The fungal mitochondrial genome project: evolution of fungal mitochondrial genomes and their gene expression.

The goal of the fungal mitochondrial genome project (FMGP) is to sequence complete mitochondrial genomes for a representative sample of the major fungal lineages; to analyze the genome structure, gene content, and conserved sequence elements of these sequences; and to study the evolution of gene expression in fungal mitochondria. By using our new sequence data for evolutionary studies, we were able to construct phylogenetic trees that provide further solid evidence that animals and fungi share a common ancestor to the exclusion of chlorophytes and protists. With a database comprising multiple mitochondrial gene sequences, the level of support for our mitochondrial phylogenies is unprecedented, in comparison to trees inferred with nuclear ribosomal RNA sequences. We also found several new molecular features in the mitochondrial genomes of lower fungi, including: (1) tRNA editing, which is the same type as that found in the mitochondria of the amoeboid protozoan Acanthamoeba castellanii; (2) two novel types of putative mobile DNA elements, one encoding a site-specific endonuclease that confers mobility on the element, and the other constituting a class of highly compact, structured elements; and (3) a large number of introns, which provide insights into intron origins and evolution. Here, we present an overview of these results, and discuss examples of the diversity of structures found in the fungal mitochondrial genome.

Mitochondrial tRNAs in the lower fungus Spizellomyces punctatus: tRNA editing and UAG 'stop' codons recognized as leucine.

The mitochondrial DNA of the chytridiomycete fungus Spizellomyces punctatusen codes only eight tRNAs, although a minimal set of 24-25 tRNAs is normally found in fungi. One of these tRNAs has a CAU anticodon and is structurally related to leucine tRNAs, which would permit the translation of the UAG 'stop' codons that occur in most of its protein genes. The predicted structures of all S. punctatus tRNAs have the common feature of containing one to three mis-pairings in the first three positions of their acceptor stems. Such mis-pairing is expected to impair proper folding and processing of tRNAs from their precursors. Five of these eight RNAs were shown to be edited at the RNA level, in the 5'portion of the molecules. These changes include both pyrimidine to purine and A to G substitutions that restore normal pairing in the acceptor stem. Editing was not found at other positions of the tRNAs, or in the mitochondrial mRNAs of S. punctatus. While tRNA editing has not been observed in other fungi, the editing pattern inS.punctatus is virtually identical to that described in the amoeboid protozoan Acanthamoeba castellanii. If this type of mitochondrial tRNA editing has originated from their common ancestor, one has to assume that it was independently lost in plants, animals and in most fungi. Alternatively, editing might have evolved independently, or the genes coding for the components of the editing machinery were laterally transferred.

Molecular phylogeny of Allomyces macrogynus: congruency between nuclear ribosomal RNA- and mitochondrial protein-based trees.

We have sequenced the nuclear and mitochondrial small subunit rRNA genes (rns) and the mitochondrial genes coding for subunits 1 and 3 of the cytochrome oxidase (cox1 and cox3, respectively) of the chytridiomycete Allomyces macrogynus. Phylogenetic trees inferred from the derived COX1 and COX3 proteins and the nuclear rns sequences show with good bootstrap support that A. macrogynus is an early diverging fungus. The trees inferred from mitochondrial rns sequences do not yield a topology that is supported by bootstrap analysis. The similarity and the relative robustness of the nuclear rns and the mitochondrial protein-derived phylogenetic trees suggest that protein sequences are of higher value than rRNA sequences for reconstructing mitochondrial evolution. In addition, our trees support a monophyletic origin of mitochondria for the range of analyzed eukaryotes.

Transient induction of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) genes in cell suspension cultures of Catharanthus roseus.

When cell suspension cultures of Catharanthus roseus are treated with autoclaved elicitor from the fungus Pythium aphanidermatum, they respond with the rapid transient induction of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) enzyme activities, followed by the accumulation of indole alkaloids (Eilert et al., 1987). In this report, we demonstrate that expression of TDC and SS enzyme activities is preceeded by the transient appearance of mRNAs for both enzymes, suggesting transcriptional control of these events. The strong transient accumulation of both TDC and SS enzyme transcripts observed in elicitor-treated cell suspension cultures contrasts with the barely detectable level of TDC transcripts and the undetectable level of SS transcripts observed in developing seedlings.

Effect of ethylene on sanguinarine production from Papaver somniferum cell cultures.

A Papaver somniferum cell line capable of producing sanguinarine equivalent to 3% of cell dry weight was used to determine if ethylene was involved in signalling the biosynthesis of this alkaloid. A 3.3-fold increase in ethylene emanation from these cell suspension cultures was observed 7 h after elicitation with a Botrytis fungal homogenate. The rate of ethylene release then decreased to near zero after 48 h, suggesting that a pulse of ethylene production may be involved in sanguinarine production. However, sanguinarine biosynthesis was not promoted when either the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), or the ethylene releasing agent, 2-chloroethylphosphonic acid (ethephon), was added to the culture. These results strongly suggest that ethylene is not intimately involved in the production of sanguinarine from Papaver somniferum cell cultures or in the transduction of the elicitation event.

Semi-continuous production of sanguinarine and dihydrosanguinarine by Papaver somniferum L. cell suspension cultures treated with fungal homogenate.

Papaver somniferum L. (opium poppy) cells were elicited with a Botrytis sp. homogenate and cultured by a semi-continuous process. Elicitation induced synthesis of sanguinarine and dihydrosanguinarine. Significant release of both alkaloids into the culture medium occurred. Medium exchange at 2-day intervals enabled product recovery from spent medium and maintained culture viability. Culture growth was not inhibited by elicitor treatment necessitating sub-culture prior to re-elicitation. Re-elicited cultures displayed an increasing sensitivity (reduced growth rate, higher alkaloid yield) to the elicitor with each successive treatment and did not survive a fourth elicitation.

Cell cultures of the wild sunflower Helianthus maximiliani schrader: growth and secondary metabolite synthesis.

Callus cultures derived from leaves, stem, sepals and ray flowers and cell suspension cultures of Helianthus maximiliani Schrader were established and analysed for their phytochemicals. Dark-grown cultures were found to synthesize small amounts of non-toxic, polycyclic diterpenoids when grown on modified MS-medium, while ß-sitosterol and palmitic acid were found in dark- and light-grown cultures. Red light irradiation did not enhance terpenoid production compared to dark- and light-grown cells.