Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

The renaissance and enlightenment of Marchantia as a model system.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades, there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Ancient Origin and Recent Innovations of RNA Polymerase IV and V.

Small RNA-mediated chromatin modification is a conserved feature of eukaryotes. In flowering plants, the short interfering (si)RNAs that direct transcriptional silencing are abundant and subfunctionalization has led to specialized machinery responsible for synthesis and action of these small RNAs. In particular, plants possess polymerase (Pol) IV and Pol V, multi-subunit homologs of the canonical DNA-dependent RNA Pol II, as well as specialized members of the RNA-dependent RNA Polymerase (RDR), Dicer-like (DCL), and Argonaute (AGO) families. Together these enzymes are required for production and activity of Pol IV-dependent (p4-)siRNAs, which trigger RNA-directed DNA methylation (RdDM) at homologous sequences. p4-siRNAs accumulate highly in developing endosperm, a specialized tissue found only in flowering plants, and are rare in nonflowering plants, suggesting that the evolution of flowers might coincide with the emergence of specialized RdDM machinery. Through comprehensive identification of RdDM genes from species representing the breadth of the land plant phylogeny, we describe the ancient origin of Pol IV and Pol V, suggesting that a nearly complete and functional RdDM pathway could have existed in the earliest land plants. We also uncover innovations in these enzymes that are coincident with the emergence of seed plants and flowering plants, and recent duplications that might indicate additional subfunctionalization. Phylogenetic analysis reveals rapid evolution of Pol IV and Pol V subunits relative to their Pol II counterparts and suggests that duplicates were retained and subfunctionalized through Escape from Adaptive Conflict. Evolution within the carboxy-terminal domain of the Pol V largest subunit is particularly striking, where illegitimate recombination facilitated extreme sequence divergence.

Identification of Arabidopsis thaliana small RNAs responsive to the fungal pathogen Botrytis cinerea at an early stage of interaction.

In plants, small RNAs (sRNAs), mainly microRNAs (miRNAs) and small interfering RNAs (siRNAs), have been described as key regulators of plant development, growth, and abiotic and biotic responses. Despite reports indicating the involvement of certain sRNAs in regulating the interaction between Botrytis cinerea (a major necrotrophic fungal phytopathogen) and host plants, there remains a lack of analysis regarding the potential regulatory roles of plant sRNAs during early stages of the interaction despite early immune responses observed then during infection. We present the first transcriptome-wide analysis of small RNA expression on the early interaction between the necrotrophic fungus Botrytis cinerea and the model plant Arabidopsis thaliana. We found that evolutionary conserved A. thaliana miRNAs were the sRNAs that accumulated the most in the presence of B. cinerea. The upregulation of miR167, miR159 and miR319 was of particular interest because these, together with their target transcripts, are involved in the fine regulation of the plant hormone signaling pathways. We also describe that miR173, which triggers the production of secondary siRNAs from TAS1 and TAS2 loci, as well as secondary siRNAs derived from these loci, is upregulated in response to B. cinerea. Thus, at an early stage of the interaction there are transcriptional changes of sRNA-guided silencing pathway genes and of a subset of sRNAs that targeted genes from the PPR gene superfamily, and these may be important mechanisms regulating the interaction between A. thaliana and B. cinerea. This work provides the basis for a better understanding of the regulation mediated by sRNAs during early B. cinerea-plant interaction and may help in the development of more effective strategies for its control.

Energy-saving pretreatments affect pelagic Sargassum composition and DNA metabarcoding reveals the microbial community involved in methane yield.

Sargassum spp. flood the Caribbean coastline, causing damage to the local economy and environment. Anaerobic digestion (AD) has been proposed as an attractive option for turning macroalgae into valuable resources. Sargassum spp. has a complex composition that affects the microbial composition involved in AD which generates a low methane yield. This study aimed to improve the methane yield of pelagic Sargassum, using different energy-saving pretreatments and identifying the microbial community associated with methane production. We applied different energy-saving pretreatments to algal biomass and assessed the methane yield using a biomethane potential (BMP) test. The microbial communities involved in the AD of the best- and worst-performing methanogenic systems were analyzed by high-throughput sequencing. The results showed that pretreatment modified the content of inorganic compounds, fibers, and the C:N ratio, which had a strong positive correlation with BMP. The water washing pretreatment resulted in the best methane yield, with an increase of 38%. DNA metabarcoding analysis revealed that the bacterial genera Marinilabiliaceae_uncultured, DMER64, Treponema, and Hydrogenispora, as well as the archaea genera Methanosarcina, RumEn_M2, Bathyarchaeia, and Methanomassiliicocus, dominated the microbial community with a high methane yield. This study is the first to demonstrate the microbial community structure involved in the AD of Sargassum spp. The pretreatments presented in this study can help overcome the limitations associated with methane yield.

Methane Production of Sargassum spp. Biomass from the Mexican Caribbean: Solid-Liquid Separation and Component Distribution.

In the last decade, Sargassum spp. seaweed species have caused massive flooding on the Caribbean Sea coasts. These seaweed species have a high content of recalcitrant compounds, such as insoluble fibers and polyphenols, which generate low methane yields in anaerobic digestion (AD). This study investigated the effect of solid-liquid separation of Sargassum biomass on biodegradability and methane yield. A biochemical methane potential (BMP) test was conducted with both fractions and raw biomass (RB). A mass balance was developed to assess the distribution of the components. The obtained liquid fraction (LF) showed high biodegradability and a high methane production rate, and it generated a methane yield of 159.7 ± 7.1 N L kg VS-1, a value that corresponds to approximately twice that achieved with RB and the solid fraction (SF). The component distribution analysis showed that about 90% of total solids (TS), volatile solids (VS), ash, carbon, and cellulose were retained in the SF. In conclusion, the LF had high biodegradability and methane yield. This suggests the potential for LFs of Sargassum biomass to be treated in large-scale high-load reactors; however, studies applied to SFs are needed because they retain a large amount of organic matter with low biodegradability.

Distinct extremely abundant siRNAs associated with cosuppression in petunia.

Cosuppression is a classical form of eukaryotic post-transcriptional gene silencing. It was first reported in transgenic petunia, where a sense transgene meant to overexpress the host Chalcone Synthase-A (CHS-A) gene caused the degradation of the homologous transcripts and the loss of flower pigmentation. In this work, we used deep sequencing technology to characterize in detail the small RNA population generated from the CHS-A sequence in cosuppressed transgenic petunia. Unexpectedly, two distinct small interfering RNAs (siRNAs) were found to vastly predominate. Our demonstration that they guide prominent cleavage events in CHS-A mRNA provides compelling and previously lacking evidence of a causative association between induction of individual siRNAs and an example of cosuppression. The preferential accumulation of these siRNAs provides new insights about sense cosuppression that may apply to other natural and engineered RNA silencing events.

Novel tephritid-specific features revealed from cytological and transcriptomic analysis of Anastrepha ludens embryonic development.

Anastrepha ludens is a major pest of fruits including citrus and mangoes in Mexico and Central America with major economic and social impacts. Despite its importance, our knowledge on its embryonic development is scarce. Here, we report the first cytological study of embryonic development in A. ludens and provide a transcriptional landscape during key embryonic stages. We established 17 stages of A. ludens embryogenesis that closely resemble the morphological events observed in Drosophila. In addition to the extended duration of embryonic development, we observed notable differences including yolk extrusion at both poles of the embryo, distinct nuclear division waves in the syncytial blastoderm and a heterochronic change during the involution of the head. Characterization of the transcriptional dynamics during syncytial blastoderm, cellular blastoderm and gastrulation, showed that approximately 9000 different transcripts are present at each stage. Even though we identified most of the transcripts with a role during embryonic development present in Drosophila, including sex determination genes, a number of transcripts were absent not only in A. ludens but in other tephritids such as Ceratitis capitata and Bactrocera dorsalis. Intriguingly, some A. ludens embryo transcripts encode proteins present in other organisms but not in other flies. Furthermore, we developed an RNA in situ hybridization protocol that allowed us to obtain the expression patterns of genes whose functions are important in establishing the embryonic body pattern. Our results revealed novel tephritid-specific features during A. ludens embryonic development and open new avenues for strategies aiming to control this important pest.

Marchantia liverworts as a proxy to plants' basal microbiomes.

Microbiomes influence plant establishment, development, nutrient acquisition, pathogen defense, and health. Plant microbiomes are shaped by interactions between the microbes and a selection process of host plants that distinguishes between pathogens, commensals, symbionts and transient bacteria. In this work, we explore the microbiomes through massive sequencing of the 16S rRNA genes of microbiomes two Marchantia species of liverworts. We compared microbiomes from M. polymorpha and M. paleacea plants collected in the wild relative to their soils substrates and from plants grown in vitro that were established from gemmae obtained from the same populations of wild plants. Our experimental setup allowed identification of microbes found in both native and in vitro Marchantia species. The main OTUs (97% identity) in Marchantia microbiomes were assigned to the following genera: Methylobacterium, Rhizobium, Paenibacillus, Lysobacter, Pirellula, Steroidobacter, and Bryobacter. The assigned genera correspond to bacteria capable of plant-growth promotion, complex exudate degradation, nitrogen fixation, methylotrophs, and disease-suppressive bacteria, all hosted in the relatively simple anatomy of the plant. Based on their long evolutionary history Marchantia is a promising model to study not only long-term relationships between plants and their microbes but also the transgenerational contribution of microbiomes to plant development and their response to environmental changes.

Conserved Peptide Upstream Open Reading Frames are Associated with Regulatory Genes in Angiosperms.

Upstream open reading frames (uORFs) are common in eukaryotic transcripts, but those that encode conserved peptides occur in less than 1% of transcripts. The peptides encoded by three plant conserved peptide uORF (CPuORF) families are known to control translation of the downstream ORF in response to a small signal molecule (sucrose, polyamines, and phosphocholine). In flowering plants, transcription factors are statistically over-represented among genes that possess CPuORFs, and in general it appeared that many CPuORF genes also had other regulatory functions, though the significance of this suggestion was uncertain (Hayden and Jorgensen, 2007). Five years later the literature provides much more information on the functions of many CPuORF genes. Here we reassess the functions of 27 known CPuORF gene families and find that 22 of these families play a variety of different regulatory roles, from transcriptional control to protein turnover, and from small signal molecules to signal transduction kinases. Clearly then, there is indeed a strong association of CPuORFs with regulatory genes. In addition, 16 of these families play key roles in a variety of different biological processes. Most strikingly, the core sucrose response network includes three different CPuORFs, creating the potential for sophisticated balancing of the network in response to three different molecular inputs. We propose that the function of most CPuORFs is to modulate translation of a downstream major ORF (mORF) in response to a signal molecule recognized by the conserved peptide and that because the mORFs of CPuORF genes generally encode regulatory proteins, many of them centrally important in the biology of plants, CPuORFs play key roles in balancing such regulatory networks.