Affiliation of Dihydrolipoyl Dehydrogenase Allozymes in Mycorrhizae of European Forest Trees and Characterization of the Enzyme of the Matt Bolete ( Xerocomus pruinatus ) and the Bay Bolete ( X . badius )

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. How to cite this paper: Schirkonyer, U. and Rothe, G.M. (2018) Affiliation of Dihydrolipoyl Dehydrogenase Allozymes in Mycorrhizae of European Forest Trees and Characterization of the Enzyme of the Matt Bolete (Xerocomus pruinatus) and the Bay Bolete (X. badius). Open Journal of Ecology, 8, 356-377. https://doi.org/10.4236/oje.2018.86022 Received: April 25, 2018 Accepted: June 12, 2018 Published: June 15, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access U. Schirkonyer, G. M. Rothe DOI: 10.4236/oje.2018.86022 357 Open Journal of Ecology


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].The hyphae of ectomycorrhizal fungi associated with the root tips of their host trees inhibit the formation of long roots and cause instead the development of coralloid branched short roots [3] [4] [5].The fungal hyphae partly grow between cells of the outer mantle of the root tips, forming a Hartig net, but also extend into the soil.This way they are able to mediate between the soil and their hosts supplying the trees with nutrients and water while obtaining organic nutrients from their hosts, especially carbohydrates.The difficulty is that in forest soils plant nutrients occur mostly in an organic form which cannot be taken up by ectomycorrhizal fungi [6].That is why they excrete various enzymes to hydrolyze the corresponding components.Proteases serve to hydrolyze proteins [7], peroxidases split humus acids, and chitins are hydrolyzed by chitinases [8].
Phosphoric acid is set free from organic soil compounds either by excreted phosphomono-and diesterases hydrolyzing e.g.inositol phosphate, sugar phosphates and polyphosphates [8] [9] or by excreting organic acids such as oxalic acid or by excretion of protons [10].Mycorrhizal roots are predominantly found within the upper soil horizon which indicates that they gain energy by use of oxygen processed in mitochondria to obtain energy in form of ATP.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 + , FADH 2 ).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].The enzyme dihydrolipoyl dehydrogenase (EC 1.8.1.4)is part of two enzyme complexes of the citric acid cycle namely pyruvate dehydrogenase (EC 1.2.4.1) and α-ketoglutarate dehydrogenase (EC 1.2.4.2).After electrophoretic separations of native mycorrhizal extracts varying dihydrolipoyl dehydrogenase isozyme patterns result.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).The fungal enzyme was studied in more detail de-termining its DNA and cDNA sequence, amino acid sequence, molecular weight, isoelectric point and the putative secondary structure.The data and results presented here were collected during my doctoral thesis in the Department of Biology at the Johannes Gutenberg-University (Mainz, Germany).

Stand Characteristics
Mycorrhizal samples and fruiting bodies were collected from European beech (Fagus sylvatica (L.)), Sessile oak (Quercus petraea (MattuschkaLiebl.)),Norway spruce (Picea abies (L.) H. Karst.) and European larch (Larix decidua (Mill.))growing in pure stands at the south-side of the Taunus Mountains situated on the southern part of the state of Hesse, Germany (Table 1).Samples were taken in April and June and in September and October 50 cm to 1 m away from a trunk at a soil depth of 5 cm.Fruiting bodies were collected in autumn.The exact sampling periods are given in Table 1.
The collected mycorrhizae were put in marked plastic bags and transported at 4˚C in a cooled box to the lab where they were put in ice water and cleaned from adhering soil and humus particles under a microscope.Then, mycorrhizae of the same species were put in 1.5 ml Safelock Eppendorf tubes and stored at −20˚C.

Fruiting Bodies of the Two Xerocomus Species
The cap of the fruiting body of X. pruinatus can reach a diameter of 10 cm.Its

Protein Extraction
Native proteins were extracted from mycorrhizal roots associated with X. pruinatus or X. badius, non mycorrhizal fine roots of seedlings, mycorrhizal roots separated into root tissues and enclosing hyphae, and fruiting bodies.Mycorrhizae were separated into hyphae and central root-tissues under an enlargement of 25 × fixing a mycorrhizal root put in ice water with a fine tweezers and separating the outer hyphae with a needle or a preparation forceps [4].A 1.5 ml Eppendorf tube was weighted, filled with a mycorrhizal sample and weighted again to determine the amount of fresh weight filled in.Then the sample was homogenized with a micropistill in fluid nitrogen.After homogenization the proteins were extracted on ice.To 100 mg of frozen and pulverized mycorrhiza 200 µl extraction medium and 7.5 mg PVPP (polyvinylpyrrolidone) were added.The extraction medium contained: 30 mg (2.5 mM) cysteine, 3.3 mg (0.2 mM) mercaptobenzothiazole, 95 mg (5 mM) Na-metabisulfite, 186 mg (5 mM) Na 2 -EDTA, 102 mg (5 mM) MgCl 2 × 6H 2 O, 39.2 mg (0.5 M) NADP, 33.2 mg (0.5 M) NAD, 14 g (14% w/v) sucrose, 0.5 g (0.5% w/v) BSA and 0.5 g (0.5% w/v) TWEEN  80 in 100 ml of 0.1 M sodium phosphate buffer of pH 7.0 (57.7 ml of 1M di-sodiumhydrogenphosphate and 42.3 ml of 1 M sodiumdihydrogen-phosphate [16].Then, the mixture was centrifuged at 4˚C for 30 min at 5000 × g.The supernatant containing the native proteins was aliquoted, snap-frozen until further use, and then frozen at −20˚C, or directly used in cellulose acetate electrophoretic separations.

Cellulose Acetate Electrophoresis
Cellulose acetate gels (Titan III, 7.6 cm × 7.6 cm, Helena Laboratories, Beaumont, Texas) were swollen under about 8˚C for 20 min in electrophoresis buffer which consisted of: 0.05 M Tris, 0.001 M Na 2 -EDTA, 0.001 M MgCl 2 and 0.18 M maleic acid, pH 7.8 (modified according to [17]).Then the two chambers of the electrophoretic device were filled with buffer and two filter paper bridges (7 cm long and 12 cm wide) were installed to connect the cellulose acetate gel with its gel side for 3 mm at each end.Then the gel was submitted for 5 min to a pre-electrophoresis at 200 V.After that the gel was taken off and 0.25 µl samples applied by use of a Super-Z-12 application kit (Helena Laboratories).Usually sample applications were repeated three times to gain enough enzyme activity.
Then the gel side of the cellulose acetate gel was again put on the platform of the electrophoresis chamber, contacted to the buffer strips, and submitted to 200 V for 30 min [18].

Visualization of Dihydrolipoyl Dehydrogenase Allozymes
Immediately after electrophoresis dihydrolipoyl dehydrogenase activities were visualized covering gels with an agar overlay.The staining solution consisted of The decolorized gels were photographed and put on a transmitted light plate to note the visible enzyme bands.Gels were then dried over night between several layers of dry tissue and then stored in welded polyethylene pockets.

PCR-RFLP Analysis
To identify mycorrhizal samples and fruiting bodies, the multicopy internal transcribed spacer (ITS) region of their ribosomal DNA (rDNA) was amplified and sequenced.The rDNA repeats, comprising the 18S rRNA gene, the ITS-1-spacer, the 5.8S rRNA, the ITS-2-spacer and the 28S rRNA gene, was amplified using the primer pair ITS1 [22] and ITS4b [23] (Table 2).Primer ITS1 binds to the 3'-end of the 18S rRNA gene and primer ITS4b binds to the 5'-end of the 28S rRNA gene.If no PCR product resulted, the primer pair ITS1F/ITS4 was used [23].The detailed analysis followed the one published by Schirkonyer et al. [15].

RNA Extraction
Total RNA was extracted from mycorrhizal roots or fruiting bodies using the "NucleoSpin  RNA-Plant"-Kit, by Machery and Nagel (Düren, Germany).An amount of 50 to 100 mg of fresh material was homogenized in a 1.5 ml Eppendorf Safe tube with a micropistil under liquid nitrogen.The resulting homogenate was pipetted on ice into a microcentrifuge tube and 350 µl "RAP"-buffer (guanidine-HCl lysis buffer) and 3.5 µl 2-mercaptoethanol added, vortexing the mixture.The resulting lysate was pipetted on a "NucleoSpin  "-filter inserted into a collecting tube and then centrifuged for 1 min at 11,000 × g at room temperature.The filtrate was transferred into a microcentrifuge tube, 350 µl ethanol (70%) added and the mixture five times pipetted up and down.The resulting lysate was loaded on a "NucleoSpin  -RNA-Plant" column and the unit centrifuged for 1 min at 11,000 × g, to bind the total RNA (and DNA) to the silica membrane.Then the column was placed into a new collecting tube, 350 µl "Membrane Desalting" buffer added to the column and the unit centrifuged for 1 min at 11,000 × g, to desalt the membrane.Afterwards, 95 µl DNase reaction mixture were applied onto the silica membrane of the column and the unit incubated at room temperature for 15 min to digest the bound DNA.Then, the silica membrane was washed adding 200 µl "RA2" buffer to the column and centrifuging the unit for 1 min at 11,000 × g.Afterwards, the column was placed into a new collecting tube adding 600 µl "RA3" solution to the column and centrifuging the unit for 1 min at 11,000 × g.The flow-through was discarded and the column placed in the collecting tube again.Then, 250 µl "RA3" buffer was added and the unit centrifuged for 2 min at 11,000 × g.After that the column was put into a nuclease-free 1.5 ml microcentrifuge tube.Total RNA was eluted from the membrane by adding 60 µl RNase-free water followed by a 1 min lasting centrifugation at 11,000 × g.

Amplification of cDNA and DNA Dihydrolipoyl Dehydrogenase-Sequences
The DNA sequence of the enzyme NADH diaphorase of Xerocomus badius and X. pruinatus associated with European beech or Norway spruce was amplified by use of the primer pair P1 and P2 (Table 3) while the primers Dia1-fw and Primer-rev1 were applied to amplify the corresponding cDNA sequences.Primers were purchased from Eurofins MWG Operon (Ebersberg, Germany).The used Primers were deduced from partial cDNA sequences published at the Genbank NCBI for the basidiomycetes Ustilago maydis, Cryptococcus neoformans, Laccaria bicolor and the diaphorase sequence of the two basidiomycetes Xerocomus badius and Xerocomus pruinatus gained via genome sequencing [24].To avoid pcr-products of the diaphorase enzymes belonging to the host trees the sequence of the plant Arabidopsis thaliana was integrated into the primer construction.Primers were designed from the aligned sequences of the above named organisms using the software Primer Premier (PREMIER Biosoft International, Palo Alto, USA).The dihydrolipoyl dehydrogenase gene sequence was amplified using a 20 µl PCR mixture contained the following components: 2 µl of a 10 × PCR-buffer, 2 µl 2 mM dNTP mix, 1 µl 10 pM Primer-fw1 or Primer-fw2, 1 µl 10 pM Primer-rev1 or Primer-rev2, 3.8 µl HPLC-H 2 O and 0.2 µl 5 U/µl polymerase.Amplifications were performed by applying the following temperature program: 1) denaturation (5 min 94˚C), 2) 35 cycles for amplification (30 sec at 94˚C, 1 min at 50˚C, 2 min at 72˚C), 3) final extension (30 sec at 94˚C, 1 min at 50˚C and 10 min at 72˚C) and 4) storage at 5˚C.

Sequencing of PCR Products and Genome Sequencing
Sequencing of PCR products and genome-sequencing of the fungi Xerocomus badius and X. pruinatus via Illumina HiSeq 2000 (Illumina 2006, San Diego, California, USA) was done by GENterprise-Genomics (Mainz, Germany).For fungal identification, BLAST searches were carried out against the public sequence databases NCBI (http://www.ncbi.nlm.nih.gov/) and UNITE (http://unite.ut.ee).Sequences were assigned to matching species names when the BLAST matches showed identities higher than 97% and scores higher than 900 bits.Table 3. Sequences of the primer pairs P1 and P2 and the primer DIA1-fw.The name suggested by UNITE, a curated database for ectomycorrhizal fungi [25], was used preferentially and that of NCBI only if there was no entry in UNITE.In extracts of the ectomycorrhizal fungi Boletus edulis, Laccaria amethystina, Russula ochroleuca, Tylopilus felleus, Xerocomus badius and Xerocomus pruinatus only one active dihydrolipoyl dehydrogenase enzyme and one corres- ponding enzyme gene was observed.We assume that the enzyme is part of the two mitochondrial enzyme complexes pyruvate dehydrogenase (EC 1.2.4.1) and alpha-ketoglutarate dehydrogenase (EC 1.2.4.2).In contrast to these results we conclude the presence of two active dihydrolipoyl dehydrogenase genes within the deciduous tree species European beach and Sessile oak.Here, each of the two mitochondrial enzyme complexes pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase may contain a slightly differing form of the enzyme dihydrolipoyl dehydrogenase.The two conifers Norway spruce and European larch which function as hosts for ectomycorrhizal fungi are expressing three dihydrolipoyl dehydrogenase gene loci.Provided the corresponding enzyme forms result from two, respectively three different enzyme loci, upon the evolution of the deciduous trees from conifers one of the corresponding enzyme genes may have been silenced.Histochemical stainings that served to visualize dihydrolipoyl dehydrogenase activities showed that the enzyme was more active in hyphae of Xerocomus badius than in those of X. pruinatus.Kinetic analyses lead to corresponding results.Differing activities were also observed between the ectomycorrhizal species Cenococcum geophilum, Scleroderma citrium, Paxillus involutus and Pisolitus tinctorius [26].

Molecular Genetic Analyses
The DNA of Xerocomus pruinatus and X. badius was analyzed by use of "The Next-Generation-Illumina sequencing-method" (Solexa/Illumina, Berlin, (performed by GENterprise-Genomics, Mainz University).After the genome had been sequenced, localized primers were deduced as described in chapter 2.11 in order to amplify the gene sequence of the dihydrolipoyl dehydrogenase gene.
The full length of the dihydrolipoyl dehydrogenase gene has a length of 1631bp (cDNA: 1370 bp + (5 Introns = 261 bp) in Xerocomus badius and 1721 (cDNA: 1460 bp + 261 bp) in X. pruinatus (cf.sequences listed at the Appendix).The DNA sequences of the dihydrolipoyl dehydrogenase gene isolated from Xerocomus pruinatus and X. badius resemble those of other fungi deposited at the NCBI-gene bank to 70% to 78% (Table 4).
Five introns, each having a length of 52 bp, could be localized comparing the full gene length with that of the cDNA length (Table 5).
The gene sequences of the dihydrolipoyl dehydrogenase enzymes existing within the ectomycorrhizal fungi Boletus edulis, Laccaria amethystina, Paxillus

Protein Structures
The cDNA sequences of the dihydrolipoyl dehydrogenases from the two Xero-comus species served to determine their amino acid sequences (Figure 4 and Figure 5).
The number of positively charged amino acid residues (Arg and Lys) within the enzyme of X. pruinatus makes 79 while it makes 68 in X. badius.The number of negatively charged amino acids (Asp and Glu) makes 46 in X. pruinatus and 48 in X. badius.Molecular weights and isoelectric points were determined by use of the software ExPASy-"Protparam" (https://web.expasy.org/protparam/)(Table 6).
The length of cDNA of the dihydrolipoyl dehydrogenase gene of the basidiomycete Coprinopsis cinerea makes 1527 bp, corresponding to 494 amino acids [27].The cDNA length of the gene of the fungus Laccaria bicolor makes 1593 bp, which equals 514 amino acids [28].The dihydrolipoyl dehydrogenase enzyme of the yeast Saccharomyces cerevisiae comprises 487 amino acids and its molecular  mass makes 51558 Da [29].In most organisms, the enzyme represents a homodimer with a monomeric molecular weight of 50 to 55 kDa (Data Bank BRENDA, https://www.brenda-enzymes.org/index.php).Crystallographic studies of the human enzyme dihydrolipoyl dehydrogenase lead to the conclusion that its amino acid sequence contains four functional domains: an NADH domain within a larger FAD domain, a central domain and a dimerization domain at its C-terminal end [30].This result was confirmed for the bacterial, fungal and plant enzyme [31].Comparing the amino acid sequences of the dihydrolipoyl dehydrogenase enzymes of several fungi with those of the two Xerocomus species, we conclude that within the latter the range from amino acid 207 to 281 represents a short NADH binding site being part of a larger FAD-binding domain.The second highly conserved region is located at the C-terminal end and ranges from amino acid 375 to 485 making the binding/dimerization domain.These domains are characteristic for pyridine nucleotide-disulfide oxidoreductases (InterPro Protein sequence analysis & classification; http://www.ebi.ac.uk/interpro/entry/IPR012999).By use of the SWISS-MODEL a fully automated protein structure homology-modelling server, accessible via ExPASy web server, the 3D-structure of the Xerocomus badius enzyme (454 amino acids) and X. pruinatus (486 amino acids) could be evaluated (Figure 6).
The amino acid chains deviate by 32 amino acids but the 3D structures are congruent.

1 ml 0. 1 M
Tris-HCl buffer, pH 8.5, 1.5 ml NADH solution (3 mg/ml), 5 drops DCIP (2,6-dichlorphenol-indophenol solution, 3 mg/ml) and 5 drops MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromid solution, 10 mg/ml)[19] [20].To that mixture 2 ml of a boiling agar solution (40 mg agar agar in 2 ml distilled water) were added, mixed and poured on the dry-tipped gel.After the agar had set, the covered cellulose acetate gels were incubated in the dark at room temperature until enzyme bands were visible.Then the overlay was washed under running tape water and afterwards put into a shaking water bath up to several hours.If the staining solution is intensively coloured and enzyme activity is low, the staining solution must be washed from the gel for several hours until the weak enzyme bands are clearly visible.In the case of light staining solutions and high enzyme activity the colour bands can be seen after a few minutes.

Figure 2 .
Figure 2. Scheme of dihydrolipoyl dehydrogenase patterns visualized on the separation medium Cellulose Acetate after electrophoresis.Myc: mycorrhizal root tips associated with Xerocomus pruinatus.Fungus: fruiting bodies, peeled off hyphae or rhizomorphae.Root: root tips of European beech seedlings grown in hydroculture or peeled fine roots without fungal hyphae.

Figure 4 .
Figure 4. Amino acid sequences of the dihydrolipoyl dehydrogenase gene of Xerocomus pruinatus.

Figure 5 .
Figure 5. Amino acid sequences of the dihydrolipoyl dehydrogenase gene of the fungus Xerocomus badius.

AppendixFigure A1 .
Figure A1.Comparison of the DNA and cDNA sequence of the enzyme NADH diaphorase in the two ectomycorrhiza fungi Xerocomus badius (Xb) and Xerocomus pruinatus (Xp) in symbiosis with beech (Fagus sylvatica, Fs) and spruce (Picea abies, Pa).

Table 1 .
Location of the investigated forest stands in the Taunus Mountains.

Table 4 .
Similarity of the genomic sequence of the dihydrolipoyl dehydrogenase of Xerocomus pruinatus and X. badius in comparison to other fungi.

Table 5 .
Position and length of the five introns of the dihydrolipoyl dehydrogenase gene in Xerocomus badius and X. pruinatus.Russula ochroleuca also include five 52 bp long introns located at the regions of the Xerocomus gene.These observations are in accordance with reports concerning the gene structure of the basidiomycetes Cryptococcus gatti, Cryptococcus neoformans, Coprinus cinerea, Ustilago maydis and Laccaria bicolor and the ascomycetes Candida albicans, C. orthopsilosis, Mycosphaerella graminicola and Trichophytum rubrum deposited at the Gene Bank (NCBI).The coding sequence of the gene of X. pruinatus deviates at 144 positions from that of X. badius.Besides the single nucleotide polymorphisms, the X. pruinatus gene contains a 48 bp long sequence at the positions 200 to 248 that could not be proved for the DNA and cDNA sequences of the gene from X. badius.Altogether, the two gene sequences deviate at 192 positions, which makes 11%.The number of single nucleotide polymorphisms of the five introns of X. badius and X. pruinatus sum up to 74 bp, corresponding to a deviation of 28.5%.Consequently, the nucleotide deviations in the five intron areas are about three times higher than those within the coding regions.The host trees European beech and Norway spruce did not influence the dihydrolipoyl dehydrogenase gene sequences in the two Xerocomus species.

Table 6 .
Number of nucleotides and number of amino acids, molecular weights and isoelectric points of the enzyme dihydrolipoyl dehydrogenase of Xerocomus pruinatus and X. badius.