Oxygen uptake kinetics during heavy submaximal exercise: Effect of sickle cell trait with or without alpha-thalassemia.

Sickle cell trait (SCT) is a genetic disease affecting the synthesis of normal hemoglobin (Hb) marked by the heterozygous presence of HbA and HbS. It is thought that exercise tolerance and aerobic capacity could be limited in SCT carriers, but that the co-existence of alpha-thalassemia with SCT (SCTAT) could improve exercise response. To examine these issues, we compared the characteristics of VO2 kinetics during a constant heavy exercise among athletes carrying either the SCT (n = 6), the SCTAT (n = 9), or the normal Hb (control group; n = 10). After determination of maximal power output (Ppeak), all subjects underwent a constant heavy cycling exercise lasting 9 min at approximately 70 % Ppeak. Pulmonary VO2 and cardio-respiratory parameters were measured breath-by-breath and the VO2 response was modelled using non-linear regression techniques. The time constant of the VO2 primary component and oxygen deficit were not significantly different among the three groups. The VO2 slow component was 28 % and 33 % higher (p < 0.05) in SCT and SCTAT than in the control groups, respectively. Altogether, athletes with the SCT and the SCTAT had higher heart rate at the beginning (+ 5.2 %) and the end (+ 7.4 %) of the slow component compared to the control group (p < 0.05). These results suggest that SCT and SCTAT subjects are not limited during the first exercise minutes, but are prone to exercise intolerance and to lower aerobic capacity thereafter, due to a higher VO2 slow component, and that alpha-thalassemia does not improve exercise response. The finding of a higher slow component in SCT and SCTAT athletes was possibly due to the loss of O2 availability to muscles, additional fiber recruitment and/or higher cardiac load with time.

Hebeloma cylindrosporum- a model species to study ectomycorrhizal symbiosis from gene to ecosystem.

The basidiomycete Hebeloma cylindrosporum has been extensively studied with respect to mycorrhiza differentiation and metabolism and also to population dynamics. Its life cycle can be reproduced in vitro and it can be genetically transformed. Combined biochemical, cytological, genetical and molecular approaches led to the characterisation of mutant strains affected in mycorrhiza formation. These studies demonstrated the role of fungal auxin as a signal molecule in mycorrhiza formation and should allow the characterisation of essential fungal genes necessary to achieve a compatible symbiotic interaction. Random sequencing of cDNAs has identified numerous key functional genes which allowed dissection of essential nitrogen assimilation pathways. H. cylindrosporum also proved to be a remarkable model species to uncover the dynamics of natural populations of ectomycorrhizal fungi and the way in which they respond and adapt to anthropogenic disturbance of the forest ecosystem. Although studies on mycorrhiza differentiation and functioning and those on the population dynamics of H. cylindrosporum have been carried out independently, they are likely to converge in a renewed molecular ecophysiology which will envisage how ectomycorrhizal symbiosis functions under varying field conditions. Contents Summary 481 I. Introduction 482 II. Taxonomy, distribution, autecology, and host range of H. cylindrosporum 482 III. The Hebeloma cylindrosporum toolbox 483 IV. Mycorrhiza differentiation 486 V. Nutritional interactions 488 VI. Genetic diversity and dynamics of H. cylindrosporum populations in P. pinaster forest ecosystems 491 VII. Future directions 494 Acknowledgements 494 References 494.

Correspondence between genet diversity and spatial distribution of above- and below-ground populations of the ectomycorrhizal fungus Hebeloma cylindrosporum.

Population studies of ectomycorrhizal fungal species have largely relied upon fruit body (the reproductive organ) sampling. Analysis of the fruit bodies alone supposes that they reflect the present and spatial organization of all below-ground genets (mycorrhizas and extramatrical mycelia). The relation between fruit bodies and ectomycorrhizas was investigated for the basidiomycete agaric Hebeloma cylindrosporum in four Pinus pinaster stands in south-west France. Genet identification was based on the comparison of polymorphisms within a hypervariable segment of the ribosomal intergenic spacer amplified by polymerase chain reaction (PCR) using a H. cylindrosporum species-specific primer. Mycorrhizas were sorted from soil samples collected underneath patches of fruit bodies or patches where fruit bodies had or had not been observed during the years prior to mycorrhiza collection. On average 65% of the 1026 mycorrhizas collected underneath fruit bodies were formed by H. cylindrosporum, whereas only 2% of the 954 collected in places from where fruit bodies were absent were formed by this species. All genotypes identified above ground were also identified below ground. In patches where one genotype formed all or more than 90% of the fruit bodies, the same genotype formed all or a large majority of the mycorrhizas. In patches occupied by several different fruiting genotypes, additional nonfruiting ones could be present on the root systems. In all cases, the mycorrhizas of one genotype were found no more than 10-20 cm away from its corresponding fruit bodies, and fruit body disappearance at a given place was associated with the disappearance of the corresponding mycorrhizas within 1 year. Although there was not a strict coincidence between the total numbers of genets present below ground and of those forming fruit bodies, fruit body analysis for H. cylindrosporum appears to reflect both the genetic diversity and the spatial structure of its below-ground populations. The results obtained also illustrate the rapid turnover of ectomycorrhizal fungal species on the root systems in the absence of any obvious major disturbance of the ecosystem.

Transcription of a nitrate reductase gene isolated from the symbiotic basidiomycete fungus Hebeloma cylindrosporum does not require induction by nitrate.

Ectomycorrhizal fungi contribute to the nitrogen nutrition of their host plants, but no information is available on the molecular control of their nitrogen metabolism. The cloning and pattern of transcriptional regulation of two nitrite reductase genes of the symbiotic basidiomycete Hebeloma cylindrosporum are presented. The genomic copy of one of these genes (nar1) was entirely sequenced; the coding region is interrupted by 12 introns. The nar1 gene, which is transcribed and codes for a putative 908-amino acid polypeptide complemented nitrate reductase-deficient mutants of H. cylindrosporum upon transformation, thus demonstrating that the gene is functional. The second gene (nar2), for which no mRNA transcripts were detected, is considered to be an ancestral, non-functional duplication of nar1. In a 462-nt partial sequence of nar2 two introns were identified at positions identical to those of introns 8 and 9 of nar1, although their respective nucleotide sequences were highly divergent; the exon sequences were much more conserved. In wild-type strains, transcription of nar1 is repressed in the presence of a high concentration of ammonium. High levels of transcription are observed in the presence of either very low nitrogen concentrations or high concentrations of nitrate or organic N sources such as urea, glycine or serine. This indicates that in H. cylindrosporum, in contrast to all nitrophilous organisms studied so far, an exogenous supply of nitrate is not required to induce transcription of a nitrate reductase gene. In contrast, repression by ammonium suggests the existence of a wide-domain regulatory gene, as already characterized in ascomycete species.

Population dynamics of the symbiotic mushroom Hebeloma cylindrosporum: mycelial persistence and inbreeding.

The pattern of colonization of a forest site by the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum Romagnesi was followed from 1993 until 1997. Fruit-bodies of this tetrapolar heterothallic species were mapped, collected and propagated as pure mycelial cultures. Isolates were analysed for their mating-types and molecular markers (rDNA polymorphism and RAPD). Dedikaryotization of the 26 isolates collected in 1993 and the separate analysis of each individual haploid nucleus established that two fully compatible genets, which occupied two nonoverlapping territories, were present. Isolates belonging to the same genet could nevertheless be distinguished from each other based on Southern hybridization using hyperpolymorphic DNA probes. A majority of the 143 isolates collected from 1994 to 1997 belonged to either of the two genets identified in 1993, whose territories extended at a rate of about 0. 45-0.60 m per year. Selfing of the two 1993 genets, rather than outcrossing, was the most likely explanation for the origin of additional genotypes identified between 1995 and 1997. The spatial distribution of fruit-bodies and genets of H. cylindrosporum suggested that only a fraction of the sampled area was favourable to colonization and that genetic diversification through meiospore dispersal may be inhibited by the presence of resident genets, possibly via a somatic incompatibility system.

The nuclear ribosomal DNA intergenic spacer as a target sequence to study intraspecific diversity of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum directly on pinus root systems.

Polymorphism of the nuclear ribosomal DNA intergenic spacer (IGS) of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was studied to evaluate whether this sequence could be used in field studies to estimate the diversity of strains forming mycorrhizas on individual Pinus pinaster root systems. This sequence was amplified by PCR from 125 haploid homokaryotic strains collected in 14 P. pinaster stands along the Atlantic coast of France by using conserved oligonucleotide primers. Restriction enzyme digestion of the amplified 3.4-kbp-long IGS allowed us to characterize 24 alleles whose frequencies differed. Nine of these alleles were found only once, whereas about 60% of the strains contained four of the alleles. Local populations could be almost as diverse as the entire population along a 150-km stretch of coastline that was examined; for example, 13 alleles were found in a single forest stand. The IGS from one strain was partially sequenced, and the sequence data were used to design oligonucleotides which allowed separate PCR amplification of three different segments of the IGS. Most polymorphisms observed among the full-length IGS regions resulted from polymorphisms in an internal ca. 1,500-bp-long sequence characterized by length variations that may have resulted from variable numbers of a T2AG3 motif. This internal polymorphic sequence could not be amplified from the genomes of nine other Hebeloma species. Analysis of this internal sequence amplified from the haploid progenies of 10 fruiting bodies collected in a 70-m2 area resulted in identification of six allelic forms and seven distinct diplotypes out of the 21 possible different combinations. Moreover, optimization of the PCR conditions resulted in amplification of this sequence from more than 80% of the DNA samples extracted from individual H. cylindrosporum infected P. pinaster mycorrhizal root tips, thus demonstrating the usefulness of this sequence for studying the below-ground diversity of mycorrhizas formed by genets belonging to the same fungal species.

Genetic transformation of the symbiotic basidiomycete fungus Hebeloma cylindrosporum.

The pAN7.1 plasmid containing the E. coli hygromycin B phosphotransferase gene was used to transform protoplasts of the ectomycorrhizal fungus Hebeloma cylindrosporum. Hygromycin-resistant transformants were selected at a frequency of one to five per micrograms of transforming DNA. Southern blot analyses revealed multiple copy integration of the transforming plasmid into the genome. The selection system was used to introduce other genes of interest by co-transformation. Two plasmids, one containing tryptophan biosynthesis genes and the other the NADP-glutamate dehydrogenase gene from the saprophytic basidiomycete Coprinus cinereus, were successfully introduced into the H. cylindrosporum genome with up to 70% efficiency of co-transformation. The hygromycin resistance phenotype was stably maintained during growth of transformants on hygromycin-free medium. All transformants retained their ability to form mycorrhizae with the habitual host plant Pinus pinaster, making them suitable for future physiological studies.

Genetic variation of nitrate reductase activity in mono- and dikaryotic populations of the ectomycorrhizal fungus, Hebeloma cylindrosporum Romagnési.

Intraspecific variability in the activity of nitrate reductase (NR) has been studied in the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnési at the interstrain and intrastrain levels, the latter within a population of 11 wild dikaryotic strains collected in'Les Landes'(SW France) at four locations less than 100 km away from one another. An attempt was made to determine whether variability within the monokaryotic and dikaryotic progeny of the HC1 fruiting strain could be used as a basis for an improvement programme involving breeding between selected monokaryons. The NR activity of the wild strains ranged from 201 to 700 nmol NO2 synthesized h-1 mg -1 fungal protein whilst that of 20 sib-monokaryons (5 per mating type) of the HCl strain varied from 51 to 510 nmol NO2 synthesized h -1 mg-1 fungal protein. Fifty controlled dikaryotic myeclia were obtained from all the compatible fusions with these monokaryons. In these, variation of NR activity was of the same order of magnitude as that recorded at the interstrain level, ranging from 72 to 689 nmol NO2 synthesized h -1 rag -1 fungal protein. Analysis of the components of the variation of NR activity in these controlled dikaryons demonstrated that the additive component of this variation accounted for less than 1 % of the total observed variation. The NR activity of any one controlled dikaryon could not therefore be predicted from the activity of its parental monokaryons However 14 of the 50 controlled dikaryons e8hibited an NR activity higher than that of the HCl parental dikaryon. These results indicate that breeding with sib-monokaryons can be used as a basis for an improvement programme of NR activity in this ectomycorrhizal Basidiomycete.

Ectomycorrhizal ability of genetically different homokaryotic and dikaryotic mycelia of Hebeloma cylindrosporum.

Ectomycorrhizas were synthesized under axenic conditions between Pinus pinaster (Ait.) Sol. and different mycelial cultures of Hebeloma cylindrosporum Romagnesi. These included a wild dikaryon and four sib-homokaryons (one for each mating type) belonging to the progeny of this fruiting strain. The homokaryotic mycelia formed typical ectomyeorrhizas showing a morphology and an ultrastructural organization similar to that of ectomyeorrhizas obtained with the parental dikaryon. The ultrastructural localization of acid phosphatase activities was also comparable for homokaryotic and dikaryotic mycorrhizas. These results demonstrate that the ectomycorrhizal ability is not restricted to the dikaryotic state of fungal life-cycle since homokaryotic mycelia also formed typical and functional ectomyeorrhizas. The formation of normal mycorrhizas by homokaryotic mycelia confirms the usefulness of H. cylindrosporum as a model for genetical studies and improvement of ectomycorrhizal fungi by means of chromosomal genetics.

IN VITRO FRUITING UNDER CONTROLLED CONDITIONS OF THE ECTOMYCORRHIZAL FUNGUS HEBELOMA CYLINDROSPORUM ASSOCIATED WITH PINUS PINASTER.

The ectomycorrhizal fungus Hebeloma cylindrosporum Romagnési produced sporulating fruit bodies under axenic conditions on a synthetic medium when cultivated with its usual host plant Pinus pinaster (Sol.). In the absence of the host plant, H. cylindrosporum only formed primordia or immature fruit bodies. In 11 Erlenmeyer flasks, the fungal fruiting started about one and half months after the inoculation of one-month-old P. pinaster plants by the mycelium of H. cylindrosporum, and a total of 144 fruit bodies appeared in 45 flasks during a 9.5-month fruiting period. At 18 °C, the mean number of basidiomes developed per flask was about 3-5. A thermal shock of ± 6 °C for 5 d did not significantly affect the mean number of basidiomes per flask but modified the time course of their rate of formation. Spores were collected from the fruit bodies and were found to be capable of germinating.