The expanding world of small RNAs in plants.

Plant genomes encode various small RNAs that function in distinct, yet overlapping, genetic and epigenetic silencing pathways. However, the abundance and diversity of small-RNA classes varies among plant species, suggesting coevolution between environmental adaptations and gene-silencing mechanisms. Biogenesis of small RNAs in plants is well understood, but we are just beginning to uncover their intricate regulation and activity. Here, we discuss the biogenesis of plant small RNAs, such as microRNAs, secondary siRNAs and heterochromatic siRNAs, and their diverse cellular and developmental functions, including in reproductive transitions, genomic imprinting and paramutation. We also discuss the diversification of small-RNA-directed silencing pathways through the expansion of RNA-dependent RNA polymerases, DICER proteins and ARGONAUTE proteins.

Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA.

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.

Bypassing reproductive barriers in hybrid seeds using chemically induced epimutagenesis.

The triploid block, which prevents interploidy hybridizations in flowering plants, is characterized by a failure in endosperm development, arrest in embryogenesis, and seed collapse. Many genetic components of triploid seed lethality have been successfully identified in the model plant Arabidopsis thaliana, most notably the paternally expressed genes (PEGs), which are upregulated in tetraploid endosperm with paternal excess. Previous studies have shown that the paternal epigenome is a key determinant of the triploid block response, as the loss of DNA methylation in diploid pollen suppresses the triploid block almost completely. Here, we demonstrate that triploid seed collapse is bypassed in Arabidopsis plants treated with the DNA methyltransferase inhibitor 5-Azacytidine during seed germination and early growth. We identified strong suppressor lines showing stable transgenerational inheritance of hypomethylation in the CG context, as well as normalized expression of PEGs in triploid seeds. Importantly, differentially methylated loci segregate in the progeny of "epimutagenized" plants, which may allow epialleles involved in the triploid block response to be identified in future studies. Finally, we demonstrate that chemically induced epimutagenesis facilitates hybridization between different Capsella species, thus potentially emerging as a strategy for producing triploids and interspecific hybrids with high agronomic interest.

Evaluation of quality and quantity of randomized controlled trials in hepatobiliary surgery: A scoping/mapping review.

AIM: To evaluate the quantity and quality of randomized controlled trials (RCTs) in hepatobiliary surgery and for identifying gaps in current evidences. METHODS: A systematic search was conducted in MEDLINE (via PubMed), Web of Science, and Cochrane Controlled Register of Trials (CENTRAL) for RCTs of hepatobiliary surgery published from inception until the end of 2023. The quality of each study was assessed using the Cochrane risk-of-bias (RoB) tool. The associations between risk of bias and the region and publication date were also assessed. Evidence mapping was performed to identify research gaps in the field. RESULTS: The study included 1187 records. The number and proportion of published randomized controlled trials (RCTs) in hepatobiliary surgery increased over time, from 13 RCTs (.0005% of publications) in 1970-1979 to 201 RCTs (.003% of publications) in 2020-2023. There was a significant increase in the number of studies with a low risk of bias in RoB domains (p < .01). The proportion of RCTs with low risk of bias improved significantly after the introduction of CONSORT guidelines (p < .001). The evidence mapping revealed a significant research focus on major and minor hepatectomy and cholecystectomy. However, gaps were identified in liver cyst surgery and hepatobiliary vascular surgery. Additionally, there are gaps in the field of perioperative management and nutrition intervention. CONCLUSION: The quantity and quality of RCTs in hepatobiliary surgery have increased over time, but there is still room for improvement. We have identified gaps in current research that can be addressed in future studies.

Epigenetic reprogramming and small RNA silencing of transposable elements in pollen.

The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that could transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with downregulation of the heterochromatin remodeler decrease in DNA methylation 1 and of many TE siRNAs. However, 21 nucleotide siRNAs from Athila retrotransposons are generated and accumulate in pollen and sperm, suggesting that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes.

DEFECTIVE EMBRYO AND MERISTEMS genes are required for cell division and gamete viability in Arabidopsis.

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

Arabidopsis retrotransposon virus-like particles and their regulation by epigenetically activated small RNA.

In Arabidopsis, LTR retrotransposons are activated by mutations in the chromatin gene DECREASE in DNA METHYLATION 1 (DDM1), giving rise to 21- to 22-nt epigenetically activated siRNA (easiRNA) that depend on RNA DEPENDENT RNA POLYMERASE 6 (RDR6). We purified virus-like particles (VLPs) from ddm1 and ddm1rdr6 mutants in which genomic RNA is reverse transcribed into complementary DNA. High-throughput short-read and long-read sequencing of VLP DNA (VLP DNA-seq) revealed a comprehensive catalog of active LTR retrotransposons without the need for mapping transposition, as well as independent of genomic copy number. Linear replication intermediates of the functionally intact COPIA element EVADE revealed multiple central polypurine tracts (cPPTs), a feature shared with HIV in which cPPTs promote nuclear localization. For one member of the ATCOPIA52 subfamily (SISYPHUS), cPPT intermediates were not observed, but abundant circular DNA indicated transposon "suicide" by auto-integration within the VLP. easiRNA targeted EVADE genomic RNA, polysome association of GYPSY (ATHILA) subgenomic RNA, and transcription via histone H3 lysine-9 dimethylation. VLP DNA-seq provides a comprehensive landscape of LTR retrotransposons and their control at transcriptional, post-transcriptional, and reverse transcriptional levels.

Polymerase IV Plays a Crucial Role in Pollen Development in Capsella.

In Arabidopsis (Arabidopsis thaliana), DNA-dependent RNA polymerase IV (Pol IV) is required for the formation of transposable element (TE)-derived small RNA transcripts. These transcripts are processed by DICER-LIKE3 into 24-nucleotide small interfering RNAs (siRNAs) that guide RNA-directed DNA methylation. In the pollen grain, Pol IV is also required for the accumulation of 21/22-nucleotide epigenetically activated siRNAs, which likely silence TEs via post-transcriptional mechanisms. Despite this proposed role of Pol IV, its loss of function in Arabidopsis does not cause a discernible pollen defect. Here, we show that the knockout of NRPD1, encoding the largest subunit of Pol IV, in the Brassicaceae species Capsella (Capsella rubella), caused postmeiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were depleted in Capsella nrpd1 microspores. In the wild-type background, the same TEs produced 21/22-nucleotide and 24-nucleotide siRNAs; these processes required Pol IV activity. Arrest of Capsella nrpd1 microspores was accompanied by the deregulation of genes targeted by Pol IV-dependent siRNAs. TEs were much closer to genes in Capsella compared with Arabidopsis, perhaps explaining the essential role of Pol IV in pollen development in Capsella. Our discovery that Pol IV is functionally required in Capsella microspores emphasizes the relevance of investigating different plant models.

Epigenetic activation of meiotic recombination near Arabidopsis thaliana centromeres via loss of H3K9me2 and non-CG DNA methylation.

Eukaryotic centromeres contain the kinetochore, which connects chromosomes to the spindle allowing segregation. During meiosis, centromeres are suppressed for inter-homolog crossover, as recombination in these regions can cause chromosome missegregation and aneuploidy. Plant centromeres are surrounded by transposon-dense pericentromeric heterochromatin that is epigenetically silenced by histone 3 lysine 9 dimethylation (H3K9me2), and DNA methylation in CG and non-CG sequence contexts. However, the role of these chromatin modifications in control of meiotic recombination in the pericentromeres is not fully understood. Here, we show that disruption of Arabidopsis thaliana H3K9me2 and non-CG DNA methylation pathways, for example, via mutation of the H3K9 methyltransferase genes KYP/SUVH4 SUVH5 SUVH6, or the CHG DNA methyltransferase gene CMT3, increases meiotic recombination in proximity to the centromeres. Using immunocytological detection of MLH1 foci and genotyping by sequencing of recombinant plants, we observe that H3K9me2 and non-CG DNA methylation pathway mutants show increased pericentromeric crossovers. Increased pericentromeric recombination in H3K9me2/non-CG mutants occurs in hybrid and inbred backgrounds and likely involves contributions from both the interfering and noninterfering crossover repair pathways. We also show that meiotic DNA double-strand breaks (DSBs) increase in H3K9me2/non-CG mutants within the pericentromeres, via purification and sequencing of SPO11-1-oligonucleotides. Therefore, H3K9me2 and non-CG DNA methylation exert a repressive effect on both meiotic DSB and crossover formation in plant pericentromeric heterochromatin. Our results may account for selection of enhancer trap Dissociation (Ds) transposons into the CMT3 gene by recombination with proximal transposon launch-pads.

miRNAs trigger widespread epigenetically activated siRNAs from transposons in Arabidopsis.

In plants, post-transcriptional gene silencing (PTGS) is mediated by DICER-LIKE 1 (DCL1)-dependent microRNAs (miRNAs), which also trigger 21-nucleotide secondary short interfering RNAs (siRNAs) via RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), DCL4 and ARGONAUTE 1 (AGO1), whereas transcriptional gene silencing (TGS) of transposons is mediated by 24-nucleotide heterochromatic (het)siRNAs, RDR2, DCL3 and AGO4 (ref. 4). Transposons can also give rise to abundant 21-nucleotide 'epigenetically activated' small interfering RNAs (easiRNAs) in DECREASED DNA METHYLATION 1 (ddm1) and DNA METHYLTRANSFERASE 1 (met1) mutants, as well as in the vegetative nucleus of pollen grains and in dedifferentiated plant cell cultures. Here we show that easiRNAs in Arabidopsis thaliana resemble secondary siRNAs, in that thousands of transposon transcripts are specifically targeted by more than 50 miRNAs for cleavage and processing by RDR6. Loss of RDR6, DCL4 or DCL1 in a ddm1 background results in loss of 21-nucleotide easiRNAs and severe infertility, but 24-nucleotide hetsiRNAs are partially restored, supporting an antagonistic relationship between PTGS and TGS. Thus miRNA-directed easiRNA biogenesis is a latent mechanism that specifically targets transposon transcripts, but only when they are epigenetically reactivated during reprogramming of the germ line. This ancient recognition mechanism may have been retained both by transposons to evade long-term heterochromatic silencing and by their hosts for genome defence.

A Th2 Cytokine Profile in Appendicular Lavage Fluid Suggests Allergy as a Possible Etiology for Acute Appendicitis.

Acute appendicitis is the most frequent surgical abdominal emergency, but its etiology remains poorly understood. Histological examination of the appendix, following its removal due to acute appendicitis, consistently shows features in common with bronchial asthma, suggesting an allergic reaction as a candidate etiologic factor. Here, we propose the concept of appendicular lavage and use it to study the levels of the Th2 cytokines IL-4, IL-5, and IL-9 in patients with a clinical diagnosis of acute appendicitis. The study group included 20 patients with a histological diagnosis of phlegmonous appendicitis, 13 patients with gangrenous appendicitis, and a control group of 8 patients with a clinical diagnosis of appendicitis but with normal histology. Cytokine levels were higher in acute appendicitis. The difference was more pronounced when comparing phlegmonous appendicitis with nonpathological appendicitis (p = 0.01) for IL-4 (48.3 vs. 21.3 pg/mL), IL-5 (29.2 vs. 8.0 pg/mL), and IL-9 (34.1 vs. 16.6 pg/mL). This Th2 cytokine profile is compatible with the hypothesis of allergy as an etiologic factor for acute appendicitis and may have important implications for the diagnosis, prevention, and treatment of this condition.

Porcelain gallbladder.

A 62-year-old female was referenced to our outpatient clinic for a single episode of right upper quadrant pain and weight loss of 3 kg in the last 6 months. No other complaints were reported. Her past medical history was unremarkable except for total hysterectomy. The upper abdominal ultrasonography and abdominal plain X-ray revealed a porcelain gallbladder.

Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signature.

BACKGROUND: Current views on the control of cell development are anchored on the notion that phenotypes are defined by networks of transcriptional activity. The large amounts of information brought about by transcriptomics should allow the definition of these networks through the analysis of cell-specific transcriptional signatures. Here we test this principle by applying an analogue to comparative anatomy at the cellular level, searching for conserved transcriptional signatures, or conserved small gene-regulatory networks (GRNs) on root hairs (RH) and pollen tubes (PT), two filamentous apical growing cells that are a striking example of conservation of structure and function in plants. RESULTS: We developed a new method for isolation of growing and mature root hair cells, analysed their transcriptome by microarray analysis, and further compared it with pollen and other single cell transcriptomics data. Principal component analysis shows a statistical relation between the datasets of RHs and PTs which is suggestive of a common transcriptional profile pattern for the apical growing cells in a plant, with overlapping profiles and clear similarities at the level of small GTPases, vesicle-mediated transport and various specific metabolic responses. Furthermore, cis-regulatory element analysis of co-regulated genes between RHs and PTs revealed conserved binding sequences that are likely required for the expression of genes comprising the apical signature. This included a significant occurrence of motifs associated to a defined transcriptional response upon anaerobiosis. CONCLUSIONS: Our results suggest that maintaining apical growth mechanisms synchronized with energy yielding might require a combinatorial network of transcriptional regulation. We propose that this study should constitute the foundation for further genetic and physiological dissection of the mechanisms underlying apical growth of plant cells.

Targeted suppression of siRNA biogenesis in Arabidopsis pollen promotes triploid seed viability.

In plants, small-interfering RNAs (siRNAs) mediate epigenetic silencing via the RNA-directed DNA methylation (RdDM) pathway, which is particularly prominent during reproduction and seed development. However, there is limited understanding of the origins and dynamics of reproductive siRNAs acting in different cellular and developmental contexts. Here, we used the RNaseIII-like protein RTL1 to suppress siRNA biogenesis in Arabidopsis pollen, and found distinct siRNA subsets produced during pollen development. We demonstrate that RTL1 expression in the late microspore and vegetative cell strongly impairs epigenetic silencing, and resembles RdDM mutants in their ability to bypass interploidy hybridization barriers in the seed. However, germline-specific RTL1 expression did not impact transgenerational inheritance of triploid seed lethality. These results reveal the existence of multiple siRNA subsets accumulated in mature pollen, and suggest that mobile siRNAs involved in the triploid block are produced in germline precursor cells after meiosis, or in the vegetative cell during pollen mitosis.

Pseudouridine guides germline small RNA transport and epigenetic inheritance.

Developmental epigenetic modifications in plants and animals are mostly reset during gamete formation but some are inherited from the germline. Small RNAs guide these epigenetic modifications but how inherited small RNAs are distinguished in plants and animals is unknown. Pseudouridine (Ψ) is the most abundant RNA modification but has not been explored in small RNAs. Here, we develop assays to detect Ψ in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs. Germline small RNAs, namely epigenetically activated small interfering RNAs (easiRNAs) in Arabidopsis pollen and Piwi-interacting RNAs in mouse testes, are enriched for Ψ. In pollen, pseudouridylated easiRNAs are transported to sperm cells from the vegetative nucleus, and PAUSED/HEN5 (PSD), the plant homolog of Exportin-t, interacts genetically with Ψ and is required for this transport. We further show that Exportin-t is required for the triploid block: small RNA dosage-dependent seed lethality that is epigenetically inherited from pollen. Thus, Ψ has a conserved role in marking inherited small RNAs in the germline.

Establishing epigenetic variation during genome reprogramming.

Transgenerational reprogramming of DNA methylation is important for transposon silencing and epigenetic inheritance. A stochastic regulation of methylation states in the germline may lead to epigenetic variation and the formation of epialleles that contribute to phenotypic variation. In Arabidopsis thaliana inbred lines, the frequency of single base variation of DNA methylation is much higher than genetic mutation and, interestingly, variable epialleles are pre-methylated in the male germline. However, these same alleles are targeted for demethylation in the pollen vegetative nucleus, by a mechanism that seems to contribute to the accumulation of small RNAs that reinforce transcriptional gene silencing in the gametes. These observations are paving the way toward understanding the extent of epigenetic reprogramming in higher plants, and the mechanisms regulating the stability of acquired epigenetic states across generations.

Epigenetic control of transposons during plant reproduction: From meiosis to hybrid seeds.

The regulation of transposable elements (TEs) requires overlapping epigenetic modifications that must be reinforced every cell division and generation. In plants, this is achieved by multiple pathways including small RNAs, DNA methylation, and repressive histone marks that act together to control TE expression and activity throughout the entire life cycle. However, transient TE activation is observed during reproductive transitions as a result of epigenome reprogramming, thus providing windows of opportunity for TE proliferation and epigenetic novelty. Ultimately, these events may originate complex TE-driven transcriptional networks or cell-to-cell communication strategies via mobile small RNAs. In this review, we discuss recent findings and current understanding of TE regulation during sexual plant reproduction, and its implications for fertility, early seed development, and epigenetic inheritance.

Pseudouridine guides germline small RNA transport and epigenetic inheritance.

Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes1. Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation2,3. In C. elegans, these inherited small RNAs have poly (UG) tails4, but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine (Ψ) is the most abundant RNA modification but has not been explored in small RNAs. Here, we develop novel assays to detect Ψ in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs and their precursors. We also detect substantial enrichment in germline small RNAs, namely epigenetically activated siRNAs (easiRNAs) in Arabidopsis pollen, and piwi-interacting piRNAs in mouse testis. In pollen, pseudouridylated easiRNAs are localized to sperm cells, and we found that PAUSED/HEN5 (PSD), the plant homolog of Exportin-t, interacts genetically with Ψ and is required for transport of easiRNAs into sperm cells from the vegetative nucleus. We further show that Exportin-t is required for the triploid block: chromosome dosage-dependent seed lethality that is epigenetically inherited from pollen. Thus, Ψ has a conserved role in marking inherited small RNAs in the germline.

Whole genome analysis of gene expression reveals coordinated activation of signaling and metabolic pathways during pollen-pistil interactions in Arabidopsis.

Plant reproduction depends on the concerted activation of many genes to ensure correct communication between pollen and pistil. Here, we queried the whole transcriptome of Arabidopsis (Arabidopsis thaliana) in order to identify genes with specific reproductive functions. We used the Affymetrix ATH1 whole genome array to profile wild-type unpollinated pistils and unfertilized ovules. By comparing the expression profile of pistils at 0.5, 3.5, and 8.0 h after pollination and applying a number of statistical and bioinformatics criteria, we found 1,373 genes differentially regulated during pollen-pistil interactions. Robust clustering analysis grouped these genes in 16 time-course clusters representing distinct patterns of regulation. Coregulation within each cluster suggests the presence of distinct genetic pathways, which might be under the control of specific transcriptional regulators. A total of 78% of the regulated genes were expressed initially in unpollinated pistil and/or ovules, 15% were initially detected in the pollen data sets as enriched or preferentially expressed, and 7% were induced upon pollination. Among those, we found a particular enrichment for unknown transcripts predicted to encode secreted proteins or representing signaling and cell wall-related proteins, which may function by remodeling the extracellular matrix or as extracellular signaling molecules. A strict regulatory control in various metabolic pathways suggests that fine-tuning of the biochemical and physiological cellular environment is crucial for reproductive success. Our study provides a unique and detailed temporal and spatial gene expression profile of in vivo pollen-pistil interactions, providing a framework to better understand the basis of the molecular mechanisms operating during the reproductive process in higher plants.