Constitutive activation of ABA receptors in Arabidopsis reveals unique regulatory circuitries.

Abscisic acid (ABA) is best known for regulating the responses to abiotic stressors. Thus, applications of ABA signaling pathways are considered promising targets for securing yield under stress. ABA levels rise in response to abiotic stress, mounting physiological and metabolic responses that promote plant survival under unfavorable conditions. ABA elicits its effects by binding to a family of soluble receptors found in monomeric and dimeric states, differing in their affinity to ABA and co-receptors. However, the in vivo significance of the biochemical differences between these receptors remains unclear. We took a gain-of-function approach to study receptor-specific functionality. First, we introduced activating mutations that enforce active ABA-bound receptor conformation. We then transformed Arabidopsis ABA-deficient mutants with the constitutive receptors and monitored suppression of the ABA deficiency phenotype. Our findings suggest that PYL4 and PYL5, monomeric ABA receptors, have differential activity in regulating transpiration and transcription of ABA biosynthesis and stress response genes. Through genetic and metabolic data, we demonstrate that PYR1, but not PYL5, is sufficient to activate the ABA positive feedback mechanism. We propose that ABA signaling - from perception to response - flows differently when triggered by different PYLs, due to tissue and transcription barriers, thus resulting in distinct circuitries.

Chromosome-level genomes of multicellular algal sisters to land plants illuminate signaling network evolution.

The filamentous and unicellular algae of the class Zygnematophyceae are the closest algal relatives of land plants. Inferring the properties of the last common ancestor shared by these algae and land plants allows us to identify decisive traits that enabled the conquest of land by plants. We sequenced four genomes of filamentous Zygnematophyceae (three strains of Zygnema circumcarinatum and one strain of Z. cylindricum) and generated chromosome-scale assemblies for all strains of the emerging model system Z. circumcarinatum. Comparative genomic analyses reveal expanded genes for signaling cascades, environmental response, and intracellular trafficking that we associate with multicellularity. Gene family analyses suggest that Zygnematophyceae share all the major enzymes with land plants for cell wall polysaccharide synthesis, degradation, and modifications; most of the enzymes for cell wall innovations, especially for polysaccharide backbone synthesis, were gained more than 700 million years ago. In Zygnematophyceae, these enzyme families expanded, forming co-expressed modules. Transcriptomic profiling of over 19 growth conditions combined with co-expression network analyses uncover cohorts of genes that unite environmental signaling with multicellular developmental programs. Our data shed light on a molecular chassis that balances environmental response and growth modulation across more than 600 million years of streptophyte evolution.

Directed Evolution of Herbicide Biosensors in a Fluorescence-Activated Cell-Sorting-Compatible Yeast Two-Hybrid Platform.

Developing sensory modules for specific molecules of interest represents a fundamental challenge in synthetic biology and its applications. A somewhat generalizable approach for this challenge is demonstrated here by evolving a naturally occurring chemically induced heterodimer into a genetically encoded sensor for herbicides. The interaction between PYRABACTIN-RESISTANT-like receptors and type-2C protein phosphatases is induced by abscisic acid─a small-molecule hormone in plants. We considered abscisic acid receptors as a potential scaffold for the development of biosensors because of past successes in their engineering, a structurally defined ligand cavity and the availability of large-scale assays for their activation. A panel of 475 receptor variants, mutated at ligand-proximal residues, was screened for activation by 37 herbicides from several classes. Twelve compounds activated at least one member of the mutant panel. To facilitate the subsequent improvement of herbicide receptors through directed evolution, we engineered a yeast two-hybrid platform optimized for sequential positive and negative selection using fluorescence-activated cell sorting. By utilizing this system, we were able to isolate receptors with low nanomolar sensitivity and a broad dynamic range in sensing a ubiquitous group of chloroacetamide herbicides. Aside from its possible applicative value, this work lays down conceptual groundwork and provides infrastructure for the future development of biosensors through directed evolution.

A Modified Yeast Two-Hybrid Platform Enables Dynamic Control of Expression Intensities to Unmask Properties of Protein-Protein Interactions.

The yeast two-hybrid (Y2H) assay is widely used for protein-protein interaction characterization due to its simplicity and accessibility. However, it may mask changes in affinity caused by mutations or ligand activation due to signal saturation. To overcome this drawback, we modified the Y2H system to have tunable protein expression by introducing a fluorescent reporter and a pair of synthetic inducible transcription factors to regulate the expression of interacting components. We found that the application of inducers allowed us to adjust the concentrations of interacting proteins to avoid saturation and observe interactions otherwise masked in the canonical Y2H assay, such as the abscisic acid-mediated increase in affinity of monomeric abscisic acid receptors to the coreceptor. When applied in future studies, our modified system may provide a more accurate characterization of protein-protein interactions.

Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo.

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA's physiological roles in vivo. We set out to design antagonists that block receptor-PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT-PYL10 complex (1.86 Å) reveals that ANT's peptidotriazole headgroup is positioned to sterically block receptor-PP2C interactions in the 4' tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA-ANT. Equilibrium dissociation constants for TAMRA-ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA-receptor function for dissecting and manipulating ABA signaling.

Evolution of Abscisic Acid Signaling Module and Its Perception.

We hereby review the perception and responses to the stress hormone Abscisic acid (ABA), along the trajectory of 500M years of plant evolution, whose understanding may resolve how plants acquired this signaling pathway essential for the colonization of land. ABA levels rise in response to abiotic stresses, coordinating physiological and metabolic responses, helping plants survive stressful environments. In land plants, ABA signaling cascade leads to growth arrest and large-scale changes in transcript levels, required for coping with environmental stressors. This response is regulated by a PYRABACTIN RESISTANCE 1-like (PYL)-PROTEIN PHOSPHATASE 2C (PP2C)-SNF1-RELATED PROTEIN KINASE 2 (SnRK2) module, that initiates phosphor-activation of transcription factors and ion channels. The enzymatic portions of this module (phosphatase and kinase) are functionally conserved from streptophyte algae to angiosperms, whereas the regulatory component -the PYL receptors, putatively evolved in the common ancestor of Zygnematophyceae and embryophyte as a constitutive, ABA-independent protein, further evolving into a ligand-activated receptor at the embryophyta. This evolutionary process peaked with the appearance of the strictly ABA-dependent subfamily III stress-triggered angiosperms' dimeric PYL receptors. The emerging picture is that the ancestor of land plants and its predecessors synthesized ABA, as its biosynthetic pathway is conserved between ancestral and current day algae. Despite this ability, it was only the common ancestor of land plants which acquired the hormonal-modulation of PYL activity by ABA. This raises several questions regarding both ABA's function in ABA-non-responsive organisms, and the evolutionary aspects of the ABA signal transduction pathway.

A ligand-independent origin of abscisic acid perception.

Land plants are considered monophyletic, descending from a single successful colonization of land by an aquatic algal ancestor. The ability to survive dehydration to the point of desiccation is a key adaptive trait enabling terrestrialization. In extant land plants, desiccation tolerance depends on the action of the hormone abscisic acid (ABA) that acts through a receptor-signal transduction pathway comprising a PYRABACTIN RESISTANCE 1-like (PYL)-PROTEIN PHOSPHATASE 2C (PP2C)-SNF1-RELATED PROTEIN KINASE 2 (SnRK2) module. Early-diverging aeroterrestrial algae mount a dehydration response that is similar to that of land plants, but that does not depend on ABA: Although ABA synthesis is widespread among algal species, ABA-dependent responses are not detected, and algae lack an ABA-binding PYL homolog. This raises the key question of how ABA signaling arose in the earliest land plants. Here, we systematically characterized ABA receptor-like proteins from major land plant lineages, including a protein found in the algal sister lineage of land plants. We found that the algal PYL-homolog encoded by Zygnema circumcarinatum has basal, ligand-independent activity of PP2C repression, suggesting this to be an ancestral function. Similarly, a liverwort receptor possesses basal activity, but it is further activated by ABA. We propose that co-option of ABA to control a preexisting PP2C-SnRK2-dependent desiccation-tolerance pathway enabled transition from an all-or-nothing survival strategy to a hormone-modulated, competitive strategy by enabling continued growth of anatomically diversifying vascular plants in dehydrative conditions, enabling them to exploit their new environment more efficiently.

Abiotic stress modulates root patterning via ABA-regulated microRNA expression in the endodermis initials.

Patterning of the root xylem into protoxylem (PX) and metaxylem is regulated by auxin-cytokinin signaling and microRNA miR165a/166b-mediated suppression of genes encoding Class III HOMEODOMAIN LEU-ZIPPER (HD-ZIPIII) proteins. We found that, in Arabidopsis, osmotic stress via core abscisic acid (ABA) signaling in meristematic endodermal cells induces differentiation of PX in radial and longitudinal axes in association with increased VND7 expression. Similarly, in tomato, ABA enhanced PX differentiation longitudinally and radially, indicating an evolutionarily conserved mechanism. ABA increased expression of miR165a/166b and reduced expression of the miR165a/166b repressor ZWILLE (ZLL) (also known as ARGONAUTE10), resulting in reduced levels of all five HD-ZIPIII RNAs. ABA treatments failed to induce additional PX files in a miR165a/166b-resistant PHB mutant, phb1-d, and in scr and shr mutants, in which miR165a/166b expression is strongly reduced. Thus, ABA regulates xylem patterning and maturation via miR165a/166b-regulated expression of HD-ZIPIII mRNAs and associated VND7 levels. In lateral root initials, ABA induced an increase in miR165a levels in endodermal precursors and inhibited their reduction in the future quiescent center specifically at pre-emergence stage. Hence, ABA-induced inhibition of lateral root is associated with reduced HD-ZIPIII levels.

GNI-A1 mediates trade-off between grain number and grain weight in tetraploid wheat.

KEY MESSAGE: Wild emmer allele of GNI-A1 ease competition among developing grains through the suppression of floret fertility and increase grain weight in tetraploid wheat. Grain yield is a highly polygenic trait determined by the number of grains per unit area, as well as by grain weight. In wheat, grain number and grain weight are usually negatively correlated. Yet, the genetic basis underlying trade-off between the two is mostly unknown. Here, we fine-mapped a grain weight QTL using wild emmer introgressions in a durum wheat background and showed that grain weight is associated with the GNI-A1 gene, a regulator of floret fertility. In-depth characterization of grain number and grain weight indicated that suppression of distal florets by the wild emmer GNI-A1 allele increases weight of proximal grains in basal and central spikelets due to alteration in assimilate distribution. Re-sequencing of GNI-A1 in tetraploid wheat demonstrated the rich allelic repertoire of the wild emmer gene pool, including a rare allele which was present in two gene copies and contained a nonsynonymous mutation in the C-terminus of the protein. Using an F2 population generated from a cross between wild emmer accessions Zavitan, which carries the rare allele, and TTD140, we demonstrated that this unique polymorphism is associated with grain weight, independent of grain number. Moreover, we showed, for the first time, that GNI-A1 proteins are transcriptional activators and that selection targeted compromised activity of the protein. Our findings expand the knowledge of the genetic basis underlying trade-off between key yield components and may contribute to breeding efforts for enhanced grain yield.

ABA signaling components in Phelipanche aegyptiaca.

Obligate root holoparasite Phelipanche aegyptiaca is an agricultural pest, which infests its hosts and feeds on the sap, subsequently damaging crop yield and quality. Its notoriously viable seed bank may serve as an ideal pest control target. The phytohormone abscisic acid (ABA) was shown to regulate P. aegyptiaca seed dormancy following strigolactones germination stimulus. Transcription analysis of signaling components revealed five ABA receptors and two co-receptors (PP2C). Transcription of lower ABA-affinity subfamily III receptors was absent in all tested stages of P. aegyptiaca development and parasitism stages. P. aegyptiaca ABA receptors interacted with the PP2Cs, and inhibited their activity in an ABA-dependent manner. Moreover, sequence analysis revealed multiple alleles in two P. aegyptiaca ABA receptors, with many non-synonymous mutations. Functional analysis of selected receptor alleles identified a variant with substantially decreased inhibitory effect of PP2Cs activity in-vitro. These results provide evidence that P. aegyptiaca is capable of biochemically perceiving ABA. In light of the possible involvement of ABA in parasitic activities, the discovery of active ABA receptors and PP2Cs could provide a new biochemical target for the agricultural management of P. aegyptiaca. Furthermore, the potential genetic loss of subfamily III receptors in this species, could position P. aegyptiaca as a valuable model in the ABA perception research field.

Optimized small-molecule pull-downs define MLBP1 as an acyl-lipid-binding protein.

Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand-binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC-MS to identify candidate binding ligands. We optimized this method using ABA-PYL interactions and show that ABA co-purifies with wild-type PYL5 but not a binding site mutant. The Kd of PYL5 for ABA is 1.1 µm, which suggests that the method has sufficient sensitivity for many ligand-protein interactions. Using this method, we surveyed a set of 37 START domain-related proteins, which resulted in the identification of ligands that co-purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co-purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein-metabolite interaction and better understand protein-ligand interactions in plants.

Defining and Exploiting Hypersensitivity Hotspots to Facilitate Abscisic Acid Agonist Optimization.

Pyrabactin resistance 1 (PYR1) and related abscisic acid (ABA) receptors are new targets for manipulating plant drought tolerance. Here, we identify and use PYR1 hypersensitive mutants to define ligand binding hotspots and show that these can guide improvements in agonist potency. One hotspot residue defined, A160, is part of a pocket that is occupied by ABA's C6 methyl or by the toluyl methyl of the synthetic agonist quinabactin (QB). A series of QB analogues substituted at the toluyl position were synthesized and provide up to 10-fold gain in activity in vitro. Furthermore, we demonstrate that hypersensitive receptors can be used to improve the sensitivity of a previously described mammalian cell ABA-regulated transcriptional circuit by three orders of magnitude. Collectively, our data show that the systematic mapping of hypersensitivity sites in a ligand-binding pocket can help guide ligand optimization and tune the sensitivity of engineered receptors.

Abscisic acid catabolism enhances dormancy release of grapevine buds.

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It was formerly proposed that dormancy is maintained by abscisic acid (ABA)-mediated repression of bud-meristem activity and that removal of this repression triggers dormancy release. It was also proposed that such removal of repression may be achieved via natural or artificial up-regulation of VvA8H-CYP707A4, which encodes ABA 8'-hydroxylase, and is the most highly expressed paralog in grapevine buds. The current study further examines these assumptions, and its experiments reveal that (a) hypoxia and ethylene, stimuli of bud dormancy release, enhance expression of VvA8H-CYP707A4 within grape buds, (b) the VvA8H-CYP707A4 protein accumulates during the natural transition to the dormancy release stage, and (c) transgenic vines overexpressing VvA8H-CYP707A4 exhibit increased ABA catabolism and significant enhancement of bud break in controlled and natural environments and longer basal summer laterals. The results suggest that VvA8H-CYP707A4 functions as an ABA degrading enzyme, and are consistent with a model in which the VvA8H-CYP707A4 level in the bud is up-regulated by natural and artificial bud break stimuli, which leads to increased ABA degradation capacity, removal of endogenous ABA-mediated repression, and enhanced regrowth. Interestingly, it also hints at sharing of regulatory steps between latent and lateral bud outgrowth.

Regulatory chromatin landscape in Arabidopsis thaliana roots uncovered by coupling INTACT and ATAC-seq.

BACKGROUND: There is a growing interest in the role of chromatin in acquiring and maintaining cell identity. Despite the ever-growing availability of genome-wide gene expression data, understanding how transcription programs are established and regulated to define cell identity remains a puzzle. An important mechanism of gene regulation is the binding of transcription factors (TFs) to specific DNA sequence motifs across the genome. However, these sequences are hindered by the packaging of DNA to chromatin. Thus, the accessibility of these loci for TF binding is highly regulated and determines where and when TFs bind. We present a workflow for measuring chromatin accessibility in Arabidopsis thaliana and define organ-specific regulatory sites and binding motifs of TFs at these sites. RESULTS: We coupled the recently described isolation of nuclei tagged in specific cell types (INTACT) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) as a genome-wide strategy to uncover accessible regulatory sites in Arabidopsis based on their accessibility to nuclease digestion. By applying this pipeline in Arabidopsis roots, we revealed 41,419 accessible sites, of which approximately half are found in gene promoters and contain the H3K4me3 active histone mark. The root-unique accessible sites from this group are enriched for root processes. Interestingly, most of the root-unique accessible sites are found in nongenic regions but are correlated with root-specific expression of distant genes. Importantly, these gene-distant sites are enriched for binding motifs of TFs important for root development as well as motifs for TFs that may play a role as novel transcriptional regulators in roots, suggesting that these accessible loci are functional novel gene-distant regulatory elements. CONCLUSIONS: By coupling INTACT with ATAC-seq methods, we present a feasible pipeline to profile accessible chromatin in plants. We also introduce a rapid measure of the experiment quality. We find that chromatin accessibility at promoter regions is strongly associated with transcription and active histone marks. However, root-specific chromatin accessibility is primarily found at intergenic regions, suggesting their predominance in defining organ identity possibly via long-range chromatin interactions. This workflow can be rapidly applied to study the regulatory landscape in other cell types, plant species and conditions.

Non-redundant functions of the dimeric ABA receptor BdPYL1 in the grass Brachypodium.

Abiotic stresses have severe detrimental effects on agricultural productivity worldwide. Abscisic acid (ABA) levels rise in response to abiotic stresses, and play a role in coordinating physiological responses. ABA elicits its effects by binding a family of soluble receptors, increasing affinity of the receptors to type 2C phosphatases (PP2Cs) leading to phosphatase inhibition. In the current study, we conducted a comprehensive analysis of the ABA signaling pathway in the cereal model grass Brachypodium distachyon. The Brachypodium genome encodes a family of 10 functionally conserved ABA receptors. The 10th in the series, BdPYL10, encodes a defective receptor and is likely a pseudogene. Combinatorial protein interaction assay further validated computational analysis, which grouped Brachypodium ABA receptors into three subfamilies, similarly to Arabidopsis classification. Brachypodium subfamily III receptors inhibited PP2C activity in vitro and complemented Arabidopsis quadruple (pyr1/pyl1/pyl2/pyl4) mutant. BdPYL1 T-DNA mutant exhibited clear ABA hyposensitivity phenotypes during seedling establishment and in mature plants. Single receptor predominance is in agreement with high transcriptional abundance of only a small Brachypodium ABA receptors subset, harboring the higher marginal significance of BdPYL1. Our findings suggest that unlike the highly redundant ABA core signaling components of Arabidopsis, Brachypodium encompasses a more compact and specialized ABA receptor apparatus. This organization may contribute to plant adaptations to ecological niches. These results lay the groundwork for targeting the prominent ABA receptors for stress perception in grasses, and reveal functional differences and commonalities between monocots and eudicots.

The Polycomb group protein CLF emerges as a specific tri-methylase of H3K27 regulating gene expression and development in Physcomitrella patens.

Packaging of eukaryotic DNA largely depends on histone modifications that affect the accessibility of DNA to transcriptional regulators, thus controlling gene expression. The Polycomb group (PcG) chromatin remodeling complex deposits a methyl group on lysine 27 of histone 3 leading to repressed gene expression. Plants encode homologs of the Enhancer of zeste (E(z)), a component of the PcG complex from Drosophila, one of which is a SET domain protein designated CURLY LEAF (CLF). Although this SET domain protein exhibits a strong correlation with the presence of the H3K27me3 mark in plants, the methyl-transferase activity and specificity of its SET domain have not been directly tested in-vivo. Using the evolutionary early-diverged land plant model species Physcomitrella patens we show that abolishment of a single copy gene PpCLF, as well as an additional member of the PcG complex, FERTILIZATION-INDEPENDENT ENDOSPERM (PpFIE), results in a specific loss of tri-methylation of H3K27. Using site-directed mutagenesis of key residues, we revealed that H3K27 tri-methylation is mediated by the SET domain of the CLF protein. Moreover, the abolishment of H3K27me3 led to enhanced expression of transcription factor genes. This in turn led to the development of fertilization-independent sporophyte-like structures, as observed in PpCLF and PpFIE null mutants. Overall, our results demonstrate the role of PpCLF as a SET protein in tri-methylation of H3K27 in-vivo and the importance of this modification in regulating the expression of transcription factor genes involved in developmental programs of P. patens.

The Putative O-Linked N-Acetylglucosamine Transferase SPINDLY Inhibits Class I TCP Proteolysis to Promote Sensitivity to Cytokinin.

Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) is a putative serine and threonine O-linked N-acetylglucosamine transferase (OGT). While SPY has been shown to suppress gibberellin signaling and to promote cytokinin (CK) responses, its catalytic OGT activity was never demonstrated and its effect on protein fate is not known. We previously showed that SPY interacts physically and functionally with TCP14 and TCP15 to promote CK responses. Here, we aimed to identify how SPY regulates TCP14/15 activities and how these TCPs promote CK responses. We show that SPY activity is required for TCP14 stability. Mutation in the putative OGT domain of SPY (spy-3) stimulated TCP14 proteolysis by the 26S proteasome, which was reversed by mutation in CULLIN1 (CUL1), suggesting a role for SKP, CUL1, F-box E3 ubiquitin ligase in TCP14 proteolysis. TCP14 proteolysis in spy-3 suppressed all TCP14 misexpression phenotypes, including the enhanced CK responses. The increased CK activity in TCP14/15-overexpressing flowers resulted from increased sensitivity to the hormone and not from higher CK levels. TCP15 overexpression enhanced the response of the CK-induced synthetic promoter pTCS to CK, suggesting that TCP14/15 affect early steps in CK signaling. We propose that posttranslational modification of TCP14/15 by SPY inhibits their proteolysis and that the accumulated proteins promote the activity of the CK phosphorelay cascade in developing Arabidopsis leaves and flowers.

Agrochemical control of plant water use using engineered abscisic acid receptors.

Rising temperatures and lessening fresh water supplies are threatening agricultural productivity and have motivated efforts to improve plant water use and drought tolerance. During water deficit, plants produce elevated levels of abscisic acid (ABA), which improves water consumption and stress tolerance by controlling guard cell aperture and other protective responses. One attractive strategy for controlling water use is to develop compounds that activate ABA receptors, but agonists approved for use have yet to be developed. In principle, an engineered ABA receptor that can be activated by an existing agrochemical could achieve this goal. Here we describe a variant of the ABA receptor PYRABACTIN RESISTANCE 1 (PYR1) that possesses nanomolar sensitivity to the agrochemical mandipropamid and demonstrate its efficacy for controlling ABA responses and drought tolerance in transgenic plants. Furthermore, crystallographic studies provide a mechanistic basis for its activity and demonstrate the relative ease with which the PYR1 ligand-binding pocket can be altered to accommodate new ligands. Thus, we have successfully repurposed an agrochemical for a new application using receptor engineering. We anticipate that this strategy will be applied to other plant receptors and represents a new avenue for crop improvement.

A single CMT methyltransferase homolog is involved in CHG DNA methylation and development of Physcomitrella patens.

C-5 DNA methylation is an essential mechanism controlling gene expression and developmental programs in a variety of organisms. Though the role of DNA methylation has been intensively studied in mammals and Arabidopsis, little is known about the evolution of this mechanism. The chromomethylase (CMT) methyltransferase family is unique to plants and was found to be involved in DNA methylation in Arabidopsis, maize and tobacco. The moss Physcomitrella patens, a model for early terrestrial plants, harbors a single homolog of the CMT protein family designated as PpCMT. Our phylogenetic analysis suggested that the CMT family is unique to embryophytes and its earliest known member PpCMT belongs to the CMT3 subfamily. Thus, P. patens may serve as a model to study the ancient functions of the CMT3 family. We have generated a ΔPpcmt deletion mutant which demonstrated that PpCMT is essential for P. patens protonema and gametophore development and is involved in CHG methylation as demonstrated at four distinct genomic loci. PpCMT protein accumulation pattern correlated with proliferating cells and was sub-localized to the nucleus as predicted from its function. Taken together, our results suggested that CHG DNA methylation mediated by CMT has been employed early in land plant evolution to control developmental programs during both the vegetative and reproductive haploid phases along the plant life cycle.

Potent and selective activation of abscisic acid receptors in vivo by mutational stabilization of their agonist-bound conformation.

Pyrabactin resistance (PYR) 1 and its relatives belong to a family of soluble abscisic acid (ABA) receptors that inhibit type 2C protein phosphatases (PP2C) when in their agonist-stabilized conformation. Given their switch-like properties, we envisioned that mutations that stabilize their agonist-bound conformation could be used to activate signaling in vivo. To identify such mutations, we subjected PYR1 to site-saturation mutagenesis at 39 highly conserved residues that participate in ABA or PP2C contacts. All 741 possible single amino acid substitutions at these sites were tested to identify variants that increase basal PYR1-PP2C interactions, which uncovered activating mutations in 10 residues that preferentially cluster in PYR1's gate loop and C-terminal helix. The mutations cause measurable but incomplete receptor activation in vitro; however, specific triple and quadruple mutant combinations were constructed that promote an agonist-bound conformation, as measured by heteronuclear single quantum coherence NMR, and lead to full receptor activation. Moreover, these mutations retain functionality when introduced into divergent family members, and can therefore be used to dissect individual receptor function in vivo, which has been problematic because of redundancy and family size. Expression of activated PYL2 in Arabidopsis seeds activates ABA signaling by a number of measures: modulation of ABA-regulated gene expression, induction of hyperdormancy, and suppression of ABA deficiency phenotypes in the aba2-1 mutant. Our results set the stage for systematic gain-of-function studies of PYR1 and related ABA receptors and reveal that, despite the large number of receptors, activation of a single receptor is sufficient to activate signaling in planta.