Article

Dynamic control of plant water use using designed ABA receptor agonists

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Abstract

Drought causes crop losses worldwide, and its impact is expected to increase as the world warms. This has motivated the development of small-molecule tools for mitigating the effects of drought on agriculture. We show here that current leads are limited by poor bioactivity in wheat, a widely grown staple crop, and in tomato. To address this limitation, we combined virtual screening, x-ray crystallography, and structure-guided design to develop opabactin (OP), an abscisic acid (ABA) mimic with up to an approximately sevenfold increase in receptor affinity relative to ABA and up to 10-fold greater activity in vivo. Studies in Arabidopsis thaliana reveal a role of the type III receptor PYRABACTIN RESISTANCE-LIKE 2 for the antitranspirant efficacy of OP. Thus, virtual screening and structure-guided optimization yielded newly discovered agonists for manipulating crop abiotic stress tolerance and water use.

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... Since the discovery of ABA receptor structures, substantial amount of research studies have been trying to either engineer PYLs or design new ABA agonists for effective manipulation of plant water use. [19][20][21][22] GAs control a variety of growth and developmental processes in higher plants, and has an essential role in modern agriculture industry. While there are at least 136 GAs identified from plants, only a few are bioactive hormones, including GA1, GA3, GA4 and GA7, and GA4 is the major bioactive GA in Arabidopsis thaliana. ...
... In addition, there are growing interests in developing specific hormone agonists to target certain receptor protein in the families. [21,22] The bottleneck is the understanding of the molecular origin of varying binding affinity and selectivity, which can not be obtained from crystallographic snapshots. In this sense, understanding the full picture of plant hormone binding and receptor activation processes might help us understand the mechanism. ...
... [114][115][116] PYR/PYL/RCAR receptors are promising targets for improving plant water use efficiency. [117][118][119][120] Notably, a series of ABA agonists covering various chemotypes [20][21][22][121][122][123][124] and a variety of engineered ABA receptors [19,125] have been discovered in recent years, in order to achieve selective activity control of PYR/PYL/RCAR receptors in multiple plant species. ...
Thesis
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Plant hormones are small molecules derived from natural metabolites that regulate plant growth, development and responses to biotic and abiotic stresses. There are nine major classes of plant hormones that have been identified, including abscisic acid, auxin, brassinosteroid, cytokinin, ethylene, giberellin, jasmonic acid, salicylic acid and strigolactone. Plant hormones are specifically recognized by their receptor proteins, and then initiate downstream protein-protein interactions to trigger a variety of hormone responses. Detailed understanding of the molecular mechanism of specific molecular recognition in plant hormone perception and signal transduction will create exciting opportunities for targeting hormone signaling pathway to control plant activities. While substantial experimental studies have provided insights into plant signaling mechanism, it remains, in many cases, challenging to fully understand those protein-ligand and protein-protein interactions at molecular level. Molecular dynamics (MD) simulation is a powerful technique for studying protein dynamics and function at an unparalleled spatial-temporal resolution. In this thesis, I have utilized MD simulations as the primary tool to study a series of protein-ligand and protein-protein interactions involved in early stage of plant hormone signal transduction. I have focused on abscisic acid (ABA) for the major part of the thesis, and also investigated six other classes of plant hormones, including auxin, brassinosteroid, cytokinin, giberellin, jasmonic acid, and strigolactone. By performing microsecond-long timescale MD simulations (aggregate 1.06 milliseconds) and Markov state model analysis, I have elucidated the molecular basis for the binding of the seven major classes of plant hormones to their receptors and the subsequent activation of plant receptors. I have reported the free energy landscapes that quantitatively characterize the thermodynamics and kinetics of receptor-hormone binding and receptor activation processes. I have also identified the key intermediate states in their binding pathways. For ABA, I have also studied the molecular mechanism of negative regulation of ABA signaling by post-translational modification of ABA receptor. To better understand the thermodynamic driving forces involved in binding affinity of plant hormones, I have investigated the role of water reorganization around the binding cavity of plant receptors on plant hormone perception. I have characterized the solvation structural and thermodynamic properties of plant receptors using MD simulations and inhomogeneous solvation theory-based hydration site analysis. Importantly, I have shown that water thermodynamics can potentially be exploited for rational agrochemical design to target plant hormone receptors. For the rest of this thesis, I have focused on the investigation of protein-protein interactions involved in plant signaling. In order to improve computational efficiency of unbiased molecular simulations of complex biomolecule dynamics such as protein-protein association, I have developed an adaptive sampling strategy that combines low-resolution experimental information with MD simulations. I have demonstrated the utility of this method in predicting the structures of dimeric ABA receptor and several other single domain proteins and complexes. Also, I have established a computational workflow for protein complex structure prediction via large-scale coarse grained MD simulations. Using this method, I have predicted a complex structure of protein phosphatase and small GTPase that play an essential role in negative regulatory network of ABA signaling. Overall, this thesis has provided fundamental insights into specific molecular recognition involved in plant hormone signal transduction, which can create new avenues for agrochemical control of plant growth and development. Furthermore, this thesis has established computational framework for understanding and engineering plant signaling.
... Chemical modulators of ABA perception have been sought as both research tools for dissecting ABA's role in plant physiology and for their potential agricultural utility (6,7). Dozens of ABA receptor agonists, which reduce transpiration and water use by inducing guard cell closure, have been developed and are being explored as chemical tools for mitigating the effects of drought on crop yields (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23), most of them either being analogs of ABA or sulfonamides similar to quinabactin (24). ABA receptor antagonists could conceivably be useful in cases where water is not limiting, for example, to increase transpiration and gas exchange under elevated CO 2 in glasshouse agriculture, as germination stimulators, and for studying the ABA dependence of physiological processes, among other applications (25)(26)(27)(28)(29)(30)(31). ...
... Together, these data suggest that higher-affinity panantagonists and/or molecules with increased bioavailability will be necessary to more efficiently block endogenous ABA signaling. We set out to address these limitations by modifying the scaffold of the synthetic ABA agonist opabactin (OP), which has an approximately sevenfold increase in both affinity and bioactivity relative to ABA (21). We describe an OP derivative called antabactin (ANT) and show that it is a high-affinity binder and antagonist of ABA receptors that disrupts ABA-mediated signaling in vivo. ...
... Discovery of ABA Receptor Antagonists by Click Chemistry Mediated Scaffold Elaboration. OP possesses a C4-nitrile substituent that is superimposable with ABA's C4′-ring ketone in ternary receptor/ ligand/complexes (21). We hypothesized that this position could be used to design high-affinity antagonists by appending moieties that block receptor-PP2C interactions ( Fig. 2A), analogously to C4′-modified ABA-based antagonists like PanMe (Fig. 1). ...
Article
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Significance Abscisic acid (ABA) is a phytohormone that plants utilize to coordinate responses to abiotic stress, modulate seed dormancy, and is central to plant development in several contexts. Chemicals that activate or block ABA signaling are useful as research tools and as potential agrochemical leads. Many successes have been reported for ABA activators (agonists), but existing ABA blockers (antagonists) are limited by modest in vivo activity. Here we report antabactin (ANT), a potent ABA blocker developed using “click chemistry”–based diversification of a known ABA activator. Structural studies reveal, ANT disrupts signaling by stabilizing ABA receptors in an unproductive form. ANT can accelerate seed germination in multiple species, making it a chemical tool for improving germination.
... Although structurally diverse, many of the ligands screened contain a carbonyl functional group that, as observed with WIN 55,212-2, can engage the Trp-lock to stabilize activated receptors ( Fig. 2 and Extended Data Fig. 3). Prior work has shown that other H-bond acceptors (e.g., nitriles and others) can function in place of a carbonyl to activate both wild-type and engineered PYR1 receptors 11,[29][30][31] (Extended Data Fig. 3). In addition, our evolved PYR1 CP sensor recognizes a ligand lacking a C=O (Fig. 2). ...
... Y2H screening of mutagenized PYR1 libraries. Selection experiments for mutant receptors that respond to new ligands were conducted as previously described 29,31 . Briefly, the PYR1 DSM mutant library was transformed into MAV99 harboring pACT-HAB1. ...
... Constructs were sequenced and transformed into the BL21 (DE3) pLysS E. coli strain for heterologous expression with IPTG (1 mM), followed by purification, using affinity chromatography, as previously reported 35 . In vitro validation of evolved sensors was performed by using recombinant sensors and ΔN-HAB1 (HAB1 Δ1-178, ref. 36 ) proteins, as previously described 29 . Ligand/receptor-dependent inhibition of PP2C activity was performed essentially as previously reported using 25 nM ΔN-HAB1 with either 25 nM titrated PYR1 4F or 50 nM titrated PYR1 WIN . ...
Article
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A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense–response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense–response applications.
... (B) The binding of ABA receptor agonists (ABA shown in cyan) leads to the formation of hydrophobic tunnels that accommodate a conserved tryptophan residue (Trp-lock-Brown side chain) from HAB1 (overlay of PDB:3R6P and PDB:3RT0) leading to inhibition of HAB1 enzymatic activity (C) ABA receptor antagonists (representative molecule shown in cyan) occlude access to the hydrophobic tunnels formed on the receptor, consequently blocking interaction with HAB1. Frackenpohl, Willms, & Dittgen, 2017;Lachia et al., 2018;Okamoto et al., 2013;Vaidya et al., 2017Vaidya et al., , 2019 have not only delineated members of subfamily III ABA receptors (PYR1, PYL1 & PYL2) as important target sites for control of plant water use but also revealed structurally diverse chemical scaffolds that can function as improved ABA mimics (Helander, Vaidya, & Cutler, 2016). In this chapter, we focus primarily on the synthetically accessible opabactin (OP) scaffold as a model ABA agonist, which was discovered using a combination of virtual screening and structure-guided optimization (Vaidya et al., 2019). ...
... Frackenpohl, Willms, & Dittgen, 2017;Lachia et al., 2018;Okamoto et al., 2013;Vaidya et al., 2017Vaidya et al., , 2019 have not only delineated members of subfamily III ABA receptors (PYR1, PYL1 & PYL2) as important target sites for control of plant water use but also revealed structurally diverse chemical scaffolds that can function as improved ABA mimics (Helander, Vaidya, & Cutler, 2016). In this chapter, we focus primarily on the synthetically accessible opabactin (OP) scaffold as a model ABA agonist, which was discovered using a combination of virtual screening and structure-guided optimization (Vaidya et al., 2019). ...
... ABA receptor protein genes were cloned into the pET28 vector and HAB1 (PP2C) with N-terminal deletion (À178 aa) was cloned to the pET11 vector to produce Hisx6 fusion proteins. Further information on construct names and clones can be found in references (Park et al., 2009;Vaidya et al., 2019). ...
Chapter
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As the climate becomes increasingly unpredictable due to global warming, plants will encounter a greater challenge to adapt to their hostile environment (e.g., drought, heat, pollution). Volatile apocarotenoids (VAs) are an integral part of this necessary adaptation. VAs are involved in diverse plant life processes such as defense against biotic or abiotic stresses and regulate various aspects of plant development. The discovery of new VAs will help enhance abiotic and biotic stress tolerance, optimize biomass and crop yield, improve root development to better search for nutrients and promote symbiotic associations. This chapter describes an optimized method, HeadSpace Solid-Phase MicroExtraction (HS-SPME) coupled to Gas Chromatography-Mass Spectrometry (GC/MS), for the sensitive, reproducible, accurate, and high-throughput detection and quantification of novel and known VAs. Further optimization of this method can be performed by (1) adapting optimal growth conditions for your plants, (2) identifying the correct SPME fiber coating chemistry for the VAs of interest, (3) adapting optimal sample HS-SPME extraction temperature and time, and the desorption time in the GC inlet, (4) identifying the correct GC column and applying the optimal GC/MS parameters for good chromatographic baseline separation of the VAs, mass spectral matching and retention index (RI) validation, and (5) performing suitable quantification and statistical analyses. With this optimized and validated analytical technique, we detected and quantified 28 VAs; 20 of these were identified for the first time in Arabidopsis.
... With the rapid development of chemicobiology and computer-aided technologies, successful cases of computer-aided drug discovery continue to emerge (William, 2009;Lavecchia and Giovanni, 2013;Cavasotto et al., 2019). For example, opabactin (Vaidya et al., 2019) and AMF4 (Cao et al., 2013(Cao et al., , 2017, the outstanding abscisic acid (ABA) receptor agonists, were obtained using computer-aided screening and computer-guided optimization, providing a potential approach for drought resistance in agricultural production systems. However, few studies have focused on the discovery of auxin receptor agonists acting as root growth promoters. ...
... As we know, plant growth regulators have been widely used in field crop research and have made great contributions to agricultural production (Motyka, 2005). Researchers have developed various plant growth regulators by virtual screening or/and the empirical modifications, such as uniconazole, which adjusts plant height to resist lodging (Ahmad et al., 2019), thidiazuron, which sheds plant leaves for mechanical harvest to reduce labor (Suttle, 1985;Ledbetter and Preece, 2004), and opabactin (Vaidya et al., 2019), which mitigates the effects of drought, reducing risks to crop yields. Combining the chemical FIGURE 1 | Computer-aided drug design and the synthesis of target compounds K-1 to K-22. ...
Article
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Deeper and longer roots allow crops to survive and flourish, but our understanding of the plant growth regulators promoting root system establishment is limited. Here, we report that, a novel auxin receptor agonist, named K-10, had a remarkable promotive effect on root growth in both Arabidopsis thaliana and Oryza sativa through the enhancement of root-related signaling responses. Using computer-aided drug discovery approaches, we developed potent lead compound by screening artificial chemicals on the basis of the auxin receptor TIR1 (Transport Inhibitor Response 1), and a series of N-(benzo[d] [1,3] dioxol-5-yl)-2-(one-benzylthio) acetamides, K-1 to K-22, were designed and synthesized. The results of bioassay showed that K-10 exhibited an excellent root growth-promoting activity far exceeding that of NAA (1-naphthylacetic acid). A further morphological investigation of the auxin related mutants ( yucQ , tir1 ) revealed that K-10 had auxin-like physiological functions and was recognized by TIR1, and K-10 significantly enhanced auxin response reporter’s ( DR5:GUS ) transcriptional activity. Consistently, transcriptome analysis showed that K-10 induced a common transcriptional response with auxin and down-regulated the expression of root growth-inhibiting genes. Further molecular docking analysis revealed that K-10 had a stronger binding ability with TIR1 than NAA. These results indicated that this class of derivatives could be a promising scaffold for the discovery and development of novel auxin receptor agonists, and the employment of K-10 may be effective for enhancing root growth and crop production.
... Dimeric receptors AtPYR1, AtPYL1 and AtPYL2 have low affinity for ABA and therefore a high dissociation constant (K D > 50 µM, 52 µM and 59 µM, respectively) [29]. However, the affinity for ABA considerably increases (and therefore, the constant drops) when the PP2C phosphatase is included in the assay, probably due to a reduction in the dissociation rate of ABA in the ternary complex [29,54]. For example, AtPYR1 exhibits a K D for ABA of 97 +/ 36 µM when measured using NMR [29]. ...
... Opabactin is a designed agonist that activates all the wheat PYL members with IC50s in the nanomolar range, including TaPYL8. However, in Arabidopsis, AtPYL8-like subfamily members are not activated by this agonist [54]. Another interesting case involves the PYL8-like members (subfamily I) of date palm (Phoenix dactylifera) that are predominantly expressed under abiotic stress conditions [65]. ...
Article
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The hormone abscisic acid (ABA) orchestrates the plant stress response and regulates sophisticated metabolic and physiological mechanisms essential for survival in a changing environment. Plant ABA receptors were described more than 10 years ago, and a considerable amount of information is available for the model plant Arabidopsis thaliana. Unfortunately, this knowledge is still very limited in crops that hold the key to feeding a growing population. In this review, we summarize genomic, genetic and structural data obtained in crop ABA receptors. We also provide an update on ABA perception in major food crops, highlighting specific and common features of crop ABA receptors.
... Consequently, new electron-withdrawing groups were introduced into the benzene ring. It is widely recognized that the chemical structure, particularly the alkyl chain length and benzene ring substituents, has a significant impact on the physiological activity of a compound (Kazuko et al. 2019;Wang et al. 2017a, b;Liu et al. 2013;Matušková et al. 2020;Vaidya et al. 2019). For instance, analogs of the plant hormone abscisic acid exhibiting different length alkyl chain or cytokinins with various substituents on the benzene ring displayed diverse bioactivities. ...
... This result was attributed to the greater sensitivity of A. thaliana and rice to the synthetic compounds and the absence of enzymes able to degrade the synthetic hormones in the plant (Chi-Linh et al. 2016). These results indicated that derivative S8 could efficiently cause GA-like effects in plants at low concentrations; therefore, it could be utilized in plant control and seed germination under harsh conditions (Vaidya et al. 2019). Parallel activity contrast studies on compounds S1-S12 (A), X represents halogen groups on the benzene ring, i.e., fluorine, chlorine, bromine, and trifluoromethyl, respectively, corresponding four lines, 2-4 represents the position of different substituents on the benzene ring, i.e., ortho, para, and meta; parallel activity contrast stud-ies between compounds S4-S6 and compounds S17-S22 (B), n represents alkyl chain length, i.e., 2, 1, 0, respectively, corresponding compounds S4-S6, S17-S19, S20-S22; Promoting rates represent the growth promoting rates of A. thaliana hypocotyl treated with compounds S1-S22 at a dosage of 0.1 μM Furthermore, genetic approaches were used to accurately and efficiently evaluate the nature of the target compounds. ...
Article
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It has previously been demonstrated that a thiourea derivative, namely 1-phenethyl-3-[3-(trifluoromethyl)phenyl]thiourea (Y21), exhibits high gibberellin-like activity. In this work, the influence of the alkyl chain length and various benzene ring substituents on the biological activity of Y21 was investigated. 1-Substituted 3-[3-(trifluoromethyl)phenyl]thiourea derivatives were designed, synthesized, and comprehensively evaluated. The biological activity analysis demonstrated that at a dose of 0.1 μM, compound S8, i.e., 1-(3-bromophenethyl)-3-[3-(trifluoromethyl)phenyl]thiourea), exhibited significantly higher promoting activity with respect to Arabidopsis thaliana hypocotyl elongation and rice germination than did gibberellin A3 (GA3) and Y21. Moreover, the physiological response of the A. thaliana mutant ga1-1 to treatment with S8 suggested that this compound possessed GA-like function. The molecular docking analysis indicated that S8 formed hydrogen bonding interactions with residues Phe238 and Ser191 of the gibberellin insensitive DWARF1 (GID1) receptor, which resulted in stronger binding with GID1 than Y21. The bioactivity of derivatives S1–S22 exhibited a clear negative monotonic association with the molecular docking-based score values. Overall, it was determined that S8 displayed excellent GA-like bioactivity and facilitated hypocotyl elongation and germination; therefore, it could be applied as a potential plant growth regulator.
... Recently, a variety of new strategies have been devised to improve plant performance under environmental stress. Numerous studies have indicated that exogenous application of various PGRs, such as hormones, hormone agonists, polyamines (PAs), nutrients, antioxidants, osmoprotectants, and others, enhances crop drought tolerance (Raza et al., 2012;Latif et al., 2016;Vaidya et al., 2019;Hassan et al., 2020;Huan et al., 2020;Veroneze-Jú nior et al., 2020). According to these studies, the application of such compounds can enhance parameters such as morphology, photosynthetic capacity, relative water content (RWC), and gas exchange attributes. ...
... The AMF ABA analogs displayed long-lasting effects on the promotion of stomatal closure and the induction of stress-responsive gene expression (Cao et al., 2017). Recently, Vaidya et al. (2019) combined virtual screening, X-ray crystallography, and structure-guided design to develop opabactin (OP), an ABA mimic with up to an approximately seven-fold increase in receptor affinity relative to ABA and up to 10fold greater activity in vivo. They demonstrated that OP has activities in multiple monocots and eudicots and addressed the Pro oats (Avena sativa L.) enhanced leaf mesophyll thickness and metaxylem area, suppressed leaf midrib thickness and phloem area, increased proline and total soluble protein contents, improved photosynthesis, growth, seed oil quality, and enhanced seed oil 1,1diphenyl-2-picryl-hydrazyl (DPPH ) free radical-scavenging activity limitations of existing sulfonamide molecules. ...
Article
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Drought is one of the main abiotic stresses that causes crop yield loss. Improving crop yield under drought stress is a major goal of crop breeding as it is critical to food security. Although the mechanism of plant drought resistance has been well studied and diverse drought-resistant genes have been identified in recent years, transferring knowledge from laboratory to field production remains a significant challenge. Recently, some new strategies have become research frontiers due to their advantages of low cost, convenience, strong field operability and/or environmental friendliness. Among them, the strategies of exogenous plant growth regulator (PGR) treatment and microbe-based plant biotechnology have been utilized to effectively improve crop drought tolerance and save yield under drought stress. However, our understanding of the mechanism of PGRs regulating plant drought resistance and plant-microbiome interaction under drought is still unclear. In this review, we summarized these two strategies reported in recent studies, focusing on mechanisms of these exogenous treatments in regulating crop drought resistance. At the end, future challenge and directions of crop drought-resistant breeding were discussed.
... Exogenous administration of ABA or synthetic ABA mimics (i.e. ABA receptor agonists) is reported to elicit a stress response in plants, which improves their adaptability, showing the relevance of its activity under stress circumstances [98,99]. ...
Article
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It is vital to ramp up crop production dramatically by 2050 due to the increasing global population and demand for food. However, with the climate change projections showing that droughts and heatwaves becoming common in much of the globe, there is a severe threat of a sharp decline in crop yields. Thus, developing crop varieties with inbuilt genetic tolerance to environmental stresses is urgently needed. Selective breeding based on genetic diversity is not keeping up with the growing demand for food and feed. However, the emergence of contemporary plant genetic engineering, genome-editing, and synthetic biology offer precise tools for developing crops that can sustain productivity under stress conditions. Here, we summarize the systems biology-level understanding of regulatory pathways involved in perception, signalling, and protective processes activated in response to unfavourable environmental conditions. The potential role of noncoding RNAs in the regulation of abiotic stress responses has also been highlighted. Further, examples of imparting abiotic stress tolerance by genetic engineering are discussed. Additionally, we provide perspectives on the rational design of abiotic stress tolerance through synthetic biology and list various bioparts that can be used to design synthetic gene circuits whose stress-protective functions can be switched on/off in response to environmental cues.
... Studies aimed at enhancing plant productivity or survival under water deficit have shown that transpiration can be limited through exogenous application of ABA and ABA agonists, transgenic or ectopic PYL expression, and down-regulation of PP2C expression Okamoto et al., 2013;Park et al., 2015;Yang et al., 2016;Cao et al., 2017;Mega et al., 2019;Vaidya et al., Overall, these findings suggest that ABA receptors are excellent candidates for manipulating crop production efficiency under drought stress conditions. ...
Article
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The role of abscisic acid (ABA) receptors, PYR1/PYL/RCAR (PYLs), is well established in ABA signaling and plant drought response, but limited research has explored the regulation of wheat PYLs in this process, especially the effects of their allelic variations on drought tolerance or grain yield. Here, we found that overexpression of a TaABFs-regulated PYL gene, TaPYL1-1B, exhibited higher ABA sensitivity, photosynthetic capacity, and water-use efficiency (WUE), all contributed to higher drought tolerance than that of wild-type plants. This heightened water-saving mechanism further increased grain yield and protected productivity during water deficit. Candidate gene association analysis revealed that a favorable allele TaPYL1-1BIn-442, carrying an MYB recognition site insertion in the promoter, is targeted by TaMYB70 and confers enhanced expression of TaPYL1-1B in drought-tolerant genotypes. More importantly, an increase in frequency of the TaPYL1-1BIn-442 allele over decades among modern Chinese cultivars and its association with high thousand-kernel weight together demonstrated that it was artificially selected during wheat improvement efforts. Taken together, our findings illuminate the role of TaPYL1-1B plays in coordinating drought tolerance and grain yield. In particular, the allelic variant TaPYL1-1BIn-442 substantially contributes to enhanced drought tolerance while maintaining high yield, and thus represents a valuable genetic target for engineering drought-tolerant wheat germplasm.
... As an important hormone, ABA plays a vital role in the drought tolerance of crops, while, due to the chemical instability, the rapid catabolism of ABA limits its application in the field. Scientists have conducted vast amounts of research to search for more stable ABA analogs, such as pyrabactin, AM1 (ABA mimic 1)/quinabactin, cyanabactin, opabactin, and AMFs [143]. Furthermore, combining chemical and genetic approaches, AMFs are applied to PYL2 overexpression transgenic plants, increasing their drought tolerance [144]. ...
Article
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Drought has become a major threat to food security, because it affects crop growth and development. Drought tolerance is an important quantitative trait, which is regulated by hundreds of genes in crop plants. In recent decades, scientists have made considerable progress to uncover the genetic and molecular mechanisms of drought tolerance, especially in model plants. This review summarizes the evaluation criteria for drought tolerance, methods for gene mining, characterization of genes related to drought tolerance, and explores the approaches to enhance crop drought tolerance. Collectively, this review illustrates the application prospect of these genes in improving the drought tolerance breeding of crop plants.
... The abscisic acid ( transduction pathway (Gupta et al. 2020). Promoting the expression of ABA receptors, SnRK2 and ABA-responding genes, inhibiting the expression of PP2C, and introducing opabactin (OP, ABA agonist) can improve drought resistance in plants (Song et al. 2016, Chang et al. 2017, Vaidya et al. 2019. ...
Article
The basic helix-loop-helix (bHLH) transcription factors (TFs) are involved in plant morphogenesis and various abiotic and biotic stress responses. However, it requires further exploration of drought-responsive bHLH family members and their detailed regulatory mechanisms in Populus. Two bHLH TF genes, PxbHLH01/02, were identified in Populus simonii × P. nigra and cloned. The aim of this study was to examine the role of bHLH TFs in drought tolerance in P. simonii × P. nigra. The results showed that the amino acid sequences of the two genes were homologous to Arabidopsis thaliana UPBEAT1 (AtUPB1) and overexpression of PxbHLH01/02 restored normal root length in the AtUPB1 insertional mutant (upb1-1). The PxbHLH01/02 gene promoter activity analysis suggested that they were involved in stress responses and hormone signaling. Furthermore, Arabidopsis transgenic lines overexpressing PxbHLH01/02 exhibited higher stress tolerance compared with the wild-type. P. simonii × P. nigra overexpressing PxbHLH02 increased drought tolerance and exhibited higher superoxide dismutase and peroxidase activities, lower H2O2 and malondialdehyde (MDA) content, and lower relative conductivity. The results of transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) suggested that the response of PxbHLH02 to drought stress was related to ABA signal transduction. Overall, the findings of this study suggest that PxbHLH02 from P. simonii × P. nigra functions as a positive regulator of drought stress responses by regulating stomatal aperture and promoting ABA signal transduction.
... Plants have long been exploited as sources of bioactive and high-value natural products (15) and, recently, applications of model-informed synthetic biology approaches have led to sophisticated engineering of crop traits including biomass and responses to environment (16)(17)(18). There is also growing interest and investment in the use of plants, particularly Nicotiana benthamiana, as photosynthetic platforms for the production of recombinant proteins and small molecules for industry and medicine (19)(20)(21), including rapid-response vaccines (22)(23)(24)(25). ...
Article
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Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterisation has remained a technical bottleneck, often requiring significant effort to optimise expression and purification protocols. To leverage the ability of biofoundries to accelerate design-built-test-learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimisation and enables rapid screening of enzyme activity. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using E. coli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification, and improved expression/folding. We next optimised automated assembly of miniaturised cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11 aa tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional assays can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.
... When HaHB4 TF was transformed into wheat and tested across a range of growing conditions, it exhibited adaptation to droughtprone environments and gave 20% more yield (Gonzelez et al. 2018). Validya et al. (2019) have developed opabactin (OP), an ABA receptor antagonist that mimics the effect of abiotic stress tolerance and water use. This OP enhances drought tolerance and hyperactivate ABA transcriptional responses in wheat. ...
Chapter
This chapter focuses on the practicality of the challenge of food security that is threatened by various climate change constraints and ensure that well integrated outputs are made over a wide spectrum of characteristics. To complement the existent genetic diversity prevalent in conventional wheat germplasm, harnessing the variation that resides in approximately 325 annual and perennial Triticeae genera and their abundant accessional strength is a valid option. Wheat productivity around the world is heavily dependent on commercial cultivars carrying genes derived from related species. Besides varietal improvement, wheat DH populations have also been used in the creation of molecular marker maps and identification of new alleles/QTLs and unveiling of their action mechanisms. A concerted effort to harness this rich readily available progenitor genome resource warrants intensive exploitation for attaining time bound food security projected goals.
... Interestingly, PB is an agonist for PYR1 and PYL1, but is an antagonist of PYL2 [35], thus explaining the differential bioactivities of PB and ABA. Subsequent research has led to the development of other synthetic agonists such as quinabactin [36] and opabactin [37] to improve selectivity and activity for certain receptors or enhance potency to trigger certain ABAmediated biological effects [34,38,39]. ...
Article
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Roots are composed of different root types and, in the dicotyledonous Arabidopsis, typically consist of a primary root that branches into lateral roots. Adventitious roots emerge from non-root tissue and are formed upon wounding or other types of abiotic stress. Here, we investigated adventitious root (AR) formation in Arabidopsis hypocotyls under conditions of altered abscisic acid (ABA) signaling. Exogenously applied ABA suppressed AR formation at 0.25 µM or higher doses. AR formation was less sensitive to the synthetic ABA analog pyrabactin (PB). However, PB was a more potent inhibitor at concentrations above 1 µM, suggesting that it was more selective in triggering a root inhibition response. Analysis of a series of phosphonamide and phosphonate pyrabactin analogs suggested that adventitious root formation and lateral root branching are differentially regulated by ABA signaling. ABA biosynthesis and signaling mutants affirmed a general inhibitory role of ABA and point to PYL1 and PYL2 as candidate ABA receptors that regulate AR inhibition.
... In Arabidopsis, 14 PYLs, at least 8 PP2Cs, three SnRK2s, and 12 members of the B2 and B3 RAF subgroups comprise a complex network in ABA sensing and signaling, which may ensure that plants precisely respond to everchanging environments. The engineering of ABA receptors is an efficient way to improve stress resistance in both Arabidopsis and crops [50][51][52] . Our findings regarding B2 and B3 RAFs in stress signaling provide additional targets (e.g., ectopic expression of stress-inducible or constitutively activated forms of RAFs in guard cells) for engineering crops resistant to harsh environmental conditions. ...
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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.
... Extensive studies have revealed that exogenous application of ABA induces the tolerance of a plant to a variety of abiotic stresses, such as drought, heat, cold, and high salinity [26][27][28], and some biotic stresses such as pathogens, and insects [29][30][31]. Many ABA analogues have been actively studied owing to their beneficial effects on plants [32,33]. Although there is no evidence to indicate that 1 ,4 -trans-diol-ABA can be biosynthesized in plants, it has been confirmed as a precursor of ABA synthesis in microorganisms [13]. ...
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1',4'-trans-diol-ABA is a key precursor of the biosynthesis of abscisic acid (ABA) biosynthesis in fungi. We successfully obtained the pure compound from a mutant of Botrytis cinerea and explored its function and possible mechanism on plants by spraying 2 mg/L 1',4'-trans-diol-ABA on tobacco leaves. Our results showed that this compound enhanced the drought tolerance of tobacco seedlings. A comparative transcriptome analysis showed that a large number of genes responded to the compound, exhibiting 1523 genes that were differentially expressed at 12 h, which increased to 1993 at 24 h and 3074 at 48 h, respectively. The enrichment analysis demonstrated that the differentially expressed genes (DEGs) were primarily enriched in pathways related to hormones and resistance. The DEGs of transcription factors were generally up-regulated and included the bHLH, bZIP, ERF, MYB, NAC, WRKY and HSF families. Moreover, the levels of expression of PYL/PYR, PP2C, SnRK2, and ABF at the ABA signaling pathway responded positively to exogenous 1',4'-trans-diol-ABA. Among them, seven ABF transcripts that were detected were significantly up-regulated. In addition, the genes involved in salicylic acid, ethylene and jasmonic acid pathways, reactive oxygen species scavenging system, and other resistance related genes were primarily induced by 1',4'-trans-diol-ABA. These findings indicated that treatment with 1',4'-trans-diol-ABA could improve tolerance to plant abiotic stress and potential biotic resistance by regulating gene expression, similar to the effects of exogenous ABA.
... ABA and indole-3-acetic acid (IAA) are important signaling molecules that regulate metabolic processes related to stress adaptation and induce stress resistance in plants (Verma et al., 2019). Numerous studies have shown that the ABA content increases under abiotic stress and induces the expression of downstream genes (Vaidya et al., 2019). The upregulated cellular defensive substance synthesis pathways supported the maintenance of homeostasis in the cellular membrane to achieve enhanced freezing resistance . ...
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Liriope spicata is an evergreen perennial ornamental groundcover with a strong freezing tolerance. However, the molecular mechanism underlying the freezing tolerance in L. spicata remains unclear. In this study, a comprehensive investigation of L. spicata freezing tolerance was conducted at the levels of physiology and biochemistry, metabolite, and transcript during the stress treatment. There were 581 unique differentially expressed metabolites (DEMs) and 10,444 unique differentially expressed genes (DEGs) between freezing treatment and normal cultured plant in leaves. Integrated analysis of metabolomics and transcriptomics showed that flavonoid biosynthesis, carbohydrate metabolism, amino acid metabolism, lipid metabolism, and signal transduction pathways were prominently enriched in response to the freezing stress in L . spicata . Now, we identified genes and metabolites involved in the flavonoid pathway, abscisic acid (ABA) biosynthesis, and the oxidative synthesis pathway of nitric oxide (NO), which may form a regulatory network and play a synergistic effect in osmotic adjustment, reactive oxygen species (ROS) homeostasis, and stomatal closure under freezing stress. These results offer a comprehensive network of flavonoids, ABA, and NO comodulating the freezing tolerance in L. spicata .
... For example, molecular docking can be used to perform in silico screens for potential agonists or antagonists that are refined further through in vitro assays and structure-guided optimization. This approach was employed with great success to design a headgroup that dramatically improves the affinity of an ABA receptor agonist, opabactin (Vaidya et al., 2019). In another example, pharmacophore models developed from crystal structures of rice D14 were used to perform in silico screening of 4.7 million compounds for potential SL signaling inhibitors (Mashita et al., 2016). ...
Article
Chemical signals known as strigolactones were discovered more than 50 years ago as host-derived germination stimulants of parasitic plants in the Orobanchaceae. Strigolactone-responsive germination is an essential adaptation of obligate parasites in this family, which depend upon a host for survival. Several species of obligate parasites, including witchweeds (Striga, Alectra spp.) and broomrapes (Orobanche, Phelipanche spp.), are highly destructive agricultural weeds that pose a significant threat to global food security. Understanding how parasites sense strigolactones and other host-derived stimulants will catalyze the development of innovative chemical and biological control methods. This review synthesizes the recent discoveries of strigolactone receptors in parasitic Orobanchaceae, their signaling mechanism, and key steps in their evolution.
... In plants, stomata regulate gaseous exchange and are integral to regulating water and photosynthesis (Hsu et al. 2021). Several approaches involving conventional and transgenic technologies have been used to improve crop growth under water-limited conditions, including the regulation of transcription factors, osmolyte biosynthesis, peptides and metabolic enzymes (Kim et al. 2017, Takahashi et al. 2018, Vaidya et al. 2019, Ahmad et al. 2021. Much of the related research has focused on decreasing the transpiration rate under drought conditions by regulating stomatal opening (Hetherington and Woodward 2003). ...
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Water scarcity is a serious agricultural problem causing significant losses to crop yield and product quality. The development of technologies to mitigate the damage caused by drought stress is essential for ensuring a sustainable food supply for the increasing global population. We herein report that the exogenous application of ethanol, an inexpensive and environmentally friendly chemical, significantly enhances drought tolerance in Arabidopsis thaliana, rice and wheat. The transcriptomic analyses of ethanol-treated plants revealed the upregulation of genes related to sucrose and starch metabolism, phenylpropanoids and glucosinolate biosynthesis, while metabolomic analysis showed an increased accumulation of sugars, glucosinolates and drought-tolerance-related amino acids. The phenotyping analysis indicated that drought-induced water loss was delayed in the ethanol-treated plants. Furthermore, ethanol treatment induced stomatal closure, resulting in decreased transpiration rate and increased leaf water contents under drought stress conditions. The ethanol treatment did not enhance drought tolerance in the mutant of ABI1, a negative regulator of abscisic acid (ABA) signaling in Arabidopsis, indicating that ABA signaling contributes to ethanol-mediated drought tolerance. The nuclear magnetic resonance analysis using 13C-labeled ethanol indicated that gluconeogenesis is involved in the accumulation of sugars. The ethanol treatment did not enhance the drought tolerance in the aldehyde dehydrogenase (aldh) triple mutant (aldh2b4/aldh2b7/aldh2c4). These results show that ABA signaling and acetic acid biosynthesis are involved in ethanol-mediated drought tolerance and that chemical priming through ethanol application regulates sugar accumulation and gluconeogenesis, leading to enhanced drought tolerance and sustained plant growth. These findings highlight a new survival strategy for increasing crop production under water-limited conditions.
... In addition to the benefit for nutrition and health, wheat provides about 21% of dietary calories and 20% of protein for humans, playing a prominent role in improving food security [2]. However, drought stress occurs frequently, which seriously restricts the production of wheat [3]. Therefore, improving wheat drought tolerance is an important approach to ensure food security [4,5]. ...
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Drought stress frequently occurs, which seriously restricts the production of wheat (Triticum aestivum L.). Leaf rolling is a typical physiological phenomenon of plants during drought stress. To understand the genetic mechanism of wheat leaf rolling, we constructed an F2 segregating population by crossing the slight-rolling wheat cultivar “Aikang 58” (AK58) with the serious-rolling wheat cultivar ″Zhongmai 36″ (ZM36). A combination of bulked segregant analysis (BSA) with Wheat 660K SNP Array was used to identify molecular markers linked to leaf rolling degree. A major locus for leaf rolling degree under drought stress was detected on chromosome 7A. We named this locus LEAF ROLLING DEGREE 1 (LERD1), which was ultimately mapped to a region between 717.82 and 720.18 Mb. Twenty-one genes were predicted in this region, among which the basic helix-loop-helix (bHLH) transcription factor TraesCS7A01G543300 was considered to be the most likely candidate gene for LERD1. The TraesCS7A01G543300 is highly homologous to the Arabidopsis ICE1 family proteins ICE/SCREAM, SCREAM2 and bHLH093, which control stomatal initiation and development. Two nucleotide variation sites were detected in the promoter region of TraesCS7A01G543300 between the two wheat cultivars. Gene expression assays indicated that TraesCS7A01G543300 was higher expressed in AK58 seedlings than that of ZM36. This research discovered a candidate gene related to wheat leaf rolling under drought stress, which may be helpful for understanding the leaf rolling mechanism and molecular breeding in wheat.
... Recently, Vaidya et al. developed opabactin by a combination of virtual screening, x-ray crystallography, and structure-guided design. Opabactin is the most potent ABA agonist developed, which showed approximately sevenfold increase in receptor affinity relative to ABA and up to 10-fold greater activity in vivo (Vaidya et al., 2019). As a known fungicide, mandipropamid ( Figure 5) was reported to interact with a hextuple PYR1 mutant (PYR1 MANDI ) and induced stomatal closure and inhibited seed germination in transgenic plants with PYR1 MANDI (Park et al., 2015). ...
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Abscisic acid (ABA) is an important plant endogenous hormone that participates in the regulation of various physiological processes in plants, including the occurrence and development of somatic embryos, seeddevelopment and dormancy. ABA is called “plant stress resistance factor”, while with the limitation of the rapid metabolic inactivation and photoisomerization inactivation of ABA for its large-scale use. Understanding the function and role of ABA in plants is of great significance to promote its application. For decades, scientists have conducted in-depth research on its mechanism of action and signaling pathways, a series of progress were achieved, and hundreds of ABA analogues (similar in structure or function) have been synthesized to develop highly active plant growth regulators and tools to elucidate ABA perception. In this review, we summarize a variety of ABA analogues, especially the ABA receptor analogues, and explore the mechanisms of ABA action and catabolism, which will facilitate the development of novel ABA analogues with high biological activities.
... carbamate-D-ring to the structure of aryloxyacetyl piperazines might improve the seed germination bioactivity of P. aegyptiaca as well. The carbamate-D-ring moiety was introduced into the aryloxyacetyl piperazines of 3f and 3g, respectively, by adopting the "scaffold hopping" strategy 38 . Compounds 4a and 4b were then successfully synthesized (Supplementary Scheme 2). ...
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The development of potent strigolactone (SL) agonists as suicidal germination inducers could be a useful strategy for controlling root parasitic weeds, but uncertainty about the SL perception mechanism impedes real progress. Here we describe small-molecule agonists that efficiently stimulate Phelipanchce aegyptiaca, and Striga hermonthica, germination in concentrations as low as 10−8 to 10−17 M. We show that full efficiency of synthetic SL agonists in triggering signaling through the Striga SL receptor, ShHTL7, depends on the receptor-catalyzed hydrolytic reaction of the agonists. Additionally, we reveal that the stereochemistry of synthetic SL analogs affects the hydrolytic ability of ShHTL7 by influencing the probability of the privileged conformations of ShHTL7. Importantly, an alternative ShHTL7-mediated hydrolysis mechanism, proceeding via nucleophilic attack of the NE2 atom of H246 to the 2′C of the D-ring, is reported. Together, our findings provide insight into SL hydrolysis and structure-perception mechanisms, and potent suicide germination stimulants, which would contribute to the elimination of the noxious parasitic weeds. Strigolactone agonists could potentially help control noxious weeds by promoting suicidal germination. Here the authors describe a series of small molecule agonists that stimulate germination via the Striga ShHTL7 receptor and show that stereochemistry and hydrolysis-independent signalling mediate potency.
... Carotenoids are isoprenoid pigments that provide precursors for the evolutionary-conserved plant hormones such as abscisic acid (ABA) and strigolactones (SLs) (Al-Babili and Bouwmeester, 2015;Hou et al., 2016;Wang et al., 2021a), as well as apocarotenoid signaling molecules, such as anchorene and zaxinone Jia et al., 2019;Wang et al., 2019;Ablazov et al., 2020). Due to the instability of authentic metabolites or restricted natural sources, several ABA and SL analogs have been developed in the past few years and some of them have been tested in the field (Samejima et al., 2016;Screpanti et al., 2016;Jamil et al., 2019Jamil et al., , 2020Jamil et al., , 2022aKountche et al., 2019;Vaidya et al., 2019). The further case is zaxinone, a candidate of novel apocarotenoid-derived phytohormones, which is required for normal rice growth and development . ...
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Global food security is a critical concern that needs practical solutions to feed the expanding human population. A promising approach is the employment of biostimulants to increase crop production. Biostimulants include compounds that boost plant growth. Recently, mimics of zaxinone (MiZax) were shown to have a promising growth-promoting effect in rice (Oryza sativa). In this study, we investigated the effect of MiZax on the growth and yield of three dicot horticultural plants, namely, tomato (Solanum lycopersicum), capsicum (Capsicum annuum), and squash (Cucurbita pepo) in different growth environments, as well as on the growth and development of the monocot date palm (Phoenix dactylifera), an important crop in the Middle East. The application of MiZax significantly enhanced plant height, flower, and branch numbers, fruit size, and total fruit yield in independent field trials from 2020 to 2021. Importantly, the amount of applied MiZax was far less than that used with the commercial compound humic acid, a widely used biostimulant in horticulture. Our results indicate that MiZax have significant application potential to improve the performance and productivity of horticultural crops.
... These molecules can modulate ABA signaling dynamically and exogenously either by enhancing drought tolerance or inhibiting ABA mediated responses in plants, respectively, (Hewage et al., 2020;Lozano-Juste et al., 2020). Major breakthroughs in this field have been the discovery of Opabactin, a potent ABA agonist for manipulating crop abiotic stress tolerance and water use (Vaidya et al., 2019) and Antabactin, a pan-receptor antagonist that disrupts ABA mediated response in Arabidopsis and different crop species (Vaidya et al., 2021). ...
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The binding of the plant phytohormone Abscisic acid (ABA) to the family of ABA receptors (PYR/PYL/RCAR) triggers plant responses to abiotic stress. Thus, the implementation of genetic or chemical strategies to modulate PYR/PYL activity might be biotechnologically relevant. We have employed the available structural information on the PYR/PYL receptors to design SlPYL1, a tomato receptor, harboring a single point mutation that displays enhanced ABA dependent and independent activity. Interestingly, crystallographic studies show that this mutation is not directly involved in ABA recognition or in the downstream phosphatase (PP2C) inhibitory interaction, rather, molecular dynamic based ensemble refinement restrained by crystallographic data indicates that it enhances the conformational variability required for receptor activation and it is involved in the stabilization of an active form of the receptor. Moreover, structural studies on this receptor have led to the identification of niacin as an ABA antagonist molecule in vivo. We have found that niacin blocks the ABA binding site by mimicking ABA receptor interactions, and the niacin interaction inhibits the biochemical activity of the receptor.
... Stress circumstances frequently result in an excess of the lethal metabolite methylglyoxal being produced in the body (MG) [98,190]. Abiotic stressors such as high salt concentrations, dry conditions, and high temperatures were made tolerable by increasing the expression of genes related with the glyoxalase pathway for MG detoxification in Oryza species [191]. Through the use of a short tandem target mimic method to knock down miR166 in rice, researchers were able to induce increased drought tolerance and developmental alterations that were similar to natural plant responses to water scarcity stress [67,192]. ...
Article
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, signaling, 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.
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ABA receptor agonists capable of improving plant performance under drought conditions have been described during the last years. However, monocot and eudicot plant species respond differently to various agonists. Here, we provide a detailed methodology to evaluate the anti-transpirant activity of ABA receptor agonists in both monocot and eudicot plant species using infrared imaging and image data analysis. Plant growth conditions, chemical application, and infrared image analysis are explained in detail to evaluate the anti-transpirant activity of ABA receptor agonists in the eudicot model Arabidopsis thaliana and in the C4-monocot model Setaria viridis.
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The endomembrane system is critical for plant growth and development and understanding its function and regulation is of great interest for plant biology research. Small-molecule targeting distinctive endomembrane components have proven powerful tools to dissect membrane trafficking in plant cells. However, unambiguous elucidation of the complex and dynamic trafficking processes requires chemical probes with enhanced precision. Determination of the mechanism of action of a compound, which is facilitated by various chemoproteomic approaches, opens new avenues for the improvement of its specificity. Moreover, rational molecule design and reverse chemical genetics with the aid of virtual screening and artificial intelligence will enable us to discover highly precise chemical probes more efficiently. The next decade will witness the emergence of more such accurate tools, which together with advanced live quantitative imaging techniques of subcellular phenotypes, will deepen our insights into the plant endomembrane system.
Chapter
Water is an essential resource for the survival of living organisms on earth. Pollution of water sources due to organic and inorganic pollutants is one of the major environmental problems in the 21st century. Although there are many conventional approaches to cleanin polluted water, they have limitations due to handling problems, high cost, relatively low effectiveness, and they cannot be used in large-scale water cleaning. In modern times, microbes and various adsorbents extracted from diverse nanoparticles are frequently utilized to generate clean water resources. The use of adsorbents is generally promoted as a remarkable approach due to their excellent adsorption capacity, affordability, simplicity in handling, and effectiveness. Biochar is a carbonaceous material produced by pyrolysis processing of wastes and is one of the major adsorbents used in the removal of pollutants. Through hydrophobic and hydrophilic interactions, biochar provide effective removal of hazardous pollutants. This chapter provides deeper insights into the production process of biochar, its interaction mechanisms with pollutants, factors that affect the adsorption capacity of biochar, its various modified types, as well as its uses to adsorb and remove various organic and inorganic pollutants from the environment.
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Agriculture is particularly vulnerable to climate change. To cope with the risks posed by climate-related stressors to agricultural production, global population growth, and changes in food preferences, it is imperative to develop new climate-smart crop varieties with increased yield and environmental resilience. Molecular genetics and genomic analyses have revealed that allelic variations in genes involved in phytohormone-mediated growth regulation have greatly improved productivity in major crops. Plant science has remarkably advanced our understanding of the molecular basis of various phytohormone-mediated events in plant life. These findings provide essential information for improving the productivity of crops growing in changing climates. In this review, we highlight the recent advances in plant hormonomics (multiple phytohormone profiling) and discuss their application to crop improvement. We present plant hormonomics as a key tool for deep physiological phenotyping, focusing on representative plant growth regulators associated with the improvement of crop productivity. Specifically, we review advanced methodologies in plant hormonomics, highlighting mass spectrometry- and nanosensor-based plant hormone profiling techniques. We also discuss the applications of plant hormonomics in crop improvement through breeding and agricultural management practices.
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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.
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Maize (Zea mays L.) underwent profound changes during domestication in root anatomy for environmental adaptation. However, the genetic mechanism of maize root anatomy domestication and plasticity remains unclear. In this study, a high-resolution mapping was performed for nine root anatomical traits using a maize-teosinte population (mexicana × Mo17) across three environments. Large genetic variations were detected for different root anatomical traits. The cortex area, stele area, aerenchyma area, xylem vessels number and cortical cell number had large variations across three environments, indicating high plasticity. Sixteen quantitative trait loci (QTL) were identified, including seven QTL with QTL × Environment interaction (EIQTL) for high plastic traits and nine QTL without QTL × Environment interaction (SQTL). Most of the root loci were consistent with shoot QTL depicting domestication signals. Combining transcriptome and genome-wide association study (GWAS) revealed that ZmPILS4 serve as a candidate gene underlying a major QTL of xylem traits. The near-isogenic lines (NILs) with lower expression of ZmPILS4 had 18-24% more IAA concentration in the root tip and 8-15% more xylem vessels. Significant domestication signal in promoter region suggested that ZmPILS4 was involved in maize domestication and adaptation. These results divulged the potential genetic basis of root anatomy plasticity and domestication.
Chapter
Ca2+ signaling is part of universal signal transduction pathways to respond to external and internal stimuli or stress and in plants plays a central role in chloroplasts, such as in the regulation of photosynthetic enzymes or the transition from light to dark. Only recently, the underlying molecular machinery, e.g., transporters and channels that enable chloroplast Ca2+ fluxes, has started to be elucidated. However, chemical tools to specifically perturb these chloroplast Ca2+ fluxes are largely lacking. Here, we describe an efficient aequorin-based system in Arabidopsis thaliana suspension cell cultures to screen for chemicals that alter light-to-dark-induced chloroplast stroma Ca2+ signals. Subsequently, the effect of the hits on chloroplast Ca2+ signals is validated in Arabidopsis seedlings. The research lays a foundation for the identification of novel proteins involved in Ca2+ transport in chloroplast stroma under light-to-dark transition and for investigating the interaction of chloroplast Ca2+ signaling with photosynthesis in general.
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The StARkin domain (derived from ‘kin of steroidogenic acute regulatory protein (StAR)’) is an evolutionarily conserved helix-grip-fold structure. StARkin domains possess a deep hydrophobic pocket capable of binding lipophilic ligands such as fatty acids, sterols, and isoprenoids. Dysregulation of StARkin proteins has profound effects on disease and development. In this review, we profile recent mechanistic and evolutionary studies, which highlight the remarkable diversity of regulatory mechanisms employed by the StARkin module. Although primarily focused on land plants, we also discuss select key advances in mammalian StARkin biology. The diversity of perspectives, systems, and approaches described here may be helpful to researchers characterizing poorly understood StARkin proteins.
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Rice (Oryza sativa L.) is one of the most important crops in the world. Since the completion of rice reference genome sequences, tremendous progress has been achieved in understanding the molecular mechanisms on various rice traits and dissecting the underlying regulatory networks. In this review, we summarize the research progress of rice biology over past decades, including omics, genome-wide association study, phytohormone action, nutrient use, biotic and abiotic responses, photoperiodic flowering, and reproductive development (fertility and sterility). For the roads ahead, cutting-edge technologies such as new genomics methods, high-throughput phenotyping platforms, precise genome-editing tools, environmental microbiome optimization, and synthetic methods will further extend our understanding of unsolved molecular biology questions in rice, and facilitate integrations of the knowledge for agricultural applications.
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Phytohormone abscisic acid (ABA) is essential for plant responses to biotic and abiotic stresses. Dimeric receptors are a class of PYR1/PYL/RCAR (pyrabactin resistance 1/PYR1-like/regulatory component of ABA receptors) ABA receptors that are important for various ABA responses. While extensive experimental and computational studies have investigated these receptors, it remains not fully understood how ABA leads to their activation and dissociation for interaction with downstream protein phosphatase 2C (PP2C). Here, we study the activation and the homodimeric association processes of the PYL2 receptor as well as its heterodimeric association with protein phosphatase 2C 16 (HAB1) using molecular dynamics simulations. Free energy landscapes from ∼223 μs simulations show that dimerization substantially constrains PYL2 conformational plasticity and stabilizes the inactive state, resulting in lower ABA affinity. Also, we establish the thermodynamic model for competitive binding between homodimeric PYL2 association and heterodimeric PYL2-HAB1 association in the absence and presence of ABA. Our results suggest that the binding of ABA destabilizes the PYL2 complex and further stabilizes PYL2-HAB1 association, thereby promoting PYL2 dissociation. Overall, this study explains several key aspects on the activation of dimeric ABA receptors, which provide new avenues for selective regulation of these receptors.
Chapter
The apocarotenoid phytohormone abscisic acid (ABA) regulates several aspects of plant development and stress responses. ABA is synthesized in response to multiple stressors and indirectly activates subfamily 2 Snf1-related kinases (SnRK2s) by receptor-mediated inhibition of clade A type IIC protein phosphatases (PP2Cs), which normally repress SnRK2 activity. The binding of ABA to its receptors triggers a change in receptor conformation that directs the formation of a receptor-ligand-PP2C complex that inhibits the activity of PP2C; this core mechanism can be harnessed for phosphatase activity-based measurements of receptor activation. In this chapter, we describe general methods for determining the effects of small molecules on ABA receptor function and supplement these with methods describing the synthesis of the high-affinity ligands opabactin (OP), which activates subfamily III and II ABA receptors, and the pan-receptor antagonist antabactin (ANT), and TAMRA-ANT fluorescent derivative of ANT. We present simple methods for quantifying receptor-ligand interactions using both PP2C inhibition and fluorescence polarization (FP) assays. Controls for determining the quality of recombinant receptors are provided, which are required for both quantitative analyses and for experimental consistency. Collectively, these methods will facilitate consistent biochemical measurements of the effects of ligand binding on ABA receptor function in vitro. Although the chapter describes ABA-specific methods, they illustrate and address a common need across receptor systems—reproducible assays that enable high throughput discovery and subsequent optimization of receptor modulators.
Article
Unfriendly environments like drought, cold or any other properties stress lead to a certain reduction of crop yield and quality. Abscisic acid (ABA), as an important plant hormone, plays a very important role in plant growth and resisting plant stress responses. The discovery of ABA receptor crystal structure is a very critical event and provide a very important role for ABA signaling pathways to be exposed to us more clearly. In this process, the screening of ABA receptor agonists played a decisive role in the analysis of ABA crystal structure. Further, various ABA receptor agonists have been screened out, and their functions have also been analyzed. Together, structural studies suggest a detailed mechanism for ABA and receptor binding and regulation of downstream ABA signal pathway, which provide new thoughts for improvement of crop yield and quality through the rational utilize the designed ABA agonists.
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The current climate situation potentiates the need for distinctive crops which have to be high yielding and resilient to abiotic and biotic stresses, particularly to drought stress. Responses to stresses are regulated and coordinated by phytohormones, whose transport and perception are commonly centered around plasma membrane (PM)-localized proteins. Localization and abundance of these are organized by endosomal trafficking, whereby the post-translational modification of PM proteins with the small protein ubiquitin is key in signaling their endocytosis and degradation. This review focuses on the endosomal degradation pathway in plants with a special focus on a protein family termed TARGET OF MYB1 (TOM1)-LIKE (TOLs), responsible for initiating sorting of ubiquitinated proteins destined for degradation. TOLs, albeit having apparent redundancies, play a role in very specific pathways where they may be relevant for fine-tuning of plant hormone signaling by means of tightly controlled protein turnover. Understanding the function of TOLs could give key insights into the mechanisms by which plants define the trade-off between stress tolerance and plant development when faced with challenging environments. Findings obtained in the model plant Arabidopsis thaliana provide a solid foundation for translational research aimed at breeding more tolerant crops.
Chapter
Gravity is a powerful element in shaping plant development, with gravitropism, the oriented growth response of plant organs to the direction of gravity, leading to each plant’s characteristic form both above and below ground. Despite being conceptually simple to follow, monitoring a plant’s directional growth responses can become complex as variation arises from both internal developmental cues as well as effects of the environment. In this protocol, we discuss approaches to gravitropism assays, focusing on automated analyses of root responses. For Arabidopsis, we recommend a simple 90° rotation using seedlings that are 5–8 days old. If images are taken at regular intervals and the environmental metadata is recorded during both seedling development and gravitropic assay, these data can be used to reveal quantitative kinetic patterns at distinct stages of the assay. The use of software that analyzes root system parameters and stores this data in the RSML format opens up the possibility for a host of root parameters to be extracted to characterize growth of the primary root and a range of lateral root phenotypes.
Chapter
Phytohormones plays crucial physiological functions in plants, where they are involved in plant development, reproduction, defense, and many other functions. Phytohormones production has been found to be regulated in response to abiotic and biotic factors affecting the plant metabolism, and therefore, biosynthesis of primary and secondary metabolites. Thus, the detection and quantification of phytohormones in different plant tissues are essential to be determined unraveling the various plant metabolic pathways and behavior. Yet phytohormones analysis is always problematic, since they are found in extremely low concentrations and have a wide range of chemical and physicochemical properties. As a result, the ideal method should start with an appropriate extraction procedure followed by quantification by highly sensitive instrumental techniques providing precise and robust results. The current chapter presents an improved extraction method based on liquid-liquid extraction from a 50-mg aliquot of plant tissue for analysis of the major classes of phytohormones. Then, mass spectrometry (MS) analysis is conducted using quadrupole/linear ion trap (QLIT) mass analyzer equipped with electrospray ionization (ESI) source after a liquid chromatographic separation step. The developed method demonstrates an appropriate feasibility addressing biological questions related to phytohormones production and regulation.
Article
While crop yields have historically increased, the drought resistance becomes a major concern in the context of global climate change. The trade-off between crop yields and drought resistance is a common phenomenon; however, the underlying molecular modulators are remained to undetermine. Through genome-wide association study, we unraveled that three nonsynonymous variants in a drought-resistant allele of ZmSRO1d-R conferred the protein plasma membrane localization and enhanced the mono-ADP-ribosyltransferase activity on ZmRBOHC, which increased ROS levels in guard cells and promoted stomatal closure. ZmSRO1d-R enhanced plant drought resilience and protected grain yields under drought, but led to yield-drag under favorable conditions. The loss-of-function mutants of ZmRBOHC showed remarkably increased yields under well-watered conditions, whereas they compromised drought resistance. Interestingly, by analyzing 189 teosinte accessions we found that the ZmSRO1d-R allele was present in teosinte but selected-against during maize domestication and modern breeding. Thus, our work found that reducing the ZmSRO1d-R allele in maize breeding programs may compromise drought resistance, while increase yields.
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Regulation of the stomatal aperture is essential for plant growth and survival in response to environmental stimuli. Opening of stomata induces uptake of CO2 for photosynthesis and transpiration, which enhances uptake of nutrients from roots. Light is the most important stimulus for stomatal opening. Under drought stress, the plant hormone abscisic acid (ABA) induces stomatal closure to prevent water loss. However, the molecular mechanisms of stomatal movements are not fully understood. In this study, we screened chemical libraries to identify compounds that affect stomatal movements in Commelina benghalensis and characterize the underlying molecular mechanisms. We identified nine compounds (SCL1-SCL9) that suppress light-induced stomatal opening by more than 50%, and two compounds (Temsirolimus and CP-100356) that induce stomatal opening in the dark. Further investigations revealed that SCL1 and SCL2 had no effect on autophosphorylation of phototropin or the activity of the inward-rectifying plasma membrane (PM) K+ channel, KAT1, but suppressed blue light-induced phosphorylation of the penultimate residue, threonine, in PM H+-ATPase, which is a key enzyme for stomatal opening. SCL1 and SCL2 had no effect on ABA-dependent responses, including seed germination and expression of ABA-induced genes. These results suggest that SCL1 and SCL2 suppress light-induced stomatal opening at least in part by inhibiting blue light-induced activation of PM H+-ATPase, but not by the ABA-signaling pathway. Interestingly, spraying of SCL1 onto dicot and monocot leaves suppressed wilting of leaves, indicating that inhibition of stomatal opening by these compounds confers tolerance to drought stress in plants.
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Drought is one of the major abiotic stresses affecting world agriculture. Breeding drought-resistant crops is one of the most important challenges for plant biologists. PYR1/PYL/RCARs, which encode the abscisic acid (ABA) receptors, play pivotal roles in ABA signaling, but how these genes function in crop drought response remains largely unknown. Here we identified 13 PYL family members in maize (ZmPYL1-13). Changes in expression of these genes under different stresses indicated that ZmPYLs played important roles in responding to multiple abiotic stresses. Transgenic analyses of ZmPYL genes in Arabidopsis showed that overexpression of ZmPYL3, ZmPYL9, ZmPYL10, and ZmPYL13 significantly enhanced the sensitivity of transgenic plants to ABA. Additionally, transgenic lines overexpressing ZmPYL8, ZmPYL9, and ZmPYL12 were more resistant to drought. Accumulation of proline and enhanced expression of drought-related marker genes in transgenic lines further confirmed the positive roles of ZmPYL genes in plant drought resistance. Association analyses with a panel of 368 maize inbred lines identified natural variants in ZmPYL8 and ZmPYL12 that were significantly associated with maize drought resistance. Our results deepen the knowledge of the function of maize PYL genes in responses to abiotic stresses, and the natural variants identified in ZmPYL genes may serve as potential molecular markers for breeding drought-resistant maize cultivars.
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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.
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Plant growth requires the influx of atmospheric CO2 through stomatal pores, and this carbon uptake for photosynthesis is inherently associated with a large efflux of water vapor. Under water deficit, plants reduce transpiration and are able to improve carbon for water exchange leading to higher water use efficiency (WUE). Whether increased WUE can be achieved without trade-offs in plant growth is debated. The signals mediating the WUE response under water deficit are not fully elucidated but involve the phytohormone abscisic acid (ABA). ABA is perceived by a family of related receptors known to mediate acclimation responses and to reduce transpiration. We now show that enhanced stimulation of ABA signaling via distinct ABA receptors can result in plants constitutively growing at high WUE in the model species Arabidopsis WUE was assessed by three independent approaches involving gravimetric analyses, (13)C discrimination studies of shoots and derived cellulose fractions, and by gas exchange measurements of whole plants and individual leaves. Plants expressing the ABA receptors RCAR6/PYL12 combined up to 40% increased WUE with high growth rates, i.e., are water productive. Water productivity was associated with maintenance of net carbon assimilation by compensatory increases of leaf CO2 gradients, thereby sustaining biomass acquisition. Leaf surface temperatures and growth potentials of plants growing under well-watered conditions were found to be reliable indicators for water productivity. The study shows that ABA receptors can be explored to generate more plant biomass per water transpired, which is a prime goal for a more sustainable water use in agriculture.
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Two commonly used methods for calculating 50% endpoint using serial dilutions are Spearman-Karber method and Reed and Muench method. To understand/apply the above formulas, moderate statistical/mathematical skills are necessary. In this paper, a simple formula/method for calculating 50% endpoints has been proposed. The formula yields essentially similar results as those of the Spearman-Karber method. The formula has been rigorously evaluated with several samples.
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Agricultural productivity is dictated by water availability and consequently drought is the major source of crop losses worldwide. The phytohormone abscisic acid (ABA) is elevated in response to water deficit and modulates drought tolerance by reducing water consumption and inducing other drought-protective responses. The recent identification of ABA receptors, elucidation of their structures and understanding of the core ABA signaling network has created new opportunities for agrochemical development. An unusually large gene family encodes ABA receptors and, until recently, it was unclear if selective or pan-agonists would be necessary for modulating water use. The recent identification of the selective agonist quinabactin has resolved this issue and defined Pyrabactin Resistance 1 (PYR1) and its close relatives as key targets for water use control. This review provides an overview of the structure and function of ABA receptors, progress in the development of synthetic agonists, and the use of orthogonal receptors to enable agrochemical control in transgenic plants.
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Foliar spray of antitranspirants triggered plant adaptations to water stress and reduced the negative effects of drought on crop production. This study characterized the influence of exogenous abscisic acid (ABA) and fulvic acid (FA) application on winter wheat (Triticum aestivum L.) by pot experiment during 2012–2013 season and field experiment during 2012–2013 and 2013–2014 seasons in the North China Plain. In the pot experiment, plants were sprayed with FA or ABA under three irrigation levels (deficit, moderate deficit, and full irrigated). Field experiments involved three parts, including concentration and frequency of ABA application as well as the combined application of ABA and FA. Results showed that, compared with non-treated wheat grown under moderate deficit condition, both ABA and FA influenced the biomass allocation above and below ground, which caused the increase in crop harvest index as well as water use efficiency. By analyzing wheat performances in grain yield and transfer ratio of total assimilate (TRA) under different ABA concentrations (ranged from 30 to 60 mg/L) as well as ABA application frequencies (two, three, and four applications), results indicated that ABA worked well with lower concentration and less application frequency. At the heading stage of wheat, spraying FA improved the non-structural carbohydrate content (NCS) in plants. ABA application at the early grain-fill stage decreased residual NCS. In water deficit conditions, combined application of FA (at the heading stage) and ABA (at the early grain-fill stage) significantly improved the TRA and yield performance, and worked better than using separately.
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Human carbonic anhydrase IX (CA IX) is highly expressed in tumor tissues and its selective inhibition provides potential target for treatment against numerous cancers. Development of potent, highly selective inhibitors against this target remains an unmet need in anti-cancer therapeutics. A series of fluorinated benzenesulfonamides with substituents on the benzene ring were designed and synthesized. Several of these exhibited a highly potent and selective inhibition profile against CA IX. Three fluorine atoms significantly increased the affinity by withdrawing electrons and lowering the pKa of the benzene sulfonamide group. The bulky ortho substituents such as cyclooctyl or even cyclododecyl groups fit to the hydrophobic pocket in the active site of CA IX but not CA II, as shown by the compound cocrystal structure with the chimeric CA IX. The strongest inhibitor of recombinant human CA IX catalytic domain produced in human cells achieved the affinity of 50 pM. However, the high affinity diminished the selectivity. The most selective compound for CA IX exhibited 10 nM affinity. The compound which showed the best balance between affinity and selectivity properties bound with 1 nM affinity. The inhibitors described in this work provide the basis for novel anticancer therapeutics targeting CA IX.
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Grain yields of eight representative semidwarf spring wheat (Triticum aestivum L.) cultivars released in northwest Mexico between 1962 and 1988 have increased linearly across years as measured in this region during 6 yr under favorable management and irrigation. To understand the physiological basis of this progress and possibly assist future selection for grain yield, leaf traits were determined during 3 yr in the same study. Stomatal conductance (g(s)), maximum photosynthetic rate (A(max)), and canopy temperature depression (CTD), averaged over the 3 yr, were closely and positively correlated with progress in the 6-yr mean yield. The correlation was greatest with g(s) (r = 0.94, P < 0.01). Compared with the overall yield increase of 27%, g(s) increased 63%, A(max) increased 23%, and canopies were 0.6°C cooler. Carbon-13 isotope discrimination was also positively associated with yield progress (r = 0.71, P < 0.05), but other leaf traits such as flag leaf area, specific leaf weight, percentage N and greeness were not, nor was crop growth rate around anthesis. The causal basis of the leaf activity interrelationships is reasonably clear, with both increased intercellular CO2 concentration and increased mesophyll activity contributing to the increase in A(max). However, causal links to the yield progress, and the accompanying increase in kernels per square meter, are not clear. It is concluded that g(s) and CTD should be further investigated as potential indirect selection criteria for yield.
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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.Cell Research advance online publication 9 July 2013; doi:10.1038/cr.2013.95.
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Abscisic acid (ABA) is an essential molecule in plant abiotic stress responses. It binds to soluble pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor receptors and stabilizes them in a conformation that inhibits clade A type II C protein phosphatases; this leads to downstream SnRK2 kinase activation and numerous cellular outputs. We previously described the synthetic naphthalene sulfonamide ABA agonist pyrabactin, which activates seed ABA responses but fails to trigger substantial responses in vegetative tissues in Arabidopsis thaliana. Here we describe quinabactin, a sulfonamide ABA agonist that preferentially activates dimeric ABA receptors and possesses ABA-like potency in vivo. In Arabidopsis, the transcriptional responses induced by quinabactin are highly correlated with those induced by ABA treatments. Quinabactin treatments elicit guard cell closure, suppress water loss, and promote drought tolerance in adult Arabidopsis and soybean plants. The effects of quinabactin are sufficiently similar to those of ABA that it is able to rescue multiple phenotypes observed in the ABA-deficient mutant aba2. Genetic analyses show that quinabactin's effects in vegetative tissues are primarily mediated by dimeric ABA receptors. A PYL2-quinabactin-HAB1 X-ray crystal structure solved at 1.98-Å resolution shows that quinabactin forms a hydrogen bond with the receptor/PP2C "lock" hydrogen bond network, a structural feature absent in pyrabactin-receptor/PP2C complexes. Our results demonstrate that ABA receptors can be chemically controlled to enable plant protection against water stress and define the dimeric receptors as key targets for chemical modulation of vegetative ABA responses.
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Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. Its action is mediated by the PYR/PYL/RCAR family of START proteins, but it remains unclear how these receptors bind ABA and, in turn, how hormone binding leads to inhibition of the downstream type 2C protein phosphatase (PP2C) effectors. Here we report crystal structures of apo and ABA-bound receptors as well as a ternary PYL2–ABA–PP2C complex. The apo receptors contain an open ligand-binding pocket flanked by a gate that closes in response to ABA by way of conformational changes in two highly conserved -loops that serve as a gate and latch. Moreover, ABA-induced closure of the gate creates a surface that enables the receptor to dock into and competitively inhibit the PP2C active site. A conserved tryptophan in the PP2C inserts directly between the gate and latch, which functions to further lock the receptor in a closed conformation. Together, our results identify a conserved gate–latch–lock mechanism underlying ABA signalling.
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Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ 2 ) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
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Water stress is one of the most important environmental factors that regulate a plant’s growth and development. In agronomic practice the effects of water stress are translated into low yield and/or reduced quality. Abscisic acid (ABA) sprays (1mM) were applied to wheat plants at different phenological stages and the effects on several physiological variables and on yield were evaluated under field conditions at different water regimes. Studies were conducted in the field across three consecutive winter–spring seasons. ABA treatments were applied at the beginning of shoot enlargement and repeated at anthesis. Exogenous ABA increased shoot dry weight and maintained a high concentration of photosynthetic pigments for a longer period of time during grain growth and maturation. Although ABA applications increased stomatal closure immediately after its application, the longer-term effect was to allow for a greater ostiolar opening of the stomatal pore which resulted in increased conductance of gases and water vapor. ABA also improved the transport of photoassimilates from the leaves and stem to the developing grains, that is, it effectively increased the sink strength of the grains. This correlated with a yield increase without significantly changing the protein quality in the grains. Thus, elevated ABA levels from exogenous application or genetic selection could help improve agricultural production of grains in arid areas where irrigation is not possible. KeywordsAbscisic acid-Wheat (Triticum aestivum L.)-Water stress-Field crops-Grain yield-Carbohydrates
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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.
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The phytohormone abscisic acid (ABA) regulates stress responses and controls numerous aspects of plant growth and development. Biosynthetic precursors and catabolites of ABA have been shown to trigger ABA responses in physiological assays, but it is not clear whether these are intrinsically active or whether they are converted into ABA in planta. In this study, we analyzed the effect of ABA precursors, conjugates, and catabolites on hormone signaling in Arabidopsis (Arabidopsis thaliana). The compounds were also tested in vitro for their ability to regulate the phosphatase moiety of ABA receptor complexes consisting of the protein phosphatase 2C ABI2 and the coreceptors RCAR1/PYL9, RCAR3/PYL8, and RCAR11/PYR1. Using mutants defective in ABA biosynthesis, we show that the physiological activity associated with ABA precursors derives predominantly from their bioconversion to ABA. The ABA glucose ester conjugate, which is the most widespread storage form of ABA, showed weak ABA-like activity in germination assays and in triggering ABA signaling in protoplasts. The ABA conjugate and precursors showed negligible activity as a regulatory ligand of the ABI2/RCAR receptor complexes. The majority of ABA catabolites were inactive in our assays. To analyze the chemically unstable 8'- and 9'-hydroxylated ABA catabolites, we used stable tetralone derivatives of these compounds, which did trigger selective ABA responses. ABA synthetic analogs exhibited differential activity as regulatory ligands of different ABA receptor complexes in vitro. The data show that ABA precursors, catabolites, and conjugates have limited intrinsic bioactivity and that both natural and synthetic ABA-related compounds can be used to probe the structural requirements of ABA ligand-receptor interactions.
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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.
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Changing environmental conditions and lessening fresh water supplies have sparked intense interest in understanding and manipulating abscisic acid (ABA) signaling, which controls adaptive responses to drought and other abiotic stressors. We recently discovered a selective ABA agonist, pyrabactin, and used it to discover its primary target PYR1, the founding member of the PYR/PYL family of soluble ABA receptors. To understand pyrabactin's selectivity, we have taken a combined structural, chemical and genetic approach. We show that subtle differences between receptor binding pockets control ligand orientation between productive and nonproductive modes. Nonproductive binding occurs without gate closure and prevents receptor activation. Observations in solution show that these orientations are in rapid equilibrium that can be shifted by mutations to control maximal agonist activity. Our results provide a robust framework for the design of new agonists and reveal a new mechanism for agonist selectivity.
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Pyrabactin is a synthetic abscisic acid (ABA) agonist that selectively inhibits seed germination. The use of pyrabactin was pivotal in the identification of the PYR1/PYL/RCAR family (PYL) of proteins as the ABA receptor. Although they both act through PYL proteins, pyrabactin and ABA share no apparent chemical or structural similarity. It remains unclear how pyrabactin functions as an ABA agonist. Here, we report the crystal structure of pyrabactin in complex with PYL1 at 2.4 A resolution. Structural and biochemical analyses revealed that recognition of pyrabactin by the pocket residues precedes the closure of switch loop CL2. Structural comparison between pyrabactin- and ABA-bound PYL1 reveals a general principle in the arrangements of function groups of the two distinct ligands. The study provides a framework for the development of novel ABA agonists that may have applicable potentials in agriculture.
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Heterotrimeric G-proteins mediate crucial and diverse signaling pathways in eukaryotes. Here, we generate and analyze microarray data from guard cells and leaves of G-protein subunit mutants of the model plant Arabidopsis thaliana, with or without treatment with the stress hormone, abscisic acid. Although G-protein control of the transcriptome has received little attention to date in any system, transcriptome analysis allows us to search for potentially uncommon yet significant signaling mechanisms. We describe the theoretical Boolean mechanisms of G-protein x hormone regulation, and then apply a pattern matching approach to associate gene expression profiles with Boolean models. We find that (1) classical mechanisms of G-protein signaling are well represented. Conversely, some theoretical regulatory modes of the G-protein are not supported; (2) a new mechanism of G-protein signaling is revealed, in which G beta regulates gene expression identically in the presence or absence of G alpha; (3) guard cells and leaves favor different G-protein modes in transcriptome regulation, supporting system specificity of G-protein signaling. Our method holds significant promise for analyzing analogous 'switch-like' signal transduction events in any organism. Molecular Systems Biology 6: 372; published online 8 June 2010; doi:10.1038/msb.2010.28
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Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
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Abscisic acid (ABA) is an important phytohormone that regulates plant stress responses. Proteins from the PYR-PYL-RCAR family were recently identified as ABA receptors. Upon binding to ABA, a PYL protein associates with type 2C protein phosphatases (PP2Cs) such as ABI1 and ABI2, inhibiting their activity; the molecular mechanisms by which PYLs mediate ABA signaling remain unknown, however. Here we report three crystal structures: apo-PYL2, (+)-ABA-bound PYL2 and (+)-ABA-bound PYL1 in complex with phosphatase ABI1. Apo-PYL2 contains a pocket surrounded by four highly conserved surface loops. In response to ABA binding, loop CL2 closes onto the pocket, creating a surface that recognizes ABI1. In the ternary complex, the CL2 loop is located near the active site of ABI1, blocking the entry of substrate proteins. Together, our data reveal the mechanisms by which ABA regulates PYL-mediated inhibition of PP2Cs.
Article
The phytohormone abscisic acid (ABA) mediates many physiological and developmental responses and its key role in plant water relations has fueled efforts to improve crop water productivity by manipulating ABA responses. ABA's core signaling components are encoded by large gene families, which has hampered functional studies using classical genetic approaches due to redundancy. Chemical approaches can complement genetic approaches and have the advantage of delivering both biological probes and potential agrochemical leads; these benefits have spawned the discovery and design of new chemical modulators of ABA signaling and biosynthesis, which have contributed to the identification of ABA receptors and helped to define PYR1 and related subfamily III receptors as key cellular targets for chemically manipulating water productivity. In this review we provide an overview of small molecules that have helped dissect both ABA signaling and metabolic pathways. We further discuss how the insights gleaned using ABA probe molecules might be translated to improvements in crop water productivity and future opportunities for development of small molecules that affect ABA metabolism and signaling.