Distinctive Patterns of Flavonoid Biosynthesis in Roots and Nodules of Datisca glomerata and Medicago spp. Revealed by Metabolomic and Gene Expression Profiles

Plants within the Nitrogen-fixing Clade (NFC) of Angiosperms form root nodule symbioses with nitrogen-fixing bacteria. Actinorhizal plants (in Cucurbitales, Fagales, Rosales) form symbioses with the actinobacteria Frankia while legumes (Fabales) form symbioses with proteobacterial rhizobia. Flavonoids, secondary metabolites of the phenylpropanoid pathway, have been shown to play major roles in legume root nodule symbioses: as signal molecules that in turn trigger rhizobial nodulation initiation signals and acting as polar auxin transport inhibitors, enabling a key step in nodule organogenesis. To explore a potentially broader role for flavonoids in root nodule symbioses across the NFC, we combined metabolomic and transcriptomic analyses of roots and nodules of the actinorhizal host Datisca glomerata and legumes of the genus Medicago. Patterns of biosynthetic pathways were inferred from flavonoid metabolite profiles and phenylpropanoid gene expression patterns in the two hosts to identify similarities and differences. Similar classes of flavonoids were represented in both hosts, and an increase in flavonoids generally in the nodules was observed, with differences in flavonoids prominent in each host. While both hosts produced derivatives of naringenin, the metabolite profile in D. glomerata indicated an emphasis on the pinocembrin biosynthetic pathway, and an abundance of flavonols with potential roles in symbiosis. Additionally, the gene expression profile indicated a decrease in expression in the lignin/monolignol pathway. In Medicago sativa, by contrast, isoflavonoids were highly abundant featuring more diverse and derived isoflavonoids than D. glomerata. Gene expression patterns supported these differences in metabolic pathways, especially evident in a difference in expression of cinnamic acid 4-hydroxylase (C4H), which was expressed at substantially lower levels in D. glomerata than in a Medicago truncatula transcriptome where it was highly expressed. C4H is a major rate-limiting step in phenylpropanoid biosynthesis that separates the pinocembrin pathway from the lignin/monolignol and naringenin-based flavonoid branches. Shikimate O-hydroxycinnamoyltransferase, the link between flavonoid biosynthesis and the lignin/monolignol pathway, was also expressed at much lower levels in D. glomerata than in M. truncatula. Our results indicate (a) a likely major role for flavonoids in actinorhizal nodules, and (b) differences in metabolic flux in flavonoid and phenylpropanoid biosynthesis between the different hosts in symbiosis

When A+B < A: Cognitive Bias in Experts' Judgment of Environmental Impact

When 'environmentally friendly' items are added to a set of conventional items, people report that the total set will have a lower environmental impact even though the actual impact increases. One hypothesis is that this "negative footprint illusion" arises because people, who are susceptible to the illusion, lack necessary knowledge of the item's actual environmental impact, perhaps coupled with a lack of mathematical skills. The study reported here addressed this hypothesis by recruiting participants ('experts') from a master's program in energy systems, who thus have bachelor degrees in energy-related fields including academic training in mathematics. They were asked to estimate the number of trees needed to compensate for the environmental burden of two sets of buildings: one set of 150 buildings with conventional energy ratings and one set including the same 150 buildings but also 50 'green' (energy-efficient) buildings. The experts reported that less trees were needed to compensate for the set with 150 conventional and 50 'green' buildings compared to the set with only the 150 conventional buildings. This negative footprint illusion was as large in magnitude for the experts as it was for a group of novices without academic training in energy-related fields. We conclude that people are not immune to the negative footprint illusion even when they have the knowledge necessary to make accurate judgments

A communal catalogue reveals Earth's multiscale microbial diversity

Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth's microbial diversity.Peer reviewe

A communal catalogue reveals Earth’s multiscale microbial diversity

Our growing awareness of the microbial world’s importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth’s microbial diversity

Rates of gene conversions between Escherichia coli ribosomal operons

Due to their universal presence and high sequence conservation, ribosomal RNA (rRNA) sequences are used widely in phylogenetics for inferring evolutionary relationships between microbes and in metagenomics for analyzing the composition of microbial communities. Most microbial genomes encode multiple copies of rRNA genes to supply cells with sufficient capacity for protein synthesis. These copies typically undergo concerted evolution that keeps their sequences identical, or nearly so, due to gene conversion, a type of intragenomic recombination that changes one copy of a homologous sequence to exactly match another. Widely varying rates of rRNA gene conversion have previously been estimated by comparative genomics methods and using genetic reporter assays. To more directly measure rates of rRNA intragenomic recombination, we sequenced the seven Escherichia coli rRNA operons in 15 lineages that were evolved for ∼13,750 generations with frequent single-cell bottlenecks that reduce the effects of selection. We identified 38 gene conversion events and estimated an overall rate of intragenomic recombination within the 16S and 23S genes between rRNA copies of 3.6 × 10-4 per genome per generation or 8.6 × 10-6 per rRNA operon per homologous donor operon per generation. This rate varied only slightly from random expectations at different sites within the rRNA genes and between rRNA operons located at different positions in the genome. Our accurate estimate of the rate of rRNA gene conversions fills a gap in our quantitative understanding of how ribosomal sequences and other multicopy elements diversify and homogenize during microbial genome evolution

A Stable Genetic Transformation System and Implications of the Type IV Restriction System in the Nitrogen-Fixing Plant Endosymbiont Frankia alni ACN14a.

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes that are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. The corresponding endosymbionts, Frankia and the rhizobia, however, are distantly related groups of bacteria, leading to questions about their symbiotic mechanisms and evolutionary history. To date, a stable system of electrotransformation has been lacking in Frankia despite numerous attempts by research groups worldwide. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation. Here we report the successful electrotransformation of the model strain F. alni ACN14a with an unmethylated, broad host-range replicating plasmid, expressing chloramphenicol-resistance for selection and GFP as a marker of gene expression. This system circumvented the type IV restriction barrier and allowed the stable maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the expression of egfp under the control of the nif gene cluster promoter was localized using fluorescence imaging, the expression of nitrogen fixation in nitrogen-limited culture was localized in Frankia vesicles but not in hyphae. The ability to separate gene expression patterns between Frankia hyphae and vesicles will enable deeper comparisons of molecular signaling and metabolic exchange between Frankia-actinorhizal and rhizobia-legume symbioses to be made, and may broaden potential applications in agriculture. Further downstream applications are possible, including gene knock-outs and complementation, to open up a range of experiments in Frankia and its symbioses. Additionally, in the transcriptome of F. alni ACN14a, type IV restriction enzymes were highly expressed in nitrogen-replete culture but their expression strongly decreased during symbiosis. The down-regulation of type IV restriction enzymes in symbiosis suggests that horizontal gene transfer may occur more frequently inside the nodule, with possible new implications for the evolution of Frankia

Contiguous Genome Sequence of Frankia sp. Strain ArI3, Isolated from Root Nodules of Alnus rubra Bong.

We report the genome sequence of Frankia sp. strain ArI3, recovered as a single contig from one run of the Oxford Nanopore Technologies (ONT) MinION instrument. The genome has a G+C content of 72%, is 7,541,222 bp long, and contains 5,427 predicted protein-coding genes