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Abscisic acid (ABA) is the primary preventing factor of seed germination, which is crucial to plant survival and propagation. ABA-induced seed germination inhibition is mainly mediated by the dimeric PYR/PYL/RCAR (PYLs) family members. However, little is known about the relevance between dimeric stability of PYLs and seed germination. Here, we reveal that stabilization of PYL dimer can relieve ABA-induced inhibition of seed germination using chemical genetic approaches. Di-nitrobensulfamide (DBSA), a computationally designed chemical probe, yields around ten-fold improvement in receptor affinity relative to ABA. DBSA reverses ABA-induced inhibition of seed germination mainly through dimeric receptors and recovers the expression of ABA-responsive genes. DBSA maintains PYR1 in dimeric state during protein oligomeric state experiment. X-ray crystallography shows that DBSA targets a pocket in PYL dimer interface and may stabilize PYL dimer by forming hydrogen networks. Our results illustrate the potential of PYL dimer stabilization in preventing ABA-induced seed germination inhibition., (© 2024. The Author(s).)

DNA methylation, also known as 5-methylcytosine, is an epigenetic modification that has crucial functions in plant growth, development and adaptation. The cellular DNA methylation level is tightly regulated by the combined action of DNA methyltransferases and demethylases. Protein complexes involved in the targeting and interpretation of DNA methylation have been identified, revealing intriguing roles of methyl-DNA binding proteins and molecular chaperones. Structural studies and in vitro reconstituted enzymatic systems have provided mechanistic insights into RNA-directed DNA methylation, the main pathway catalysing de novo methylation in plants. A better understanding of the regulatory mechanisms will enable locus-specific manipulation of the DNA methylation status. CRISPR-dCas9-based epigenome editing tools are being developed for this goal. Given that DNA methylation patterns can be stably transmitted through meiosis, and that large phenotypic variations can be contributed by epimutations, epigenome editing holds great promise in crop breeding by creating additional phenotypic variability on the same genetic material., (© 2024. Springer Nature Limited.)

Nicotinamide adenine dinucleotide (NAD + ) is a redox cofactor and signal central to cell metabolisms. Disrupting NAD homeostasis in plant alters growth and stress resistance, yet the underlying mechanisms remain largely unknown. Here, by combining genetics with multi-omics, we discover that NAD + deficiency in qs-2 caused by mutation in NAD + biosynthesis gene-Quinolinate Synthase retards growth but induces biosynthesis of defense compounds, notably aliphatic glucosinolates that confer insect resistance. The elevated defense in qs-2 is resulted from activated jasmonate biosynthesis, critically hydroperoxidation of α-linolenic acid by the 13-lipoxygenase (namely LOX2), which is escalated via the burst of chloroplastic ROS-singlet oxygen ( 1 O 2 ). The NAD + deficiency-mediated JA induction and defense priming sequence in plants is recapitulated upon insect infestation, suggesting such defense mechanism operates in plant stress response. Hence, NAD homeostasis is a pivotal metabolic checkpoint that may be manipulated to navigate plant growth and defense metabolism for stress acclimation., (© 2024. The Author(s).)

A contribution of DNA methylation to defense against invading nucleic acids and maintenance of genome integrity is uncontested; however, our understanding of the extent of involvement of this epigenetic mark in genome-wide gene regulation and plant developmental control is incomplete. Here, we knock out all five known DNA methyltransferases in Arabidopsis, generating DNA methylation-free plants. This quintuple mutant exhibits a suite of developmental defects, unequivocally demonstrating that DNA methylation is essential for multiple aspects of plant development. We show that CG methylation and non-CG methylation are required for a plethora of biological processes, including pavement cell shape, endoreduplication, cell death, flowering, trichome morphology, vasculature and meristem development, and root cell fate determination. Moreover, we find that DNA methylation has a strong dose-dependent effect on gene expression and repression of transposable elements. Taken together, our results demonstrate that DNA methylation is dispensable for Arabidopsis survival but essential for the proper regulation of multiple biological processes., (© 2022. The Author(s).)

Plants cannot move, so they must endure abiotic stresses such as drought, salinity and extreme temperatures. These stressors greatly limit the distribution of plants, alter their growth and development, and reduce crop productivity. Recent progress in our understanding of the molecular mechanisms underlying the responses of plants to abiotic stresses emphasizes their multilevel nature; multiple processes are involved, including sensing, signalling, transcription, transcript processing, translation and post-translational protein modifications. This improved knowledge can be used to boost crop productivity and agricultural sustainability through genetic, chemical and microbial approaches., (© 2021. Springer Nature Limited.)

Phosphate, a key plant nutrient, is perceived through inositol polyphosphates (InsPs) by SPX domain-containing proteins. SPX1 an inhibit the PHR2 transcription factor to maintain Pi homeostasis. How SPX1 recognizes an InsP molecule and represses transcription activation by PHR2 remains unclear. Here we show that, upon binding InsP 6 , SPX1 can disrupt PHR2 dimers and form a 1:1 SPX1-PHR2 complex. The complex structure reveals that SPX1 helix α1 can impose a steric hindrance when interacting with the PHR2 dimer. By stabilizing helix α1, InsP 6 allosterically decouples the PHR2 dimer and stabilizes the SPX1-PHR2 interaction. In doing so, InsP 6 further allows SPX1 to engage with the PHR2 MYB domain and sterically block its interaction with DNA. Taken together, our results suggest that, upon sensing the surrogate signals of phosphate, SPX1 inhibits PHR2 via a dual mechanism that attenuates dimerization and DNA binding activities of PHR2., (© 2021. The Author(s).)

In plants, RNA-directed DNA methylation (RdDM) is a well-known de novo DNA methylation pathway that involves two plant-specific RNA polymerases, Pol IV and Pol V. In this study, we discovered and characterized an RdDM factor, RDM15. Through DNA methylome and genome-wide siRNA analyses, we show that RDM15 is required for RdDM-dependent DNA methylation and siRNA accumulation at a subset of RdDM target loci. We show that RDM15 contributes to Pol V-dependent downstream siRNA accumulation and interacts with NRPE3B, a subunit specific to Pol V. We also show that the C-terminal tudor domain of RDM15 specifically recognizes the histone 3 lysine 4 monomethylation (H3K4me1) mark. Structure analysis of RDM15 in complex with the H3K4me1 peptide showed that the RDM15 tudor domain specifically recognizes the monomethyllysine through an aromatic cage and a specific hydrogen bonding network; this chemical feature-based recognition mechanism differs from all previously reported monomethyllysine recognition mechanisms. RDM15 and H3K4me1 have similar genome-wide distribution patterns at RDM15-dependent RdDM target loci, establishing a link between H3K4me1 and RDM15-mediated RdDM in vivo. In summary, we have identified and characterized a histone H3K4me1-specific binding protein as an RdDM component, and structural analysis of RDM15 revealed a chemical feature-based lower methyllysine recognition mechanism.

The phytohormone abscisic acid (ABA) is crucial for plant responses to environmental challenges. The SNF1-regulated protein kinase 2s (SnRK2s) are key components in ABA-receptor coupled core signaling, and are rapidly phosphorylated and activated by ABA. Recent studies have suggested that Raf-like protein kinases (RAFs) participate in ABA-triggered SnRK2 activation. In vitro kinase assays also suggest the existence of autophosphorylation of SnRK2s. Thus, how SnRK2 kinases are quickly activated during ABA signaling still needs to be clarified. Here, we show that both B2 and B3 RAFs directly phosphorylate SnRK2.6 in the kinase activation loop. This transphosphorylation by RAFs is essential for SnRK2 activation. The activated SnRK2s then intermolecularly trans-phosphorylate other SnRK2s that are not yet activated to amplify the response. High-order Arabidopsis mutants lacking multiple B2 and B3 RAFs show ABA hyposensitivity. Our findings reveal a unique initiation and amplification mechanism of SnRK2 activation in ABA signaling in higher plants.

Histone 3 Lys 27 trimethylation (H3K27me3)-mediated epigenetic silencing plays a critical role in multiple biological processes. However, the H3K27me3 recognition and transcriptional repression mechanisms are only partially understood. Here, we report a mechanism for H3K27me3 recognition and transcriptional repression. Our structural and biochemical data showed that the BAH domain protein AIPP3 and the PHD proteins AIPP2 and PAIPP2 cooperate to read H3K27me3 and unmodified H3K4 histone marks, respectively, in Arabidopsis. The BAH-PHD bivalent histone reader complex silences a substantial subset of H3K27me3-enriched loci, including a number of development and stress response-related genes such as the RNA silencing effector gene ARGONAUTE 5 (AGO5). We found that the BAH-PHD module associates with CPL2, a plant-specific Pol II carboxyl terminal domain (CTD) phosphatase, to form the BAH-PHD-CPL2 complex (BPC) for transcriptional repression. The BPC complex represses transcription through CPL2-mediated CTD dephosphorylation, thereby causing inhibition of Pol II release from the transcriptional start site. Our work reveals a mechanism coupling H3K27me3 recognition with transcriptional repression through the alteration of Pol II phosphorylation states, thereby contributing to our understanding of the mechanism of H3K27me3-dependent silencing.

Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raf-like kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana. B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants.

The widespread agricultural problem of pre-harvest sprouting (PHS) could potentially be overcome by improving seed dormancy. Here, we report that miR156, an important grain yield regulator, also controls seed dormancy in rice. We found that mutations in one MIR156 subfamily enhance seed dormancy and suppress PHS with negligible effects on shoot architecture and grain size, whereas mutations in another MIR156 subfamily modify shoot architecture and increase grain size but have minimal effects on seed dormancy. Mechanistically, mir156 mutations enhance seed dormancy by suppressing the gibberellin (GA) pathway through de-represssion of the miR156 target gene Ideal Plant Architecture 1 (IPA1), which directly regulates multiple genes in the GA pathway. These results provide an effective method to suppress PHS without compromising productivity, and will facilitate breeding elite crop varieties with ideal plant architectures.

Broomcorn millet (Panicum miliaceum L.) is the most water-efficient cereal and one of the earliest domesticated plants. Here we report its high-quality, chromosome-scale genome assembly using a combination of short-read sequencing, single-molecule real-time sequencing, Hi-C, and a high-density genetic map. Phylogenetic analyses reveal two sets of homologous chromosomes that may have merged ~5.6 million years ago, both of which exhibit strong synteny with other grass species. Broomcorn millet contains 55,930 protein-coding genes and 339 microRNA genes. We find Paniceae-specific expansion in several subfamilies of the BTB (broad complex/tramtrack/bric-a-brac) subunit of ubiquitin E3 ligases, suggesting enhanced regulation of protein dynamics may have contributed to the evolution of broomcorn millet. In addition, we identify the coexistence of all three C 4 subtypes of carbon fixation candidate genes. The genome sequence is a valuable resource for breeders and will provide the foundation for studying the exceptional stress tolerance as well as C 4 biology.

Heterochromatin is a tightly packed form of chromatin that is associated with DNA methylation and histone 3 lysine 9 methylation (H3K9me). Here, we identify an H3K9me2-binding protein, Agenet domain (AGD)-containing p1 (AGDP1), in Arabidopsis thaliana. Here we find that AGDP1 can specifically recognize the H3K9me2 mark by its three pairs of tandem AGDs. We determine the crystal structure of the Agenet domain 1 and 2 cassette (AGD12) of Raphanus sativus AGDP1 in complex with an H3K9me2 peptide. In the complex, the histone peptide adopts a unique helical conformation. AGD12 specifically recognizes the H3K4me0 and H3K9me2 marks by hydrogen bonding and hydrophobic interactions. In addition, we find that AGDP1 is required for transcriptional silencing, non-CG DNA methylation, and H3K9 dimethylation at some loci. ChIP-seq data show that AGDP1 preferentially occupies long transposons and is associated with heterochromatin marks. Our findings suggest that, as a heterochromatin-binding protein, AGDP1 links H3K9me2 to DNA methylation in heterochromatin regions.

DNA methylation is a conserved epigenetic modification that is important for gene regulation and genome stability. Aberrant patterns of DNA methylation can lead to plant developmental abnormalities. A specific DNA methylation state is an outcome of dynamic regulation by de novo methylation, maintenance of methylation and active demethylation, which are catalysed by various enzymes that are targeted by distinct regulatory pathways. In this Review, we discuss DNA methylation in plants, including methylating and demethylating enzymes and regulatory factors, and the coordination of methylation and demethylation activities by a so-called methylstat mechanism; the functions of DNA methylation in regulating transposon silencing, gene expression and chromosome interactions; the roles of DNA methylation in plant development; and the involvement of DNA methylation in plant responses to biotic and abiotic stress conditions.

Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.

Epigenetic variation has been proposed to facilitate adaptation to changing environments, but evidence that natural epialleles contribute to adaptive evolution has been lacking. Here we identify a retrotransposon, named "NMR19" (naturally occurring DNA methylation variation region 19), whose methylation and genomic location vary among Arabidopsis thaliana accessions. We classify NMR19 as NMR19-4 and NMR19-16 based on its location, and uncover NMR19-4 as an epiallele that controls leaf senescence by regulating the expression of PHEOPHYTIN PHEOPHORBIDE HYDROLASE (PPH). We find that the DNA methylation status of NMR19-4 is stably inherited and independent of genetic variation. In addition, further analysis indicates that DNA methylation of NMR19-4 correlates with local climates, implying that NMR19-4 is an environmentally associated epiallele. In summary, we discover a novel epiallele, and provide mechanistic insights into its origin and potential function in local climate adaptation.

An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumour DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive 'liquid biopsy' for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation marker panel by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumour DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1,098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P < 0.001) and was highly correlated with tumour burden, treatment response, and stage. Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P < 0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC.

Drought stress is a major threat to crop production, but effective methods to mitigate the adverse effects of drought are not available. Here, we report that adding fluorine atoms in the benzyl ring of the abscisic acid (ABA) receptor agonist AM1 optimizes its binding to ABA receptors by increasing the number of hydrogen bonds between the compound and the surrounding amino acid residues in the receptor ligand-binding pocket. The new chemicals, known as AMFs, have long-lasting effects in promoting stomatal closure and inducing the expression of stress-responsive genes. Application of AMFs or transgenic overexpression of the receptor PYL2 in Arabidopsis and soybean plants confers increased drought resistance. The greatest increase in drought resistance is achieved when AMFs are applied to the PYL2-overexpression transgenic plants. Our results demonstrate that the combining of potent chemicals with transgenic overexpression of an ABA receptor is very effective in helping plants combat drought stress.

The phytohormone abscisic acid (ABA) is important for growth, development and stress responses in plants. Recent research has identified ABA receptors and signalling components that regulate seed germination and stomatal closure. However, proteins that regulate ABA signalling remain poorly understood. Here we use a forward-genetic screen to identify rbm25-1 and rbm25-2, two Arabidopsis mutants with increased sensitivity to growth inhibition by ABA. Using RNA-seq, we found that RBM25 controls the splicing of many pre-mRNAs. The protein phosphatase 2C HAB1, a critical component in ABA signalling, shows a dramatic defect in pre-mRNA splicing in rbm25 mutants. Ectopic expression of a HAB1 complementary DNA derived from wild-type mRNAs partially suppresses the rbm25-2 mutant phenotype. We suggest that RNA splicing is of particular importance for plant response to ABA and that the splicing factor RBM25 has a critical role in this response.

DNA methylation is a reversible epigenetic mark regulating genome stability and function in many eukaryotes. In Arabidopsis, active DNA demethylation depends on the function of the ROS1 subfamily of genes that encode 5-methylcytosine DNA glycosylases/lyases. ROS1-mediated DNA demethylation plays a critical role in the regulation of transgenes, transposable elements and some endogenous genes; however, there have been no reports of clear developmental phenotypes in ros1 mutant plants. Here we report that, in the ros1 mutant, the promoter region of the peptide ligand gene EPF2 is hypermethylated, which greatly reduces EPF2 expression and thereby leads to a phenotype of overproduction of stomatal lineage cells. EPF2 gene expression in ros1 is restored and the defective epidermal cell patterning is suppressed by mutations in genes in the RNA-directed DNA methylation pathway. Our results show that active DNA demethylation combats the activity of RNA-directed DNA methylation to influence the initiation of stomatal lineage cells.

Thylakoid assembly 8 (THA8) is a pentatricopeptide repeat (PPR) RNA-binding protein required for the splicing of the transcript of ycf3, a gene involved in chloroplast thylakoid-membrane biogenesis. Here we report the identification of multiple THA8-binding sites in the ycf3 intron and present crystal structures of Brachypodium distachyon THA8 either free of RNA or bound to two of the identified RNA sites. The apostructure reveals a THA8 monomer with five tandem PPR repeats arranged in a planar fold. The complexes of THA8 bound to the two short RNA fragments surprisingly reveal asymmetric THA8 dimers with the bound RNAs at the dimeric interface. RNA binding induces THA8 dimerization, with a conserved G nucleotide of the bound RNAs making extensive contacts with both monomers. Together, these results establish a new model of RNA recognition by RNA-induced formation of an asymmetric dimer of a PPR protein.

The phytohormone abscisic acid (ABA) functions through a family of fourteen PYR/PYL receptors, which were identified by resistance to pyrabactin, a synthetic inhibitor of seed germination. ABA activates these receptors to inhibit type 2C protein phosphatases, such as ABI1, yet it remains unclear whether these receptors can be antagonized. Here we demonstrate that pyrabactin is an agonist of PYR1 and PYL1 but is unexpectedly an antagonist of PYL2. Crystal structures of the PYL2-pyrabactin and PYL1-pyrabactin-ABI1 complexes reveal the mechanism responsible for receptor-selective activation and inhibition, which enables us to design mutations that convert PYL1 to a pyrabactin-inhibited receptor and PYL2 to a pyrabactin-activated receptor and to identify new pyrabactin-based ABA receptor agonists. Together, our results establish a new concept of ABA receptor antagonism, illustrate its underlying mechanisms and provide a rational framework for discovering novel ABA receptor ligands.