Suppression subtractive hybridization (SSH) was employed to investigate bioluminescence in Panellus stipticus (Bull.) P. Karst. by detecting proteins differentially expressed in bioluminescent and luminescent strains. Comparisons of luminescent and non-luminescent monokaryon cultures of North American strains revealed differences in transcript levels of proteins responsible for post-translational modification (PTM) of enzymes. A similar comparison of a luminescent strain of P. stipticus from North America with a non-luminescent European strain revealed the presence of extracellular manganese superoxide dismutase (MnSOD) in the luminescent form, in addition to proteins involved in PTM. The application of MnSOD-specific inhibitors to luminescent mycelium resulted in the rapid loss of luminescence. The relevance to luminescence of proteins involved in PTM is discussed, together with a possible role for MnSOD that considers the potential for SODs to form stable complexes with catechols revealed in previously published research. In light of the recent discovery that hispidine may be the precursor of fungal luciferin, we consider a hypothetical mechanism for fungal luminescence in which the ο-hydroquinone moiety of a hispidine derivative ligates with the extracellular form of MnSOD producing a semiquinone-radical complex, with the resultant semiquinonato complex potentially reacting with molecular oxygen or other reactive oxygen species to produce sufficiently excited intermediates to emit light on relaxation.
Approximately 71 species of luminous fungi have been recognized distributed among four evolutionarily distinct euagaric lineages [
The precise molecular nature of fungal bioluminescence remains unresolved today, with the nature of the light emitter, the enzymes involved and the biochemical mechanism not fully resolved. Two alternative concepts for the cellular organization and molecular mechanism of fungal luminescence are currently being investigated. According to the first concept luminescence occurs in the absence of a specialized enzyme by oxidation of the energized state of the luciferin involving an active oxygen species such as the superoxide anion [
The second concept is based on evidence for the enzymatic nature of bioluminescence, whereby bioluminescence is mediated by a luciferase enzyme acting on the luciferin light-emitter [
Comprehensive reviews on the current state of knowledge of fungal bioluminescence have been published recently that reflect the current impasse involving alternate proposals for the mechanism of fungal bioluminescence [
Presumed dikaryon strains studied were G20.4 (DAOM 242555) a luminous culture isolated on 16 August 2010 from clustered basidiomes on a decaying stump of Betula papyrifera near Almonte, Ontario, Canada, and DAOM F2118 a non-luminous dikaryon strain isolated in 1931 from a stump of Quercus pedunculata in Darmstadt, Germany (leg. Kallenback and Macrae). Dikaryon cultures were isolated by plating portions of the pileus interior onto Difco™ potato dextrose agar (PDA-BD Diagnostics, Sparks, MD, USA). Single-spore isolates of P. stipticus were obtained by careful extraction under microscopic observation of individual germinating basidiospores released from freshly collected pilei suspended over PDA. The following four monokaryon single-spore cultures were studied, all isolated from the same source as G20.4: ssp5 (DAOM 242556) and ssp6 (DAOM 242557) luminous single-spore strains, and ssp7 (DAOM 242558) and ssp9 (DAOM 242559) non-lu- minous strains. All cultures cited are deposited in the culture collection of Agriculture and Agri-Food Canada, ECORC, Ottawa, Canada (DAOM).
Luminescence (relative units) of 2-wk old colonies growing on PDA in 3.5cm Petri dishes was determined with a GloMax®20/20 luminometer (Promega, Madison, WI, USA). Baseline intensity of the luminometer was 0 - 200 RU. Differences in luminescence between the luminescent and non-luminescent cultures were 5 - 6 orders of magnitude (
Total RNA was isolated from mycelium harvested from 2-wk old cultures using the Total RNA Isolation NucleoSpin® RNA Plant Kit (Macherey-Nagel, Düren, Germany) using Lysis buffer RAP and following the manufacturer’s instructions. Total RNA was measured with a NanoDrop® ND-1000 spectrophotometer (Thermo- Scientific, Wilmington, DE, USA). For samples with low total RNA (<2 μg), cDNA was synthesized from total RNA using the SMARTer™ Pico PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA). For higher concentrations of total RNA, mRNA was isolated using the GenElute™ mRNA Miniprep Kit (Sigma-Aldrich, Saint Louis, USA), and mRNA isolation repeated once to enhance mRNA purification. Bidirectional suppression subtractive hybridization (SSH) was carried out using Clontech PCR-Select™ cDNA Subtraction Kit (Clontech) following the manufacturer’s directions, starting with first strand cDNA synthesis for isolated mRNA, and with adaptor ligation for synthesized cDNA. SSH was performed separately on 1) North American (DAOM 242555) and European dikaryon isolates (DAOM F2118)―luminous and non-luminous strains respectively; 2) North American monokaryon isolates ssp5 (luminous) and ssp7 (non-luminous); and 3) ssp6 (luminous) and ssp9 (non-luminous) isolates.
Strain | Luminescence (relative units) |
---|---|
non-luminescent | |
ssp71 | 276 |
ssp91 | 397 |
DAOM F21182 | 208 |
luminescent | |
ssp51 | 13145771 |
ssp61 | 137839120 |
DAOM 2425553 | 156469480 |
1Single spore isolates derived from DAOM 242555; 2European isolate in herb. DAOM; 3North American isolate in herb. DAOM.
Subtracted cDNA was cloned with the TOPO TA Cloning® kit (Invitrogen, Carlsbad, CA, USA) using One Shot® TOP10 electrocompetent cells, and transformation performed by electroporation with a Bio-Rad Gene Pulser Xcell™ electroporator in 0.1 cm cuvettes at 1.8kV. Amplification of cloned cDNA was performed with the PCR-Select™ primers (M13F, M13R) using a Labnet Multigene thermocycler (Labnet Intl., Edison, NJ). PCR reactions were prepared in 10μL volume containing the following mix: 1 μL 10X Titanium Taq Buffer (Clontech, Mountain View, CA), 0.5 μL 2 mM dNTP, 0.32 μL each of 5 μM upper and lower primers, 7.76 μL sterile distilled water and 0.1 μL Titanium Taq Polymerase (Clontech). Sequencing reactions were prepared using the ABI Prism® BigDye™ Terminator reaction kit (v3.1, Applied Biosystems Inc., Foster City, CA) in 10 μL volume and 1/8 dilution using 5X sequencing buffer. The cycle sequencing reaction contained the following mix: 1.75 μL 5X Sequencing Buffer, 0.5 μL BigDye V3.1 Mix, 0.5 μL of 3.2 μM primer (T3 or T7), 6.25 μL sterile distilled water, 1.0 μL (10 - 40 ng) PCR template, and employed the following amplification protocol: 25 cycles each of 30 sec denaturation at 96˚C, 15 sec annealing at 50˚C, and 4 min extension at 60˚C. Sequences were obtained using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Sequence segments of vector origin were identified and removed using the program VecScreen (http://www.ncbi.nlm.nih.gov/tools/vecscreen/). The resulting clean gene fragments were identified by blastx analysis (http://blast.ncbi.nlm.nih.gov/) on the GenBank non-redundant protein-sequence database using the conceptually translated nucleotide sequence. Acceptable identifications contained conserved domains and/or had highly significant sequence similarity (E value < 1e−10) to known genes of other basidiomycetes. Sequence fragments representing genes with possible relevance for luminescence were deposited in GenBank and accession numbers cited in Tables 2-4.
The following SOD inhibitors were used to determine the metal cofactor of SOD: 5 mM sodium azide, 5 mM hydrogen peroxide and 3 mM potassium ferricyanide according to standard procedures for cofactor determination of Roberts and Hirst [
Forward SSH (DAOM 242555) luminous | Reverse SSH (DAOM F2118) nonluminous |
---|---|
56 clones | 31 clones |
Manganese superoxide dismutase (JZ516005)2 | (none) |
Cop9 signalosome complex (KF846070) | |
Sentrin/sumo-specific protease (KF846078) | |
RNB domain-containing protein (KF846068) | |
AB-hydrolase YheT (KF846067) | |
UPF0041 unknown protein family (KF846072) | |
3 unidentified or hypothetical proteins |
1GenBank accession numbers in brackets; 2sequence extended to N-terminus (KJ842128).
Forward SSH (ssp5) luminous | Reverse SSH (ssp7) nonluminous |
---|---|
115 clones | 173 clones |
Oligosaccaryltransferase (KF846077) | Cullin NEDD8 ubiquitin protein ligase (KF846071) |
PRO41-like protein(KF846075) | Rho GDP dissociation inhibitor (KF846079) |
14 unidentified or hypothetical proteins | Pepidylprolyl cis-trans isomerase (KF846073) |
Serine-threonine protein phosphatase (KF846080) | |
Metallo-dependent phosphatase (KF846076) | |
Dienelactone hydrolase (KF846074) | |
Syntaxin (KF846081) | |
Phospholipase/carboxylesterase (KF846069) | |
Cyclohydrolase (KF846072) | |
39 unidentified or hypothetical proteins |
1GenBank accession numbers in brackets.
Forward SSH (ssp6) luminous | Reverse SSH (ssp9) nonluminous |
---|---|
66 clones | 21 clones |
E3 ubiquitin protein ligase (JZ516004) | (2 unidentified sequence fragments) |
8 unidentified and hypothetical proteins |
1GenBank accession numbers in brackets.
The SOD sequence fragment obtained by SSH was extended using primers designed from within the 3’ UTR region of the fragment obtained by SSH and from within the N-terminus region of an alignment of Genbank MnSOD sequences. PCR was performed as above using primers SOD-F (5’-CTC CCY TAC GCC TAY GAT G-3’) and SOD-R1 (5’-AGC TTG GAA CCC TCG ACA A-3’) with the following amplification protocol: 3 min at 95˚C; 35 cycles of (60 s at 94˚C, 75 s at 65˚C, 105 s at 72˚C); 10 min at 72˚C. Sequencing was performed as above using primers SOD-F and SOD-R2 (CC TCG ACA AAG CGT GTC TG) and the sequence deposited in Genbank (KJ842128). Subcellular targeting of the MnSOD was investigated using Target P1.1 (http://www.cbs.dtu.dk/services/TargetP) and SecretomeP 2.0 (http://www.cbs.dtu.dk/services/SecretomeP).
Three bidirectional SSH were performed providing differing although not necessarily contradictory results. The first SSH compared luminescent and non-luminescent dikaryon strains from North America and Europe respectively (
The second SSH compared the expressed cDNA in two monokaryon isolates (
The third SSH compared the other two monokaryon strains (
It is perhaps not surprising that different results were observed from the three different SSH trials. We might expect only few detectable differences in metabolism of the single-spore monokaryon cultures, which were isolated from clustered fruiting bodies occurring on the same birch stump. However, one or a few genetic differences occurring between monokaryon pairs could lead to a cascade of metabolic differences, compounded by the loss of the mediating effect of paired dikaryon nuclei, and result in the loss of luminescence. Notably, in all three experiments we found differences in transcript levels of enzymes involved in PTM of proteins that would affect metabolism. In each experiment there were differences in the regulation of proteins involved in ubiquitination which can signal degradation, alter location, affect activity and promote or prevent protein interactions. The cop9 signalosome protein up-regulated in the luminous dikaryon isolate has deneddylation activity which is in opposition to the activity of nedd8 ubiquitin protein ligase up-regulated in the nonluminous ssp7 strain. The observation that MnSOD was up-regulated in the North American luminous strain was notable considering the well documented antioxidant activity of SODs in catalyzing the dismutation of superoxide (
The active cation of the up-regulated SOD in North American luminous Panellus was determined by examining the effect of inhibitors differentially affecting SODs (
JZ516005) contained the C-terminal conserved domain of the Sod_Fe_C family (pfam02777) together with 87 bases of the 3’ UTR. This gene fragment most closely resembled MnSOD in Agaricus bisporus (embl/CAB85688) having 85% similarity with a 2-aa residue modification and 3 residue deletion in the C-terminal conserved domain. The extended cDNA sequence obtained from primers designed for MnSOD comprised 189 aa residues. Protein ligands characteristic of MnSOD were located at positions 26 (histidine), 71 (histidine), 159 (aspartic acid) and 163 (histidine) of the translated protein. The upregulated MnSOD protein sequence from P. stipticus most closely matched a sequence for Laccaria bicolor (XP_001887049) with 85% similarity. Protein sequence similarities were only 53% and 51% with human and Saccharomyces cerevisiae MnSOD respectively, which could indicate functional differences for MnSODs from different organisms. Using the MnSOD primers to sequence genomic DNA resulted in a sequence of 840 bp with three introns at locations 169-217 (49 bp), 407-467 (61 bp) and 606-691 (76 bp) of the alignment. Signal peptides for protein sorting were not detected in the MnSOD sequence; however, non-classical secretion of the protein was predicted in analysis by SecretomeP (SecP score = 0.716).
Proteins involved in ubiquitination were differentially expressed between luminescent and non-luminescent forms in every SSH subtraction, impacting the activities or cellular localization of proteins involved in luminescence. The finding of an extracellular isoform of MnSOD in the luminescent North American form of Panellus stipticus indicates that ubiquitine targeting of this MnSOD to the cell surface in luminescent mushrooms could be a determining factor for fungal luminescence. The association of luminescence with non-mitochondrial membranes was observed by Kamzolkina et al. [
SODs reduce oxidative stress by catalyzing the dismutation of superoxide ion producing H2O2 as represented by the following half-reactions:
Mn3+-SOD + O2∙− → Mn2+-SOD + O2 (1)
Mn2+-SOD + O2∙− + 2H+ → Mn3+-SOD + H2O2 (2)
In human MnSOD removal of O2∙− involves product inhibition and reduction of O2∙− may occur by “prompt protonation” (2) or involve an Mn3-peroxo complex in an “inhibited pathway” (3), (4) which slows catalysis:
Mn2+-SOD + O2∙− → Mn3+(OO2−)-SOD (3)
Mn3+(OO2−)-SOD + 2H+ → Mn3+-SOD + H2O2 (4)
Few organisms have been investigated for product inhibition of MnSOD activity. In the only fungi studied (Saccharomyces cerevisiae, Candida albicans) [
MnSOD isoforms preferentially bound to cell walls and metabolically distinct from other isoforms may occur widely in eukaryotes [
In previous studies (unpublished) we observed the darkening of growth media in luminescent cultures of mushroom species (Armillaria borealis, A. mellea, P. stipticus) grown on solid and liquid media, in time producing dark exudates which could be due to the accumulation and polymerization of enzymatically oxidized phenols or quinhydrones. Similar dark metabolites were not observed in non-luminous cultures of the same species grown under the same conditions. This observation suggests a role for the hydroquinone/semiquinone/quinone oxido- reductase triad in luminescence. Rapp et al. [
Among luciferin precursors that have been proposed, Nakamura in a personal communication cited by Shimomura [
The following are the part-reactions we propose for luminescence in fungi:
1) one-electron oxidation of a substituted hydroquinone (Q) to produce the semiquinone anion radical in complex with Mn-SOD:
Q + Mn3+ → Mn2+-Q∙−
Purtov et al. [
2) non-enzymatic, one-electron oxidation of the Mn-SOD complexed semiquinone radical by a reactive oxygen species such as a hydroxyl radical to produce the excited state of the semiquinone luciferin (Q*):
Q∙− + OH∙ → Q* + OH−
The resulting semiquinone radicals would be oxidized by OH∙ yielding the quinone product Q* in an electronically exited state [
3) relaxation of the peroxidized (excited) state of the luciferin to produce energy with resultant light emission:
Q* → Q + hν
Generation of the electronically exited state during the reaction of p-benzoquinone with OH• (H2O2) was assumed by Brunmark and Cadenas [
Shimomura [
Hydroquinones may act as shuttles, moving protons from the inner surface of the cytoplasmic membrane to the outer surface, similar to the action of 2,4-dinitrophenol in mitochondria. The H+ is transported to the outer surface where the hydroquinone is oxidized and the electrons are passed back to metalloproteins. Excess hydroxide ions from the luminescence reaction may accept protons producing H2O, and at the same time quinones/ hydroquinones can be condensed or polymerised outside of the cell forming quinolic polymers or quinhydrones. The participation of hydroquinones in luminescence as luciferin precursors may explain the correlation of mushroom luminescence with ligninolytic fungi [
1) The literature on mushroom bioluminescence is reviewed supporting the following, alternate hypotheses for the bioluminescence mechanism in fungi: i) luminescence occurs in the absence of a specialized enzyme and involves the oxidation of an energized state of the fungal luciferin by a reactive oxygen species such as the superoxide anion; or ii) bioluminescence is mediated by a specialized enzyme (the luciferase) acting on the luciferin light-emitter.
2) Differential transcript levels for proteins responsible for post-translational modification (PTM) of enzymes were observed in three separate experiments involving suppression subtractive hybridizations (SSHs) contrasting bioluminescent and non-luminescent strains of Panellus stipticus. Notably, differential transcript levels were observed in all three experiments for proteins involved in ubiquitination which can be determinants for cell localization of proteins or for degradation of proteins in the proteasome.
3) SSH and sequence analysis evidenced that an extracellular isoform of MnSOD was observed in a luminous North American dikaryon isolate of Panellus stipticus which was not observed in a nonluminous European isolate.
4) A hypothetical mechanism for mushroom bioluminescence was proposed whereby the o-hydroquinone moiety of a hispidine derivative ligates with an excreted isoform of MnSOD producing a semiquinone radical complex which reacts with molecular oxygen or other reactive oxygen species producing an excited intermediate which produces light on relaxation. This hypothesis takes into consideration previously published research indicating that mushroom luminescence occurs on mushroom surface tissues [
The senior author especially thanks N.S. Manukovskii for many hours of discussions on the biochemistry of mushrooms and possible mechanisms for luminescence.
Galina A. Vydryakova,John Bissett, (2016) Differential Regulation of Proteins and a Possible Role for Manganese Superoxide Dismutase in Bioluminescence of Panellus stipticus Revealed by Suppression Subtractive Hybridization. Advances in Microbiology,06,613-626. doi: 10.4236/aim.2016.69061