Abstract

Mycorrhizal roots of the deciduous trees European beech (Fagus sylvatica (L.)) and Sessile oak (Quercus petraea (MattuschkaLiebl.)) and the conifers Norway spruce (Picea abies (L.) H. Karst.) and European larch (Larix decidua (Mill.)) associated with the ectomycorrhizal fungi matt bolete (Xerocomus pruinatus (Fries 1835)) or bay bolete (X. badius (Fries 1818)) were analysed with respect to the occurrence of dihydrolipoyl dehydrogenase (EC 1.8.1.4) allozymes. In root tissues of the two deciduous trees, two gene loci could be visualized after cellulose acetate electrophoresis while three loci were expressed in root tissues of the two coniferous species. The two fungal species and further ectomycorrhizal fungi expressed exclusively one dihydrolipoyl dehydrogenase gene. In Xerocomus pruinatus and X. badius, the dihydrolipoyl dehydrogenase gene consists of 1460 bp and 1370 bp, respectively, including five introns each consisting of 52 bp. Their DNA sequences correspond to 70 to 90% to other fungal dihydrolipoyl dehydrogenase genes. One monomer of the dimeric dihydrolipoyl dehydrogenase enzyme consists of 486 (X. pruinatus) or 454 (X. badius) amino acids which sum up to a molecular mass of 55 kDa (X. pruinatus), respectively 52 kDa (X. badius). The number of positively charged amino acid residues makes 79 (X. pruinatus) and 68 (X. badius) and the number of negatively charged amino acid residues was calculated to make 46 (X. pruinatus) and 48 (X. badius); isoelectric points make 9.99 (X. pruinatus) and 9.68 (X. badius). Calculated three dimensional structures reveal a short NADH binding site being part of a larger FAD-binding site and a binding/dimerization domain.

Highlights

  • Most European forest trees form at their root tips, a symbiosis with ectomycorrhizal fungi belonging to the ascomycota, basidiomycota or mitosporic fungi [1] [2]

  • In Xerocomus pruinatus and X. badius, the dihydrolipoyl dehydrogenase gene consists of 1460 bp and 1370 bp, respectively, including five introns each consisting of 52 bp

  • In this study we investigated mycorrhizal roots of several European forest trees in order to allocate the various dihydrolipoyl dehydrogenase enzymes to the root tissues and the hyphae of the mycorrhizal fungi matte bolete (Xerocomus pruinatus) and bay bolete (X. badius)

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Summary

Introduction

Most European forest trees form at their root tips, a symbiosis with ectomycorrhizal fungi belonging to the ascomycota, basidiomycota or mitosporic fungi [1] [2]. Mitochondria take up pyruvate, some amino acids and several fatty acids from the cytoplasm and transfer these metabolites to the citrate cycle where they are used to provide the basis for several molecular syntheses and to gain energy (GTP) and reduction equivalents (NADH + H+, FADH2). The latter are transmitted to the respiration chain where they are oxidized (NAD+, FAD) and the arising electrons are transferred to oxygen while the protons set free are used for the generation of an electrochemical gradient providing the energy to synthesize ATP via the membrane bound enzyme ATP synthase [11].

Stand Characteristics
Mycorrhizae with Xerocomus Species
Protein Extraction
Cellulose Acetate Electrophoresis
Visualization of Dihydrolipoyl Dehydrogenase Allozymes
DNA Extraction
PCR-RFLP Analysis
RNA Extraction
2.11. Amplification of cDNA and DNA Dihydrolipoyl Dehydrogenase-Sequences
2.12. Sequencing of PCR Products and Genome Sequencing
Allozymes and Affiliation of Dihydrolipoyl Dehydrogenases
Molecular Genetic Analyses
Protein Structures
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