Cellular metabolites have emerged as noncanonical RNA caps. Despite its early discovery as an RNA cap, the dephospho-CoA (dpCoA) cap remains largely uncharacterized because of a lack of detection technologies. Here we use biochemical and structural analysis to identify Arabidopsis NUDT11 as a specific decapping enzyme toward dpCoA-RNA. Leveraging this specificity, we develop biochemical and transcriptomic methods to quantify and profile dpCoA-RNA across the genome, revealing that dpCoA-RNAs exist across species and exhibit tissue-specific and/or condition-specific variations. In Arabidopsis, dpCoA-RNAs possess distinct transcription start sites and respond more rapidly to high light intensity as compared to 7-methylguanosine (m7G)-capped RNAs. Moreover, Arabidopsis dpCoA-RNAs can reach up to 15% of m7G-capped RNAs in abundance and are associated with translating ribosomes. We further demonstrate that an in vitro transcribed dpCoA-RNA is translated in human cells. This study uncovers a dynamic dpCoA cap that may potentially influence gene expression and establishes a toolkit for future investigations.

In the anthers of flowering plants, the innermost meiocytes and surrounding somatic cells (tapetum) are differentiated from the same precursor archesporial cells, and these cells acquire distinct cell identities after specification. However, the underlying mechanism regulating meiocyte identity is elusive. Here we demonstrate a conserved surveillance mechanism governed by Arabidopsis zinc-finger proteins C3H14 and C3H15, which redundantly regulate mRNA homoeostasis to ensure meiocyte identity. The meiocytes of Atc3h14 Atc3h15 display meiotic arrest accompanied by ectopic accumulation of mRNAs essential for archesporial cell differentiation and tapetum development. These meiocytes ultimately undergo reactive oxygen species bursts and programmed cell death in synchrony with the tapetum. Moreover, C3H14/C3H15 interacts with processing-body proteins and the CCR4-NOT deadenylase complex, and is required for eliminating unwanted transcripts. Consistently, CRISPR-Cas9-induced mutations in AtC3H14/15 paralogues in soybean and rice caused similar defects in meiocyte identity, indicating that the regulation of meiocyte identity by post-transcriptional RNA elimination is conserved in flowering plants.

Histone demethylases serve essential functions in plant growth and development across various species. However, their roles in Glycine max remain largely unexplored. This study identified GmJMJ19 and GmJMJ20, encoding JmjC domain-containing proteins. They exhibit circadian rhythmic expression patterns and interact with LUX ARRHYTHMO 2 (GmLUX2) both in vitro and in vivo. Although GmJMJ19/20 bind to histones, they lack conventional histone demethylase activity. Rather, GmJMJ19/20 function as endopeptidases that specifically cleave histone H3 peptides at unmethylated lysine 27 residues, in a manner independent of Fe²⁺ and α-ketoglutarate, the cofactors required for typical JmjC enzymes. Structural modeling supports the occlusion of the catalytic pocket that prevents access to methylated substrates. The simultaneous knockout of GmJMJ19/20 significantly delays flowering time. Comparative transcriptomic analyses reveal that the GmJM19/20-GmLUX2 module enhances GmFULc expression via independent of the canonical evening complex. Haplotype analysis suggests that GmJMJ19 underwent selection during domestication, potentially contributing to soybean geographical adaptation.

Meiotic crossover (CO) exchanges genetic information between homologs, thereby promoting genetic diversity among offspring. COs are non-randomly distributed across chromosomes, tending to occur in euchromatin, but rarely in heterochromatin. In plants, H3 lysine 27 monomethylation (H3K27me1) is crucial for maintaining heterochromatin condensation and genome stability in somatic cells; however, its role in germline cells remains to be determined. Here, we demonstrate that the plant-specific H3K27 mono-methyltransferases ATXR5/6 (ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5/6) play an important role in inhibiting CO formation in meiotic heterochromatin. In atxr5 atxr6, both ZMM-dependent Type I COs and ZMM-independent Type II COs are significantly increased. We further observed decondensation, decreased H3K27me1 signals, and specifically compromised non-CG methylation in atxr5 atxr6 meiotic heterochromatin. Unexpectedly, in contrast to their roles in somatic cells, where ATXR5/6 primarily regulate heterochromatin condensation and gene silencing without influencing DNA methylation, in meiocytes, ATXR5/6 mainly function in suppressing recombination and preserving heterochromatic DNA methylation without directly regulating gene expression. Moreover, loss of Type II CO regulator MMS AND UV SENSITIVE 81 (MUS81) leads to pericentromeric fragmentation and polyad formation during meiosis in the absence of ATXR5/6, indicating that MUS81 is critical for resolving atypical recombination intermediates in pericentromeric heterochromatin. Taken together, our results provide insights into the roles of ATXR5/6 in repressing meiotic recombination within heterochromatin by regulating chromosome compaction and modifications.

Small RNAs, including ta-siRNAs, play crucial roles in various processes in plants. Efforts have been made for decades to elucidate the biogenesis and function of ta-siRNAs. Though the key proteins involved in ta-siRNA biogenesis have been identified, the subcellular localization where ta-siRNAs are processed remains largely unexplored. Remarkably, non-coding TAS transcripts were reported to be bound by ribosomes, the machinery responsible for protein translation. Utilizing edited TAS genes in Arabidopsis, a combination of sRNA-seq, mRNA-seq, RIP-seq, and degradome-seq was employed to investigate the role of ribosomes in ta-siRNA biogenesis in this study. In the two-hit model, deletion of ribosome-binding regions resulted in a decrease in the abundance of intact TAS3 transcripts but did not significantly affect ta-siRNAs production or the efficiency of miRNA-guided cleavage. Conversely, the deletion of ribosome-binding regions led to a significant reduction in ta-siRNA abundance without affecting mRNA levels in the one-hit model. These findings indicate that in the two-hit model, ribosomes primarily stabilize TAS transcripts, while in the one-hit model, they suppress miRNA cleavage but facilitate subsequent processing. Collectively, this study proposes a model that ribosomes play distinct roles in the one-hit and two-hit models of ta-siRNA biogenesis, and provides a new angle to investigate the tangled connection between small RNAs, including miRNA and ta-siRNA, and translation.

Photosynthesis requires chloroplasts, in which most proteins are nucleus-encoded and produced via cytoplasmic translation. The translation initiation factor eIF5B gates the transition from initiation (I) to elongation (E), and the Kozak motif is associated with translation efficiency, but their relationship is previously unknown. Here, with ribosome profiling, we determined the genome-wide I-E transition efficiencies. We discovered that the most prevalent Kozak motif is associated with high I-E transition efficiency in Arabidopsis, rice, and wheat, thus implicating the potential of the Kozak motif in facilitating the I-E transition. Indeed, the effects of Kozak motifs in promoting translation depend on HOT3/eIF5B1 in Arabidopsis. HOT3 preferentially promotes the translation of photosynthesis-associated nuclear genes in a Kozak motif-dependent manner, which explains the chloroplast defects and reduced photosynthesis activity of hot3 mutants. Our study linked the Kozak motif to eIF5B-mediated I-E transition during translation and uncovered the function of HOT3 in the cytoplasmic translational control of chloroplast biogenesis and photosynthesis. Cytoplasmic mRNA translation contributes to the production of most chloroplast proteins. Here, the authors revealed that an interplay between Kozak motifs and HOT3/eIF5B1 controls the translation of nucleus-encoded chloroplast proteins.

Reciprocal exchanges of DNA between homologous chromosomes during meiosis, or crossovers (COs), shuffle genetic information in gametes and progeny. In many eukaryotes, the majority of COs (class I COs) are sensitive to a phenomenon called interference, which influences the occurrence of closely spaced double COs. Class I COs depend on a group of factors called ZMM (Zip, Msh, Mer) proteins including HEI10 (Human Enhancer of Invasion-10). However, how these proteins are recruited to class I CO sites is unclear. Here, we show that HEI10 forms foci on chromatin via a liquid–liquid phase separation (LLPS) mechanism that relies on residue Ser70. A HEI10 S70F allele results in LLPS failure and a defect in class I CO formation. We further used immunoprecipitation–mass spectrometry to identify RPA1a (Replication Protein A 1) as a HEI10 interacting protein. Surprisingly, we find that RPA1a also undergoes phase separation and its ubiquitination and degradation are directly regulated by HEI10. We also show that HEI10 is required for the condensation of other class I CO factors. Thus, our results provide mechanistic insight into how meiotic class I CO formation is controlled by HEI10 coupling LLPS and ubiquitination.

Meiotic recombination requires the specific RecA homolog DMC1 recombinase to stabilize strand exchange intermediates in most eukaryotes. Normal DMC1 levels are crucial for its function, yet the regulatory mechanisms of DMC1 stability are unknown in any organism. Here, we show that the degradation of Arabidopsis DMC1 by the 26S proteasome depends on F-box proteins RMF1/2-mediated ubiquitination. Furthermore, RMF1/2 interact with the Skp1 ortholog ASK1 to form the ubiquitin ligase complex SCFRMF1/2. Genetic analyses demonstrate that RMF1/2, ASK1 and DMC1 act in the same pathway downstream of SPO11-1 dependent meiotic DNA double strand break formation and that the proper removal of DMC1 is crucial for meiotic crossover formation. Moreover, six DMC1 lysine residues were identified as important for its ubiquitination but not its interaction with RMF1/2. Our results reveal mechanistic insights into how the stability of a key meiotic recombinase that is broadly conserved in eukaryotes is regulated.

Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S ribosomal RNA (rRNA) 3 & PRIME; end mat-uration during late -stage 40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the genome- wide effects of eIF5B have not been studied at the single-nucleotide resolution in any organism, and 18S rRNA 3 & PRIME; end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat stress acclimation by translational regula-tion, but its molecular function remained unknown. Here, we show that HOT3 is a late -stage ribosome biogenesis factor that facilitates 18S rRNA 3 & PRIME; end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3 & PRIME; end maturation or metabolism. We quantitatively defined processing hotspots and identified adenylation as the prevalent nontemplated RNA addition at the 3 & PRIME; ends of pre -18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNA interference to generate RDR1-and DCL2/4-dependent risiRNAs mainly from a 3 & PRIME; portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome -free fractions and were not responsi-ble for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late 40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, messenger RNA (mRNA) translation initiation, and siRNA biogenesis in plants.

Small RNAs are short non-coding nucleotides and regulate various biological processes in eukaryotes. During past decades, numerous studies have demonstrated the biogenesis of small RNAs and their modes of action in regulating gene expression at the transcriptional and post-transcriptional level. Based on the involvement of genes in biogenesis and function, small RNAs can be classified into three categories, miRNA, hc-siRNA, and phasiRNA. With the continuous advances in cell sorting/isolation technology and next-generation sequencing, recent discoveries provided evidence that all three categories play important roles in reproductive development in plants including model plants and crops. This review summarizes the recent findings on small RNAs, their potential targets, and genes involved in small RNA biogenesis in plant reproductive development and mainly focuses on gametogenesis.

Light initiates chloroplast biogenesis in Arabidopsis by eliminating PHYTOCHROME-INTERACTING transcription FACTORs (PIFs), which in turn de-represses nuclear photosynthesis genes, and synchronously, generates a nucleus-to-plastid (anterograde) signal that activates the plastid-encoded bacterial-type RNA polymerase (PEP) to transcribe plastid photosynthesis genes. However, the identity of the anterograde signal remains frustratingly elusive. The main challenge has been the difficulty to distinguish regulators from the plethora of necessary components for plastid transcription and other essential chloroplast functions, such as photosynthesis. Here, we show that the genome-wide induction of nuclear photosynthesis genes is insufficient to activate the PEP. PEP inhibition is imposed redundantly by multiple PIFs and requires PIF3's activator activity. Among the nuclear-encoded components of the PEP holoenzyme, we identify four light-inducible, PIF-repressed sigma factors as anterograde signals. Together, our results elucidate that light-dependent inhibition of PIFs activates plastid photosynthesis genes via sigma factors as anterograde signals in parallel with the induction of nuclear photosynthesis genes.

Meiotic recombination is initiated by the SPORULATION 11 (SPO11)–triggered formation of double-strand breaks (DSBs) that usually occur in open chromatin with active transcriptional features in many eukaryotes. However, gene transcription at DSB sites appears to be detrimental for repair, but the regulatory mechanisms governing transcription at meiotic DSB sites are largely undefined in plants. Here, we demonstrate that the largest DNA polymerase epsilon subunit POL2A interacts with SU(VAR)3 to 9 homologs SUVH2 and SUVH9. N-SIM (structured illumination microscopy) observation shows that the colocalization of SUVH2 with the meiotic DSB marker γ-H2AX is dependent on POL2A. RNA-seq of male meiocytes demonstrates that POL2A and SUVH2 jointly repress the expression of 865 genes, which have several known characteristics associated with meiotic DSB sites. Bisulfite-seq and small RNA-seq of male meiocytes support the idea that the silencing of these genes by POL2A and SUVH2/9 is likely independent of CHH methylation or 24-nt siRNA accumulation. Moreover, pol2a suvh2 suvh9 triple mutants have more severe defects in meiotic recombination and fertility compared with either pol2a or suvh2 suvh9 . Our results not only identify a epigenetic regulatory mechanism for gene silencing in male meiocytes but also reveal roles for DNA polymerase and SUVH2/9 beyond their classic functions in mitosis.

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.

In plants, RNA-directed DNA methylation (RdDM) uses small interfering RNAs (siRNAs) to target transposable elements (TEs) but usually avoids genes. RNA polymerase IV (Pol IV) shapes the landscape of DNA methylation through its pivotal role in siRNA biogenesis. However, how Pol IV is recruited to specific loci, particularly how it avoids genes, is poorly understood. Here, we identified a Pol IV–interacting protein, ZMP (zinc finger, mouse double-minute/switching complex B, Plus-3 protein), which exerts a dual role in regulating siRNA biogenesis and DNA methylation at specific genomic regions. ZMP is required for siRNA biogenesis at some pericentromeric regions and prevents Pol IV from targeting a subset of TEs and genes at euchromatic loci. As a chromatin-associated protein, ZMP prefers regions with depleted histone H3 lysine 4 (H3K4) methylation abutted by regions with H3K4 methylation, probably monitoring changes in local H3K4 methylation status to regulate Pol IV’s chromatin occupancy. Our findings uncover a mechanism governing the specificity of RdDM.

Meiosis is a specialized cell division that generates gametes and is essential for sexual reproduction. Studying meiosis in plants, like the model flowering plant Arabidopsis thaliana, contributes to our understanding of the fundamental biology of reproductive biology and has practical implications for improving economically important crop species. In this chapter, we provide a detailed protocol for capillary collection of Arabidopsis male meiocytes followed by total RNA extraction, RNA-Seq, and bioinformatics analysis of small-RNAs (sRNAs) including analysis of sRNA cluster that correlate with genomic features.

Male sterility enables hybrid crop breeding to increase yields and has been extensively studied. But thermo-sensitive female sterility, which is an ideal property that may enable full mechanization in hybrid rice breeding, has rarely been investigated due to the absence of such germplasm. Here we identify the spontaneous thermo-sensitive female sterility 1 (tfs1) mutation that confers complete sterility under regular/high temperature and partial fertility under low temperature as a point mutation in ARGONAUTE7 (AGO7). AGO7 associates with miR390 to form an RNA-Induced Silencing Complex (RISC), which triggers the biogenesis of small interfering RNAs (siRNAs) from TRANS-ACTING3 (TAS3) loci by recruiting SUPPRESSOR OF GENE SILENCING (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) to TAS3 transcripts. These siRNAs are known as tasiR-ARFs as they act in trans to repress auxin response factor genes. The mutant TFS1 (mTFS1) protein is compromised in its ability to load the miR390/miR390* duplex and eject miR390* during RISC formation. Furthermore, tasiR-ARF levels are reduced in tfs1 due to the deficiency in RDR6 but not SGS3 recruitment by mTFS1 RISC under regular/high temperature, while low temperature partially restores mTFS1 function in RDR6 recruitment and tasiR-ARF biogenesis. A miR390 mutant also exhibits female sterility, suggesting that female fertility is controlled by the miR390-AGO7 module. Notably, the tfs1 allele introduced into various elite rice cultivars endows thermo-sensitive female sterility. Moreover, field trials confirm the utility of tfs1 as a restorer line in fully mechanized hybrid rice breeding.

Scientists have developed many approaches based on PCR or next-generation sequencing to localize and characterize integrated T-DNAs in transgenic plants generated by Agrobacterium tumefaciens-mediated T-DNA transfer. However, none of these methods has the robust ability to handle all transgenic plants with diversified T-DNA patterns. Utilizing the valuable information in the whole-genome sequencing data of transgenic plants, we have developed a comprehensive approach (T-LOC) to localize and characterize T-DNA integration sites (TISs). We evaluated the performance of T-LOC on genome sequencing data from 48 transgenic rice (Oryza sativa) plants that provide real and unbiased resources of T-DNA integration patterns. T-LOC discovered 75 full TISs and reported a diversified pattern of T-DNA integration: the ideal single-copy T-DNA between two borders, multiple-copy of T-DNAs in tandem or inverted repeats, truncated partial T-DNAs with or without the selection hygromycin gene, the inclusion of T-DNA backbone, the integration at the genome repeat region, and the concatenation of multiple ideal or partial T-DNAs. In addition, we reported that DNA fragments from the two A. tumefaciens plasmids can be fused with T-DNA and integrated into the plant genome. Besides, T-LOC characterizes the genomic changes at TISs, including deletion, duplication, accurate repair, and chromosomal rearrangement. Moreover, we validated the robustness of T-LOC using PCR, Sanger sequencing, and Nanopore sequencing. In summary, T-LOC is a robust approach to studying the TISs independent of the integration pattern and can recover all types of TISs in transgenic plants.

MicroRNAs (miRNAs) are endogenous 20-24-nucleotide non-coding RNAs that play important regulatory roles in many biological processes in eukaryotes. miRNAs modulate the expression of target genes at the post-transcriptional level by transcript cleavage or translational inhibition. The identification of miRNA target genes has been extensively investigated in Arabidopsis and rice, but an in-depth global analysis of miRNA-mediated target regulation is still lacking in maize. Here, we report a transcriptome-wide identification of miRNA targets by analyzing parallel analysis of RNA ends (PARE) datasets derived from nine different tissues at five developmental stages of the maize (Zea mays L.) B73 cultivar. In total, 246 targets corresponding to 60 miRNAs from 25 families were identified, including transcription factors and other genes. In addition, PARE analysis revealed that miRNAs guide specific target transcript cleavage in a tissue-preferential manner. Primary transcripts of MIR159c and MIR169e were found to be cleaved by mature miR159 and miR169, respectively, indicating a negative-feedback regulatory mechanism in miRNA biogenesis. Moreover, several miRNA-target gene pairs involved in seed germination were identified and experimentally validated. Our PARE analyses generated a wide and detailed miRNA-target interaction atlas, which provides a valuable resource for investigating the roles of miRNAs and their targets in maize.