Enzymatic basis for C-lignin monomer biosynthesis in the seed coat of Cleome hassleriana.

C-lignin is a linear polymer of caffeyl alcohol, found in the seed coats of several exotic plant species, with promising properties for generation of carbon fibers and high value chemicals. In the ornamental plant Cleome hassleriana, guaiacyl (G) lignin is deposited in the seed coat for the first 6-12 days after pollination, after which G-lignin deposition ceases and C-lignin accumulates, providing an excellent model system to study C-lignin biosynthesis. We performed RNA sequencing of seed coats harvested at 2-day intervals throughout development. Bioinformatic analysis identified a complete set of lignin biosynthesis genes for Cleome. Transcript analysis coupled with kinetic analysis of recombinant enzymes in Escherichia coli revealed that the switch to C-lignin formation was accompanied by down-regulation of transcripts encoding functional caffeoyl CoA- and caffeic acid 3-O-methyltransferases (CCoAOMT and COMT) and a form of cinnamyl alcohol dehydrogenase (ChCAD4) with preference for coniferaldehyde as substrate, and up-regulation of a form of CAD (ChCAD5) with preference for caffealdehyde. Based on these analyses, blockage of lignin monomer methylation by down-regulation of both O-methyltransferases (OMTs) and methionine synthase (for provision of C1 units) appears to be the major factor in diversion of flux to C-lignin in the Cleome seed coat, although the change in CAD specificity also contributes based on the reduction of C-lignin levels in transgenic Cleome with down-regulation of ChCAD5. Structure modeling and mutational analysis identified amino acid residues important for the preference of ChCAD5 for caffealdehyde.

Benchmarking Metagenomic Classifiers on Simulated Ancient and Modern Metagenomic Data.

Taxonomic profiling of ancient metagenomic samples is challenging due to the accumulation of specific damage patterns on DNA over time. Although a number of methods for metagenome profiling have been developed, most of them have been assessed on modern metagenomes or simulated metagenomes mimicking modern metagenomes. Further, a comparative assessment of metagenome profilers on simulated metagenomes representing a spectrum of degradation depth, from the extremity of ancient (most degraded) to current or modern (not degraded) metagenomes, has not yet been performed. To understand the strengths and weaknesses of different metagenome profilers, we performed their comprehensive evaluation on simulated metagenomes representing human dental calculus microbiome, with the level of DNA damage successively raised to mimic modern to ancient metagenomes. All classes of profilers, namely, DNA-to-DNA, DNA-to-protein, and DNA-to-marker comparison-based profilers were evaluated on metagenomes with varying levels of damage simulating deamination, fragmentation, and contamination. Our results revealed that, compared to deamination and fragmentation, human and environmental contamination of ancient DNA (with modern DNA) has the most pronounced effect on the performance of each profiler. Further, the DNA-to-DNA (e.g., Kraken2, Bracken) and DNA-to-marker (e.g., MetaPhlAn4) based profiling approaches showed complementary strengths, which can be leveraged to elevate the state-of-the-art of ancient metagenome profiling.