Characterization of organelle DNA degradation mediated by DPD1 exonuclease in the rice genome-edited line.

Mitochondria and plastids, originated as ancestral endosymbiotic bacteria, contain their own DNA sequences. These organelle DNAs (orgDNAs) are, despite the limited genetic information they contain, an indispensable part of the genetic systems but exist as multiple copies, making up a substantial amount of total cellular DNA. Given this abundance, orgDNA is known to undergo tissue-specific degradation in plants. Previous studies have shown that the exonuclease DPD1, conserved among seed plants, degrades orgDNAs during pollen maturation and leaf senescence in Arabidopsis. However, tissue-specific orgDNA degradation was shown to differ among species. To extend our knowledge, we characterized DPD1 in rice in this study. We created a genome-edited (GE) mutant in which OsDPD1 and OsDPD1-like were inactivated. Characterization of this GE plant demonstrated that DPD1 was involved in pollen orgDNA degradation, whereas it had no significant effect on orgDNA degradation during leaf senescence. Comparison of transcriptomes from wild-type and GE plants with different phosphate supply levels indicated that orgDNA had little impact on the phosphate starvation response, but instead had a global impact in plant growth. In fact, the GE plant showed lower fitness with reduced grain filling rate and grain weight in natural light conditions. Taken together, the presented data reinforce the important physiological roles of orgDNA degradation mediated by DPD1.

Transgenerational increases in DNA methylation in Arabidopsis plants defective in active DNA demethylation.

Spontaneous gain or loss of DNA methylation occurs in plant and animal genomes, and DNA methylation changes can lead to meiotically stable epialleles that generate heritable phenotypic diversity. However, it is unclear whether transgenerational epigenetic stability may be regulated by any cellular factors. Here, we examined spontaneously occurring variations in DNA methylation in wild-type and ros1 mutant Arabidopsis plants that were propagated for ten generations from single-seed descent. We found that the ros1 mutant, which is defective in active DNA demethylation, showed an increased transgenerational epimutation rate. The ros1 mutation led to more spontaneously gained methylation than lost methylation at individual cytosines, compared to the wild type which had similar numbers of spontaneously gained and lost methylation cytosines. Consistently, transgenerational differentially methylated regions were also biased toward hypermethylation in the ros1 mutant. Our results reveal a genetic contribution of the ROS1 DNA demethylase to transgenerational epigenetic stability and suggest that ROS1 may have an unexpected surveillance function in preventing transgenerational DNA methylation increases.

Identification of AtALKBH1A and AtALKBH1D as DNA N6-adenine demethylases in Arabidopsis thaliana.

DNA N6-methyladenine (6 mA) has recently been discovered as a novel DNA modification in animals and plants. In mammals, AlkB homolog 1 (ALKBH1) has been identified as a DNA 6 mA demethylase. ALKBH1 tightly controls the DNA 6 mA methylation level of mammalian genomes and plays important role in regulating gene expression. DNA 6 mA methylation has also been reported to exist in plant genomes, however, the plant DNA 6 mA demethylases and their function remain largely unknown. Here we identify homologs of ALKBH1 as DNA 6 mA demethylases in Arabidopsis. We discover that there are four homologs of ALKBH1, AtALKBH1A, AtALKBH1B, AtALKBH1C and AtALKBH1D, in Arabidopsis. In vitro enzymatic activity studies reveal that AtALKBH1A and 1D can efficiently erase DNA 6 mA methylation. Loss of function of AtALKBH1A and AtALKBH1D causes elevated DNA 6 mA methylation levels in vivo. atalkbh1a/1d mutant displays delayed seed gemination. Based on our RNA-seq data, we find some regulators of seed gemination are dysregulated in atalkbh1a/1d, and the dysregulation is correlated with changes of DNA 6 mA methylation levels. This study identifies plant DNA 6 mA demethylases and reports the function of DNA 6 mA methylation in regulating seed germination.

Population epigenetics: DNA methylation in the plant omics era.

DNA methylation plays an important role in many biological processes. The mechanisms underlying the establishment and maintenance of DNA methylation are well understood thanks to decades of research using DNA methylation mutants, primarily in Arabidopsis (Arabidopsis thaliana) accession Col-0. Recent genome-wide association studies (GWASs) using the methylomes of natural accessions have uncovered a complex and distinct genetic basis of variation in DNA methylation at the population level. Sequencing following bisulfite treatment has served as an excellent method for quantifying DNA methylation. Unlike studies focusing on specific accessions with reference genomes, population-scale methylome research often requires an additional round of sequencing beyond obtaining genome assemblies or genetic variations from whole-genome sequencing data, which can be cost prohibitive. Here, we provide an overview of recently developed bisulfite-free methods for quantifying methylation and cost-effective approaches for the simultaneous detection of genetic and epigenetic information. We also discuss the plasticity of DNA methylation in a specific Arabidopsis accession, the contribution of DNA methylation to plant adaptation, and the genetic determinants of variation in DNA methylation in natural populations. The recently developed technology and knowledge will greatly benefit future studies in population epigenomes.

Molecular basis of chromatin remodelling by DDM1 involved in plant DNA methylation.

Eukaryotic gene regulation occurs at the chromatin level, which requires changing the chromatin structure by a group of ATP-dependent DNA translocases-namely, the chromatin remodellers1. In plants, chromatin remodellers function in various biological processes and possess both conserved and plant-specific components2-5. DECREASE IN DNA METHYLATION 1 (DDM1) is a plant chromatin remodeller that plays a key role in the maintenance DNA methylation6-11. Here we determined the structures of Arabidopsis DDM1 in complex with nucleosome in ADP-BeFx-bound, ADP-bound and nucleotide-free conformations. We show that DDM1 specifically recognizes the H4 tail and nucleosomal DNA. The conformational differences between ADP-BeFx-bound, ADP-bound and nucleotide-free DDM1 suggest a chromatin remodelling cycle coupled to ATP binding, hydrolysis and ADP release. This, in turn, triggers conformational changes in the DDM1-bound nucleosomal DNA, which alters the nucleosome structure and promotes DNA sliding. Together, our data reveal the molecular basis of chromatin remodelling by DDM1.

Brassinosteroid-dependent phosphorylation of PHOSPHATE STARVATION RESPONSE2 reduces its DNA-binding ability in rice.

Brassinosteroids (BRs) are widely used as plant growth regulators in modern agriculture. Understanding how BRs regulate nutrient signaling is crucial for reducing fertilizer usage. Here we elucidate that the central BR signaling inhibitor GSK3/SHAGGY-LIKE KINASE2 (GSK2) interacts directly with and phosphorylates PHOSPHATE STARVATION RESPONSE2 (OsPHR2), the key regulator of phosphate (Pi) signaling, to suppress its transcription factor activity in rice (Oryza sativa). We identify a critical phosphorylation site at serine residue S269 of OsPHR2 and demonstrate that phosphorylation by GSK2 or phosphor-mimic mutation of S269 substantially impairs the DNA-binding activity of OsPHR2, and thus diminishes expression of OsPHR2-induced genes and reduces Pi levels. Like BRs, Pi starvation noticeably induces GSK2 instability. We further show that this site-specific phosphorylation event is conserved in Arabidopsis (Arabidopsis thaliana), but varies among the PHR-family members, being present only in most land plants. These results unveil a distinctive post-transcriptional regulatory mechanism in Pi signaling by which BRs promote Pi acquisition, with a potential contribution to the environmental adaptability of plants during their evolution.

Molecular mechanisms of the RNA polymerases in plant RNA-directed DNA methylation.

In plants, two atypical DNA-dependent RNA polymerases, RNA polymerase IV (Pol IV) and Pol V, and an RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) together produce noncoding RNAs (ncRNAs) to guide the plant-specific RNA-directed DNA methylation (RdDM). Although both Pol IV and Pol V have evolved from the canonical Pol II, they have adapted to different roles in RdDM. The mechanisms of their adaptation are key to understanding plant DNA methylation and the divergent evolution of polymerases. In this review, we summarize insights that have emerged from recent structural studies of Pol IV, Pol V, and RDR2 and discuss their structural features critical for efficient ncRNA production in RdDM.

DNA conformational dynamics in the context-dependent non-CG CHH methylation by plant methyltransferase DRM2.

DNA methylation provides an important epigenetic mechanism that critically regulates gene expression, genome imprinting, and retrotransposon silencing. In plants, DNA methylation is prevalent not only in a CG dinucleotide context but also in non-CG contexts, namely CHG and CHH (H = C, T, or A) methylation. It has been established that plant non-CG DNA methylation is highly context dependent, with the +1- and +2-flanking sequences enriched with A/T nucleotides. How DNA sequence, conformation, and dynamics influence non-CG methylation remains elusive. Here, we report structural and biochemical characterizations of the intrinsic substrate preference of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a plant DNA methyltransferase responsible for establishing all cytosine methylation and maintaining CHH methylation. Among nine CHH motifs, the DRM2 methyltransferase (MTase) domain shows marked substrate preference toward CWW (W = A or T) motifs, correlating well with their relative abundance in planta. Furthermore, we report the crystal structure of DRM2 MTase in complex with a DNA duplex containing a flexible TpA base step at the +1/+2-flanking sites of the target nucleotide. Comparative structural analysis of the DRM2-DNA complexes provides a mechanism by which flanking nucleotide composition impacts DRM2-mediated DNA methylation. Furthermore, the flexibility of the TpA step gives rise to two alternative DNA conformations, resulting in different interactions with DRM2 and consequently temperature-dependent shift of the substrate preference of DRM2. Together, this study provides insights into how the interplay between the conformational dynamics of DNA and temperature as an environmental factor contributes to the context-dependent CHH methylation by DRM2.

Base Excision DNA Repair in Plants: Arabidopsis and Beyond.

Base excision DNA repair (BER) is a key pathway safeguarding the genome of all living organisms from damage caused by both intrinsic and environmental factors. Most present knowledge about BER comes from studies of human cells, E. coli, and yeast. Plants may be under an even heavier DNA damage threat from abiotic stress, reactive oxygen species leaking from the photosynthetic system, and reactive secondary metabolites. In general, BER in plant species is similar to that in humans and model organisms, but several important details are specific to plants. Here, we review the current state of knowledge about BER in plants, with special attention paid to its unique features, such as the existence of active epigenetic demethylation based on the BER machinery, the unexplained diversity of alkylation damage repair enzymes, and the differences in the processing of abasic sites that appear either spontaneously or are generated as BER intermediates. Understanding the biochemistry of plant DNA repair, especially in species other than the Arabidopsis model, is important for future efforts to develop new crop varieties.

Structure and mechanism of the plant RNA polymerase V.

In addition to the conserved RNA polymerases I to III (Pols I to III) in eukaryotes, two atypical polymerases, Pols IV and V, specifically produce noncoding RNA in the RNA-directed DNA methylation pathway in plants. Here, we report on the structures of cauliflower Pol V in the free and elongation conformations. A conserved tyrosine residue of NRPE2 stacks with a double-stranded DNA branch of the transcription bubble to potentially attenuate elongation by inducing transcription stalling. The nontemplate DNA strand is captured by NRPE2 to enhance backtracking, thereby increasing 3'-5' cleavage, which likely underpins Pol V's high fidelity. The structures also illuminate the mechanism of Pol V transcription stalling and enhanced backtracking, which may be important for Pol V's retention on chromatin to serve its function in tethering downstream factors for RNA-directed DNA methylation.

Molecular basis of the plant ROS1-mediated active DNA demethylation.

Active DNA demethylation plays a crucial role in eukaryotic gene imprinting and antagonizing DNA methylation. The plant-specific REPRESSOR OF SILENCING 1/DEMETER (ROS1/DME) family of enzymes directly excise 5-methyl-cytosine (5mC), representing an efficient DNA demethylation pathway distinct from that of animals. Here, we report the cryo-electron microscopy structures of an Arabidopsis ROS1 catalytic fragment in complex with substrate DNA, mismatch DNA and reaction intermediate, respectively. The substrate 5mC is flipped-out from the DNA duplex and subsequently recognized by the ROS1 base-binding pocket through hydrophobic and hydrogen-bonding interactions towards the 5-methyl group and Watson-Crick edge respectively, while the different protonation states of the bases determine the substrate preference for 5mC over T:G mismatch. Together with the structure of the reaction intermediate complex, our structural and biochemical studies revealed the molecular basis for substrate specificity, as well as the reaction mechanism underlying 5mC demethylation by the ROS1/DME family of plant-specific DNA demethylases.

Factor of DNA methylation 1 affects woodland strawberry plant stature and organ size via DNA methylation.

RNA-directed DNA methylation (RdDM) is an epigenetic process that directs silencing to specific genomic regions and loci. The biological functions of RdDM are not well studied in horticultural plants. Here, we isolated the ethyl methane-sulfonate-induced mutant reduced organ size (ros) producing small leaves, flowers, and fruits in woodland strawberry (Fragaria vesca) due to reduced cell numbers compared with that in the wild-type (WT). The candidate mutation causes a premature stop codon in FvH4_6g28780, which shares high similarity to Arabidopsis (Arabidopsis thaliana) Factor of DNA Methylation1 (FDM1) encoding an RdDM pathway component and was named FveFDM1. Consistently, the fvefdm1CR mutants generated by CRISPR/Cas9 also produced smaller organs. Overexpressing FveFDM1 in an Arabidopsis fdm1-1 fdm2-1 double mutant restored DNA methylation at the RdDM target loci. FveFDM1 acts in a protein complex with its homolog Involved in De Novo 2 (FveIDN2). Furthermore, whole-genome bisulfite sequencing revealed that DNA methylation, especially in the CHH context, was remarkably reduced throughout the genome in fvefdm1. Common and specific differentially expressed genes were identified in different tissues of fvefdm1 compared to in WT tissues. DNA methylation and expression levels of several gibberellic acid (GA) biosynthesis and cell cycle genes were validated. Moreover, the contents of GA and auxin were substantially reduced in the young leaves of fvefdm1 compared to in the WT. However, exogenous application of GA and auxin could not recover the organ size of fvefdm1. In addition, expression levels of FveFDM1, FveIDN2, Nuclear RNA Polymerase D1 (FveNRPD1), Domains Rearranged Methylase 2 (FveDRM2), and cell cycle genes were greatly induced by GA treatment. Overall, our work demonstrated the critical roles of FveFDM1 in plant growth and development via RdDM-mediated DNA methylation in horticultural crops.

Plant DNA Methylation: An Epigenetic Mark in Development, Environmental Interactions, and Evolution.

DNA methylation is an epigenetic modification of the genome involved in the regulation of gene expression and modulation of chromatin structure. Plant genomes are widely methylated, and the methylation generally occurs on the cytosine bases through the activity of specific enzymes called DNA methyltransferases. On the other hand, methylated DNA can also undergo demethylation through the action of demethylases. The methylation landscape is finely tuned and assumes a pivotal role in plant development and evolution. This review illustrates different molecular aspects of DNA methylation and some plant physiological processes influenced by this epigenetic modification in model species, crops, and ornamental plants such as orchids. In addition, this review aims to describe the relationship between the changes in plant DNA methylation levels and the response to biotic and abiotic stress. Finally, we discuss the possible evolutionary implications and biotechnological applications of DNA methylation.

A diRNA-protein scaffold module mediates SMC5/6 recruitment in plant DNA repair.

In eukaryotes, the STRUCTURAL MAINTENANCE OF CHROMOSOME 5/6 (SMC5/6) complex is critical to maintaining chromosomal structures around double-strand breaks (DSBs) in DNA damage repair. However, the recruitment mechanism of this conserved complex at DSBs remains unclear. In this study, using Arabidopsis thaliana as a model, we found that SMC5/6 localization at DSBs is dependent on the protein scaffold containing INVOLVED IN DE NOVO 2 (IDN2), CELL DIVISION CYCLE 5 (CDC5), and ALTERATION/DEFICIENCY IN ACTIVATION 2B (ADA2b), whose recruitment is further mediated by DNA-damage-induced RNAs (diRNAs) generated from DNA regions around DSBs. The physical interactions of protein components including SMC5-ADA2b, ADA2b-CDC5, and CDC5-IDN2 result in formation of the protein scaffold. Further analysis indicated that the DSB localization of IDN2 requires its RNA-binding activity and ARGONAUTE 2 (AGO2), indicating a role for the AGO2-diRNA complex in this process. Given that most of the components in the scaffold are conserved, the mechanism presented here, which connects SMC5/6 recruitment and small RNAs, will improve our understanding of DNA repair mechanisms in eukaryotes.

Epigenetic Marks, DNA Damage Markers, or Both? The Impact of Desiccation and Accelerated Aging on Nucleobase Modifications in Plant Genomic DNA.

Modifications of DNA nucleobases are present in all forms of life. The purpose of these modifications in eukaryotic cells, however, is not always clear. Although the role of 5-methylcytosine (m5C) in epigenetic regulation and the maintenance of stability in plant genomes is becoming better understood, knowledge pertaining to the origin and function of oxidized nucleobases is still scarce. The formation of 5-hydroxymetylcytosine (hm5C) in plant genomes is especially debatable. DNA modifications, functioning as regulatory factors or serving as DNA injury markers, may have an effect on DNA structure and the interaction of genomic DNA with proteins. Thus, these modifications can influence plant development and adaptation to environmental stress. Here, for the first time, the changes in DNA global levels of m5C, hm5C, and 8-oxo-7,8-dihydroguanine (8-oxoG) measured by ELISA have been documented in recalcitrant embryonic axes subjected to desiccation and accelerated aging. We demonstrated that tissue desiccation induces a similar trend in changes in the global level of hm5C and 8-oxoG, which may suggest that they both originate from the activity of reactive oxygen species (ROS). Our study supports the premise that m5C can serve as a marker of plant tissue viability whereas oxidized nucleobases, although indicating a cellular redox state, cannot.

The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication.

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. We found that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed histone H3 lysine 27 monomethylation in plants depends on H3.1, TSK, and DNA polymerase theta (Pol θ). This work reveals an H3.1-specific function during replication and a common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone monomethyltransferases and reading of the H3.1 variant.

Glycation damage to organelles and their DNA increases during maize seedling development.

Shoot development in maize begins when meristematic, non-pigmented cells at leaf base stop dividing and proceeds toward the expanded green cells of the leaf blade. During this transition, promitochondria and proplastids develop into mature organelles and their DNA becomes fragmented. Changes in glycation damage during organelle development were measured for protein and DNA, as well as the glycating agent methyl glyoxal and the glycation-defense protein DJ-1 (known as Park7 in humans). Maize seedlings were grown under normal, non-stressful conditions. Nonetheless, we found that glycation damage, as well as defenses against glycation, follow the same developmental pattern we found previously for reactive oxygen species (ROS): as damage increases, damage-defense measures decrease. In addition, light-grown leaves had more glycation and less DJ-1 compared to dark-grown leaves. The demise of maize organellar DNA during development may therefore be attributed to both oxidative and glycation damage that is not repaired. The coordination between oxidative and glycation damage, as well as damage-response from the nucleus is also discussed.

Optimization of T-DNA configuration with UBIQUITIN10 promoters and tRNA-sgRNA complexes promotes highly efficient genome editing in allotetraploid tobacco.

KEY MESSAGE: Combination of UBIQUITIN10 promoter-directed CAS9 and tRNA-gRNA complexes in gene-editing assay induces 80% mutant phenotype with a knockout of the four allelic copies in the T0 generation of allotetraploid tobaccos. While gene-editing methodologies, such as CRISPR-Cas9, have been developed and successfully used in many plant species, their use remains challenging, because they most often rely on stable or transient transgene expression. Regrettably, in all plant species, transformation causes epigenetic effects such as gene silencing and variable transgene expression. Here, UBIQUITIN10 promoters from several plant species were characterized and showed their capacity to direct high levels of transgene expression in transient and stable transformation assays, which in turn was used to improve the selection process of regenerated transformants. Furthermore, we compared various sgRNAs delivery systems and showed that the combination of UBIQUITIN10 promoters and tRNA-sgRNA complexes produced 80% mutant phenotype with a complete knockout of the four allelic copies, while the remaining 20% exhibited weaker phenotype, which suggested partial allelic knockout, in the T0 generation of the allotetraploid Nicotiana tabacum. These data provide valuable information to optimize future designs of gene editing constructs for plant research and crop improvement and open the way for valuable gene editing projects in non-model Solanaceae species.

The Arabidopsis ATR-SOG1 signaling module regulates pleiotropic developmental adjustments in response to 3'-blocked DNA repair intermediates.

Base excision repair and active DNA demethylation produce repair intermediates with DNA molecules blocked at the 3'-OH end by an aldehyde or phosphate group. However, both the physiological consequences of these accumulated single-strand DNAs break with 3'-blocked ends (DNA 3'-blocks) and the signaling pathways responding to unrepaired DNA 3'-blocks remain unclear in plants. Here, we investigated the effects of DNA 3'-blocks on plant development using the zinc finger DNA 3'-phosphoesterase (zdp) AP endonuclease2 (ape2) double mutant, in which 3'-blocking residues are poorly repaired. The accumulation of DNA 3'-blocked triggered diverse developmental defects that were dependent on the ATM and RAD3-related (ATR)-suppressor of gamma response 1 (SOG1) signaling module. SOG1 mutation rescued the developmental defects of zdp ape2 leaves by preventing cell endoreplication and promoting cell proliferation. However, SOG1 mutation caused intensive meristematic cell death in the radicle of zdp ape2 following germination, resulting in rapid termination of radicle growth. Notably, mutating FORMAMIDOPYRIMIDINE DNA GLYCOSYLASE (FPG) in zdp ape2 sog1 partially recovered its radicle growth, demonstrating that DNA 3'-blocks generated by FPG caused the meristematic defects. Surprisingly, despite lacking a functional radicle, zdp ape2 sog1 mutants compensated the lack of root growth by generating anchor roots having low levels of DNA damage response. Our results reveal dual roles of SOG1 in regulating root establishment when seeds germinate with excess DNA 3'-blocks.

The transcription factor ORA59 exhibits dual DNA binding specificity that differentially regulates ethylene- and jasmonic acid-induced genes in plant immunity.

Jasmonic acid (JA) and ethylene (ET) signaling modulate plant defense against necrotrophic pathogens in a synergistic and interdependent manner, while JA and ET also have independent roles in certain processes, e.g. in responses to wounding and flooding, respectively. These hormone pathways lead to transcriptional reprogramming, which is a major part of plant immunity and requires the roles of transcription factors. ET response factors are responsible for the transcriptional regulation of JA/ET-responsive defense genes, of which ORA59 functions as a key regulator of this process and has been implicated in the JA-ET crosstalk. We previously demonstrated that Arabidopsis (Arabidopsis thaliana) GDSL LIPASE 1 (GLIP1) depends on ET for gene expression and pathogen resistance. Here, promoter analysis of GLIP1 revealed ERELEE4 as the critical cis-element for ET-responsive GLIP1 expression. In a yeast one-hybrid screening, ORA59 was isolated as a specific transcription factor that binds to the ERELEE4 element, in addition to the well-characterized GCC box. We found that ORA59 regulates JA/ET-responsive genes through direct binding to these elements in gene promoters. Notably, ORA59 exhibited a differential preference for GCC box and ERELEE4, depending on whether ORA59 activation is achieved by JA and ET, respectively. JA and ET induced ORA59 phosphorylation, which was required for both activity and specificity of ORA59. Furthermore, RNA-seq and virus-induced gene silencing analyses led to the identification of ORA59 target genes of distinct functional categories in JA and ET pathways. Our results provide insights into how ORA59 can generate specific patterns of gene expression dynamics through JA and ET hormone pathways.