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Mating has profound physiological and behavioral consequences for female insects. During copulation, female insects typically receive not only sperm, but a complex ejaculate containing hundreds of proteins and other molecules from male reproductive tissues, primarily the reproductive accessory glands. The post-mating phenotypes affected by male accessory gland (MAG) proteins include egg development, attraction to oviposition hosts, mating, attractiveness, sperm storage, feeding, and lifespan. In the Mexican fruit fly, Anastrepha ludens, mating increases egg production and the latency to remating. However, previous studies have not found a clear relationship between injection of MAG products and oviposition or remating inhibition in this species. We used RNA-seq to study gene expression in mated, unmated, and MAG-injected females to understand the potential mating- and MAG-regulated genes and pathways in A. ludens. Both mating and MAG-injection regulated transcripts and pathways related to egg development. Other transcripts regulated by mating included those with orthologs predicted to be involved in immune response, musculature, and chemosensory perception, whereas those regulated by MAG-injection were predicted to be involved in translational control, sugar regulation, diet detoxification, and lifespan determination. These results suggest new phenotypes that may be influenced by seminal fluid molecules in A. ludens. Understanding these influences is critical for developing novel tools to manage A. ludens. This article is protected by copyright. All rights reserved.

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.

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.

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.

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.

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.

Paramutation is an exception among eukaryotes, in which epigenetic information is conserved through mitosis and meiosis. It has been studied for over 70 years in maize, but the mechanisms involved are largely unknown. Previously described actors of paramutation encode components of the RNA-dependent DNA-methylation (RdDM) pathway all involved in the biogenesis of 24-nt small RNAs. However, no actor of paramutation have been identified in the effector complex of RdDM. Here, through a combination of reverse genetics, immunolocalization and immunoprecipitation (siRNA-IP) we found that ARGONAUTE104 (AGO104), AGO105 and AGO119 are members of the RdDM effector complex in maize and bind siRNAs produced from the tandem repeats required for paramutation at theb1locus. We also showed that AGO104 is an effector of theb1paramutation in maize.Author summaryReprogramming of epigenetic information has been described in both plants and mammals. Here, we show that maizeARGONAUTE (AGO) AGO104andAGO105/AGO119, respectively the close homologs ofA. thaliana AGO9andAGO4, are required to enable paramutation at theb1locus in maize. Paramutation is an epigenetic phenomenon that is stable over many generations (both mitotically and meiotically). A classic example is thebooster1(b1) gene in maize, where the weakly expressedBooster’(B’) allele stably decreases the expression of theBooster-Intense(B-I) allele, and changes it into a newB’allele. This newB’allele will in turn changeB-Iinto newB’in subsequent crosses. Previous research demonstrated that paramutation requires several proteins involved in the biosynthesis of small interfering RNAs (siRNAs) all related to the RNA-dependent DNA-methylation (RdDM) pathway. Yet, few members of the RdDM were functionally identified in maize. Here, we identify two new members of the maize RdDM pathway, and provide evidence that they are also involved in paramutation at theb1locus.

With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them., National Science Foundation 1916797 David W Ehrhardt, Kenneth D Birnbaum, Seung Yon Rhee; National Science Foundation 2052590 Seung Yon Rhee

Plant hormones are master regulators of developmental and genetic mechanisms to deal with diverse environmental cues. Upon phosphate (Pi) limitation, vascular plants modify phytohormone metabolism to coordinate diverse mechanisms to overcome such stress. However, the transcriptional program underlying the hormonal signaling in response to Pi scarcity in early branches of land plant phylogeny remains unclear. Therefore, we explored the transcriptional dynamics of key genes involved in auxin, cytokinin, ethylene, jasmonate, gibberellin, and abscisic acid metabolism in the early divergent land plant Marchantia polymorpha upon Pi starvation. Our RNAseq approach revealed major changes in genes associated with auxin and ethylene biosynthesis. Genes involved in cytokinin synthesis are repressed. Interestingly, genes involved in auxin and ethylene signaling, such as MpARF1 and MpARF2, are upregulated. In contrast, MpARRb is down-regulated. Moreover, genes involved in the synthesis of jasmonates were highly upregulated, but those related to signaling did not change in expression. Our data suggest that auxin and ethylene act as positive regulators of rhizoid development under Pi-limited conditions, whereas cytokinin may act as a negative regulator. The transcriptional behavior of some hormone-related genes in Marchantia is similar to those described in controlling root hair development in arabidopsis, maize, and rice, upon Pi scarcity.

Prickly pear (cactus fruit) is an important crop in Mexico, it helps to the establishment of Mexican antique civilization. As a ripening model, it has a unique character because include morphospecies with contrasting characteristics, making easier their interpretation. The main commercialization problems for prickly pear include the presence of seeds and spines (gloquids), it would be interesting to idenfied the genetic factors involve in their synthesis of these structures, through genetic expression in order to design some strategy for their control. In this study we have been used different molecular and bioinformatics strategies for the identification of the factors related to prickly pear development. In proteomics assays using three morphospecies with contrasting development and fruit ripening, it was possible to identified the minimal number of required peptides for fruit development and ripening delay, also were identified peptides associated to regulatory functions in transcendental metabolic functions. In the miRNA transcriptome, were identified 255 molecules associated to the fruit development and other important process, such as adaptation to extremal growth conditions, were cactus plants growth. Seven families of miRNAs were associated to fruit development and plant adaptation, and the pathway of miRNA participation was determined. The expression assays shown the effects of miRNAs on fruit development in prickly pear.

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.

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.

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.

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. Transcritpomic 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-expresser plants. Additionally, we detected up-regulation of transcripts involved in photosynthesis that 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 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.

Bryophytes (liverworts, hornworts and mosses) comprise the three earliest diverging lineages of land plants (embryophytes). Marchantia polymorpha, a complex thalloid Marchantiopsida liverwort that has been developed into a model genetic system, occupies a key phylogenetic position. Therefore, M. polymorpha is useful in studies aiming to elucidate the evolution of gene regulation mechanisms in plants. In this study, we used computational, transcriptomic, small RNA and degradome analyses to characterize microRNA (miRNA)-mediated pathways of gene regulation in M. polymorpha. The data have been integrated into the open access ContigViews-miRNA platform for further reference. In addition to core components of the miRNA pathway, 129 unique miRNA sequences, 11 of which could be classified into seven miRNA families that are conserved in embryophytes (miR166a, miR390, miR529c, miR171-3p, miR408a, miR160 and miR319a), were identified. A combination of computational and degradome analyses allowed us to identify and experimentally validate 249 targets. In some cases, the target genes are orthologous to those of other embryophytes, but in other cases, the conserved miRNAs target either paralogs or members of different gene families. In addition, the newly discovered Mpo-miR11707.1 and Mpo-miR11707.2 are generated from a common precursor and target MpARGONAUTE1 (LW1759). Two other newly discovered miRNAs, Mpo-miR11687.1 and Mpo-miR11681.1, target the MADS-box transcription factors MpMADS1 and MpMADS2, respectively. Interestingly, one of the pentatricopeptide repeat (PPR) gene family members, MpPPR_66 (LW9825), the protein products of which are generally involved in various steps of RNA metabolism, has a long stem-loop transcript that can generate Mpo-miR11692.1 to autoregulate MpPPR_66 (LW9825) mRNA. This study provides a foundation for further investigations of the RNA-mediated silencing mechanism in M. polymorpha as well as of the evolution of this gene silencing pathway in embryophytes.

While Marchantia polymorpha has been utilized as a model system to investigate fundamental biological questions for over almost two centuries, there is renewed interest in M. polymorpha as a model genetic organism in the genomics era. Here we outline community guidelines for M. polymorpha gene and transgene nomenclature, and we anticipate that these guidelines will promote consistency and reduce both redundancy and confusion in the scientific literature.

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&ndash, SPX1 and STOP1&ndash, 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.

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 plantArabidopsis thaliana. However, far less is known about DNA methylation regulation and functions in basal land plants. Here we focus on the liverwortMarchantia polymorpha, an emerging model species that represents a basal lineage of land plants. We identified MpMET, theM. polymorphaorthologue of theMETHYLTRANSFERASE 1(MET1) gene required for maintenance of methylation at CG sites in angiosperms. We generated Mpmetmutants using the CRISPR/Cas9 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 MpMETis required for maintaining differentiated cellular identities in the gametophyte. Numerous TEs were up-regulated, even though non-CG methylation was highly increased at TEs in the Mpmetmutants. Closer inspection of CHG methylation revealed features unique toM. polymorpha. Methylation of CCG sites inM. polymorphadoes not depend onMET1, unlike inA. thalianaandPhyscomitrella patens. Furthermore, unlikeA. thaliana,M. polymorphashows higher methylation level at CAG sites than at other CHG contexts and CAG/CTG sites are mostly methylated asymmetrically. Interestingly, CAG and CTG methylation reached comparable levels and symmetry upon loss of CG methylation. Our results highlight the diversity of non-CG methylation regulatory mechanisms in plants.

Plants' ability to respond to environmental stimuli and developmental cues depends upon changes in gene expression. In eukaryotes, genetic information encoded by DNA is packed in a highly regulated and dynamic nucleoprotein complex known as chromatin that is subject to epigenetic modifications. Historically, several biological phenomena relying on epigenetic mechanisms were first characterized in plants. Seminal discoveries such as paramutation and silencing of transposable elements were made in maize (Zea mays). Later rice (Oryza sativa) was selected as a good model for monocotyledons owing to its relatively small genome size and well-annotated sequenced genome. In the past few years an increasing number of epigenetic and epigenomic studies were performed in both maize and rice. In this chapter we will first compare the basic knowledge acquired on epigenetic regulation in rice and maize versus Arabidopsis, then we will describe cereals-specific aspects of epigenetic regulations.

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.

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., 陸上植物の祖先の特徴をもつ苔類ゼニゴケの全ゲノム構造を解明. 京都大学プレスリリース. 2017-10-06.

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.

In plants, l-ascorbic acid (AsA) is a functional enzyme cofactor, a major antioxidant, and a modulator of several biological processes including photosynthesis, photo-protection, cell wall growth and expansion, tolerance to environmental stresses, and synthesis of other molecules. One of the major roles of AsA in plants is detoxifying reactive oxygen species (ROS) such as singlet oxygen or peroxide radicals. ROS are produced when plants undergo biotic or abiotic stresses and if accumulated in high concentrations, can cause damage to macromolecules such as nucleic acids, membrane lipids, and proteins. Until now, little study has been done on ascorbate metabolism in liverworts. Bryophytes (liverworts, hornworts, and mosses) comprise the earliest diverging land plant lineages that came about approximately 360–450 million years ago between the Ordovician and Devonian periods. The ancient liverwort Marchantia polymorpha is an emergent model system specifically suited to use in the study of the evolution of different biosynthetic pathways. In this chapter, basal levels of both reduced and oxidized AsA in M. polymorpha are reported. Comparative and functional genomics experiments in combination with precursor feeding experiment are also discussed in order to provide valuable insights on the evolution of the AsA biosynthetic pathways.

It has been argued that the evolution of plant genome size is principally unidirectional and increasing owing to the varied action of wholegenome duplications (WGDs) and mobile element proliferation 1 . However, extreme genome size reductions have been reported in the angiosperm family tree. Here we report the sequence of the 82megabase genome of the carnivorous bladderwort plant Utricularia gibba. Despite its tiny size, the U. gibba genome accommodates a typical number of genes for a plant, with the main difference from other plant genomes arising from a drastic reduction in non-genic DNA. Unexpectedly, we identified at least three rounds of WGD in U. gibba since common ancestry with tomato (Solanum) and grape (Vitis). The compressed architecture of the U. gibba genome indicates that a small fraction of intergenic DNA, with few or no active retrotransposons, is sufficient to regulate and integrate all the processes required for the development and reproduction of a complex organism. Like other carnivorous plants, Utricularia (Lentibulariaceae) species derive nitrogen and phosphorus supplements by trapping and digesting prey organisms 2,3 . Lentibulariaceae are asterid angiosperms closely related to the model plants snapdragon (Antirrhinum) and monkey

MicroRNAs (miRNAs) are endogenous non-coding RNAs that play important regulatory roles in animals and plants by targeting mRNAs for cleavage or transla‐ tional repression. Small RNAs are classified into different types by their biogenesis and mode of action, such as miRNAs, siRNAs, piRNAs, and snoRNAs. In the case of miRNAs, this specific type regulates gene expression in plants and animals by targeting mRNAs for cleavage and translational repression, respectively. Diverse miRNAs regulate plant development, metabolism, and responses to biotic and abiotic stresses. The identification of miRNAs has been accomplished in diverse species, organs and developmental or diverse biotic and abiotic stress conditions. Novel massive sequencing techniques and further bioinformatics analysis have allowed the identification of hundreds of miRNAs in Arabidopsis thaliana, Oryza sativa, Malus domestica, Zea mays, Solanum lycopersicum, and other plants. Functional characteriza‐ tion of a given miRNA in a specific biological context has shown their role in the finetuning mechanisms of posttranscriptional gene regulation. In this chapter, besides making a summary of genome-wide miRNA profiling in plants, we describe how gain and loss of function approaches influence plant phenotypes that affect development, physiology or stress responses, pointing to miRNAs as effective tools for the generation of new plant phenotypes that improve plant productivity and conserva‐ tion.

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.

Summary The colonization of the land by plants, sometime before 470 million years ago, was accompanied by the evolution tissue systems [1, 2, 3]. Specialized structures with diverse functions—from nutrient acquisition to reproduction—derived from single cells in the outermost layer (epidermis) were important sources of morphological innovation at this time [2, 4, 5]. In extant plants, these structures may be unicellular extensions, such as root hairs or rhizoids [6, 7, 8, 9], or multicellular structures, such as asexual propagules or secretory hairs (papillae) [10, 11, 12]. Here, we show that a ROOTHAIR DEFECTIVE SIX-LIKE (RSL) class I basic helix-loop-helix transcription factor positively regulates the development of the unicellular and multicellular structures that develop from individual cells that expand out of the epidermal plane of the liverwort Marchantia polymorpha; mutants that lack MpRSL1 function do not develop rhizoids, slime papillae, mucilage papillae, or gemmae. Furthermore, we discovered that RSL class I genes are also required for the development of multicellular axillary hairs on the gametophyte of the moss Physcomitrella patens. Because class I RSL proteins also control the development of rhizoids in mosses and root hairs in angiosperms [13, 14], these data demonstrate that the function of RSL class I genes was to control the development of structures derived from single epidermal cells in the common ancestor of the land plants. Class I RSL genes therefore controlled the generation of adaptive morphological diversity as plants colonized the land from the water., Highlights • Class I RSL genes control the development of structures derived from single cells • Class I RSL function is conserved among land plants • Class I RSL controlled epidermal development in the land plant common ancestor • These genes controlled the development of the first plant rooting systems, The colonization of the land by plants was accompanied by the evolution of specialized cells and structures that develop from single cells. Proust et al. show that RSL class I basic helix-loop-helix transcription factors controlled the development of specialized structures from single epidermal cells in the last common ancestor of the land plants.

As sessile organisms, plants have to cope with the ever-changing environment as well as with numerous forms of stress. To react to these external cues, plants have evolved a suite of response mechanisms operating at many different levels, ranging from physiological to molecular processes that provide the organism with a wide phenotypic plasticity, allowing for fine tuning of the reactions to these adverse circumstances. During the past decade, non-coding RNAs (ncRNAs) have emerged as key regulatory molecules, which contribute to a significant portion of the transcriptome in eukaryotes and are involved in the control of transcriptional and post-transcriptional gene regulatory pathways. Although accumulated evidence supports an important role for ncRNAs in plant response and adaptation to abiotic stress, their mechanism(s) of action still remains obscure and a functional characterization of the ncRNA repertoire in plants is still needed. Moreover, common features in the biogenesis of different small ncRNAs, and in some cases, cross talk between different gene regulatory pathways may add to the complexity of these pathways and could play important roles in modulating stress responses. Here we review the various ncRNAs that have been reported to participate in the response to abiotic stress in plants, focusing on their importance in plant adaptation and evolution. Understanding how ncRNAs work may reveal novel mechanisms involved in the plant responses to the environment.

Ben Scheres and colleagues report that in the growing tip of plant roots, a gene regulatory network that includes the plant homologue of Retinoblastoma regulates the divisions of long-term stem cells to replenish tissue and to protect the root stem cell niche., Quiescent long-term somatic stem cells reside in plant and animal stem cell niches. Within the Arabidopsis root stem cell population, the Quiescent Centre (QC), which contains slowly dividing cells, maintains surrounding short-term stem cells and may act as a long-term reservoir for stem cells. The RETINOBLASTOMA-RELATED (RBR) protein cell-autonomously reinforces mitotic quiescence in the QC. RBR interacts with the stem cell transcription factor SCARECROW (SCR) through an LxCxE motif. Disruption of this interaction by point mutation in SCR or RBR promotes asymmetric divisions in the QC that renew short-term stem cells. Analysis of the in vivo role of quiescence in the root stem cell niche reveals that slow cycling within the QC is not needed for structural integrity of the niche but allows the growing root to cope with DNA damage., Author Summary In the plant Arabidposis thaliana, root meristems (in the growing tip of the root) contain slowly dividing cells that act as an organizing center for the root stem cells that surround them. This centre is called the quiescent centre (QC). In this study, we show that the slow rate of division in the QC is regulated by the interaction between two proteins: Retinoblastoma homolog (RBR) and SCARECROW (SCR), a transcription factor that controls stem cell maintenance. RBR and SCR regulate quiescence in the QC by repressing an asymmetric cell division that generates short-term stem cells. Here we genetically manipulate the cells in the QC to alter their quiescence by regulating the RBR/SCR interaction to demonstrate that quiescence is not needed for the organizing capacity of the QC but instead provides cells with a higher resistance to genotoxic stress, allowing stem cells in the QC to survive even if more rapidly cycling stem cells are damaged. A role for mitotic quiescence has been reported in animal stem cells, in which Rb has been implicated. These findings indicate that it might serve a similar role in plant stem cells.

Paramutation is a well-studied epigenetic phenomenon in which trans communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the b1 locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1) are the tandem repeats sufficient for paramutation, 2) do they need to be in an allelic position to mediate paramutation, and 3) is there an association between the ability to mediate paramutation and repeat DNA methylation levels? Paramutation was achieved using multiple transgenes containing the b1 tandem repeats, including events with tandem repeats of only one half of the repeat unit (413 bp), demonstrating that these sequences are sufficient for paramutation and an allelic position is not required for the repeats to communicate. Furthermore, the transgenic tandem repeats increased the expression of a reporter gene in maize, demonstrating the repeats contain transcriptional regulatory sequences. Transgene-mediated paramutation required the mediator of paramutation1 gene, which is necessary for endogenous paramutation, suggesting endogenous and transgene-mediated paramutation both require an RNA-mediated transcriptional silencing pathway. While all tested repeat transgenes produced small interfering RNAs (siRNAs), not all transgenes induced paramutation suggesting that, as with endogenous alleles, siRNA production is not sufficient for paramutation. The repeat transgene-induced silencing was less efficiently transmitted than silencing induced by the repeats of endogenous b1 alleles, which is always 100% efficient. The variability in the strength of the repeat transgene-induced silencing enabled testing whether the extent of DNA methylation within the repeats correlated with differences in efficiency of paramutation. Transgene-induced paramutation does not require extensive DNA methylation within the transgene. However, increased DNA methylation within the endogenous b1 repeats after transgene-induced paramutation was associated with stronger silencing of the endogenous allele., Author Summary Paramutation is a fascinating process in which genes communicate to efficiently establish changes in their expression that are stably transmitted to future generations without any changes in DNA sequences. While paramutation was first described in the 1950s and extensively studied through the 1960s, its underlying mechanism remained mysterious for many years. Over the past ten years paramutation at the b1 locus in maize was shown to require transcribed, non-coding tandem repeats located 100 kb upstream of b1. These repeats generate small RNAs, and mutations in multiple genes mediating small RNA silencing at the transcriptional level prevent paramutation. While underlying mechanisms are shared, current models for RNA-mediated transcriptional silencing that are based on experiments with S. pombe and Arabidopsis do not explain many aspects of paramutation. In this manuscript we used a transgenic approach to demonstrate that the b1 non-coding tandem repeats are sufficient to send and respond to the paramutation signals and that this occurs even when the repeats are not at their normal chromosomal location.

The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization. The expression of 9775 genes was quantified in wild-type ovules, additionally detecting >2200 new transcripts mapping to antisense or intergenic regions. A quantitative comparison of global expression in wild-type and sporocyteless (spl) individuals resulted in 1301 genes showing 25-fold reduced or null activity in ovules lacking a female gametophyte, including those encoding 92 signalling proteins, 75 transcription factors, and 72 RNA-binding proteins not reported in previous studies based on microarray profiling. A combination of independent genetic and molecular strategies confirmed the differential expression of 28 of them, showing that they are either preferentially active in the female gametophyte, or dependent on the presence of a female gametophyte to be expressed in sporophytic cells of the ovule. Among 18 genes encoding pentatricopeptide-repeat proteins (PPRs) that show transcriptional activity in wild-type but not spl ovules, CIHUATEOTL (At4g38150) is specifically expressed in the female gametophyte and necessary for female gametogenesis. These results expand the nature of the transcriptional universe present in the ovule of Arabidopsis, and offer a large-scale quantitative reference of global expression for future genomic and developmental studies.

Epigenetic regulation is essential for growth and development in eukaryotic organisms (Henikoff et al., 2008; Suzuki and Bird, 2008) and is also responsible for the establishment, maintenance, and reversal of non-genetic cellular memory that records developmental and environmental cues, including those arising from biotic and abiotic stress (Bonasio et al., 2010). A series of recent stimulating papers, show that biotic stress can trigger a transgenerational epigenetic response in plants, where DNA methylation seems to play a central role. Plants sense and respond to environmental cues by a repertoire of mechanisms that regulate gene expression in order to maximize chances of survival in hostile environments. In addition to preformed defense traits, plants have evolved inducible defenses against microbial pathogens, herbivores, and even other plants that involve the regulation of gene expression for the synthesis of defensive secondary metabolites and specific proteins (Walling, 2000; Howe and Jander, 2008; Mithofer and Boland, 2012). Plants rely on the innate immunity of each cell and on systemic signals emanating from infection sites (Jones and Dangl, 2006). Plant hormones play essential roles during systemic defense signaling (Robert-Seilaniantz et al., 2011). Salicylic acid (SA) primarily triggers resistance against biotrophic and hemibiotrophic pathogens whereas a combination of jasmonic acid (JA) and ethylene (ET) signaling activates resistance against necrotrophic pathogens (Glazebrook, 2005; Robert-Seilaniantz et al., 2011). SA acts as an endogenous signal involved in systemic acquired resistance (SAR), an inducible resistance against a broad spectrum of pathogens including viruses, bacteria, and fungi that cause necrosis through rapid programmed cell death of infected cells, known as the hypersensitive response (Durrant and Dong, 2004). SAR induces resistance in the systemic (uninoculated) plant organs in response to local infection (Vlot et al., 2009). Recent evidence shows that NON-EXPRESSOR of PATHOGENESIS-RELATED GENES 3 (NPR3) and NPR4 are SA receptors that bind SA with different affinities and regulate degradation of the transcription cofactor NPR1 in a SA-dependent manner (Fu et al., 2012). In addition to their role during plant development, JA and JA-related compounds, including methyl-jasmonate (MeJA) and jasmonate-isoleucine conjugate (JA-Ile), play essential roles during endogenous regulation of plant resistance to mechanical wounding and herbivory by modulating global changes in gene expression (Creelman and Mullet, 1997; Sheard et al., 2010). Priming (or sensitization) refers to the enhanced ability for the quicker and more effective activation of specific cellular defense responses upon previous exposure to biotic or abiotic stress (Conrath et al., 2002, 2006). Defense pathways and priming can also be induced by application of chemical stimuli including the non-protein amino acid beta-amino-butyric acid (BABA) (Zimmerli et al., 2000). In addition to plant hormones, small RNAs and genes involved in the biogenesis of small RNAs are also components of the plant defense strategies against herbivory and microbial pathogens (Pandey et al., 2008; Ruiz-Ferrer and Voinnet, 2009; Katiyar-Agarwal and Jin, 2010). Here we comment on a series of papers that provide evidence on transgenerational epigenetic effects during biotic stress. Rasmann et al. (2012) report transgenerational priming responses (TPR) in tomato and Arabidopsis induced by caterpillar herbivory (Helicoverpa zea and Pieris rapae) and application of MeJA. TPR results in a reduction of up to ~40% in caterpillar weight gain and this effect persists after one stress free generation in Arabidopsis (Rasmann et al., 2012). Experiments with the coronatine insentitive1 (coi1-1) and the triple dicer-like2/dicer-like3/dicer-like4 (dcl2/dcl3/dcl4) mutants show that the TPR depends on JA perception and on components of the RNA-dependent DNA methylation pathway involved in de novo DNA methylation (RdDM) (Law and Jacobsen, 2010; Rasmann et al., 2012). In order to assay transgenerational effects in response to the biotrophic pathogen Pseudomonas syringae pv tomato DC3000 (PstDC3000) in Arabidopsis, Luna et al. (2012) recurrently inoculated a set of parental lines (P0) with increasing doses of PstDC3000. Fitness of progeny (P1) from PstDC3000 infected plants did not differ statistically from control plants progeny (C1) (Luna et al., 2012). However, P1 plants showed a statistically significant reduction in the colonization and disease symptoms after inoculation with: (1) the oomycete pathogen Hyaloperonospora arabidopsidis and (2) PstDC3000-lux (a bioluminescent strain of PstDC3000). Once established, this TPR can be maintained over one stress-free generation. Similar experiments employing parental lines in an npr1-1 mutant background showed that colonization reduction by H. arabidopsidis depends on a functional NPR1 gene. Consistent with an NPR1-dependent priming of SA-inducible defense, a faster and stronger induction of SA-inducible defense genes such as PATHOGENESIS-RELATED GENE 1 (PR1), WRKY6, WRKY53, and WRKY70 was also observed in P1 plants (Luna et al., 2012). Antagonistic effects between JA and SA pathways were confirmed by inoculating P1 plants with the nectrotrophic fungus Alternaria brassicicola (an inducer of JA-dependent defense response). P1 plants contained similar endogenous levels of JA, JA-Ile, JA-precursor, and SA to C1 plants, but showed increased hyphal colonization and a reduction in the expression of JA-inducible genes, including PLANT DEFENSINE 1.2 (PDF1.2). The priming effects on SA and JA inducible genes correlate with the NPR1-dependent deposition of epigenetic marks characteristic of active chromatin (H3K9ac; acetylation of Lys-9 on Histone 3) in the case of PR-1, WRKY6 and WRKY53; and repressive chromatin (H3K27me3; trimethylation of Lys-27 on Histone 3) in the case of PDF1.2. Although a role for chromatin modifications mediating the TPR wasn't ruled out, assays with the domains rearranged methyltransferase 1 and 2/cytosine methyltransferase 3 (drm1/drm2/cmt3) triple mutant, affected in DNA methylation, showed that this mutants mimics the TPR observed in P1 plants and indicates that components of the RdDM pathway are required for TPR (Luna et al., 2012). Similar experiments from Slaughter et al. (2012) using either BABA or an avirulent Pst strain also showed a TPR against PstDC3000 and H. arabidopsidis, and an IBS1 (IMPAIRED BABA-INDUCED STERILITY RESPONSE 1)-dependent increased expression of PR1, PR2, and PR5. However, in sharp contrast to Luna et al. and Rasmann et al., the TPR was only maintained in the immediate progeny which might be the result of differences in experimental approaches (recurrent vs. single inoculation/stimulation) or actually represent biological differences in the molecular mechanism(s) involved (Slaughter et al., 2012). High resolution genomewide profiling of the DNA methylation landscape in Arabidopsis from Dowen et al. (2012) shows that global disruption of establishment and maintenance of DNA methylation in a set of mutants including drm1/drm2/cmt3 and methyltransferase 1 (met1) enhances resistance to bacteria and induces widespread dynamic changes in methylation. Distinct patterns of differentially methylated regions (DMR) can be observed when wild type plants are exposed to SA or either virulent or avirulent Pst strains. SA treatment uncovered a class of 21-nt siRNAs particularly evident at transposable elements (TEs)–associated DMRs and in many cases, SA-induced DMR associate with reprogramming of TEs and neighboring genes (Dowen et al., 2012). Taken together, this set of papers unveiled a TPR during biotic stress in plants where DNA methylation and components of the RdDM pathway play a major role. Some open questions: what is the molecular nature of the TPR signal? What cells are responsible for sensing and transmitting the biotic stress memory? What is the impact of plant's age and intensity of the biotic stress on the TPR? Can the TPR behave as a paramutation-like phenomena?

Paramutation is the epigenetic transfer of information between alleles that leads to the heritable change of expression of one allele. Paramutation at the b1 locus in maize requires seven noncoding tandem repeat ( b1TR ) sequences located ∼100 kb upstream of the transcription start site of b1 , and mutations in several genes required for paramutation implicate an RNA-mediated mechanism. The mediator of paramutation ( mop1 ) gene, which encodes a protein closely related to RNA-dependent RNA polymerases, is absolutely required for paramutation. Herein, we investigate the potential function of mop1 and the siRNAs that are produced from the b1TR sequences. Production of siRNAs from the b1TR sequences depends on a functional mop1 gene, but transcription of the repeats is not dependent on mop1 . Further nuclear transcription assays suggest that the b1TR sequences are likely transcribed predominantly by RNA polymerase II. To address whether production of b1TR -siRNAs correlated with paramutation, we examined siRNA production in alleles that cannot undergo paramutation. Alleles that cannot participate in paramutation also produce b1TR -siRNAs, suggesting that b1TR -siRNAs are not sufficient for paramutation in the tissues analyzed. However, when b1TR -siRNAs are produced from a transgene expressing a hairpin RNA, b1 paramutation can be recapitulated. We hypothesize that either the b1TR -siRNAs or the dsRNA template mediates the trans -communication between the alleles that establishes paramutation. In addition, we uncovered a role for mop1 in the biogenesis of a subset of microRNAs (miRNAs) and show that it functions at the level of production of the primary miRNA transcripts.

Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA–mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V–like complexes could provide maize with a greater diversification of RNA–mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state—a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV–like) and potentially processes downstream (Pol-V–like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes.

In the ovules of most sexual flowering plants female gametogenesis is initiated from a single surviving gametic cell, the functional megaspore, formed after meiosis of the somatically derived megaspore mother cell (MMC)1,2. Because some mutants and certain sexual species exhibit more than one MMC2-4, and many others are able to form gametes without meiosis (by apomixis)5, it has been suggested that somatic cells in the ovule are competent to respond to a local signal likely to play an important function in determination6. Here we show that the Arabidopsis protein ARGONAUTE9 (AGO9) controls female gamete formation by restricting the specification of gametophyte precursors in a dosage-dependent, non-cell-autonomous manner. Mutations in AGO9 lead to the differentiation of multiple gametic cells that are able to initiate gametogenesis. The AGO9 protein is not expressed in the gamete lineage; instead, it is expressed in cytoplasmic foci of somatic companion cells. Mutations in SUPPRESSOR OF GENE SILENCING3 and RNA-DEPENDENT RNA POLYMERASE6 exhibit an identical defect to ago9 mutants, indicating that the movement of small RNA (sRNA) silencing out of somatic companion cells is necessary for controlling the specification of gametic cells. AGO9 preferentially interacts with 24 nucleotide (nt) sRNAs derived from transposable elements (TEs), and its activity is necessary to silence TEs in female gametes and their accessory cells. Our results show that AGO9-dependent sRNA silencing is crucial to specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA–dependent silencing in plant gametes.

Small RNAs from plants are known to be highly complex and abundant, with this complexity proportional to genome size. Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPENDENT RNA POLYMERASE 2 ( RDR2 ) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 ( mop1 ) gene is a predicted ortholog of RDR2 . The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wild-type maize and in the isogenic mop1-1 loss-of-function mutant by using Illumina's sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis , in mop1-1 , the 24-nucleotide (nt) endogenous heterochromatic short-interfering siRNAs were dramatically reduced, resulting in an enrichment of miRNAs and transacting siRNAs. In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic ≈22-nt class of small RNAs, suggesting a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24-nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced.

Although many miRNAs are deeply conserved within each kingdom, none are known to be conserved between plants and animals. We identified Arabidopsis thaliana miR854 and miR855, two microRNAs (miRNAs) with multiple binding sites in the 3′ untranslated region (3′UTR) of OLIGOURIDYLATE binding PROTEIN1b (At UBP1b), forming miRNA:mRNA interactions similar to those that cause translational repression/mRNA cleavage in animals. At UBP1b encodes a member of a heterogeneous nuclear RNA binding protein (hnRNP) family. The 3′UTR of At UBP1b is sufficient to repress reporter protein expression in tissues expressing miR854 or miR855 (rosette leaves and flowers, respectively) but not where both miRNAs are absent (cauline leaves). Intergenic regions containing sequences closely resembling miR854 are predicted to fold into stable miRNA precursors in animals, and members of the miR854 family are expressed in Caenorhabditis elegans, Mus musculus, and Homo sapiens, all with imperfect binding sites in the 3′UTR of genes encoding the T cell Intracellular Antigen-Related protein, an hnRNP of the UBP1 family. Potential binding sites for miR854 are absent from UBP1-like genes in fungi lacking the miRNA biogenetic machinery. Our results indicate that plants and animals share miRNAs of the miR854 family, suggesting a common origin of these miRNAs as regulators of basal transcriptional mechanisms.

Background We have further characterized floral organ-localized gene expression in the inflorescence of Arabidopsis thaliana by comparison of massively parallel signature sequencing (MPSS) data. Six libraries of RNA sequence tags from immature inflorescence tissues were constructed and matched to their respective loci in the annotated Arabidopsis genome. These signature libraries survey the floral transcriptome of wild-type tissue as well as the floral homeotic mutants, apetala1, apetala3, agamous, a superman/apetala1 double mutant, and differentiated ovules dissected from the gynoecia of wild-type inflorescences. Comparing and contrasting these MPSS floral expression libraries enabled demarcation of transcripts enriched in the petals, stamens, stigma-style, gynoecia, and those with predicted enrichment within the sepal/sepal-petals, petal-stamens, or gynoecia-stamens. Results By comparison of expression libraries, a total of 572 genes were found to have organ-enriched expression within the inflorescence. The bulk of characterized organ-enriched transcript diversity was noted in the gynoecia and stamens, whereas fewer genes demonstrated sepal or petal-localized expression. Validation of the computational analyses was performed by comparison with previously published expression data, in situ hybridizations, promoter-reporter fusions, and reverse transcription PCR. A number of well-characterized genes were accurately delineated within our system of transcript filtration. Moreover, empirical validations confirm MPSS predictions for several genes with previously uncharacterized expression patterns. Conclusion This extensive MPSS analysis confirms and supplements prior microarray floral expression studies and illustrates the utility of sequence survey-based expression analysis in functional genomics. Spatial floral expression data accrued by MPSS and similar methods will be advantageous in the elucidation of more comprehensive genetic regulatory networks governing floral development.