Publications

Epigenetic memory marks determine epiallele stability at loci targeted by de novo DNA methylation

Targeted insertion of regulatory elements enables translational enhancement in rice

In-locus editing of agronomically-important genes to optimize their spatiotemporal expression is becoming an important breeding approach. Compared to intensive studies on mRNA transcription, manipulating protein translation by genome editing has not been well exploited. Here, we found that precise knock-in of a regulating element into the 5'UTR of a target gene could efficiently increase its protein abundance in rice. We firstly screened a translational enhancer (AMVE) from alfalfa mosaic virus using protoplast-based luciferase assays with an 8.5-folds enhancement. Then the chemically modified donor of AMVE was synthesized and targeted inserted into the 5'UTRs of two genes (WRKY71 and SKC1) using CRISPR/Cas9. Following the in-locus AMVE knock-in, we observed up to a 2.8-fold increase in the amount of WRKY71 protein. Notably, editing of SKC1, a sodium transporter, significantly increased salt tolerance in T2 seedlings, indicating the expected regulation of AMVE knock-in. These data demonstrated the feasibility of such in-locus editing to enhance protein expression, providing a new approach to manipulating protein translation for crop breeding.

Cold‐regulated gene expression and freezing tolerance in an <i>Arabidopsis thaliana</i> mutant

Low temperature is an important environmental factor influencing plant growth and development. In this study, we report the characterization of a genetic locus, HOS2 , which is defined by three Arabidopsis thaliana mutants. The hos2–1 , hos2–2 and hos2–3 mutations result in enhanced expression of RD29A and other stress genes under low temperature treatment. Gene expression in response to osmotic stress or ABA is not affected in the hos2 mutants. Genetic analysis indicates that the hos2 mutations are recessive and in a nuclear gene. Compared with the wild‐type plants, the hos2–1 mutant plants are less capable of developing freezing tolerance when treated with low non‐freezing temperatures. However, the hos2–1 mutation does not impair the vernalization response. These results indicate that HOS2 is a negative regulator of low temperature signal transduction important for plant cold acclimation.

Coupling of H3K27me3 recognition with transcriptional repression through the BAH-PHD-CPL2 complex in<i>Arabidopsis</i>

SUMMARY 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 new 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 ) and 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.

A single amino acid substitution in the <i>Arabidopsis</i> FIERY1/HOS2 protein confers cold signaling specificity and lithium tolerance

Summary Low temperature induces the expression of many plant genes through undefined signaling pathways. To gain insight into cold signal transduction mechanisms, we isolated Arabidopsis mutants that exhibited altered regulation of low temperature‐induced gene expression. One such mutant, hos2 , was shown previously to have an enhanced induction of stress‐responsive genes by cold, whereas the expression of these genes under osmotic stress or the phytohormone absciscic acid (ABA) treatments was not affected. Here we further define the targets of HOS2 by examining the regulation of upstream cold‐specific CBF transcription factor genes. It was found that the transcript levels of CBF2 and CBF3 were significantly higher in hos2 mutant plants than in the wild type under cold treatments, suggesting that HOS2 may act upstream of CBFs. The HOS2 gene was cloned using a map‐based strategy. Surprisingly, HOS2 is identical to the FIERY1 gene that we had described previously. FIERY1 is a general negative regulator that controls cold, osmotic stress, and ABA signal transduction and possesses inositol polyphosphate 1‐phosphatase activity. The hos2 mutation rendered the HOS2/FIERY1 recombinant protein completely inactive in the cold but did not substantially affect its activity at warm temperatures. Interestingly, the hos2 mutant protein is extremely tolerant to Li + . This study provides a unique example of a single amino acid substitution in a critical regulator that can lead to conditional changes in protein functions and distinct plant phenotypes. The results reinforce the notion that phosphoinositols are important second messengers in cold signal transduction, and shed light on how the diversity of plant tolerance to cold and other abiotic stresses may evolve due to variations in a common molecular switch.

Stalk cell polar ion transport provide for bladder‐based salinity tolerance in <i>Chenopodium quinoa</i>

Chenopodium quinoa uses epidermal bladder cells (EBCs) to sequester excess salt. Each EBC complex consists of a leaf epidermal cell, a stalk cell, and the bladder. Under salt stress, sodium (Na+ ), chloride (Cl- ), potassium (K+ ) and various metabolites are shuttled from the leaf lamina to the bladders. Stalk cells operate as both a selectivity filter and a flux controller. In line with the nature of a transfer cell, advanced transmission electron tomography, electrophysiology, and fluorescent tracer flux studies revealed the stalk cell's polar organization and bladder-directed solute flow. RNA sequencing and cluster analysis revealed the gene expression profiles of the stalk cells. Among the stalk cell enriched genes, ion channels and carriers as well as sugar transporters were most pronounced. Based on their electrophysiological fingerprint and thermodynamic considerations, a model for stalk cell transcellular transport was derived.

A novel mitochondrial protein is required for cell wall integrity, auxin accumulation and root elongation in Arabidopsis under salt stress

Maintenance of root elongation is beneficial for the growth and survival of plants under salt stress, but currently the cellular components involved in the regulation of root growth under high salinity are not fully understood. In this study, we identified an Arabidopsis mutant, rres1, which exhibited reduced root elongation under treatment of a variety of salts, including NaCl, NaNO3, KCl, and KNO3. RRES1 encodes a novel mitochondrial protein and its molecular function is still unknown. Under salt stress, the root meristem length was shorter in the rres1 mutant compared to the wild type, which was correlated with a reduced auxin accumulation in the mutant. Reactive oxygen species (ROS), as important signals that regulate root elongation, were accumulated to higher levels in the rres1 mutant than the wild type after salt treatment. Measurement of monosaccharides in the cell wall showed that arabinose and xylose contents were decreased in the rres1 mutant under salt stress, and application of boric acid, which is required for the crosslinking of pectic polysaccharide rhamnogalacturonan-II (RG-II), largely rescued the root growth arrest of the rres1 mutant, suggesting that RRES1 participates in the maintenance of cell wall integrity under salt stress. GUS staining assay indicated that the RRES1 gene was expressed in leaves and weakly in root tip under normal conditions, but its expression was dramatically increased in leaves and roots after salt treatment. Together, our study reveals a novel mitochondrial protein that regulates root elongation under salt stress via the modulation of cell wall integrity, auxin accumulation, and ROS homeostasis.

Linking genes of unknown function with abiotic stress responses by high‐throughput phenotype screening

Over 13% of all genes in the Arabidopsis thaliana genome encode for proteins classified as having a completely unknown function, with the function of &gt;30% of the Arabidopsis proteome poorly characterized. Although empirical data in the form of mRNA and proteome profiling experiments suggest that many of these proteins play an important role in different biological processes, their functional characterization remains one of the major challenges in modern biology. To expand the annotation of genes with unknown function involved in the response of Arabidopsis to different environmental stress conditions, we selected 1007 such genes and tested the response of their corresponding homozygous T‐ DNA insertional mutants to salinity, oxidative, osmotic, heat, cold and hypoxia stresses. Depending on the specific abiotic stresses tested, 12–31% of mutants had an altered stress‐response phenotype. Interestingly, 832 out of 1007 mutants showed tolerance or sensitivity to more than one abiotic stress treatment, suggesting that genes of unknown function could play an important role in abiotic stress‐response signaling, or general acclimation mechanisms. Further analysis of multiple stress‐response phenotypes within different populations of mutants revealed interesting links between acclimation to heat, cold and oxidative stresses, as well as between sensitivity to ABA , osmotic, salinity, oxidative and hypoxia stresses. Our findings provide a significant contribution to the biological characterization of genes with unknown function in Arabidopsis and demonstrate that many of these genes play a key role in the response of plants to abiotic stresses.

Cold Transiently Activates Calcium-Permeable Channels in Arabidopsis Mesophyll Cells

Abstract Living organisms are capable of discriminating thermal stimuli from noxious cold to noxious heat. For more than 30 years, it has been known that plant cells respond to cold with a large and transient depolarization. Recently, using transgenic Arabidopsis (Arabidopsis thaliana) expressing the calcium-sensitive protein aequorin, an increase in cytosolic calcium following cold treatment was observed. Applying the patch-clamp technique to Arabidopsis mesophyll protoplasts, we could identify a transient plasma membrane conductance induced by rapid cooling. This cold-induced transient conductance was characterized as an outward rectifying 33 pS nonselective cation channel. The permeability ratio between calcium and cesium was 0.7, pointing to a permeation pore &amp;gt;3.34 Å (ø of cesium). Our experiments thus provide direct evidence for the predicted but not yet measured cold-activated calcium-permeable channel in plants.

Roles of DEMETER in regulating DNA methylation in vegetative tissues and pathogen resistance

Abstract DNA methylation is an epigenetic mark important for genome stability and gene expression. In Arabidopsis thaliana , the 5‐methylcytosine DNA glycosylase/demethylase DEMETER (DME) controls active DNA demethylation during the reproductive stage; however, the lethality of loss‐of‐function dme mutations has made it difficult to assess DME function in vegetative tissues. Here, we edited DME using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR‐associated protein 9 and created three weak dme mutants that produced a few viable seeds. We also performed central cell‐specific complementation in a strong dme mutant and combined this line with mutations in the other three Arabidopsis demethylase genes to generate the dme ros1 dml2 dml3 ( drdd ) quadruple mutant. A DNA methylome analysis showed that DME is required for DNA demethylation at hundreds of genomic regions in vegetative tissues. A transcriptome analysis of the drdd mutant revealed that DME and the other three demethylases are important for plant responses to biotic and abiotic stresses in vegetative tissues. Despite the limited role of DME in regulating DNA methylation in vegetative tissues, the dme mutants showed increased susceptibility to bacterial and fungal pathogens. Our study highlights the important functions of DME in vegetative tissues and provides valuable genetic tools for future investigations of DNA demethylation in plants.

In vitro reconstitution of an abscisic acid signalling pathway

Interaction network of core ABA signaling components in maize

ROS3 is an RNA-binding protein required for DNA demethylation in Arabidopsis

A catalog of gene editing sites and genetic variations in editing sites in model organisms

CRISPR-Cas systems require a protospacer adjacent motif (PAM), which plays an essential role in self/non-self discrimination in their natural context, to cleave DNA for genome editing. Unfortunately, common genetic variation is distributed throughout genomes, which can block recognition of target sites by Cas proteins. However, little information is available about the distribution of editing sites in model organisms and how often common variation overlaps with those PAM sites. Herein, we characterized six representative Cas proteins (Cas9, Cas12a, Cas12b, Cas12i, Cas12j and Cas12l) genomic editing sites in ten model organisms (yeast, flatworms, flies, zebrafish, mice, humans, rice, maize, Arabidopsis and tomato). We demonstrated that there were more than 34 editing sites per kilobase on average in these genomes. In each genome, 91.69–99.83% and 95.4–99.73% of genes had at least one unique editing site in exon and promoter, respectively. Depending on publicly available genomic diversity data, we identified the variations (SNPs and InDels) in editing sites in humans and rice, indicating the risk in the application of CRISPR/Cas technology. Finally, using CCR5 and BCL11A as examples, we revealed variation site was a factor that must be considered when designing sgRNA. Our findings not only revealed the distribution characteristics of editing sites of six representative Cas proteins in ten model organism genomes but also shed light on the adverse effect of variation sites on target site recognition. Our current work will serve as a reminder of the risks of CRISPR application.

Active DNA demethylation by oxidation and repair

Involvement of Multiple Gene-Silencing Pathways in a Paramutation-like Phenomenon in Arabidopsis

Paramutation is an epigenetic phenomenon that has been observed in a number of multicellular organisms. The epigenetically silenced state of paramutated alleles is not only meiotically stable but also "infectious" to active homologous alleles. The molecular mechanism of paramutation remains unclear, but components involved in RNA-directed DNA methylation (RdDM) are required. Here, we report a multi-copy pRD29A-LUC transgene in Arabidopsis thaliana that behaves like a paramutation locus. The silent state of LUC is induced by mutations in the DNA glycosylase gene ROS1. The silent alleles of LUC are not only meiotically stable but also able to transform active LUC alleles into silent ones, in the absence of ros1 mutations. Maintaining silencing at the LUC gene requires action of multiple pathways besides RdDM. Our study identified specific factors that are involved in the paramutation-like phenomenon and established a model system for the study of paramutation in Arabidopsis.

The Plant Cuticle Is Required for Osmotic Stress Regulation of Abscisic Acid Biosynthesis and Osmotic Stress Tolerance in<i>Arabidopsis</i>

Abstract Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 gene expression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxygenase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol) treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly that CED1 encodes a putative α/β hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cutin biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling.

Regulation of ion homeostasis under salt stress

Protein–protein interactions of tandem affinity purification‐tagged protein kinases in rice

Summary Forty‐one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP‐tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety‐five percent of the TAP‐tagged kinases were recovered. Fifty‐six percent of the TAP‐tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP‐tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14‐3‐3 proteins or cyclins.