Marchantia polymorpha GOLDEN2-LIKE transcriptional factor; a central regulator of chloroplast and plant vegetative development.

The GOLDEN2-LIKE (GLK) transcription factors act as a central regulatory node involved in both developmental processes and environmental responses. Marchantia polymorpha, a basal terrestrial plant with strategic evolutionary position, contains a single GLK representative that possesses an additional domain compared to spermatophytes. We analyzed the role of MpGLK in chloroplast biogenesis and development by altering its levels, preforming transcriptomic profiling and conducting chromatin immunoprecipitation. Decreased MpGLK levels impair chloroplast differentiation and disrupt the expression of photosynthesis-associated nuclear genes, while overexpressing MpGLK leads to ectopic chloroplast biogenesis. This demonstrates the MpGLK functions as a bona fide GLK protein, likely representing an ancestral GLK architecture. Altering MpGLK levels directly regulates the expression of genes involved in Chl synthesis and degradation, similar to processes observed in eudicots, and causes various developmental defects in Marchantia, including the formation of dorsal structures such as air pores and gemma cups. MpGLK, also directly activates MpMAX2 gene expression, regulating the timing of gemma cup development. Our study shows that MpGLK functions as a master regulator, potentially coupling chloroplast development with vegetative reproduction. This illustrates the complex regulatory networks governing chloroplast function and plant development communication and highlight the evolutionary conservation of GLK-mediated regulatory processes across plant species.

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.

RETINOBLASTOMA-RELATED interactions with key factors of the RNA-directed DNA methylation (RdDM) pathway and its influence on root development.

MAIN CONCLUSION: Our study presents evidence for a novel mechanism for RBR function in transcriptional gene silencing by interacting with key players of the RdDM pathway in Arabidopsis and several plant clades. Transposable elements and other repetitive elements are silenced by the RNA-directed DNA methylation pathway (RdDM). In RdDM, POLIV-derived transcripts are converted into double-stranded RNA (dsRNA) by the activity of RDR2 and subsequently processed into 24 nucleotide short interfering RNAs (24-nt siRNAs) by DCL3. 24-nt siRNAs serve as guides to direct AGO4-siRNA complexes to chromatin-bound POLV-derived transcripts generated from the template/target DNA. The interaction between POLV, AGO4, DMS3, DRD1, RDM1 and DRM2 promotes DRM2-mediated de novo DNA methylation. The Arabidopsis Retinoblastoma protein homolog (RBR) is a master regulator of the cell cycle, stem cell maintenance, and development. We in silico predicted and explored experimentally the protein-protein interactions (PPIs) between RBR and members of the RdDM pathway. We found that the largest subunits of POLIV and POLV (NRPD1 and NRPE1), the shared second largest subunit of POLIV and POLV (NRPD/E2), RDR1, RDR2, DCL3, DRM2, and SUVR2 contain canonical and non-canonical RBR binding motifs and several of them are conserved since algae and bryophytes. We validated experimentally PPIs between Arabidopsis RBR and several of the RdDM pathway proteins. Moreover, seedlings from loss-of-function mutants in RdDM and RBR show similar phenotypes in the root apical meristem. We show that RdDM and SUVR2 targets are up-regulated in the 35S:AmiGO-RBR background.

Stem transcriptome screen for selection in wild and cultivated pitahaya (Selenicereus undatus): an epiphytic cactus with edible fruit.

Dragon fruit, pitahaya or pitaya are common names for the species in the Hylocereus group of Selenicereus that produce edible fruit. These Neotropical epiphytic cacti are considered promising underutilized crops and are currently cultivated around the world. The most important species, S. undatus, has been managed in the Maya domain for centuries and is the focus of this article. Transcriptome profiles from stems of wild and cultivated plants of this species were compared. We hypothesized that differences in transcriptomic signatures could be associated with genes related to drought stress. De novo transcriptome assembly and the analysis of differentially expressed genes (DEGs) allowed us to identify a total of 9,203 DEGs in the Hunucmá cultivar relative of wild Mozomboa plants. Of these, 4,883 represent up-regulated genes and 4,320, down-regulated genes. Additionally, 6,568 DEGs were identified from a comparison between the Umán cultivar and wild plants, revealing 3,286 up-regulated and 3,282 down-regulated genes. Approximately half of the DEGs are shared by the two cultivated plants. Differences between the two cultivars that were collected in the same region could be the result of differences in management. Metabolism was the most representative functional category in both cultivars. The up-regulated genes of both cultivars formed a network related to the hormone-mediated signaling pathway that includes cellular responses to auxin stimulus and to hormone stimulus. These cellular reactions have been documented in several cultivated plants in which drought-tolerant cultivars modify auxin transport and ethylene signaling, resulting in a better redistribution of assimilates.

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.

AGO104 is a RdDM effector of paramutation at the maize b1 locus.

Although paramutation has been well-studied at a few hallmark loci involved in anthocyanin biosynthesis in maize, the cellular and molecular mechanisms underlying the phenomenon remain largely unknown. Previously described actors of paramutation encode components of the RNA-directed DNA-methylation (RdDM) pathway that participate in the biogenesis of 24-nucleotide small interfering RNAs (24-nt siRNAs) and long non-coding RNAs. In this study, we uncover an ARGONAUTE (AGO) protein as an effector of the RdDM pathway that is in charge of guiding 24-nt siRNAs to their DNA target to create de novo DNA methylation. We combined immunoprecipitation, small RNA sequencing and reverse genetics to, first, validate AGO104 as a member of the RdDM effector complex and, then, investigate its role in paramutation. We found that AGO104 binds 24-nt siRNAs involved in RdDM, including those required for paramutation at the b1 locus. We also show that the ago104-5 mutation causes a partial reversion of the paramutation phenotype at the b1 locus, revealed by intermediate pigmentation levels in stem tissues. Therefore, our results place AGO104 as a new member of the RdDM effector complex that plays a role in paramutation at the b1 locus in maize.

An Automated High-Throughput Phenotyping System for Marchantia polymorpha.

High-throughput phenotyping (HTP) allows automation of fast and precise acquisition and analysis of digital images for the detection of key traits in real time. HTP improves characterization of the growth and development of plants in controlled environments in a nondestructive fashion. Marchantia polymorpha has emerged as a very attractive model for studying the evolution of the physiological, cellular, molecular, and developmental adaptations that enabled plants to conquer their terrestrial environments. The availability of the M. polymorpha genome in combination with a full set of functional genomic tools including genetic transformation, homologous recombination, and genome editing has allowed the inspection of its genome through forward and reverse genetics approaches. The increasing number of mutants has made it possible to perform informative genome-wide analyses to study the phenotypic consequences of gene inactivation. Here we present an HTP protocol for M. polymorpha that will aid current efforts to quantify numerous morphological parameters that can potentially reveal genotype-to-phenotype relationships and relevant connections between individual traits.

The small RNA-mediated gene silencing machinery is required in Arabidopsis for stimulation of growth, systemic disease resistance, and suppression of the nitrile-specifier gene NSP4 by Trichoderma atroviride.

Trichoderma atroviride is a root-colonizing fungus that confers multiple benefits to plants. In plants, small RNA (sRNA)-mediated gene silencing (sRNA-MGS) plays pivotal roles in growth, development, and pathogen attack. Here, we explored the role of core components of Arabidopsis thaliana sRNA-MGS pathways during its interaction with Trichoderma. Upon interaction with Trichoderma, sRNA-MGS-related genes paralleled the expression of Arabidopsis defense-related genes, linked to salicylic acid (SA) and jasmonic acid (JA) pathways. SA- and JA-related genes were primed by Trichoderma in leaves after the application of the well-known pathogen-associated molecular patterns flg22 and chitin, respectively. Defense-related genes were primed in roots as well, but to different extents and behaviors. Phenotypical characterization of mutants in AGO genes and components of the RNA-dependent DNA methylation (RdDM) pathway revealed that different sets of sRNA-MGS-related genes are essential for (i) the induction of systemic acquired resistance against Botrytis cinerea, (ii) the activation of the expression of plant defense-related genes, and (iii) root colonization by Trichoderma. Additionally, plant growth induced by Trichoderma depends on functional RdDM. Profiling of DNA methylation and histone N-tail modification patterns at the Arabidopsis Nitrile-Specifier Protein-4 (NSP4) locus, which is responsive to Trichoderma, showed altered epigenetic modifications in RdDM mutants. Furthermore, NSP4 is required for the induction of systemic acquired resistance against Botrytis and avoidance of enhanced root colonization by Trichoderma. Together, our results indicate that RdDM is essential in Arabidopsis to establish a beneficial relationship with Trichoderma. We propose that DNA methylation and histone modifications are required for plant priming by the beneficial fungus against B. cinerea.

Physiological stabilization, community characterization, and nitrogen degradation dynamics in an anammox consortium from estuarine sediments.

Anammox is a cost-effective and sustainable process for nitrogen removal; however, the production of a physiologically stable inoculum is a critical point in the start-up process. In this work, estuarine sediments were used as incubation seeds to obtain cultures with stable anammox activity. Assays were performed in batch cultures fed with stoichiometric amounts of ammonium and nitrite, analyzing physiological response variables and the microbial community. Estuarine sediments showed a stable anammox process after 90 days, consuming ammonium and nitrite simultaneously with concomitant generation of N2 and nitrate in stoichiometric amounts. In kinetic assays, substrates were fully consumed after 210 hr, exhibiting N2 and nitrate yields of 0.85 and 0.10, respectively. The microbial community analysis using PCR-DGGE indicated the presence of uncultured anammox bacteria and members of the genus Candidatus Jettenia. The results evidenced the achievement of anammox cultures, although their start-up and kinetic characteristics were less favorable than those recorded in man-made systems. PRACTITIONER POINTS: Estuarine sediments were used as incubation seeds to obtain cultures with stable anammox activity. The sediments were fed with stoichiometric amounts of ammonium and nitrite, analyzing the physiological response variables and the microbial community. Sediments showed a stable anammox process after 90 days, converting the substrates into N2 and nitrate according to stoichiometry. Anammox cultures were achieved although their start-up and kinetic characteristics were less favorable than those recorded in man-made systems. Microbial community analysis using PCR-DGGE indicated the presence of uncultured anaerobic ammonia-oxidizing bacterium and members of genus Candidatus Jettenia.

Transcriptional and Morpho-Physiological Responses of Marchantia polymorpha upon Phosphate Starvation.

Phosphate (Pi) is a pivotal nutrient that constraints plant development and productivity in natural ecosystems. Land colonization by plants, more than 470 million years ago, evolved adaptive mechanisms to conquer Pi-scarce environments. However, little is known about the molecular basis underlying such adaptations at early branches of plant phylogeny. To shed light on how early divergent plants respond to Pi limitation, we analyzed the morpho-physiological and transcriptional dynamics of Marchantia polymorpha upon Pi starvation. Our phylogenomic analysis highlights some gene networks present since the Chlorophytes and others established in the Streptophytes (e.g., PHR1-SPX1 and STOP1-ALMT1, respectively). At the morpho-physiological level, the response is characterized by the induction of phosphatase activity, media acidification, accumulation of auronidins, reduction of internal Pi concentration, and developmental modifications of rhizoids. The transcriptional response involves the induction of MpPHR1, Pi transporters, lipid turnover enzymes, and MpMYB14, which is an essential transcription factor for auronidins biosynthesis. MpSTOP2 up-regulation correlates with expression changes in genes related to organic acid biosynthesis and transport, suggesting a preference for citrate exudation. An analysis of MpPHR1 binding sequences (P1BS) shows an enrichment of this cis regulatory element in differentially expressed genes. Our study unravels the strategies, at diverse levels of organization, exerted by M. polymorpha to cope with low Pi availability.

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.

Mechanisms underlying the enhanced biomass and abiotic stress tolerance phenotype of an Arabidopsis MIOX over-expresser.

Myo-inositol oxygenase (MIOX) is the first enzyme in the inositol route to ascorbate (L-ascorbic acid, AsA, vitamin C). We have previously shown that Arabidopsis plants constitutively expressing MIOX have elevated foliar AsA content and displayed enhanced growth rate, biomass accumulation, and increased tolerance to multiple abiotic stresses. In this work, we used a combination of transcriptomics, chromatography, microscopy, and physiological measurements to gain a deeper understanding of the underlying mechanisms mediating the phenotype of the AtMIOX4 line. Transcriptomic analysis revealed increased expression of genes involved in auxin synthesis, hydrolysis, transport, and metabolism, which are supported by elevated auxin levels both in vitro and in vivo, and confirmed by assays demonstrating their effect on epidermal cell elongation in the AtMIOX4 over-expressers. Additionally, we detected up-regulation of transcripts involved in photosynthesis and this was validated by increased efficiency of the photosystem II and proton motive force. We also found increased expression of amylase leading to higher intracellular glucose levels. Multiple gene families conferring plants tolerance/expressed in response to cold, water limitation, and heat stresses were found to be elevated in the AtMIOX4 line. Interestingly, the high AsA plants also displayed up-regulation of transcripts and hormones involved in defense including jasmonates, defensin, glucosinolates, and transcription factors that are known to be important for biotic stress tolerance. These results overall indicate that elevated levels of auxin and glucose, and enhanced photosynthetic efficiency in combination with up-regulation of abiotic stresses response genes underly the higher growth rate and abiotic stresses tolerance phenotype of the AtMIOX4 over-expressers.

DNA methylation in Marchantia polymorpha.

Methylation of DNA is an epigenetic mechanism for the control of gene expression. Alterations in the regulatory pathways involved in the establishment, perpetuation and removal of DNA methylation can lead to severe developmental alterations. Our understanding of the mechanistic aspects and relevance of DNA methylation comes from remarkable studies in well-established angiosperm plant models including maize and Arabidopsis. The study of plant models positioned at basal lineages opens exciting opportunities to expand our knowledge on the function and evolution of the components of DNA methylation. In this Tansley Insight, we summarize current progress in our understanding of the molecular basis and relevance of DNA methylation in the liverwort Marchantia polymorpha.

Loss of CG Methylation in Marchantia polymorpha Causes Disorganization of Cell Division and Reveals Unique DNA Methylation Regulatory Mechanisms of Non-CG Methylation.

DNA methylation is an epigenetic mark that ensures silencing of transposable elements (TEs) and affects gene expression in many organisms. The function of different DNA methylation regulatory pathways has been largely characterized in the model plant Arabidopsis thaliana. However, far less is known about DNA methylation regulation and functions in basal land plants. Here we focus on the liverwort Marchantia polymorpha, an emerging model species that represents a basal lineage of land plants. We identified MpMET, the M. polymorpha ortholog of the METHYLTRANSFERASE 1 (MET1) gene required for maintenance of methylation at CG sites in angiosperms. We generated Mpmet mutants using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein9) system, which showed a significant loss of CG methylation and severe morphological changes and developmental defects. The mutants developed many adventitious shoot-like structures, suggesting that MpMET is required for maintaining differentiated cellular identities in the gametophyte. Even though numerous TEs were up-regulated, non-CG methylation was generally highly increased at TEs in the Mpmet mutants. Closer inspection of CHG methylation revealed features unique to M. polymorpha. Methylation of CCG sites in M. polymorpha does not depend on MET1, unlike in A. thaliana and Physcomitrella patens. Our results highlight the diversity of non-CG methylation regulatory mechanisms in plants.

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.

Negative regulation of conserved RSL class I bHLH transcription factors evolved independently among land plants.

Basic helix-loop-helix transcription factors encoded by RSL class I genes control a gene regulatory network that positively regulates the development of filamentous rooting cells - root hairs and rhizoids - in land plants. The GLABRA2 transcription factor negatively regulates these genes in the angiosperm Arabidopsis thaliana. To find negative regulators of RSL class I genes in early diverging land plants we conducted a mutant screen in the liverwort Marchantia polymorpha. This identified FEW RHIZOIDS1 (MpFRH1) microRNA (miRNA) that negatively regulates the RSL class I gene MpRSL1. The miRNA and its mRNA target constitute a feedback mechanism that controls epidermal cell differentiation. MpFRH1 miRNA target sites are conserved among liverwort RSL class I mRNAs but are not present in RSL class I mRNAs of other land plants. These findings indicate that while RSL class I genes are ancient and conserved, independent negative regulatory mechanisms evolved in different lineages during land plant evolution.

Transcriptional landscapes of Axolotl (Ambystoma mexicanum).

The axolotl (Ambystoma mexicanum) is the vertebrate model system with the highest regeneration capacity. Experimental tools established over the past 100 years have been fundamental to start unraveling the cellular and molecular basis of tissue and limb regeneration. In the absence of a reference genome for the Axolotl, transcriptomic analysis become fundamental to understand the genetic basis of regeneration. Here we present one of the most diverse transcriptomic data sets for Axolotl by profiling coding and non-coding RNAs from diverse tissues. We reconstructed a population of 115,906 putative protein coding mRNAs as full ORFs (including isoforms). We also identified 352 conserved miRNAs and 297 novel putative mature miRNAs. Systematic enrichment analysis of gene expression allowed us to identify tissue-specific protein-coding transcripts. We also found putative novel and conserved microRNAs which potentially target mRNAs which are reported as important disease candidates in heart and liver.

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 Role of microRNAs in Animal Cell Reprogramming.

Our concept of cell reprogramming and cell plasticity has evolved since John Gurdon transferred the nucleus of a completely differentiated cell into an enucleated Xenopus laevis egg, thereby generating embryos that developed into tadpoles. More recently, induced expression of transcription factors, oct4, sox2, klf4, and c-myc has evidenced the plasticity of the genome to change the expression program and cell phenotype by driving differentiated cells to the pluripotent state. Beyond these milestone achievements, research in artificial cell reprogramming has been focused on other molecules that are different than transcription factors. Among the candidate molecules, microRNAs (miRNAs) stand out due to their potential to control the levels of proteins that are involved in cellular processes such as self-renewal, proliferation, and differentiation. Here, we review the role of miRNAs in the maintenance and differentiation of mesenchymal stem cells, epimorphic regeneration, and somatic cell reprogramming to induced pluripotent stem cells.