped, motile, and had terminal spores; cells from the mid-to-late exponential growth phase stained gram-variable but had a gram-positive cell wall structure when viewed by transmission electron microscopy. Strain A3 used a number of carbohydrates as carbon sources, including xylan, but did not utilize microcrystalline cellulose. Fermentation end products were ethanol, acetate, lactate, CO₂, and H₂. The temperature optimum for growth was between 70 and 75° C, and growth occurred in the range of 50–75° C. The pH range for growth was 4.7–8.8, with an optimum at pH 7.0. Strain A3 was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, and vancomycin at 100 mg/l but was not sensitive to chloramphenicol and neomycin at 10 mg/l, which indicates that strain A3 belongs to the eubacteria. Addition of 50.66 kPa H₂ or 2% NaCl did not affect growth. The isolate grew in the presence of exogenously added 4% (w/v) ethanol. The G+C ratio was 37 mol%. 16S rDNA studies revealed that strain A3 belongs to the genus Thermoanaerobacter. Genotypic and phenotypic differences between strain A3 and other related species indicate that strain A3 can be assigned to a new species, and the name Thermoanaerobacter mathranii is proposed. Received: 7 October 1996 / Accepted: 14 March 1997     

Differential profiles of soluble proteins during the initiation of morphogenesis in Candida albicans

Candida albicans is a dimorphic fungus that can grow either as yeast or as mycelia. The mycelial form may be required for tissue penetration and therefore may have a role in pathogenesis. The protein profiles of the cell-free S100 fraction from budding yeast cells and germ tube-forming cells (an early stage of the transition between yeast and mycelia) were evaluated using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Yeast growth or germ tube formation was induced in carbon-starved cells at 37° C by either glucose, galactose or N-acetylglucosamine at pH 4.5 or pH 6.7. More than 400 constitutively synthesised polypeptides were identified on 2-D PAGE by silver staining. A few polypeptides which seem to reflect the release from carbon starvation were detected, but no polypeptides unique to either morphology were observed. Fractionation of S100 preparations by polyethylenimine or heparin-agarose affinity chromatography, which have been used to detect DNA-binding proteins, revealed several proteins that were synthesised on the resumption of cell growth or in response to pH difference. Heparin-agarose also bound novel polypeptides in the size range 130–200 kDa that were preferentially synthesised in germ tube-forming cells. These results suggest that any protein factors that might exert a regulatory role early in germ tube formation are of low abundance, and that a minor group of soluble proteins involved in C. albicans morphogenesis may be differentially synthesised. Received: 11 March 1996 / Accepted: 10 July 1996     

Genome plasticity during seed germination in Festuca arundinacea

 Feulgen/DNA cytophotometric determinations were carried out on early prophases in the meristems of seedlings obtained by germinating seeds of different accessions of Festuca arundinacea at 10°C, 20°C, or 30°C. Feulgen/DNA contents increased significantly with the increase in the temperature of seed germination. In each accession, the greater the increase in absorption in seedlings obtained at 30°C, the lower the absorption in seedlings obtained at 10°C. In contrast, Feulgen/DNA contents did not undergo changes when the temperature was altered at developmental stages other than seed germination. The results of molecular hybridizations (slot blots) indicated that the redundancy of repeated DNA sequences belonging to two families having C_ot ranges of 0–2×10^-1 and 2×10^-1 −2×10⁰, respectively, was significantly higher in the genome of seedlings obtained at 30°C than in that of seedlings obtained at 10°C. When centrifuged to equilibrium in CsCl density gradients, the DNA extracted from seedlings obtained at 30°C formed a heavier and a lighter shoulder with buoyant densities of 1.707 g/ml and 1.692 g/ml, respectively, in addition to the main band (1.701 g/ml). Only a less apparent shoulder banding at 1.706 g/ml was formed by the DNA extracted from seedlings obtained at 10°C. After seed germination in the presence of [³H]-thymidine for 24 h at 30°C, most of radioactivity was found in the guanine + cytosine- or adenine+thymine-enriched DNA fractions, which formed the two shoulders in the density profile. In contrast, only guanine+cytosine-enriched fractions, which formed the heavier shoulder, were preferentially labelled in the DNA from seedlings obtained at 10°C. These results prove that fluid domains do exist in the nuclear DNA of F. arundinacea. These DNA domains are capable of rapid, quantitative alterations, which represent the direct responses of the genome to developmental and environmental stimuli. Seed germination appears to be a limited, specific period in development within which the adaptive response to temperature variations can be put into effect. Received: 9 August 1996 / Accepted: 23 August 1996     

Thermal tolerance and acclimation response of larvae of the sub-Antarctic beetle Hydromedion sparsutum (Coleoptera: Perimylopidae)

Hydromedion sparsutum is a locally abundant herbivorous beetle on the sub-Antarctic island of South Georgia, often living in close association with the tussock grass Parodiochloa flabellata. Over a 4-day period in mid-summer when the air temperature varied from 0 to 20°C, the temperature in the leaf litter 5–10 cm deep at the base of tussock plants (the microhabitat of H. sparsutum) was consistently within the range of 5–7.5°C. Experiments were carried out to assess the ability of H. sparsutum larvae collected from this thermally stable environment to acclimate when maintained at lower (0°C) and higher (15°C) temperatures. The mean supercooling points (freezing temperature) of larvae collected in January and acclimated at 0°C for 3 and 6 weeks and 15°C for 3 weeks were all within the range of −2.6 to −4.6°C. Larvae in all treatment groups were freeze tolerant. Acclimation at 0°C significantly increased survival in a 15-min exposure at −8°C (from 27 to 96%) and −10°C (from 0 to 63%) compared with the field-fresh and 15°C-treated larvae. Similarly, survival of 0°C-acclimated larvae in a 72-h exposure at −6°C increased from 20 to 83%. Extending the acclimation period at 0°C to 6 weeks did not produce any further increase in cold tolerance. The concentrations of glucose and trehalose in larval body fluids increased significantly with low temperature acclimation. Larvae maintained at 15°C for 3 weeks (none survived for 6 weeks) were less able to survive 1-h exposures between 30 and 35°C than the 0°C-treated samples. Whilst vegetation and snow cover are an effective buffer against low winter temperatures in many polar insects, the inability of H. sparsutum larvae to acclimate or survive at 15°C suggests that protection against high summer temperatures is equally important for this species. Accepted: 2 August 1999     

Pressure and temperature effects on growth and viability of the hyperthermophilic archaeon Thermococcus peptonophilus

We studied the effects of high temperatures and elevated hydrostatic pressures on the physiological behavior and viability of the extremely thermophilic deep-sea archaeon Thermococcus peptonophilus. Maximal growth rates were observed at 30 and 45 MPa although no significant increases in cell yields were detected. Growth at 60 MPa was slower. The optimal growth temperature shifted from 85° C at 30 MPa to 90–95° C at 45 MPa. Cell viability during the stationary phase was also enhanced under high pressure. A trend towards barophily at pressures greater than those encountered in situ at the sea floor was demonstrated at increasing growth temperatures. The viability of cells during starvation, at high temperature (90, 95° C), and at low temperature (10° C) was enhanced at 30 and 45 MPa as compared to atmospheric pressure. These results show that the extremely thermophilic archaeon T. peptonophilus is a barophile. Received: 21 October 1996 / Accepted: 5 February 1997     

Effects of constant and fluctuating temperature on time to budburst in Betula pubescens and its relation to bud respiration

 Effects of fluctuating and constant temperatures on budburst time, and respiration in winter buds were studied in Betula pubescens Ehrh. Dormant seedlings were chilled at 0°C for 4 months and then allowed to sprout in long days (LD, 24 h) at constant temperatures of 6, 9, 12, 15, 18 and 21°C, and at diurnally fluctuating temperatures (12/12 h, LD 24 h) with means of 9, 12, 15 and 18°C. No difference in thermal time requirements for budburst was found between plants receiving constant and fluctuating temperatures. The base temperature for thermal time accumulation was estimated to 1°C. Respiration in post-dormant (dormancy fully released) excised winter buds from an adult tree increased exponentially with temperature and was 20 times as high at 30°C than at 0°C. However, respiration in buds without scales was 30% higher at 0°C, and it was 2.7 times higher at 24°C than in intact buds. Thus, the tight bud scales probably constrain respiration and growth and are likely to delay budburst in spring. Arrhenius plots of the respiration data were biphasic with breaks at 13–15°C. However, this phase transition is unlikely to be associated with chilling sensitivity since the present species is hardy and adapted to a boreal climate. Received: 10 January 1997 / Accepted: 23 June 1997     

Differential Induction of the Chaperonin GroEL and the Co-Chaperonin GroES by Heat,Acid, and UV-Irradiation in Lactococcus lactis subsp. lactis

Microsequencing of a polypeptide with MW of 14.5 and pI of 5.0 induced by heat treatment at 42°C and 50°C in Lactococcus lactis subsp. lactis revealed that it corresponds to the co-chaperonin GroES. Quantitative analysis of analytical 2-D gels showed a relative induction of 12- and 11-fold after 30 min of heat adaptation at 42°C and 50°C, respectively. GroES is also induced by an acid shift from pH 7 to pH 5.5 and by UV_254 nm-irradiation, with relative induction factors of 3.8 and 2.3, respectively. To our knowledge this is the first report showing induction of GroES by mild acid treatment. Contrasting to the relative induction of the groEL gene product, the second protein encoded by the groESL operon, GroES shows significantly higher induction under all stress situations. Received: 3 June 1996 / Accepted: 5 July 1996     

Adaptation and acclimation of growth and photosynthesis of five Antarctic red algae to low temperatures

Temperature requirements for growth, photosynthesis and dark respiration were determined for five Antarctic red algal species. After acclimation, the stenothermal species Gigartina skottsbergii and Ballia callitricha grew at 0 or up to 5 °C, respectively; the eurythermal species Kallymenia antarctica, Gymnogongrus antarcticus and Phyllophora ahnfeltioides grew up to 10 °C. The temperature optima of photosynthesis were between 10 and 15 °C in the stenothermal species and between 15 and 25 °C in the eurythermal species, irrespective of the growth temperature. This shows that the temperature optima for photosynthesis are located well below the optima from species of other biogeographical regions, even from the Arctic. Respiratory rates rose with increasing temperatures. In contrast to photosynthesis, no temperature optimum was evident between 0 and 25 °C. Partial acclimation of photosynthetic capacity to growth temperature was found in two species. B. callitricha and Gymnogongrus antarcticus acclimate to 0 °C, and 5 and 0 °C, respectively. But acclimation did in no case lead to an overall shift in the temperature optimum of photosynthesis. B. callitricha and Gymnogongrus antarcticus showed acclimation of respiration to 5 °C, and P. ahnfeltioides to 5 and 10 °C, resulting in a temperature independence of respiration when measured at growth temperature. With respect to the acclimation potential of the species, no distinction can be made between the stenothermal versus the eurythermal group. (Net)photosynthetic capacity:respiration (P:R) ratios showed in all species highest values at 0 °C and decreased continuously to values lower than 1.0 at 25 °C. In turn, the low P:R ratios at higher temperatures are assumed to determine the upper temperature growth limit of the studied species. Estimated daily carbon balance reached values between 4.1 and 30.7 mg C g⁻¹ FW day⁻¹ at 0 °C, 16:8 h light/dark cycle, 12–40 μmol m⁻² s⁻¹. Received: 4 November 1999 / Accepted: 7 March 2000