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.

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.

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.

Reactive oxygen nanobiocatalysts: activity-mechanism disclosures, catalytic center evolutions, and changing states.

Benefiting from low costs, structural diversities, tunable catalytic activities, feasible modifications, and high stability compared to the natural enzymes, reactive oxygen nanobiocatalysts (RONBCs) have become dominant materials in catalyzing and mediating reactive oxygen species (ROS) for diverse biomedical and biological applications. Decoding the catalytic mechanism and structure-reactivity relationship of RONBCs is critical to guide their future developments. Here, this timely review comprehensively summarizes the recent breakthroughs and future trends in creating and decoding RONBCs. First, the fundamental classification, activity, detection method, and reaction mechanism for biocatalytic ROS generation and elimination have been systematically disclosed. Then, the merits, modulation strategies, structure evolutions, and state-of-art characterisation techniques for designing RONBCs have been briefly outlined. Thereafter, we thoroughly discuss different RONBCs based on the reported major material species, including metal compounds, carbon nanostructures, and organic networks. In particular, we offer particular insights into the coordination microenvironments, bond interactions, reaction pathways, and performance comparisons to disclose the structure-reactivity relationships and mechanisms. In the end, the future challenge and perspectives for RONBCs are also carefully summarised. We envision that this review will provide a comprehensive understanding and guidance for designing ROS-catalytic materials and stimulate the wide utilisation of RONBCs in diverse biomedical and biological applications.

Correction: Reactive oxygen nanobiocatalysts: activity-mechanism disclosures, catalytic center evolutions, and changing states.

Correction for 'Reactive oxygen nanobiocatalysts: activity-mechanism disclosures, catalytic center evolutions, and changing states' by Sujiao Cao et al., Chem. Soc. Rev., 2023, https://doi.org/10.1039/d3cs00087g.

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.

Emerging 2D Materials for Electrocatalytic Applications: Synthesis, Multifaceted Nanostructures, and Catalytic Center Design.

Currently, the development of advanced 2D nanomaterials has become an interdisciplinary subject with extensive studies due to their extraordinary physicochemical performances. Beyond graphene, the emerging 2D-material-derived electrocatalysts (2D-ECs) have aroused great attention as one of the best candidates for heterogeneous electrocatalysis. The tunable physicochemical compositions and characteristics of 2D-ECs enable rational structural engineering at the molecular/atomic levels to meet the requirements of different catalytic applications. Due to the lack of instructive and comprehensive reviews, here, the most recent advances in the nanostructure and catalytic center design and the corresponding structure-function relationships of emerging 2D-ECs are systematically summarized. First, the synthetic pathways and state-of-the-art strategies in the multifaceted structural engineering and catalytic center design of 2D-ECs to promote their electrocatalytic activities, such as size and thickness, phase and strain engineering, heterojunctions, heteroatom doping, and defect engineering, are emphasized. Then, the representative applications of 2D-ECs in electrocatalytic fields are depicted and summarized in detail. Finally, the current breakthroughs and primary challenges are highlighted and future directions to guide the perspectives for developing 2D-ECs as highly efficient electrocatalytic nanoplatforms are clarified. This review provides a comprehensive understanding to engineer 2D-ECs and may inspire many novel attempts and new catalytic applications across broad fields.

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.

Transition Metal and Metal-Nx Codoped MOF-Derived Fenton-Like Catalysts: A Comparative Study on Single Atoms and Nanoparticles.

To deal with the ever-growing toxic benzene-derived compounds in the water system, extensive efforts have been dedicated for catalytic degradation of pollutants. However, the activities and efficiencies of the transition metal-based nanoparticles or single-atom sites are still ambiguous in Fenton-like reactions. Herein, to compare the Fenton-like catalytic efficiencies of the nanoparticles and single atoms, the free-standing nanofibrous catalyst comprising Co nanocrystals and Co-Nx codoped carbon nanotubes (CNTs) or bare Co-Nx doped CNTs is fabricated. It is noteworthy that all these nanofibrous catalysts exhibit efficient activities, mesoporous structures, and conductive carbon networks, which allow a feasible validation of the catalytic effects. Benefiting from the maximized atomic utilization, the atomic Co-Nx centers exhibit much higher reaction kinetic constant (κ = 0.157 min-1 ) and mass activity toward the degradation of bisphenol A, far exceeding the Co nanocrystals (κ = 0.082 min-1 ). However, for the volume activities, the single-atom catalyst does not show apparent advantages compared to the nanocrystal-based catalyst. Overall, this work not only provides a viable pathway for comparing Fenton-like catalytic effects of transition metal-based nanoparticles or single atoms but also opens up a new avenue for developing prominent catalysts for organic pollutants' degradation.

BICELLULAR POLLEN 1 is a modulator of DNA replication and pollen development in Arabidopsis.

During male gametogenesis in Arabidopsis, the haploid microspore undergoes an asymmetric division to produce a vegetative and a generative cell, the latter of which continues to divide symmetrically to form two sperms. This simple system couples cell cycle with cell fate specification. Here we addressed the role of DNA replication in male gametogenesis using a mutant bicellular pollen 1 (bice1), which produces bicellular, rather than tricellular, pollen grains as in the wild-type plant at anthesis. The mutation prolonged DNA synthesis of the generative cell, which resulted in c. 40% of pollen grains arrested at the two-nucleate stage. The extended S phase did not impact the cell fate of the generative cell as shown by cell-specific markers. BICE1 encodes a plant homolog of human D123 protein that is required for G1 progression, but the underlying mechanism is unknown. Here we showed that BICE1 interacts with MCM4 and MCM7 of the pre-replication complex. Consistently, double mutations in BICE1 and MCM4, or MCM7, also led to bicellular pollen and condensed chromosomes. These suggest that BICE1 plays a role in modulating DNA replication via interaction with MCM4 and MCM7.

The Ca2+ /calmodulin2-binding transcription factor TGA3 elevates LCD expression and H2 S production to bolster Cr6+ tolerance in Arabidopsis.

Heavy metal (HM) contamination on agricultural land not only reduces crop yield but also causes human health concerns. As a plant gasotransmitter, hydrogen sulfide (H2 S) can trigger various defense responses and help reduce accumulation of HMs in plants; however, little is known about the regulatory mechanisms of H2 S signaling. Here, we provide evidence to answer the long-standing question about how H2 S production is elevated in the defense of plants against HM stress. During the response of Arabidopsis to chromium (Cr6+ ) stress, the transcription of L-cysteine desulfhydrase (LCD), the key enzyme for H2 S production, was enhanced through a calcium (Ca2+ )/calmodulin2 (CaM2)-mediated pathway. Biochemistry and molecular biology studies demonstrated that Ca2+ /CaM2 physically interacts with the bZIP transcription factor TGA3, a member of the 'TGACG'-binding factor family, to enhance binding of TGA3 to the LCD promoter and increase LCD transcription, which then promotes the generation of H2 S. Consistent with the roles of TGA3 and CaM2 in activating LCD expression, both cam2 and tga3 loss-of-function mutants have reduced LCD abundance and exhibit increased sensitivity to Cr6+ stress. Accordingly, this study proposes a regulatory pathway for endogenous H2 S generation, indicating that plants respond to Cr6+ stress by adjusting the binding affinity of TGA3 to the LCD promoter, which increases LCD expression and promotes H2 S production. This suggests that manipulation of the endogenous H2 S level through genetic engineering could improve the tolerance of grains to HM stress and increase agricultural production on soil contaminated with HMs.

Single-molecule targeted accessibility and methylation sequencing of centromeres, telomeres and rDNAs in Arabidopsis.

The short read-length of next-generation sequencing makes it challenging to characterize highly repetitive regions (HRRs) such as centromeres, telomeres and ribosomal DNAs. Based on recent strategies that combined long-read sequencing and exogenous enzymatic labelling of open chromatin, we developed single-molecule targeted accessibility and methylation sequencing (STAM-seq) in plants by further integrating nanopore adaptive sampling to investigate the HRRs in wild-type Arabidopsis and DNA methylation mutants that are defective in CG- or non-CG methylation. We found that CEN180 repeats show higher chromatin accessibility and lower DNA methylation on their forward strand, individual rDNA units show a negative correlation between their DNA methylation and accessibility, and both accessibility and CHH methylation levels are lower at telomere compared to adjacent subtelomeric region. Moreover, DNA methylation-deficient mutants showed increased chromatin accessibility at HRRs, consistent with the role of DNA methylation in maintaining heterochromatic status in plants. STAM-seq can be applied to study accessibility and methylation of repetitive sequences across diverse plant species.

Molecular mechanism of HNTX-I activates the intermediate-conductance Ca2+-activated K+ (IK) channels.

The IK channel, a potassium ion channel regulated by calcium ions and voltages in a bidirectional manner, has been implicated in a range of diseases. However, there are currently few compounds available that can target the IK channel with high potency and specificity. Hainantoxin-I (HNTX-I) is the first peptide activator of IK channel discovered so far, but its activity is not ideal, and the underlying mechanism interaction between HNTX-I toxin and IK channel remains unclear. Thus, our study aimed to enhance the potency of IK channel activating peptides derived from HNTX-I and elucidate the molecular mechanism underlying the interaction between HNTX-I and the IK channel. By employing virtual alanine scanning mutagenesis, we generated 11 HNTX-I mutants using site-directed mutagenesis to pinpoint specific residues crucial for the HNTX-I and IK channel interaction. Subsequently, we identified key residues on the IK channel that are involved in the interaction with HNTX-I. Additionally, molecular docking was employed to guide the molecular engineering process and clarify the binding interface between HNTX-I and the IK channel. Our results demonstrate that HNTX-I primarily acts on the IK channel via the N-terminal amino acid, and its interaction with the IK channel is mediated by electrostatic and hydrophobic interactions, specifically the amino acid residues at positions 1, 3, 5, and 7 on HNTX-I. This study provides valuable insights into the peptide toxins that may serve as potential templates for the development of activators with enhanced potency and selectivity for the IK channel.

Integrated single-nucleus and spatial transcriptomics captures transitional states in soybean nodule maturation.

Legumes form symbiosis with rhizobium leading to the development of nitrogen-fixing nodules. By integrating single-nucleus and spatial transcriptomics, we established a cell atlas of soybean nodules and roots. In central infected zones of nodules, we found that uninfected cells specialize into functionally distinct subgroups during nodule development, and revealed a transitional subtype of infected cells with enriched nodulation-related genes. Overall, our results provide a single-cell perspective for understanding rhizobium-legume symbiosis.

Single-nucleus transcriptomes reveal spatiotemporal symbiotic perception and early response in Medicago.

Establishing legume-rhizobial symbiosis requires precise coordination of complex responses in a time- and cell type-specific manner. Encountering Rhizobium, rapid changes of gene expression levels in host plants occur in the first few hours, which prepare the plants to turn off defence and form a symbiotic relationship with the microbes. Here, we applied single-nucleus RNA sequencing to characterize the roots of Medicago truncatula at 30 min, 6 h and 24 h after nod factor treatment. We found drastic global gene expression reprogramming at 30 min in the epidermis and cortex and most of these changes were restored at 6 h. Moreover, plant defence response genes are activated at 30 min and subsequently suppressed at 6 h in non-meristem cells. Only in the cortical cells but not in other cell types, we found the flavonoid synthase genes required to recruit rhizobia are highly expressed 30 min after inoculation with nod factors. A gene module enriched for symbiotic nitrogen fixation genes showed that MtFER (MtFERONIA) and LYK3 (LysM domain receptor-like kinase 3) share similar responses to symbiotic signals. We further found that MtFER can be phosphorylated by LYK3 and it participates in rhizobial symbiosis. Our results expand our understanding of dynamic spatiotemporal symbiotic responses at the single-cell level.

Genome-wide characterization of nascent RNA processing in plants.

Following transcription initiation, RNA polymerase II (Pol II) elongates through the genic region and terminates after the polyadenylation signal. This process is accompanied by splicing, 3' cleavage, and polyadenylation, to eventually form a mature mRNA. Recent advances in short-read and long-read high-throughput sequencing methods have shed light on the global landscape of these co-transcriptional events at nucleotide resolution. In this mini review, we summarize recent developments in genome-wide approaches that broadened our understanding of nascent RNA processing in plants.

An atlas of plant full-length RNA reveals tissue-specific and monocots-dicots conserved regulation of poly(A) tail length.

Poly(A) tail is a hallmark of eukaryotic messenger RNA and its length plays an essential role in regulating mRNA metabolism. However, a comprehensive resource for plant poly(A) tail length has yet to be established. Here, we applied a poly(A)-enrichment-free, nanopore-based method to profile full-length RNA with poly(A) tail information in plants. Our atlas contains over 120 million polyadenylated mRNA molecules from seven different tissues of Arabidopsis, as well as the shoot tissue of maize, soybean and rice. In most tissues, the size of plant poly(A) tails shows peaks at approximately 20 and 45 nucleotides, while the poly(A) tails in pollen exhibit a distinct pattern with strong peaks centred at 55 and 80 nucleotides. Moreover, poly(A) tail length is regulated in a gene-specific manner-mRNAs with short half-lives in general have long poly(A) tails, while mRNAs with long half-lives are featured with relatively short poly(A) tails that peak at ~45 nucleotides. Across species, poly(A) tails in the nucleus are almost twice as long as in the cytoplasm. Our comprehensive dataset lays the groundwork for future functional and evolutionary studies on poly(A) tail length regulation in plants.