Two ARGONAUTE proteins loaded with transposon-derived small RNAs are associated with the reproductive cell lineage in Arabidopsis.

In sexually propagating organisms, genetic, and epigenetic mutations are evolutionarily relevant only if they occur in the germline and are hence transmitted to the next generation. In contrast to most animals, plants are considered to lack an early segregating germline, implying that somatic cells can contribute genetic information to progeny. Here we demonstrate that 2 ARGONAUTE proteins, AGO5 and AGO9, mark cells associated with sexual reproduction in Arabidopsis (Arabidopsis thaliana) throughout development. Both AGOs are loaded with dynamically changing small RNA populations derived from highly methylated, pericentromeric, long transposons. Sequencing of single stem cell nuclei revealed that many of these transposons are co-expressed within an AGO5/9 expression domain in the shoot apical meristem (SAM). Co-occurrence of transposon expression and specific ARGONAUTE (AGO) expression in the SAM is reminiscent of germline features in animals and supports the existence of an early segregating germline in plants. Our results open the path to investigating transposon biology and epigenome dynamics at cellular resolution in the SAM stem cell niche.

Salicylic acid and RNA interference mediate antiviral immunity of plant stem cells.

Stem cells are essential for the development and organ regeneration of multicellular organisms, so their infection by pathogenic viruses must be prevented. Accordingly, mammalian stem cells are highly resistant to viral infection due to dedicated antiviral pathways including RNA interference (RNAi). In plants, a small group of stem cells harbored within the shoot apical meristem generate all postembryonic above-ground tissues, including the germline cells. Many viruses do not proliferate in these cells, yet the molecular bases of this exclusion remain only partially understood. Here, we show that a plant-encoded RNA-dependent RNA polymerase, after activation by the plant hormone salicylic acid, amplifies antiviral RNAi in infected tissues. This provides stem cells with RNA-based virus sequence information, which prevents virus proliferation. Furthermore, we find RNAi to be necessary for stem cell exclusion of several unrelated RNA viruses, despite their ability to efficiently suppress RNAi in the rest of the plant. This work elucidates a molecular pathway of great biological and economic relevance and lays the foundations for our future understanding of the unique systems underlying stem cell immunity.

A versatile CRISPR-based system for lineage tracing in living plants.

Individual cells give rise to diverse cell lineages during the development of multicellular organisms. Understanding the contribution of these lineages to mature organisms is a central question of developmental biology. Several techniques to document cell lineages have been used, from marking single cells with mutations that express a visible marker to generating molecular bar codes by CRISPR-induced mutations and subsequent single-cell analysis. Here, we exploit the mutagenic activity of CRISPR to allow lineage tracing within living plants with a single reporter. Cas9-induced mutations are directed to correct a frameshift mutation that restores expression of a nuclear fluorescent protein, labelling the initial cell and all progenitor cells with a strong signal without modifying other phenotypes of the plants. Spatial and temporal control of Cas9 activity can be achieved using tissue-specific and/or inducible promoters. We provide proof of principle for the function of lineage tracing in two model plants. The conserved features of the components and the versatile cloning system, allowing for easy exchange of promoters, are expected to make the system widely applicable.

Arabidopsis shoot stem cells display dynamic transcription and DNA methylation patterns.

In plants, aerial organs originate continuously from stem cells in the center of the shoot apical meristem. Descendants of stem cells in the subepidermal layer are progenitors of germ cells, giving rise to male and female gametes. In these cells, mutations, including insertions of transposable elements or viruses, must be avoided to preserve genome integrity across generations. To investigate the molecular characteristics of stem cells in Arabidopsis, we isolated their nuclei and analyzed stage-specific gene expression and DNA methylation in plants of different ages. Stem cell expression signatures are largely defined by developmental stage but include a core set of stem cell-specific genes, among which are genes implicated in epigenetic silencing. Transiently increased expression of transposable elements in meristems prior to flower induction correlates with increasing CHG methylation during development and decreased CHH methylation, before stem cells enter the reproductive lineage. These results suggest that epigenetic reprogramming may occur at an early stage in this lineage and could contribute to genome protection in stem cells during germline development.

Parental environmental effects are common and strong, but unpredictable, in Arabidopsis thaliana.

The phenotypes of plants can be influenced by the environmental conditions experienced by their parents. However, there is still much uncertainty about how common and how predictable such parental environmental effects really are. We carried out a comprehensive experimental test for parental effects, subjecting plants of multiple Arabidopsis thaliana genotypes to 24 different biotic or abiotic stresses, or combinations thereof, and comparing their offspring phenotypes in a common environment. The majority of environmental stresses caused significant parental effects, with -35% to +38% changes in offspring fitness. The expression of parental effects was strongly genotype-dependent, and multiple environmental stresses often acted nonadditively when combined. The direction and magnitude of parental effects were unrelated to the direct effects on the parents: Some environmental stresses did not affect the parents but caused substantial effects on offspring, while for others, the situation was reversed. Our study demonstrates that parental environmental effects are common and often strong in A. thaliana, but they are genotype-dependent, act nonadditively, and are difficult to predict. We should thus be cautious with generalizing from simple studies with single plant genotypes and/or only few individual environmental stresses. A thorough and general understanding of parental effects requires large multifactorial experiments.

DNA methylation analysis in plants: review of computational tools and future perspectives.

Genome-wide DNA methylation studies have quickly expanded due to advances in next-generation sequencing techniques along with a wealth of computational tools to analyze the data. Most of our knowledge about DNA methylation profiles, epigenetic heritability and the function of DNA methylation in plants derives from the model species Arabidopsis thaliana. There are increasingly many studies on DNA methylation in plants-uncovering methylation profiles and explaining variations in different plant tissues. Additionally, DNA methylation comparisons of different plant tissue types and dynamics during development processes are only slowly emerging but are crucial for understanding developmental and regulatory decisions. Translating this knowledge from plant model species to commercial crops could allow the establishment of new varieties with increased stress resilience and improved yield. In this review, we provide an overview of the most commonly applied bioinformatics tools for the analysis of DNA methylation data (particularly bisulfite sequencing data). The performances of a selection of the tools are analyzed for computational time and agreement in predicted methylated sites for A. thaliana, which has a smaller genome compared to the hexaploid bread wheat. The performance of the tools was benchmarked on five plant genomes. We give examples of applications of DNA methylation data analysis in crops (with a focus on cereals) and an outlook for future developments for DNA methylation status manipulations and data integration.

RETINOBLASTOMA-RELATED PROTEIN controls the transition to autotrophic plant development.

Seedling establishment is a crucial phase during plant development when the germinating heterotrophic embryo switches to autotrophic growth and development. Positive regulators of embryonic development need to be turned off, while the cell cycle machinery is activated to allow cell cycle entry and organ primordia initiation. However, it is not yet understood how the molecular mechanisms responsible for the onset of cell division, metabolism changes and cell differentiation are coordinated during this transition. Here, we demonstrate that the Arabidopsis thaliana RETINOBLASTOMA-RELATED protein (RBR) ortholog of the animal tumor suppressor retinoblastoma (pRB) not only controls the expression of cell cycle-related genes, but is also required for persistent shut-down of late embryonic genes by increasing their histone H3K27 trimethylation. Seedlings with reduced RBR function arrest development after germination, and stimulation with low amounts of sucrose induces transcription of late embryonic genes and causes ectopic cell division. Our results suggest a model in which RBR acts antagonistically to sucrose by negatively regulating the cell cycle and repressing embryonic genes. Thus, RBR is a positive regulator of the developmental switch from embryonic heterotrophic growth to autotrophic growth. This establishes RBR as a new integrator of metabolic and developmental decisions.

Histone deacetylase AtHDA7 is required for female gametophyte and embryo development in Arabidopsis.

Histone modifications are involved in the regulation of many processes in eukaryotic development. In this work, we provide evidence that AtHDA7, a HISTONE DEACETYLASE (HDAC) of the Reduced Potassium Dependency3 (RPD3) superfamily, is crucial for female gametophyte development and embryogenesis in Arabidopsis (Arabidopsis thaliana). Silencing of AtHDA7 causes degeneration of micropylar nuclei at the stage of four-nucleate embryo sac and delay in the progression of embryo development, thereby bringing the seed set down in the Athda7-2 mutant. Furthermore, AtHDA7 down- and up-regulation lead to a delay of growth in postgermination and later developmental stages. The Athda7-2 mutation that induces histone hyperacetylation significantly increases the transcription of other HDACs (AtHDA6 and AtHDA9). Moreover, silencing of AtHDA7 affects the expression of ARABIDOPSIS HOMOLOG OF SEPARASE (AtAESP), previously demonstrated to be involved in female gametophyte and embryo development. However, chromatin immunoprecipitation analysis with acetylated H3 antibody provided evidence that the acetylation levels of H3 at AtAESP and HDACs does not change in the mutant. Further investigations are essential to ascertain the mechanism by which AtHDA7 affects female gametophyte and embryo development.

Arabidopsis RETINOBLASTOMA-RELATED is required for stem cell maintenance, cell differentiation, and lateral organ production.

Several genes involved in the regulation of postembryonic organ initiation and growth have been identified. However, it remains largely unclear how developmental cues connect to the cell cycle. RETINOBLASTOMA RELATED (RBR) is a plant homolog of the tumor suppressor Retinoblastoma (pRb), which is a key regulator of the cell cycle. Using inducible RNA interference (RNAi) against Arabidopsis thaliana RBR (RBRi), we reduced RBR expression levels at different stages of plant development. Conditional reduction or loss of RBR function disrupted cell division patterns, promoted context-dependent cell proliferation, and negatively influenced establishment of cell differentiation. Several lineages of toti- and pluripotent cells, including shoot apical meristem stem cells, meristemoid mother cells, and procambial cells, failed to produce appropriately differentiated cells. Meristem activity was altered, leading to a disruption of the CLAVATA-WUSCHEL feedback loop and inhibition of lateral organ formation. Release of RBR from RNAi downregulation restored meristem activity. Gene profiling analyses soon after RBRi induction revealed that a change in RBR homeostasis is perceived as a stress, even before genes regulated by RBR-E2F become deregulated. The results establish RBR as a key cell cycle regulator required for coordination of cell division, differentiation, and cell homeostasis.

Epigenetic regulation in the shoot apical meristem.

Epigenetic mechanisms form the basis of cellular memory, developmental decisions, and the cellular immune system that defends against transposons and viruses. Organs develop from the shoot apical meristem (SAM) to shape the plant's areal phenotype, and stem cells in the SAM serve as a functional germline. While many details on the regulation of stem cell pool size, organ initiation, and patterning at the meristem periphery are known, we know surprisingly little about the molecular characteristics of SAM cells, including their epigenome and how it changes during development. Here, we summarize information on epigenetic regulation of selected genes necessary for stem cell maintenance. As recent evidence suggests that SAM stem cells might be a hotspot of transposon activation, we discuss this aspect of epigenetic control in the meristem and speculate on mechanisms that maintain the flexibility of SAM stem cells in response to developmental or environmental cues.

Chromatin during plant regeneration: Opening towards root identity?

Plants show exceptional developmental plasticity and the ability to reprogram cell identities during regeneration. Although regeneration has been used in plant propagation for decades, we only recently gained detailed cellular and molecular insights into this process. Evidently, not all cell types have the same regeneration potential, and only a subset of regeneration-competent cells reach pluripotency. Pluripotent cells exhibit transcriptional similarity to root stem cells. In different plant regeneration systems, transcriptional reprogramming involves transient release of chromatin repression during pluripotency establishment and its restoration during organ or embryo differentiation. Incomplete resetting of the epigenome leads to somaclonal variation in regenerated plants. As single-cell technologies advance, we expect novel, exciting insights into epigenome dynamics during the establishment of pluripotency.

Preparing Chromatin and RNA from Rare Cell Types with Fluorescence-Activated Nuclear Sorting (FANS).

The application of fluorescent tags to generate cell type-specific translational and transcriptional reporter lines is routine in plants, but separation of different cell types for downstream analyses is hampered by the presence of cell walls and tight connections between cells. Enzymatic removal of cell walls induces a wound response, dedifferentiation, or reprogramming of the resulting protoplasts. Their osmotic and mechanical instability and their large size range are challenging for FACS, a flow -sorting procedure based on differential expression of fluorescent tags. In contrast, plant nuclei are relatively robust and easy to isolate. Here, we describe a protocol for fluorescence-activated nuclear sorting (FANS) that allows efficient purification of very few fluorescence-tagged nuclei from a large background of non-labeled tissue. Purified nuclei are suitable for genome, epigenome, transcriptome, or proteome analyses. We describe in detail how to analyze nuclear RNA and DNA methylation from sorted nuclei representing the limited number of stem cells in the shoot apical meristem of Arabidopsis.

A Pragmatic Approach to Getting Published: 35 Tips for Early Career Researchers.

It is trite to say "publish or perish," yet many early career researchers are often at a loss on how to best get their work published. With strong competition and many manuscripts submitted, it is difficult to convince editors and reviewers to opt for acceptance. A pragmatic approach to publishing may increase one's odds of success. Here, we - a group of postdocs in the field of plant science - present specific recommendations for early career scientists on advanced levels. We cannot provide a recipe-like set of instructions with success guaranteed, but we come from a broad background in plant science, with experience publishing in a number of journals of varying topics and impact factors. We provide tips, tricks, and tools for collaboration, journal selection, and achieving acceptance.

Epigenetic responses to stress: triple defense?

Stressful conditions for plants can originate from numerous physical, chemical and biological factors, and plants have developed a plethora of survival strategies including developmental and morphological adaptations, specific signaling and defense pathways as well as innate and acquired immunity. While it has become clear in recent years that many stress responses involve epigenetic components, we are far from understanding the mechanisms and molecular interactions. Extending our knowledge is fundamental, not least for plant breeding and conservation biology. This review will highlight recent insights into epigenetic stress responses at the level of signaling, chromatin modification, and potentially heritable consequences.

Emerging roles of RETINOBLASTOMA-RELATED proteins in evolution and plant development.

RETINOBLASTOMA-RELATED (RBR) proteins are plant homologs of the human tumor suppressor pRB. Similar to their animal counterparts they have roles in cell cycle regulation and differentiation. We discuss recent findings of the evolution of RBR functions ranging from a molecular ruler and metabolic integrator in algae to a coordinator of differentiation in gametophytes. Genetic analysis and manipulation of protein levels during gametophytic and post-embryonic plant development are now providing new insights into the function of RBR in stem cell maintenance, cell specification and differentiation. We briefly explain interactions of RBR with chromatin-modifying complexes that appear to be a central underlying molecular mechanism during developmental transitions.

The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose.

We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75 degrees C. The K(m) values at 70 degrees C were 109 nM for trehalose and 114 nM for maltose; also, a high K(m) (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while d-galactose, d-fucose, l-rhamnose, l-arabinose, and d-mannose were not competitive inhibitors. In the recently published genome of T. thermophilus HB27, two gene clusters designated malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) and two monocistronic genes designated malK1 (TTC0211) and malK2 (TTC0611) are annotated as trehalose/maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the malE1 and malE2 genes, lacking the sequence encoding the signal peptides, were expressed in Escherichia coli. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (K(d) values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either malF1 or malK1 were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in malK2 or malF2 showed no growth defect on any of these sugars. Therefore, malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and malK1 encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90 degrees C for MalE1 and 105 degrees C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.