Publications

Abstract Nuclear factor Y (NF-Y) is a ubiquitous transcription factor composed of three distinct subunits (NF-YA, NF-YB, and NF-YC). We found that the Arabidopsis thaliana NFYA5 transcript is strongly induced by drought stress in an abscisic acid (ABA)-dependent manner. Promoter:β-glucuronidase analyses showed that NFYA5 was highly expressed in vascular tissues and guard cells and that part of the induction by drought was transcriptional. NFYA5 contains a target site for miR169, which targets mRNAs for cleavage or translational repression. We found that miR169 was downregulated by drought stress through an ABA-dependent pathway. Analysis of the expression of miR169 precursors showed that miR169a and miR169c were substantially downregulated by drought stress. Coexpression of miR169 and NFYA5 suggested that miR169a was more efficient than miR169c at repressing the NFYA5 mRNA level. nfya5 knockout plants and plants overexpressing miR169a showed enhanced leaf water loss and were more sensitive to drought stress than wild-type plants. By contrast, transgenic Arabidopsis plants overexpressing NFYA5 displayed reduced leaf water loss and were more resistant to drought stress than the wild type. Microarray analysis indicated that NFYA5 is crucial for the expression of a number of drought stress–responsive genes. Thus, NFYA5 is important for drought resistance, and its induction by drought stress occurs at both the transcriptional and posttranscriptional levels.

Abscisic acid (ABA) is the most important hormone for plants to resist drought and other abiotic stresses. ABA binds directly to the PYR/PYL family of ABA receptors, resulting in inhibition of type 2C phosphatases (PP2C) and activation of downstream ABA signaling. It is envisioned that intervention of ABA signaling by small molecules could help plants to overcome abiotic stresses such as drought, cold and soil salinity. However, chemical instability and rapid catabolism by plant enzymes limit the practical application of ABA itself. Here we report the identification of a small molecule ABA mimic (AM1) that acts as a potent activator of multiple members of the family of ABA receptors. In Arabidopsis, AM1 activates a gene network that is highly similar to that induced by ABA. Treatments with AM1 inhibit seed germination, prevent leaf water loss, and promote drought resistance. We solved the crystal structure of AM1 in complex with the PYL2 ABA receptor and the HAB1 PP2C, which revealed that AM1 mediates a gate-latch-lock interacting network, a structural feature that is conserved in the ABA-bound receptor/PP2C complex. Together, these results demonstrate that a single small molecule ABA mimic can activate multiple ABA receptors and protect plants from water loss and drought stress. Moreover, the AM1 complex crystal structure provides a structural basis for designing the next generation of ABA-mimicking small molecules.

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.

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.

Over 13% of all genes in the Arabidopsis thaliana genome encode for proteins classified as having a completely unknown function, with the function of >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.

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.

Abstract Drought is one of the most important environmental constraints limiting plant growth and agricultural productivity. To understand the underlying mechanism of drought tolerance and to identify genes for improving this important trait, we conducted a gain-of-function genetic screen for improved drought tolerance in Arabidopsis thaliana. One mutant with improved drought tolerance was isolated and designated as enhanced drought tolerance1. The mutant has a more extensive root system than the wild type, with deeper roots and more lateral roots, and shows a reduced leaf stomatal density. The mutant had higher levels of abscisic acid and Pro than the wild type and demonstrated an increased resistance to oxidative stress and high levels of superoxide dismutase. Molecular genetic analysis and recapitulation experiments showed that the enhanced drought tolerance is caused by the activated expression of a T-DNA tagged gene that encodes a putative homeodomain-START transcription factor. Moreover, overexpressing the cDNA of the transcription factor in transgenic tobacco also conferred drought tolerance associated with improved root architecture and reduced leaf stomatal density. Therefore, we have revealed functions of the homeodomain-START factor that were gained upon altering its expression pattern by activation tagging and provide a key regulator that may be used to improve drought tolerance in plants.

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.

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.

Abstract The 5-methylcytosine DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1)-mediated active DNA demethylation is critical for shaping the genomic DNA methylation landscape in Arabidopsis . Whether and how the stability of ROS1 may be regulated by post-translational modifications is unknown. Using a methylation-sensitive PCR (CHOP-PCR)-based forward genetic screen for Arabidopsis DNA hypermethylation mutants, we identified the SUMO E3 ligase SIZ1 as a critical regulator of active DNA demethylation. Dysfunction of SIZ1 leads to hyper-methylation at approximately one thousand genomic regions. SIZ1 physically interacts with ROS1 and mediates the SUMOylation of ROS1. The SUMOylation of ROS1 is reduced in siz1 mutant plants. Compared to that in wild type plants, the protein level of ROS1 is significantly decreased, even though there is an increased level of ROS1 transcripts in siz1 mutant plants. Our results suggest that SIZ1 positively regulates active DNA demethylation by promoting the stability of ROS1 protein through SUMOylation. Short Summary The 5-methylcytosine DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) is indispensable for proper DNA methylation landscape in Arabidopsis . Whether and how the stability of ROS1 may be regulated by post-translational modifications is unknown. Here, we show that SIZ1-mediated SUMOylation of ROS1 enhances its stability and positively regulates active DNA demethylation.

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 >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.

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.

Additional file 5: Table S4. Distribution of the indicated TF targets in each hub.