Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Lost in space: what single-cell RNA sequencing cannot tell you.

Plant scientists are rapidly integrating single-cell RNA sequencing (scRNA-seq) into their workflows. Maximizing the potential of scRNA-seq requires a proper understanding of the spatiotemporal context of cells. However, positional information is inherently lost during scRNA-seq, limiting its potential to characterize complex biological systems. In this review we highlight how current single-cell analysis pipelines cannot completely recover spatial information, which confounds biological interpretation. Various strategies exist to identify the location of RNA, from classical RNA in situ hybridization to spatial transcriptomics. Herein we discuss the possibility of utilizing this spatial information to supervise single-cell analyses. An integrative approach will maximize the potential of each technology, and lead to insights which go beyond the capability of each individual technology.

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely (i) not triggered by microRNAs, (ii) not loaded by AGO18 proteins, and (iii) not capable of mediating cis-cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Bookend: precise transcript reconstruction with end-guided assembly.

We developed Bookend, a package for transcript assembly that incorporates data from different RNA-seq techniques, with a focus on identifying and utilizing RNA 5' and 3' ends. We demonstrate that correct identification of transcript start and end sites is essential for precise full-length transcript assembly. Utilization of end-labeled reads present in full-length single-cell RNA-seq datasets dramatically improves the precision of transcript assembly in single cells. Finally, we show that hybrid assembly across short-read, long-read, and end-capture RNA-seq datasets from Arabidopsis thaliana, as well as meta-assembly of RNA-seq from single mouse embryonic stem cells, can produce reference-quality end-to-end transcript annotations.

Innate, translation-dependent silencing of an invasive transposon in Arabidopsis.

Co-evolution between hosts' and parasites' genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral-like silencing response. To investigate this response's activation, we studied the single-copy element EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full-length genomic flGAG-POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG-POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5'OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto-unrecognized "translation-dependent silencing" (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern.

Plant development: Suspensors as a battlefield for parental tug-of-war?

Parental contributions to zygotes can influence early embryogenesis and may regulate the distribution of maternal resources to progeny. A new study in Arabidopsis thaliana has demonstrated that signaling components from maternal sporophytic tissues and paternal gametes converge in zygotes to promote elongation of the extraembryonic suspensor, which supports the developing embryo proper.

The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation.

Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only a few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant, ectopic, and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3, and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady-state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

Repression of CHROMOMETHYLASE 3 prevents epigenetic collateral damage in Arabidopsis.

DNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Our results are also consistent with CMT3-induced ectopic methylation of promoters or bodies of genes undergoing transcriptional activation reducing their expression. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants.

Gene expression variation in Arabidopsis embryos at single-nucleus resolution.

Soon after fertilization of egg and sperm, plant genomes become transcriptionally activated and drive a series of coordinated cell divisions to form the basic body plan during embryogenesis. Early embryonic cells rapidly diversify from each other, and investigation of the corresponding gene expression dynamics can help elucidate underlying cellular differentiation programs. However, current plant embryonic transcriptome datasets either lack cell-specific information or have RNA contamination from surrounding non-embryonic tissues. We have coupled fluorescence-activated nuclei sorting together with single-nucleus mRNA-sequencing to construct a gene expression atlas of Arabidopsis thaliana early embryos at single-cell resolution. In addition to characterizing cell-specific transcriptomes, we found evidence that distinct epigenetic and transcriptional regulatory mechanisms operate across emerging embryonic cell types. These datasets and analyses, as well as the approach we devised, are expected to facilitate the discovery of molecular mechanisms underlying pattern formation in plant embryos. This article has an associated 'The people behind the papers' interview.

Antagonistic activities of cotranscriptional regulators within an early developmental window set FLC expression level.

Quantitative variation in expression of the Arabidopsis floral repressor FLC influences whether plants overwinter before flowering, or have a rapid cycling habit enabling multiple generations a year. Genetic analysis has identified activators and repressors of FLC expression but how they interact to set expression level is poorly understood. Here, we show that antagonistic functions of the FLC activator FRIGIDA (FRI) and the repressor FCA, at a specific stage of embryo development, determine FLC expression and flowering. FRI antagonizes an FCA-induced proximal polyadenylation to increase FLC expression and delay flowering. Sector analysis shows that FRI activity during the early heart stage of embryo development maximally delays flowering. Opposing functions of cotranscriptional regulators during an early embryonic developmental window thus set FLC expression levels and determine flowering time.

Plant Epigenetics: Propelling DNA Methylation Variation across the Cell Cycle.

DNA methylation is reconfigured during male reproduction in plants, but little is known regarding the mechanisms controlling these epigenetic dynamics. New research highlights how the cell cycle can influence DNA methylation dynamics observed during male gametogenesis and may induce epigenetic variation in clonally propagated plants.

A transposon surveillance mechanism that safeguards plant male fertility during stress.

Although plants are able to withstand a range of environmental conditions, spikes in ambient temperature can impact plant fertility causing reductions in seed yield and notable economic losses1,2. Therefore, understanding the precise molecular mechanisms that underpin plant fertility under environmental constraints is critical to safeguarding future food production3. Here, we identified two Argonaute-like proteins whose activities are required to sustain male fertility in maize plants under high temperatures. We found that MALE-ASSOCIATED ARGONAUTE-1 and -2 associate with temperature-induced phased secondary small RNAs in pre-meiotic anthers and are essential to controlling the activity of retrotransposons in male meiocyte initials. Biochemical and structural analyses revealed how male-associated Argonaute activity and its interaction with retrotransposon RNA targets is modulated through the dynamic phosphorylation of a set of highly conserved, surface-located serine residues. Our results demonstrate that an Argonaute-dependent, RNA-guided surveillance mechanism is critical in plants to sustain male fertility under environmentally constrained conditions, by controlling the mutagenic activity of transposons in male germ cells.

Epigenetic reprogramming rewires transcription during the alternation of generations in Arabidopsis.

Alternation between morphologically distinct haploid and diploid life forms is a defining feature of most plant and algal life cycles, yet the underlying molecular mechanisms that govern these transitions remain unclear. Here, we explore the dynamic relationship between chromatin accessibility and epigenetic modifications during life form transitions in Arabidopsis. The diploid-to-haploid life form transition is governed by the loss of H3K9me2 and DNA demethylation of transposon-associated cis-regulatory elements. This event is associated with dramatic changes in chromatin accessibility and transcriptional reprogramming. In contrast, the global loss of H3K27me3 in the haploid form shapes a chromatin accessibility landscape that is poised to re-initiate the transition back to diploid life after fertilisation. Hence, distinct epigenetic reprogramming events rewire transcription through major reorganisation of the regulatory epigenome to guide the alternation of generations in flowering plants.

Each pollen grain from a flowering plant houses sperm, which contain half of the genes needed to make a new plant, and a companion or vegetative cell (VC) that serves to deliver sperm to the egg. The genes in the vegetative cell and those in the sperm are identical to the genes of the plant they come from, so how can this set of identical genetic information produce such different cells? Both DNA and histones, the proteins that pack and order DNA, can be chemically modified locally through a process called methylation. The location of these modifications can affect how genetic information in the DNA is read to make different types of cells. The use of processes like methylation to regulate whether genes are switched on or off is called epigenetics. So what role does epigenetics play in plant pollen? To answer this question, Borg et al. examined the epigenetics of pollen in Arabidopsis thaliana, a widely studied plant and common weed. In vegetative cells, DNA methylation is lost together with a different methylation mark (H3K9me2), which unlocks several genes needed for pollen to transport sperm. By contrast, sperm loses an entirely different methylation mark, called H3K27me3, which unlocks a different set of genes that help to prepare development of a new plant once sperm fertilizes the egg. Through these different set of epigenetic changes, activity increases at different groups of genes that are important for shaping the function of each pollen cell type. These results reveal how the loss of DNA and histone methylation are important for plants to reproduce sexually via pollen. This offers insights into the evolution of plants and other related life forms. Learning about plant reproduction may also help to increase food production by improving crop yields.

Application of expansion microscopy on developing Arabidopsis seeds.

Expansion microscopy (ExM) improves image resolution of specimens without requirements of sophisticated techniques or equipment. Probes or proteins are anchored onto an acrylamide gel matrix which is then expanded with osmotic pressure. As the physical distance between two signal points increases, previously confounded signals can be resolved while their relative spatial locations are retained. ExM has been successfully applied to several animal tissues, but its application to plant tissues was only recently demonstrated. Here we provide a detailed ExM protocol for plant tissues using fluorescent immunostaining of developing Arabidopsis thaliana (Arabidopsis) seeds as an example. This modified ExM protocol enables expansion of ovule/seed samples, and preserves the majority of fluorescent protein signals in the expanded samples. The fluorescent immunostaining observed using this protocol demonstrates the feasibility of detecting cellular events and subcellular structures in expanded plant samples. This ExM protocol variant for plants can serve as a guideline for applying ExM to various plant tissues and help increase the resolution of corresponding microscopy based studies.

Chromatin regulates expression of small RNAs to help maintain transposon methylome homeostasis in Arabidopsis.

BACKGROUND: Eukaryotic genomes are partitioned into euchromatic and heterochromatic domains to regulate gene expression and other fundamental cellular processes. However, chromatin is dynamic during growth and development and must be properly re-established after its decondensation. Small interfering RNAs (siRNAs) promote heterochromatin formation, but little is known about how chromatin regulates siRNA expression. RESULTS: We demonstrate that thousands of transposable elements (TEs) produce exceptionally high levels of siRNAs in Arabidopsis thaliana embryos. TEs generate siRNAs throughout embryogenesis according to two distinct patterns depending on whether they are located in euchromatic or heterochromatic regions of the genome. siRNA precursors are transcribed in embryos, and siRNAs are required to direct the re-establishment of DNA methylation on TEs from which they are derived in the new generation. Decondensed chromatin also permits the production of 24-nt siRNAs from heterochromatic TEs during post-embryogenesis, and siRNA production from bipartite-classified TEs is controlled by their chromatin states. CONCLUSIONS: Decondensation of heterochromatin in response to developmental, and perhaps environmental, cues promotes the transcription and function of siRNAs in plants. Our results indicate that chromatin-mediated siRNA transcription provides a cell-autonomous homeostatic control mechanism to help reconstitute pre-existing chromatin states during growth and development including those that ensure silencing of TEs in the future germ line.

Small RNA In Situ Hybridizations on Sections of Arabidopsis Embryos.

Small RNAs mediate posttranscriptional gene silencing in plants and animals. This often occurs in specific cell or tissue types and can be necessary for their differentiation. Determining small RNA (sRNA) localization patterns at cellular resolution can therefore provide information on the corresponding gene regulatory processes they are involved in. Recent improvements with in situ hybridization methods have allowed them to be applied to sRNAs. Here we describe an in situ hybridization protocol to detect sRNAs from sections of early staged Arabidopsis thaliana (Arabidopsis) embryos.

Profiling Transcriptomes of Manually Dissected Arabidopsis Embryos.

Genome-wide characterization of RNA populations in early flowering plant embryos can yield insights into the gene regulatory processes functioning during this formative phase of development. However, early embryonic transcriptomes are technically challenging to profile because of the low amount of RNA obtainable and potential RNA contamination from surrounding nonembryonic tissues. Here we provide a detailed protocol for collecting early Arabidopsis thaliana (Arabidopsis) embryos, generating mRNA sequencing (mRNA-seq) libraries, and basic data processing and quality controls of the resulting mRNA-seq data.