Leaf surface traits: overlooked determinants of birch resistance to herbivores and foliar micro-fungi?

Previous studies on the mechanisms of birch resistance to herbivores and foliar micro-fungi (both pathogenic and endophytic) have focused mainly on the role of internal leaf chemistry. In the present study, we examined genetic correlations between leaf surface traits (glandular trichome density and total concentrations of surface flavonoid aglycones) and occurrence of three species of foliar micro-fungi, one pathogenic rust (Melampsoridium betulinum) and two endophytic fungi (Fusicladium sp. and Melanconium sp.), and performance of autumnal moth larvae (Epirrita autumnata) in two birch species, Betula pubescens ssp. czerepanovii and B. pendula. The performance of autumnal moth larvae on B. pubescens ssp. czerepanovii was negatively correlated with density of glandular trichomes (RGR: r=–0.855; pupal mass: r=–0.709). In addition, rust infection was negatively correlated with trichome density in B. pendula (r=–0.675) and with epicuticular flavonoid aglycones in B. pubescens ssp. czerepanovii (r=–0.855). The frequency of the endophytic fungus Fusicladium sp., was related to epicuticular flavonoid aglycones (r=–0.782), while another endophytic fungus, Melanconium sp., showed no associations with any of the studied variables in B. pubescens ssp. czerepanovii. Our results indicate that leaf surface traits may be at least as important determinants of herbivore performance and micro-fungi abundance in birch as leaf internal chemistry.     

Simulated acid rain affects birch leaf endophyte populations

Endophytes were frequently isolated from mountain birch (Betula pubescens var. tortuosa (Ledeb.) Nyman) leaves at a subarctic site where natural air pollution is low. We tested whether simulated acid rain had any influence on the occurrence of endophytes. Dry controls with only ambient rain and irrigated controls treated with spring water of pH 6 were compared with acid treatments at pH 3 and pH 4, prepared by adding both sulphuric and nitric acids. Treatments began in 1985 and leaf samples were taken twice during the summer of 1992. Leaves were surface sterilized, five leaf disks from each leaf placed on malt extract agar, and growing colonies were counted and identified. The most frequently isolated endophyte from birch leaves was a Fusicladium anamorph of Venturia sp. (88% of all the isolates in July and 75% of all the isolates in August), followed by a sterile mycelium and Melanconium sp. The number of endophytes isolated and the species number increased from July to August. Endophytes were most frequently isolated from the basal part of the midrib. The percentage of colonization by endophytes was similar in short and long shoots. More endophytes were isolated from leaves of branches taken at 1 m height than at 2 m height. The stronger acid rain treatment (pH 3) reduced by approximately 25% the number of isolated endophytes in August. Treatments did not have any effect on species composition of endophyte assemblages in birch leaves. Offprint requests to: Marjo L. Helander.     

Altitudinal patterns in host suitability for forest insects

Conspecific trees growing at high and low-elevations encounter different growing conditions and may vary in their suitability as hosts for herbivorous insects. Mountain tree populations may be more resistant to herbivory if low temperatures constrain growth more than they constrain photosynthesis, resulting in increased secondary metabolism (temperature hypothesis). Alternatively, mountain trees may be fertilized by atmospheric nitrogen deposition and become more palatable to insects (atmospheric deposition hypothesis). We evaluated these two hypotheses by comparing high- and low-elevation trees with insect bioassays and analyses of foliar nitrogen and condensed tannin. Contrary to the temperature hypothesis, high-elevation foliage had higher leaf nitrogen (six of six tree species) and allowed higher growth rates of Lymantria dispar larvae (five of six tree species). The nitrogen deposition hypothesis was broadly supported by measurements from two mountains showing that high-elevation trees tended to have higher leaf nitrogen, lower leaf tannins, and support higher insect growth performance than conspecific trees from lower elevations. The deposition hypothesis was further supported by fertilization studies showing that simulated atmospheric nitrogen deposition changed the foliar chemistry of valley trees to resemble that of high-elevation trees. Predictions that the altitudinal gradient in foliar chemistry and host suitability should be steepest on mountains receiving more deposition were largely not supported, but interpretations are complicated by lack of replication among mountains. In the northeastern United States, increased host suitability of high-elevation trees seems sufficient to influence the population dynamics and community composition of herbivores. Atmospheric nitrogen deposition offers a promising hypothesis to explain and predict some important spatial patterns in herbivory. Received: 21 September 1997 / Accepted: 12 June 1998     

Environmental conditions and host genotype direct genetic diversity of Venturia ditricha,a fungal endophyte of birch trees

We investigated whether genetic variation of a common foliar endophyte of birch trees, Venturia ditricha, is affected by environmental conditions or host genotype. Fungal samples were collected from 10 half-sibling families of mountain birch (Betula pubescens ssp. czerepanovii) grown in two environmental conditions with different daily average temperatures: a forested river valley and an adjacent open tundra (altitudinal difference 180 m). Genetic analysis of V. ditricha isolates was done using random amplified microsatellite polymerase chain reaction. We found that host genotypes, along with prevailing environmental conditions, influence the probability of infection by particular endophyte genotypes. The most susceptible host genotypes were highly infected with genetically similar endophyte genotypes, whereas the most resistant trees were poorly infected and they were infected by genetically dissimilar endophytes. Our results also showed environment-host genotype interactions, suggesting that the susceptibility of the host to a particular endophyte genotype may change in natural environments when environmental conditions are changed. It appears that a particular endophyte genotype needs to find the right host genotype for a successful infection. There are many host genotypes in natural stands; this means, from the point of view of the fungus, the environment is heterogeneous. Thus, under the influence of birch tree genotypes, genetically differentiated subgroups of the endophytic fungus may be formed in different environments.     

The role of foliar microfungi in mountain birch - insect herbivore relationships

We studied the effects of epiphytic and endophytic phyllosphere fungi and pathogenic birch rust fungus infection of mountain birch Betula pubescens ssp czerepanovit trees on the larval performance of leaf beetle Phratora potaris We assessed the effects of epiphytic fungi by growing larvae on leaves from trees with manipulated fungal densities We also monitored larval perfonnance and endophytic fungal densities among tree groups classified by herbivory or rust fungus densities The differences in expenmentally manipulated epiphytic fungal densities did not affect larval relative growth rates (RGR) of the species, instead we found significant tree effects Phratora polaris RGR was higher on trees with high level of herbivory than on trees with low herbivory, nevertheless, endophyte densities between these groupings did not differ In the rust fungus expenment, P polarts performance was lowest on trees with low infection compared to no and high infection trees We also did not find correlations among tree-specific endophyte densities and P polaris performance on high and low herbivory trees and trees classified by rust fungus infection Although antagonism among fungi and induction of tree defences cannot be excluded, we suggest that epiphytic and endophytic fungi of mountain birch have negligible effects on P polaris larval performance under natural conditions, probably due to mountain birch variability and a loose ecological connection between mountain birch and its epi- and endophytes Mountain birch and pathogenic birch rust have a more tightly linked relationship, which may also affect insect herbivores Still, leaf properties may play an important role and the effects will depend on the relative timing of the rust infection, herbivore development and changes in leaf quality     

Stem respiration of ponderosa pines grown in contrasting climates: implications for global climate change

We examined the effects of climate and allocation patterns on stem respiration in ponderosa pine (Pinus ponderosa) growing on identical substrate in the cool, moist Sierra Nevada mountains and the warm, dry, Great Basin Desert. These environments are representative of current climatic conditions and those predicted to accompany a doubling of atmospheric CO₂, respectively, throughout the range of many western north American conifers. A previous study found that trees growing in the desert allocate proportionally more biomass to sapwood and less to leaf area than montane trees. We tested the hypothesis that respiration rates of sapwood are lower in desert trees than in montane trees due to reduced stem maintenance respiration (physiological acclimation) or reduced construction cost of stem tissue (structural acclimation). Maintenance respiration per unit sapwood volume at 15°C did not differ between populations (desert: 6.39 ± 1.14 SE μmol m⁻³ s⁻¹, montane: 6.54 ± 1.13 SE μmol m⁻³ s⁻¹, P = 0.71) and declined with increasing stem diameter (P = 0.001). The temperature coefficient of respiration (Q ₁₀) varied seasonally within both environments (P = 0.05). Construction cost of stem sapwood was the same in both environments (desert: 1.46 ± 0.009 SE g glucose g⁻¹ sapwood, montane: 1.48 ± 0.009 SE glucose g⁻¹ sapwood, P = 0.14). Annual construction respiration calculated from construction cost, percent carbon and relative growth rate was greater in montane populations due to higher growth rates. These data provide no evidence of respiratory acclimation by desert trees. Estimated yearly stem maintenance respiration was greater in large desert trees than in large montane trees because of higher temperatures in the desert and because of increased allocation of biomass to sapwood. By analogy, these data suggest that under predicted increases in temperature and aridity, potential increases in aboveground carbon gain due to enhanced photosynthetic rates may be partially offset by increases in maintenance respiration in large trees growing in CO₂-enriched atmospheres. Received: 4 November 1996 / Accepted: 23 January 1997     

Alternative fire resistance strategies in savanna trees

Bark properties (mainly thickness) are usually presented as the main explanation for tree survival in intense fires. Savanna fires are mild, frequent, and supposed to affect tree recruitment rather than adult survival: trunk profile and growth rate of young trees between two successive fires can also affect survival. These factors and fire severity were measured on a sample of 20 trees near the recruitment stage of two savanna species chosen for their contrasted fire resistance strategies (Crossopteryx febrifuga and Piliostigma thonningii). Crossopteryx has a higher intrinsic resistance to fire (bark properties) than Piliostigma: a 20-mm-diameter stem of Crossopteryx survives exposure to 650°C, while Piliostigma needs a diameter of at least 40 mm to survive. Crossopteryx has a thicker trunk than Piliostigma: for two trees of the same height, the basal diameter of Crossopteryx will be 1.6 times greater. Piliostigma grows 2.26 times faster than Crossopteryx between two successive fires. The two species have different fire resistance strategies: one relies on resistance of aboveground structures to fire, while the other relies on its ability to quickly re-build aboveground structures. Crossopteryx is able to recruit in almost any fire conditions while Piliostigma needs locally or temporarily milder fire conditions. In savannas, fire resistance is a complex property which cannot be assessed simply by measuring only one of its components, such as bark thickness. Bark properties, trunk profile and growth rate define strategies of fire resistance. Fire resistance may interact with competition: we suggest that differences in fire resistance strategies have important effects on the structure and dynamics of savanna ecosystems. Received: 16 August 1996 / Accepted: 4 January 1997     

Populus tremuloides photosynthesis and crown architecture in response to elevated CO2 and soil N availability

We tested the hypothesis that elevated CO₂ would stimulate proportionally higher photosynthesis in the lower crown of Populus trees due to less N retranslocation, compared to tree crowns in ambient CO₂. Such a response could increase belowground C allocation, particularly in trees with an indeterminate growth pattern such as Populus tremuloides. Rooted cuttings of P. tremuloides were grown in ambient and twice ambient (elevated) CO₂ and in low and high soil N availability (89 ± 7 and 333 ± 16 ng N g⁻¹ day⁻¹ net mineralization, respectively) for 95 days using open-top chambers and open-bottom root boxes. Elevated CO₂ resulted in significantly higher maximum leaf photosynthesis (A _max) at both soil N levels. A _max was higher at high N than at low N soil in elevated, but not ambient CO₂. Photosynthetic N use efficiency was higher at elevated than ambient CO₂ in both soil types. Elevated CO₂ resulted in proportionally higher whole leaf A in the lower three-quarters to one-half of the crown for both soil types. At elevated CO₂ and high N availability, lower crown leaves had significantly lower ratios of carboxylation capacity to electron transport capacity (V _cmax/J _max) than at ambient CO₂ and/or low N availability. From the top to the bottom of the tree crowns, V _cmax/J _max increased in ambient CO₂, but it decreased in elevated CO₂ indicating a greater relative investment of N into light harvesting for the lower crown. Only the mid-crown leaves at both N levels exhibited photosynthetic down regulation to elevated CO₂. Stem biomass segments (consisting of three nodes and internodes) were compared to the total A _leaf for each segment. This analysis indicated that increased A _leaf at elevated CO₂ did not result in a proportional increase in local stem segment mass, suggesting that C allocation to sinks other than the local stem segment increased disproportionally. Since C allocated to roots in young Populus trees is primarily assimilated by leaves in the lower crown, the results of this study suggest a mechanism by which C allocation to roots in young trees may increase in elevated CO₂. Received: 12 August 1996 / Accepted: 12 November 1996     

Foliar nutrients shape fungal endophyte communities in Western white pine (Pinus monticola) with implications for white-tailed deer herbivory

Asymptomatic fungal endophytes colonize tissues of woody plants worldwide, with largely unknown ecological effects. Using culture-based methods and ITS1-5.8S-ITS2 rDNA sequence analysis, we investigated differences between foliar endophyte communities in disease-resistant hybrid and wild-type Pinus monticola (Western white pine) trees with observed variation in tree growth, vigor, and browsing damage by white-tailed deer (Odocoileus virginianus). We isolated 69 phylotypes of endophytic fungi in at least 39 genera, including 26 that have not previously been reported in P. monticola. Principal components analysis revealed that endophyte communities differed between browsed seedlings, unbrowsed seedlings, and unbrowsed adult trees. Sulfur, nitrate and calcium concentrations correlated with endophyte community differences among tree groups based on a distance-based redundancy analysis. Our results indicate that foliar nutrient variation influences endophyte community assembly and deer herbivory in P. monticola on a small landscape scale (80 hectares).     

Homeostatic responses in the gut of Porcellio scaber (Isopoda: Oniscidea) optimize litter degradation

In order to examine the influence of differences in food conditions on gut characteristics in Porcellio scaber, pH-manipulated and microbially inoculated leaf litter from three different tree species were offered. Microbial activity was clearly influenced by the pH levels of the leaves. Analyses of the pH levels in the gut indicated the ability of P. scaber to buffer the pH value in the intestinal tract to about 5.5–6.0 in the anterior hindgut, and to about 6.0–6.5 in the posterior hindgut. The pH levels of the gut sections remained in this range, within a range of food pH from 4.0 to 7.5, no matter what kind of leaves the animals were fed. Homeostatic responses to changes in food pH guarantee optimized digestion of leaf litter. However, when the pH level of the litter dropped below 3.5, P. scaber was not able to maintain the pH conditions in the gut. Furthermore, microorganisms colonizing the litter biased the pH level in the anterior hindgut where digestive processes mainly take place. These results indicate a decline of litter quality with regard to the nutrition of terrestrial isopods, caused by acidification and consequently reduced microbial activity. Accepted: 19 July 1997     

The birch tube-maker Acrobasis betulella in a fragmented habitat: the importance of patch isolation and edges

We examined the response of the birch tube-maker Acrobasis betulella Hulst (Lepidoptera: Pyralidae) to habitat patch isolation and edges. Density of A.␣betulella larva was higher on distant islands than on islands close to the shore. Following experimental removal of all larvae from the islands, adults were able to recolonize even the most distant islands, and larval density was again positively correlated with the degree of isolation. Larval density was not correlated with island size or the amount of birch present on the islands. Larvae on more distant islands did not have lower mortality than those in less isolated sites. Larvae were found more often on edges than in the interior of birch stands and developed faster on edges. This positive edge effect, coupled with the ability of the adults to disperse to even our most distant islands, suggests that A. betulella would be favored in an environment fragmented at the same scale as our island system. Received: 15 April 1996 / Accepted: 30 September 1996     

Fungal endophyte communities in two tropical forests of southern India: diversity and host affiliation

Fifteen tree species from a tropical dry thorn forest and fifteen tree species from a tropical dry deciduous forest in the Mudumalai Wildlife Sanctuary, Nilgiri Biosphere Reserve, southern India, were surveyed for their foliar endophyte communities during the dry and wet seasons. Surface sterilized leaf segments of uniform dimension were plated on nutrient agar and culturable endophytes growing from the segments were identified. Endophyte diversity was greater in the dry thorn forest than in the dry deciduous forest in the dry season. Although the isolation frequency of culturable endophytes increased for both forests during the wet season, the assemblages were represented not by any unique fungal species but by the commonly occurring ones. Furthermore, although individual leaves were densely colonized by endophytes, only a few species of endophytes colonized the whole leaves; and, only a few fungal species dominated the foliar endophytic communities and were common for both forests during both dry and wet seasons. Thus, even under wet conditions that favour dispersal and infection by fungi, the endophyte diversity increased only marginally, an indication that certain tropical forests are not hyperdiverse with reference to fungal endophytes. This should be considered when using culturable endophyte diversity as a surrogate for estimating global fungal diversity.     

Discrimination of sinusoidally frequency-modulated sound signals mimicking species-specific communication calls in the FM-bat Phyllostomus discolor

In the lesser spear-nosed bat, Phyllostomus discolor, maternal directive calls are characterized by an individual type of sinusoidal frequency modulation (= SFM) pattern. Beside modulation frequency, modulation depth, carrier frequency, and number of modulation cycles per call contribute to the mother's vocal signature. Since juvenile P. discolor learn to adapt their isolation calls to the corresponding call characteristics of the own mother or even to playback of a computer-stored directive call, if hand-reared in the absence of conspecifics, the bats' auditory system ought to be able to resolve interindividual differences in communication call structure. However, quantitative psychoacoustic data on the discrimination of SFM signals in this species are not available. Thus, in the present study, lesser spear-nosed bats were trained in a two-alternative forced-choice procedure to discriminate between two alternatingly presented SFM sound signals differing in modulation frequency. Other characteristics of acoustic stimuli were identical and designed to mimick the fundamental of species-specific calls. By gradually reducing the difference in modulation frequency between both stimuli within the behavioural relevant range until the animals' performance dropped below the 75%-correct level, a considerable auditory spectro-temporal resolution has been revealed. Particularly in comparison to the overall interindividual variation of this call parameter (minimal modulation frequency = 49 Hz, maximum = 100 Hz), the determined average difference limen for modulation frequency of 2.42 ± 0.29 Hz seems substantial and sufficient for labelling individuals. Accepted: 30 November 1996     

Pyrosequencing Reveals a Highly Diverse and Cultivar-Specific Bacterial Endophyte Community in Potato Roots

In this study, we examined the bacterial endophyte community of potato (Solanum tuberosum) cultivar/clones using two different molecular-based techniques (bacterial automated ribosomal intergenic spacer analysis (B-ARISA) and pyrosequencing). B-ARISA profiles revealed a significant difference in the endophytic community between cultivars (perMANOVA, p < 0.001), and canonical correspondence analysis showed a significant correlation between the community structure and plant biomass (p = 0.001). Pyrosequencing detected, on average, 477 ± 71 bacterial operational taxonomic units (OTUs, 97% genetic similarity) residing within the roots of each cultivar, with a Chao estimated total OTU richness of 1,265 ± 313. Across all cultivars, a total of 238 known genera from 15 phyla were identified. Interestingly, five of the ten most common genera (Rheinheimera, Dyadobacter, Devosia, Pedobacter, and Pseudoxanthomonas) have not, to our knowledge, been previously reported as endophytes of potato. Like the B-ARISA analysis, the endophytic communities differed between cultivar/clones (∫-libshuff, p < 0.001) and exhibited low similarities on both a presence/absence (0.145 ± 0.019) and abundance (0.420 ± 0.081) basis. Seventeen OTUs showed a strong positive (r > 0.600) or negative (r < −0.600) correlation with plant biomass, suggesting a possible link between plant production and endophyte abundance. This study represents one of the most comprehensive assessments of the bacterial endophytic communities to date, and similar analyses in other plant species, cultivars, or tissues could be utilized to further elucidate the potential contribution(s) of endophytic communities to plant physiology and production.     

Short latency vestibular evoked potentials in the Japanese quail (Coturnix coturnix japonica)

Short-latency vestibular-evoked potentials to pulsed linear acceleration were characterized in the quail. Responses occurred within 8 ms following the onset of stimuli and were composed of a series of positive and negative peaks. The latencies and amplitudes of the first four peaks were quantitatively characterized. Mean latencies at 1.0 g ms⁻¹ ranged from 1265 ± 208 μs (P1, N = 18) to 4802 ± 441 μs (N4, N = 13). Amplitudes ranged from 3.72 ± 1.51 μV (P1/N1, N = 18) to 1.49 ± 0.77 μV (P3/N3, N = 16). Latency-intensity (LI) slopes ranged from −38.7 ± 7.3 μs dB⁻¹ (P1, N = 18) to −71.6 ± 21.9 μs dB⁻¹ (N3, N = 15) and amplitude-intensity (AI) slopes ranged from 0.20 ± 0.08 μV dB⁻¹ (P1/N1, N = 18) to 0.07 ± 0.04 μV dB⁻¹ (P3/N3, N = 11). The mean response threshold across all animals was −21.83 ± 3.34 dB re: 1.0 g ms⁻¹ (N = 18). Responses remained after cochlear extirpation showing that they could not depend critically on cochlear activity. Responses were eliminated by destruction of the vestibular end organs, thus showing that responses depended critically and specifically on the vestibular system. The results demonstrate that the responses are vestibular and the findings provide a scientific basis for using vestibular responses to evaluate vestibular function through ontogeny and senescence in the quail. Accepted: 18 January 1997     

Fragmented environment affects birch leaf endophytes

The effect of environmental fragmentation on the species distribution and frequency of horizontally transmitted endophytic fungi in Betula pubescens and Betula pendula leaves was studied in an archipelago in southwestern Finland. The study system consisted of 14 islands, ranging in size and distance to the mainland, and five mainland sites. Endophytic fungi were grown out from surface-sterilized leaves. The frequency of endophytic fungi mainly depended on the size of the island, explaining 32-35% of the variation, and the distance to the mainland explaining 29-35% of the variation. The birch trees on the largest islands near the mainland had the highest endophyte frequencies. Fusicladium betulae, Gnomonia setacea and Melanconium betulinum were the most commonly isolated fungi. Foliar endophytes of birch trees are able to disperse to fairly fragmented areas, but their frequencies seem to depend on environmental isolation and size of the island.     

Cold tolerance and dehydration in Enchytraeidae from Svalbard

When cooled in contact with moisture, eight species of arctic Enchytraeidae from Svalbard were killed by freezing within minutes or hours at −3 and −5 °C; an exception was Enchytraeus kincaidi which survived for up to 2 days. When the temperature approached 0 °C the enchytraeids apparently tried to escape from the moist soil. The supercooling capacity of the enchytraeids was relatively low, with mean supercooling points of −5 to −8 °C. In contrast, specimens of several species were extracted from soil cores that had been frozen in their intact state at −15 °C for up to 71 days. Compared to freezing in a moist environment, higher survival rates were obtained during cooling at freezing temperatures in dry soil. Survival was recorded in species kept at −3 °C for up to 35 days, and in some species kept at −6 °C for up to 17 days. Slow warming greatly increased survival rates at −6 °C . The results strongly suggest that arctic enchytraeids avoid freezing by dehydration at subzero temperatures. In agreement with this, weight losses of up to ca. 42% of fresh weight were recorded in Mesenchytraeus spp. and of up to 55% in Enchytraeus kincaidi at water vapour pressures above ice at −3 to −6 °C. All specimens survived dehydration under these conditions. Accepted: 12 December 1997     

Isolation of endophytes from leaves of <Emphasis Type="Italic">Neolitsea sericea</Emphasis> in broadleaf and conifer stands

Endophytic fungi were isolated from leaves of Neolitsea sericea, a major lauraceous tree in the laurel forests of southern Kyushu, collected from the understory layer of broadleaf and conifer stands. Cytosphaera sp. and a species of Ascomycetes in leaf blade segments, plus a xylariaceous species and Phomopsis spp. in petiole segments, were isolated at relatively high frequency. In general, isolation frequencies of endophytes were higher in petiole than blade segments. In blade segments, patterns of endophyte isolation were quite different among stands, while relatively similar in petiole segments. Significant effects of sampling sites or canopy vegetation were rarely detected. the understory layer of laurel forests. Neolitsea sericea is an evergreen broadleaf lauraceous tree, widely distributed in areas of eastern Asia. In southern Kyushu, it is one of the most common trees, growing both as canopy and understory species. In this study, endophytes were isolated from the leaves of N. sericea growing in the understory layer of conifer and broadleaf forest stands to survey the endophytes of N. sericea leaves and to examine the effect of the canopy layer on endophytic mycobiota in understory plants.     

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53 269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80° C; ammonia-dependent reaction, 90° C). Received: 30 October 1996 / Accepted: 13 January 1997     

The effect of a thiamin derivative on exercise performance

The purpose of this study was to investigate the effect of a thiamin derivative, thiamin tetrahydrofurfuryl disulfide (TTFD), on oxygen uptake (˙VO₂), lactate accumulation and cycling performance during exercise to exhaustion. Using a randomized, double-blind, cross-over design with a 10-day washout between trials, 14 subjects ingested either 1 g · day⁻¹ of TTFD or a placebo (PL) for 4 days. On day 3, subjects performed a progressive exercise test to exhaustion on a cycle ergometer for the determination of ˙VO_2submax, ˙VO_2peak, lactate concentration ([La⁻ ]), lactate threshold (Th_La) and heart rate ( f _c). On day 4, subjects performed a maximal 2000-m time trial on a cycle ergometer. A one-way analysis of variance (ANOVA) with repeated measures was used to determine significant differences between trials. There were no significant differences detected between trials for serial measures of ˙VO_2submax, [La⁻] or f _c. Likewise, ˙VO_2peak [PL 4.06 (0.19) TTFD 4.12 (0.19) l · min⁻¹, P = 0.83], Th_La [PL 2.47 (0.17), TTFD 2.43 (0.16) l · min⁻¹, P = 0.86] and 2000-m performance time [PL 204.5 (5.5), TTFD 200.9 (4.3) s, P = 0.61] were not significantly different between trials. The results of this study suggest that thiamin derivative supplementation does not influence high-intensity exercise performance. Accepted: 19 December 1996