Jeffrey F. D. Dean

A fungal metabolite mediates degradation of non‐phenolic lignin structures and synthetic lignin by laccase

Lignin peroxidase is generally considered to be a primary catalyst for oxidative depolymerization of lignin by white‐rot fungi. However, some white‐rot fungi lack lignin peroxidase. Instead, many produce laccase, even though the redox potentials of known laccases are two to directly oxidize non‐phenolic components of lignin. Pycnoporus cinnabarinus is one example of a laccase‐producing fungus that degrades lignin very efficiently. To overcome the redox potential barrier, P. cinnabarinus produces a metabolite, 3‐hydroxyanthranilate that can mediate the oxidation of non‐phenolic substrates by laccase. This is the first description of how laccase might function in a biological system for the complete depolymerization of lignin.

Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies

BackgroundThe size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination.ResultsWe develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome.ConclusionsIn addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied.

Laccase and the deposition of lignin in vascular plants

Laccase (p-diphenolrOi oxidoreductase, EC 1.10.3.2) was the first enzyme used to polymerize a synthetic lignin (DHP) in vitro. Based on that work, laccase was proposed to perform the same function in vivo. However, various biochemical and histological studies eventually led most researchers to believe that peroxidases, not laccases, were principally responsible for monolignol polymerization in vascular plants. Recent evidence from several laboratories necessitates a re-evaluation of the relative roles played by oxidases and peroxidases in lignin deposition. In this paper, we review the current literature regarding plant laccases, äs well äs selected fungal laccases. From this body of evidence, we have developed a model for the evolution of lignin äs a structural polymer, and we describe how this model may be used to develop new questions regarding the origins of lignin and vascular plants.

Gene structure and molecular analysis of the laccase-like multicopper oxidase (LMCO) gene family in Arabidopsis thaliana

Completed genome sequences have made it clear that multicopper oxidases related to laccase are widely distributed as multigene families in higher plants. Laccase-like multicopper oxidase (LMCO) sequences culled from GenBank and the Arabidopsis thaliana genome, as well as those from several newly cloned genes, were used to construct a gene phylogeny that clearly divided plant LMCOs into six distinct classes, at least three of which predate the evolutionary divergence of angiosperms and gymnosperms. Alignments of the predicted amino acid sequences highlighted regions of variable sequence flanked by the highly conserved copper-binding domains that characterize members of this enzyme family. All of the predicted proteins contained apparent signal sequences. The expression of 13 of the 17 LMCO genes in A. thaliana was assessed in different tissues at various stages of development using RT-PCR. A diversity of expression patterns was demonstrated with some genes being expressed in a constitutive fashion, while others were only expressed in specific tissues at a particular stage of development. Only a few of the LMCO genes were expressed in a pattern that could be considered consistent with a major role for these enzymes in lignin deposition. These results are discussed in the context of other potential physiological functions for plant LMCOs, such as iron metabolism and wound healing.

Extracellular laccases and peroxidases from sycamore maple (Acer pseudoplatanus) cell-suspension cultures

We have investigated the abilities of extracellular enzymes from dark-grown cell-suspension cultures of sycamore maple (Acer pseudoplatanus L.) to oxidize monolignols, the precursors for lignin biosynthesis in plants, as well as a variety of other lignin-related compounds. Laccase and peroxidase both exist as a multiplicity of isoenzymes in filtrates of spent culture medium, but their abilities to produce water-insoluble, dehydrogenation polymers (DHPs) from the monolignols (in the presence of hydrogen peroxide for the peroxidase reaction) appear identical whether or not the enzymes are purified from the concentrated filtrates or left in a crude mixture. The patterns of bonds formed in these DHPs are identical to those found in DHPs synthesized using horseradish peroxidase or fungal laccase, and many of these bonds are found in the natural lignins extracted from different plant sources. On the other hand, sycamore maple laccase is very much less active on phenolic substrates containing multiple aromatic rings than is sycamore maple peroxidase. We suggst that whereas laccase may function during the early stages of lignification to polymerize monolignols into oligo-lignols, cell-wall peroxidases may function when H2O2 is produced during the later stages of xylem cell development or in response to environmental stresses.

Forest tree biotechnology

The past year has seen the fruits of biotechnological manipulation of forest trees approach commercial application. Advances in somatic embryogenesis have brought mass clonal propagation of the top commercial trees closer to reality, and efficient gene transfer systems have been developed for a number of conifers and hardwoods. Radical alterations in the quantity and quality of lignin in wood have been shown to be possible in softwoods and hardwoods through identification of naturally occurring mutants, as well as by engineering the lignin biosynthetic pathway with transgenes. The potential environmental and social impacts of the release of transgenic trees have become an increasingly contentious issue that will require more attention if we are to use these technologies to their full advantage.

SAGE Analysis of Transcriptome Responses in Arabidopsis Roots Exposed to 2,4,6-Trinitrotoluene

Serial analysis of gene expression was used to profile transcript levels in Arabidopsis roots and assess their responses to 2,4,6-trinitrotoluene (TNT) exposure. SAGE libraries representing control and TNT-exposed seedling root transcripts were constructed, and each was sequenced to a depth of roughly 32,000 tags. More than 19,000 unique tags were identified overall. The second most highly induced tag (27-fold increase) represented a glutathione S-transferase. Cytochrome P450 enzymes, as well as an ABC transporter and a probable nitroreductase, were highly induced by TNT exposure. Analyses also revealed an oxidative stress response upon TNT exposure. Although some increases were anticipated in light of current models for xenobiotic metabolism in plants, evidence for unsuspected conjugation pathways was also noted. Identifying transcriptome-level responses to TNT exposure will better define the metabolic pathways plants use to detoxify this xenobiotic compound, which should help improve phytoremediation strategies directed at TNT and other nitroaromatic compounds.

Laccase-mediated formation of the phenoxazinone derivative, cinnabarinic acid

The phenoxazinone chromophore occurs in a variety of biological systems, including numerous pigments and certain antibiotics. It also appears to form as part of a mechanism to protect mammalian tissue from oxidative damage. During cultivation of the basidiomycete, Pycnoporus cinnabarinus, a red pigment was observed to accumulate in the culture medium. It was identified as the phenoxazinone derivative, cinnabarinic acid (CA). Laccase was the predominant extracellular phenoloxidase activity in P. cinnabarinus cultures. In vitro studies showed that CA was formed after oxidation of the precursor, 3‐hydroxyanthranilic acid (3‐HAA), by laccases. Moreover, oxidative coupling of 3‐HAA to form CA was also demonstrated for the mammalian counterpart of laccase, the blue copper oxidase, ceruloplasmin.

Microarray analysis and scale-free gene networks identify candidate regulators in drought-stressed roots of loblolly pine (P. taeda L.).

BackgroundGlobal transcriptional analysis of loblolly pine (Pinus taeda L.) is challenging due to limited molecular tools. PtGen2, a 26,496 feature cDNA microarray, was fabricated and used to assess drought-induced gene expression in loblolly pine propagule roots. Statistical analysis of differential expression and weighted gene correlation network analysis were used to identify drought-responsive genes and further characterize the molecular basis of drought tolerance in loblolly pine.ResultsMicroarrays were used to interrogate root cDNA populations obtained from 12 genotype × treatment combinations (four genotypes, three watering regimes). Comparison of drought-stressed roots with roots from the control treatment identified 2445 genes displaying at least a 1.5-fold expression difference (false discovery rate = 0.01). Genes commonly associated with drought response in pine and other plant species, as well as a number of abiotic and biotic stress-related genes, were up-regulated in drought-stressed roots. Only 76 genes were identified as differentially expressed in drought-recovered roots, indicating that the transcript population can return to the pre-drought state within 48 hours. Gene correlation analysis predicts a scale-free network topology and identifies eleven co-expression modules that ranged in size from 34 to 938 members. Network topological parameters identified a number of central nodes (hubs) including those with significant homology (E-values ≤ 2 × 10-30) to 9-cis-epoxycarotenoid dioxygenase, zeatin O-glucosyltransferase, and ABA-responsive protein. Identified hubs also include genes that have been associated previously with osmotic stress, phytohormones, enzymes that detoxify reactive oxygen species, and several genes of unknown function.ConclusionPtGen2 was used to evaluate transcriptome responses in loblolly pine and was leveraged to identify 2445 differentially expressed genes responding to severe drought stress in roots. Many of the genes identified are known to be up-regulated in response to osmotic stress in pine and other plant species and encode proteins involved in both signal transduction and stress tolerance. Gene expression levels returned to control values within a 48-hour recovery period in all but 76 transcripts. Correlation network analysis indicates a scale-free network topology for the pine root transcriptome and identifies central nodes that may serve as drivers of drought-responsive transcriptome dynamics in the roots of loblolly pine.

Towards decoding the conifer giga-genome.

Several new initiatives have been launched recently to sequence conifer genomes including pines, spruces and Douglas-fir. Owing to the very large genome sizes ranging from 18 to 35 gigabases, sequencing even a single conifer genome had been considered unattainable until the recent throughput increases and cost reductions afforded by next generation sequencers. The purpose of this review is to describe the context for these new initiatives. A knowledge foundation has been acquired in several conifers of commercial and ecological interest through large-scale cDNA analyses, construction of genetic maps and gene mapping studies aiming to link phenotype and genotype. Exploratory sequencing in pines and spruces have pointed out some of the unique properties of these giga-genomes and suggested strategies that may be needed to extract value from their sequencing. The hope is that recent and pending developments in sequencing technology will contribute to rapidly filling the knowledge vacuum surrounding their structure, contents and evolution. Researchers are also making plans to use comparative analyses that will help to turn the data into a valuable resource for enhancing and protecting the world’s conifer forests.