A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis.

RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nt small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30 to 40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5'-adenine preference. P4RNAs exhibit transcription start sites similar to Pol II products and are featured with 5'-monophosphates and 3'-misincorporated nucleotides. The 3'-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a "one precursor, one siRNA" model for the biogenesis of 24-nt siRNAs in Arabidopsis.

Light controls mesophyll-specific post-transcriptional splicing of photoregulatory genes by AtPRMT5.

Light plays a central role in plant growth and development, providing an energy source and governing various aspects of plant morphology. Previous study showed that many polyadenylated full-length RNA molecules within the nucleus contain unspliced introns (post-transcriptionally spliced introns, PTS introns), which may play a role in rapidly responding to changes in environmental signals. However, the mechanism underlying post-transcriptional regulation during initial light exposure of young, etiolated seedlings remains elusive. In this study, we used FLEP-seq2, a Nanopore-based sequencing technique, to analyze nuclear RNAs in Arabidopsis (Arabidopsis thaliana) seedlings under different light conditions and found numerous light-responsive PTS introns. We also used single-nucleus RNA sequencing (snRNA-seq) to profile transcripts in single nucleus and investigate the distribution of light-responsive PTS introns across distinct cell types. We established that light-induced PTS introns are predominant in mesophyll cells during seedling de-etiolation following exposure of etiolated seedlings to light. We further demonstrated the involvement of the splicing-related factor A. thaliana PROTEIN ARGININE METHYLTRANSFERASE 5 (AtPRMT5), working in concert with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a critical repressor of light signaling pathways. We showed that these two proteins orchestrate light-induced PTS events in mesophyll cells and facilitate chloroplast development, photosynthesis, and morphogenesis in response to ever-changing light conditions. These findings provide crucial insights into the intricate mechanisms underlying plant acclimation to light at the cell-type level.

Plant 22-nt siRNAs mediate translational repression and stress adaptation.

Small interfering RNAs (siRNAs) are essential for proper development and immunity in eukaryotes1. Plants produce siRNAs with lengths of 21, 22 or 24 nucleotides. The 21- and 24-nucleotide species mediate cleavage of messenger RNAs and DNA methylation2,3, respectively, but the biological functions of the 22-nucleotide siRNAs remain unknown. Here we report the identification and characterization of a group of endogenous 22-nucleotide siRNAs that are generated by the DICER-LIKE 2 (DCL2) protein in plants. When cytoplasmic RNA decay and DCL4 are deficient, the resulting massive accumulation of 22-nucleotide siRNAs causes pleiotropic growth disorders, including severe dwarfism, meristem defects and pigmentation. Notably, two genes that encode nitrate reductases-NIA1 and NIA2-produce nearly half of the 22-nucleotide siRNAs. Production of 22-nucleotide siRNAs triggers the amplification of gene silencing and induces translational repression both gene specifically and globally. Moreover, these 22-nucleotide siRNAs preferentially accumulate upon environmental stress, especially those siRNAs derived from NIA1/2, which act to restrain translation, inhibit plant growth and enhance stress responses. Thus, our research uncovers the unique properties of 22-nucleotide siRNAs, and reveals their importance in plant adaptation to environmental stresses.

Genome evolution and initial breeding of the Triticeae grass Leymus chinensis dominating the Eurasian Steppe.

Leymus chinensis, a dominant perennial grass in the Eurasian Steppe, is well known for its remarkable adaptability and forage quality. Hardly any breeding has been done on the grass, limiting its potential in ecological restoration and forage productivity. To enable genetic improvement of the untapped, important species, we obtained a 7.85-Gb high-quality genome of L. chinensis with a particularly long contig N50 (318.49 Mb). Its allotetraploid genome is estimated to originate 5.29 million years ago (MYA) from a cross between the Ns-subgenome relating to Psathyrostachys and the unknown Xm-subgenome. Multiple bursts of transposons during 0.433-1.842 MYA after genome allopolyploidization, which involved predominantly the Tekay and Angela of LTR retrotransposons, contributed to its genome expansion and complexity. With the genome resource available, we successfully developed a genetic transformation system as well as the gene-editing pipeline in L. chinensis. We knocked out the monocot-specific miR528 using CRISPR/Cas9, resulting in the improvement of yield-related traits with increases in the tiller number and growth rate. Our research provides valuable genomic resources for Triticeae evolutionary studies and presents a conceptual framework illustrating the utilization of genomic information and genome editing to accelerate the improvement of wild L. chinensis with features such as polyploidization and self-incompatibility.

Cell type-specific cytonuclear coevolution in three allopolyploid plant species.

Cytonuclear disruption may accompany allopolyploid evolution as a consequence of the merger of different nuclear genomes in a cellular environment having only one set of progenitor organellar genomes. One path to reconcile potential cytonuclear mismatch is biased expression for maternal gene duplicates (homoeologs) encoding proteins that target to plastids and/or mitochondria. Assessment of this transcriptional form of cytonuclear coevolution at the level of individual cells or cell types remains unexplored. Using single-cell (sc-) and single-nucleus (sn-) RNAseq data from eight tissues in three allopolyploid species, we characterized cell type-specific variations of cytonuclear coevolutionary homoeologous expression and demonstrated the temporal dynamics of expression patterns across development stages during cotton fiber development. Our results provide unique insights into transcriptional cytonuclear coevolution in plant allopolyploids at the single-cell level.

MCIBox: a toolkit for single-molecule multi-way chromatin interaction visualization and micro-domains identification.

The emerging ligation-free three-dimensional (3D) genome mapping technologies can identify multiplex chromatin interactions with single-molecule precision. These technologies not only offer new insight into high-dimensional chromatin organization and gene regulation, but also introduce new challenges in data visualization and analysis. To overcome these challenges, we developed MCIBox, a toolkit for multi-way chromatin interaction (MCI) analysis, including a visualization tool and a platform for identifying micro-domains with clustered single-molecule chromatin complexes. MCIBox is based on various clustering algorithms integrated with dimensionality reduction methods that can display multiplex chromatin interactions at single-molecule level, allowing users to explore chromatin extrusion patterns and super-enhancers regulation modes in transcription, and to identify single-molecule chromatin complexes that are clustered into micro-domains. Furthermore, MCIBox incorporates a two-dimensional kernel density estimation algorithm to identify micro-domains boundaries automatically. These micro-domains were stratified with distinctive signatures of transcription activity and contained different cell-cycle-associated genes. Taken together, MCIBox represents an invaluable tool for the study of multiple chromatin interactions and inaugurates a previously unappreciated view of 3D genome structure.

Multiplex CRISPR-Cas9 editing of DNA methyltransferases in rice uncovers a class of non-CG methylation specific for GC-rich regions.

DNA methylation in the non-CG context is widespread in the plant kingdom and abundant in mammalian tissues such as the brain and pluripotent cells. Non-CG methylation in Arabidopsis thaliana is coordinately regulated by DOMAINS REARRANGED METHYLTRANSFERASE (DRM) and CHROMOMETHYLASE (CMT) proteins but has yet to be systematically studied in major crops due to difficulties in obtaining genetic materials. Here, utilizing the highly efficient multiplex CRISPR-Cas9 genome-editing system, we created single- and multiple-knockout mutants for all the nine DNA methyltransferases in rice (Oryza sativa) and profiled their whole-genome methylation status at single-nucleotide resolution. Surprisingly, the simultaneous loss of DRM2, CHROMOMETHYLASE3 (CMT2), and CMT3 functions, which completely erases all non-CG methylation in Arabidopsis, only partially reduced it in rice. The regions that remained heavily methylated in non-CG contexts in the rice Os-dcc (Osdrm2/cmt2/cmt3a) triple mutant had high GC contents. Furthermore, the residual non-CG methylation in the Os-dcc mutant was eliminated in the Os-ddccc (Osdrm2/drm3/cmt2/cmt3a/cmt3b) quintuple mutant but retained in the Os-ddcc (Osdrm2/drm3/cmt2/cmt3a) quadruple mutant, demonstrating that OsCMT3b maintains non-CG methylation in the absence of other major methyltransferases. Our results showed that OsCMT3b is subfunctionalized to accommodate a distinct cluster of non-CG-methylated sites at highly GC-rich regions in the rice genome.

Correction: ARGONAUTE10 promotes the degradation of miR165/6 through the SDN1 and SDN2 exonucleases in Arabidopsis.

[This corrects the article DOI: 10.1371/journal.pbio.2001272.].

Soybean DICER-LIKE2 Regulates Seed Coat Color via Production of Primary 22-Nucleotide Small Interfering RNAs from Long Inverted Repeats.

In plants, 22-nucleotide small RNAs trigger the production of secondary small interfering RNAs (siRNAs) and enhance silencing. DICER-LIKE2 (DCL2)-dependent 22-nucleotide siRNAs are rare in Arabidopsis (Arabidopsis thaliana) and are thought to function mainly during viral infection; by contrast, these siRNAs are abundant in many crops such as soybean (Glycine max) and maize (Zea mays). Here, we studied soybean 22-nucleotide siRNAs by applying CRISPR-Cas9 to simultaneously knock out the two copies of soybean DCL2, GmDCL2a and GmDCL2b, in the Tianlong1 cultivar. Small RNA sequencing revealed that most 22-nucleotide siRNAs are derived from long inverted repeats (LIRs) and disappeared in the Gmdcl2a/2b double mutant. De novo assembly of a Tianlong1 reference genome and transcriptome profiling identified an intronic LIR formed by the chalcone synthase (CHS) genes CHS1 and CHS3 This LIR is the source of primary 22-nucleotide siRNAs that target other CHS genes and trigger the production of secondary 21-nucleotide siRNAs. Disruption of this process in Gmdcl2a/2b mutants substantially increased CHS mRNA levels in the seed coat, thus changing the coat color from yellow to brown. Our results demonstrated that endogenous LIR-derived transcripts in soybean are predominantly processed by GmDCL2 into 22-nucleotide siRNAs and uncovered a role for DCL2 in regulating natural traits.

Interplay of phytohormones and epigenetic regulation: A recipe for plant development and plasticity.

Both phytohormone signaling and epigenetic mechanisms have long been known to play crucial roles in plant development and plasticity in response to ambient stimuli. Indeed, diverse signaling pathways mediated by phytohormones and epigenetic processes integrate multiple upstream signals to regulate various plant traits. Emerging evidence indicates that phytohormones and epigenetic processes interact at multiple levels. In this review, we summarize the current knowledge of the interplay between phytohormones and epigenetic processes from the perspective of phytohormone biology. We also review chemical regulators used in epigenetic studies and propose strategies for developing novel regulators using multidisciplinary approaches.

Safeguard DCL2-Dependent 22-nt siRNA generation by DCL1.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are crucial for plant growth and development via mediating post-transcriptional gene silencing. In wild-type Arabidopsis, DICER-LIKE 2 (DCL2)-dependent 22-nt siRNAs are rare, whereas DCL1 and DCL4-dependent 21-nt miRNAs and siRNAs are highly abundant. DCL4 naturally inhibits DCL2 in producing abundant 22-nt siRNAs from endogenous transcripts, but whether DCL1 suppresses endogenous 22-nt siRNA production and the extent of repression are still unknown. Here, we report that DCL1 and DCL2 cleaved both miRNA precursors and coding transcript-derived double-stranded RNAs. In a dcl1 dcl4 double mutant, massive 22-nt siRNAs were produced from endogenous protein-coding genes (genic siRNAs). Compared with wild-type, the 22-nt genic siRNAs derived from the Nitrate Reductase 1 (NIA1), NIA2, DIACYLGLYCEROL ACYLTRANSFERASES 3 (DGAT3), SUPPRESSOR OF MAX2 1-LIKE 5 (SMXL5), and SMXL4 in dcl1 dcl4 increased up to 95%. Our analysis further indicated that the 22-nt genic siRNAs in dcl1 dcl4 were mainly loaded into ARGONAUTE 1 (AGO1) or AGO2. Thus, our results demonstrated that both DCL1 and DCL4 safeguard post-transcriptional gene silencing, preventing the production of DCL2-dependent 22-nt genic siRNAs from disrupting plant growth and development.

MicroRNA482/2118, a miRNA superfamily essential for both disease resistance and plant development.

MicroRNAs (miRNAs) are a class of 21-24 nucleotides (nt) noncoding small RNAs ubiquitously distributed across the plant kingdom. miR482/2118, one of the conserved miRNA superfamilies originating from gymnosperms, has divergent main functions in core-angiosperms. It mainly regulates NUCLEOTIDE BINDING SITE-LEUCINE-RICH REPEAT (NBS-LRR) genes in eudicots, functioning as an essential component in plant disease resistance; in contrast, it predominantly targets numerous long noncoding RNAs (lncRNAs) in monocot grasses, which are vital for plant reproduction. Usually, miR482/2118 is 22-nt in length, which can trigger the production of phased small interfering RNAs (phasiRNAs) after directed cleavage. PhasiRNAs instigated from target genes of miR482/2118 enhance their roles in corresponding biological processes by cis-regulation on cognate genes and expands their function to other pathways via trans activity on different genes. This review summarizes the origin, biogenesis, conservation, and evolutionary characteristics of the miR482/2118 superfamily and delineates its diverse functions in disease resistance, plant development, stress responses, etc.

Genome-wide Hi-C analyses in wild-type and mutants reveal high-resolution chromatin interactions in Arabidopsis.

Chromosomes form 3D structures that are critical to the regulation of cellular and genetic processes. Here, we present a study of global chromatin interaction patterns in Arabidopsis thaliana. Our genome-wide approach confirmed interactions that were previously observed by other methods as well as uncovered long-range interactions such as those among small heterochromatic regions embedded in euchromatic arms. We also found that interactions are correlated with various epigenetic marks that are localized in active or silenced chromatin. Arabidopsis chromosomes do not contain large local interactive domains that resemble the topological domains described in animals but, instead, contain relatively small interactive regions scattered around the genome that contain H3K27me3 or H3K9me2. We generated interaction maps in mutants that are defective in specific epigenetic pathways and found altered interaction patterns that correlate with changes in the epigenome. These analyses provide further insights into molecular mechanisms of epigenetic regulation of the genome.

Induced Mutation in GmCOP1b Enhances the Performance of Soybean under Dense Planting Conditions.

CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is the key photomorphogenic inhibitor that has been extensively studied in higher plants. Nevertheless, its role has not been documented in the economically important soybean. Here we investigated the functions of two COP1 homologous genes, GmCOP1a and GmCOP1b, by analyzing Gmcop1a and Gmcop1b mutants with indels using CRISPR in soybean. We revealed that, although both genes are required for skotomorphogenesis in the dark, the GmCOP1b gene seems to play a more prominent role than GmCOP1a in promoting stem elongation under normal light conditions. Consistently, the bZIP transcriptional factors STF1/2, which repress stem elongation in soybean, accumulated to the highest level in the Gmcop1a1b double mutant, followed by the Gmcop1b and Gmcop1a mutants. Furthermore, the Gmcop1b mutants showed reduced shade response and enhanced performance under high-density conditions in field trials. Taken together, this study provides essential genetic resources for elucidating functional mechanisms of GmCOP1 and breeding of high yield soybean cultivars for future sustainable agriculture.

The antagonistic MYB paralogs RH1 and RH2 govern anthocyanin leaf markings in Medicago truncatula.

Patterned leaf coloration in plants generates remarkable diversity in nature, but the underlying mechanisms remain largely unclear. Here, using Medicago truncatula leaf marking as a model, we show that the classic M. truncatula leaf anthocyanin spot trait depends on two R2R3 MYB paralogous regulators, RED HEART1 (RH1) and RH2. RH1 mainly functions as an anthocyanin biosynthesis activator that specifically determines leaf marking formation depending on its C-terminal activation motif. RH1 physically interacts with the M. truncatula bHLH protein MtTT8 and the WDR family member MtWD40-1, and this interaction facilitates RH1 function in leaf anthocyanin marking formation. RH2 has lost transcriptional activation activity, due to a divergent C-terminal domain, but retains the ability to interact with the same partners, MtTT8 and MtWD40-1, as RH1, thereby acting as a competitor in the regulatory complex and exerting opposite effects. Moreover, our results demonstrate that RH1 can activate its own expression and that RH2-mediated competition can repress RH1 expression. Our findings reveal the molecular mechanism of the antagonistic gene paralogs RH1 and RH2 in determining anthocyanin leaf markings in M. truncatula, providing a multidimensional paralogous-antagonistic regulatory paradigm for fine-tuning patterned pigmentation.

An Online Database for Exploring Over 2,000 Arabidopsis Small RNA Libraries.

Small RNAs (sRNAs) play a wide range of important roles in plants, from maintaining genome stability and enhancing disease resistance to regulating developmental processes. Over the past decade, next-generation sequencing technologies have allowed us to explore the sRNA populations with unprecedented depth and accuracy. The community has accumulated a tremendous amount of sRNA sequencing (sRNA-seq) data from various genotypes, tissues, and treatments. However, it has become increasingly challenging to access these "big data" and extract useful information, particularly for researchers lacking sophisticated bioinformatics tools and expensive computational resources. Here, we constructed an online website, Arabidopsis Small RNA Database (ASRD, http://ipf.sustech.edu.cn/pub/asrd), that allows users to easily explore the information from publicly available Arabidopsis (Arabidopsis thaliana) sRNA libraries. Our database contains ∼2.3 billion sRNA reads, representing ∼250 million unique sequences from 2,024 sRNA-seq libraries. We downloaded the raw data for all libraries and reprocessed them with a unified pipeline so that the normalized abundance of any particular sRNA or the sum of abundances of sRNAs from a genic or transposable element region can be compared across all libraries. We also integrated an online Integrative Genomics Viewer browser into our Web site for convenient visualization. ASRD is a free, web-accessible, and user-friendly database that supports the direct query of over 2,000 Arabidopsis sRNA-seq libraries. We believe this resource will help plant researchers take advantage of the vast next-generation sequencing datasets available in the public domain.

Large-scale comparative epigenomics reveals hierarchical regulation of non-CG methylation in Arabidopsis.

Genome-wide characterization by next-generation sequencing has greatly improved our understanding of the landscape of epigenetic modifications. Since 2008, whole-genome bisulfite sequencing (WGBS) has become the gold standard for DNA methylation analysis, and a tremendous amount of WGBS data has been generated by the research community. However, the systematic comparison of DNA methylation profiles to identify regulatory mechanisms has yet to be fully explored. Here we reprocessed the raw data of over 500 publicly available Arabidopsis WGBS libraries from various mutant backgrounds, tissue types, and stress treatments and also filtered them based on sequencing depth and efficiency of bisulfite conversion. This enabled us to identify high-confidence differentially methylated regions (hcDMRs) by comparing each test library to over 50 high-quality wild-type controls. We developed statistical and quantitative measurements to analyze the overlapping of DMRs and to cluster libraries based on their effect on DNA methylation. In addition to confirming existing relationships, we revealed unanticipated connections between well-known genes. For instance, MET1 and CMT3 were found to be required for the maintenance of asymmetric CHH methylation at nonoverlapping regions of CMT2 targeted heterochromatin. Our comparative methylome approach has established a framework for extracting biological insights via large-scale comparison of methylomes and can also be adopted for other genomics datasets.

MS23, a master basic helix-loop-helix factor, regulates the specification and development of the tapetum in maize.

Successful male gametogenesis involves orchestration of sequential gene regulation for somatic differentiation in pre-meiotic anthers. We report here the cloning of Male Sterile23 (Ms23), encoding an anther-specific predicted basic helix-loop-helix (bHLH) transcription factor required for tapetal differentiation; transcripts localize initially to the precursor secondary parietal cells then predominantly to daughter tapetal cells. In knockout ms23-ref mutant anthers, five instead of the normal four wall layers are observed. Microarray transcript profiling demonstrates a more severe developmental disruption in ms23-ref than in ms32 anthers, which possess a different bHLH defect. RNA-seq and proteomics data together with yeast two-hybrid assays suggest that MS23 along with MS32, bHLH122 and bHLH51 act sequentially as either homo- or heterodimers to choreograph tapetal development. Among them, MS23 is the earliest-acting factor, upstream of bHLH51 and bHLH122, controlling tapetal specification and maturation. By contrast, MS32 is constitutive and independently regulated and is required later than MS23 in tapetal differentiation.