Pro197Ser and the new Trp574Leu mutations together with enhanced metabolism contribute to cross-resistance to ALS inhibiting herbicides in Sinapis alba.

White mustard, (Sinapis alba), a problematic broadleaf weed in many Mediterranean countries in arable fields has been detected as resistant to tribenuron-methyl in Tunisia. Greenhouse and laboratory studies were conducted to characterize Target-Site Resistance (TSR) and the Non-Target Site Resistance (NTSR) mechanisms in two suspected white mustard biotypes. Herbicide dose-response experiments confirmed that the two S. alba biotypes were resistant to four dissimilar acetolactate synthase (ALS)-pinhibiting herbicide chemistries indicating the presence of cross-resistance mechanisms. The highest resistance factor (>144) was attributed to tribenuron-methyl herbicide and both R populations survived up to 64-fold the recommended field dose (18.7 g ai ha-1). In this study, the metabolism experiments with malathion (a cytochrome P450 inhibitor) showed that malathion reduced resistance to tribenuron-methyl and imazamox in both populations, indicating that P450 may be involved in the resistance. Sequence analysis of the ALS gene detected target site mutations in the two R biotypes, with amino acid substitutions Trp574Leu, the first report for the species, and Pro197Ser. Molecular docking analysis showed that ALSPro197Ser enzyme cannot properly bind to tribenuron-methyl's aromatic ring due to a reduction in the number of hydrogen bonds, while imazamox can still bind. However, Trp574Leu can weaken the binding affinity between the mutated ALS enzyme and both herbicides with the loss of crucial interactions. This investigation provides substantial evidence for the risk of evolving multiple resistance in S. alba to auxin herbicides while deciphering the TSR and NTSR mechanisms conferring cross resistance to ALS inhibitors.

Alternative splicing as a driver of natural variation in abscisic acid response.

Abscisic acid (ABA) is a crucial player in plant responses to the environment. It accumulates under stress, activating downstream signaling to implement molecular responses that restore homeostasis. Natural variance in ABA sensitivity remains barely understood, and the ABA pathway has been mainly studied at the transcriptional level, despite evidence that posttranscriptional regulation, namely, via alternative splicing, contributes to plant stress tolerance. Here, we identified the Arabidopsis accession Kn-0 as less sensitive to ABA than the reference Col-0, as shown by reduced effects of the hormone on seedling establishment, root branching, and stomatal closure, as well as by decreased induction of ABA marker genes. An in-depth comparative transcriptome analysis of the ABA response in the two variants revealed lower expression changes and fewer genes affected for the least ABA-sensitive ecotype. Notably, Kn-0 exhibited reduced levels of the ABA-signaling SnRK2 protein kinases and lower basal expression of ABA-reactivation genes, consistent with our finding that Kn-0 contains less endogenous ABA than Col-0. ABA also markedly affected alternative splicing, primarily intron retention, with Kn-0 being less responsive regarding both the number and magnitude of alternative splicing events, particularly exon skipping. We find that alternative splicing introduces a more ecotype-specific layer of ABA regulation and identify ABA-responsive splicing changes in key ABA pathway regulators that provide a functional and mechanistic link to the differential sensitivity of the two ecotypes. Our results offer new insight into the natural variation of ABA responses and corroborate a key role for alternative splicing in implementing ABA-mediated stress responses.

Transcription factors HB21/40/53 trigger inflorescence arrest through abscisic acid accumulation at the end of flowering.

Flowers, and hence, fruits and seeds, are produced by the activity of the inflorescence meristem after the floral transition. In plants with indeterminate inflorescences the final number of flowers produced by the inflorescence meristem is determined by the length of the flowering period, which ends with inflorescence arrest. Inflorescence arrest depends on many different factors, such as the presence of seeds, the influence of the environment, or endogenous factors such as phytohormone levels and age, which modulate inflorescence meristem activity. The FRUITFULL-APETALA2 (FUL-AP2) pathway plays a major role in regulating the end of flowering, likely integrating both endogenous cues and those related to seed formation. Among AP2 targets, HOMEOBOX PROTEIN21 (HB21) has been identified as a putative mediator of AP2 function in the control of inflorescence arrest. HB21 is a homeodomain leucine zipper transcription factor involved in establishing axillary bud dormancy. Here we characterized the role of HB21 in the control of the inflorescence arrest at the end of flowering in Arabidopsis (Arabidopsis thaliana). HB21, together with HB40 and HB53, are upregulated in the inflorescence apex at the end of flowering, promoting floral bud arrest. We also show that abscisic acid (ABA) accumulation occurs in the inflorescence apex in an HB-dependent manner. Our work suggests a physiological role of ABA in floral bud arrest at the end of flowering, pointing to ABA as a regulator of inflorescence arrest downstream of the HB21/40/53 genes.

Engineering plants using diverse CRISPR-associated proteins and deregulation of genome-edited crops.

The CRISPR/Cas system comprises RNA-guided nucleases, the target specificity of which is directed by Watson-Crick base pairing of target loci with single guide (sg)RNA to induce the desired edits. CRISPR-associated proteins and other engineered nucleases are opening new avenues of research in crops to induce heritable mutations. Here, we review the diversity of CRISPR-associated proteins and strategies to deregulate genome-edited (GEd) crops by considering them to be close to natural processes. This technology ensures yield without penalties, advances plant breeding, and guarantees manipulation of the genome for desirable traits. DNA-free and off-target-free GEd crops with defined characteristics can help to achieve sustainable global food security under a changing climate, but need alignment of international regulations to operate in existing supply chains.

Abscisic acid mimic-fluorine derivative 4 alleviates water deficit stress by regulating ABA-responsive genes, proline accumulation, CO2 assimilation, water use efficiency and better nutrient uptake in tomato plants.

Water deficit represents a serious limitation for agriculture and both genetic and chemical approaches are being used to cope with this stress and maintain plant yield. Next-generation agrochemicals that control stomatal aperture are promising for controlling water use efficiency. For example, chemical control of abscisic acid (ABA) signaling through ABA-receptor agonists is a powerful method to activate plant adaptation to water deficit. Such agonists are molecules able to bind and activate ABA receptors and, although their development has experienced significant advances in the last decade, few translational studies have been performed in crops. Here, we describe protection by the ABA mimic-fluorine derivative 4 (AMF4) agonist of the vegetative growth in tomato plants subjected to water restriction. Photosynthesis in mock-treated plants is markedly impaired under water deficit conditions, whereas AMF4 treatment notably improves CO2 assimilation, the relative plant water content and growth. As expected for an antitranspirant molecule, AMF4 treatment diminishes stomatal conductance and transpiration in the first phase of the experiment; however, when photosynthesis declines in mock-treated plants as stress persists, higher photosynthetic and transpiration parameters are recorded in agonist-treated plants. Additionally, AMF4 increases proline levels over those achieved in mock-treated plants in response to water deficit. Thus water deficit and AMF4 cooperate to upregulate P5CS1 through both ABA-independent and ABA-dependent pathways, and therefore, higher proline levels are produced Finally, analysis of macronutrients reveals higher levels of Ca, K and Mg in AMF4- compared to mock-treated plants subjected to water deficit. Overall, these physiological analyses reveal a protective effect of AMF4 over photosynthesis under water deficit and enhanced water use efficiency after agonist treatment. In summary, AMF4 treatment is a promising approach for farmers to protect the vegetative growth of tomatoes under water deficit stress.

Structure-guided engineering of a receptor-agonist pair for inducible activation of the ABA adaptive response to drought.

Strategies to activate abscisic acid (ABA) receptors and boost ABA signaling by small molecules that act as ABA receptor agonists are promising biotechnological tools to enhance plant drought tolerance. Protein structures of crop ABA receptors might require modifications to improve recognition of chemical ligands, which in turn can be optimized by structural information. Through structure-based targeted design, we have combined chemical and genetic approaches to generate an ABA receptor agonist molecule (iSB09) and engineer a CsPYL1 ABA receptor, named CsPYL15m, which efficiently binds iSB09. This optimized receptor-agonist pair leads to activation of ABA signaling and marked drought tolerance. No constitutive activation of ABA signaling and hence growth penalty was observed in transformed Arabidopsis thaliana plants. Therefore, conditional and efficient activation of ABA signaling was achieved through a chemical-genetic orthogonal approach based on iterative cycles of ligand and receptor optimization driven by the structure of ternary receptor-ligand-phosphatase complexes.

Tribenuron-methyl metabolism and the rare Pro197Phe double mutation together with 2,4-D metabolism and reduced absorption can evolve in Papaver rhoeas with multiple and cross herbicide resistance to ALS inhibitors and auxin mimics.

Multiple resistance mechanisms to ALS inhibitors and auxin mimics in two Papaver rhoeas populations were investigated in wheat fields from Portugal. Dose-response trials, also with malathion (a cytochrome P450 inhibitor), cross-resistance patterns for ALS inhibitors and auxin mimics, alternative herbicides tests, 2,4-D and tribenuron-methyl absorption, translocation and metabolism experiments, together with ALS activity, gene sequencing and enzyme modelling and ligand docking were carried out. Results revealed two different resistant profiles: one population (R1) multiple resistant to tribenuron-methyl and 2,4-D, the second (R2) only resistant to 2,4-D. In R1, several target-site mutations in Pro197 and enhanced metabolism (cytochrome P450-mediated) were responsible of tribenuron-methyl resistance. For 2,4-D, reduced transport was observed in both populations, while cytochrome P450-mediated metabolism was also present in R1 population. Moreover, this is the first P. rhoeas population with enhanced tribenuron-methyl metabolism. This study reports the first case for P. rhoeas of the amino acid substitution Pro197Phe due to a double nucleotide change. This double mutation could cause reduced enzyme sensitivity to most ALS inhibitors according to protein modelling and ligand docking. In addition, this study reports a P. rhoeas population resistant to 2,4-D, apparently, with reduced transport as the sole resistance mechanism.

Structure-Based Modulation of the Ligand Sensitivity of a Tomato Dimeric Abscisic Acid Receptor Through a Glu to Asp Mutation in the Latch Loop.

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.

Hydrotropism: Analysis of the Root Response to a Moisture Gradient.

Plant adaptation to environmental stress generated by low water availability requires continuous search for moisture niches in the soil. Thus, roots have evolved a hydrotropic response to sense differences in water potential of the soil, and through asymmetric growth, roots can bend to avoid lower water potential sites. Different experimental systems have been devised for hydrotropism assays, which usually rely on air moisture or split agar assays. This latter system uses plates containing an osmolyte only in a region of the plate in order to generate a water potential gradient. Seedlings are placed on the agar plate containing normal medium (NM) so that their root tips are near the junction between NM and the region supplemented with the osmolyte. As a result, a hydrotropic response is elicited to avoid the low water potential medium, which is reflected in the root curvature angle.

Evaluation of the Anti-transpirant Activity of ABA Receptor Agonists in Monocot and Eudicot Plants.

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.

PYL1- and PYL8-like ABA Receptors of Nicotiana benthamiana Play a Key Role in ABA Response in Seed and Vegetative Tissue.

To face the challenges of climate change and sustainable food production, it is essential to develop crop genome editing techniques to pinpoint key genes involved in abiotic stress signaling. The identification of those prevailing abscisic acid (ABA) receptors that mediate plant-environment interactions is quite challenging in polyploid plants because of the high number of genes in the PYR/PYL/RCAR ABA receptor family. Nicotiana benthamiana is a biotechnological crop amenable to genome editing, and given the importance of ABA signaling in coping with drought stress, we initiated the analysis of its 23-member family of ABA receptors through multiplex CRISPR/Cas9-mediated editing. We generated several high-order mutants impaired in NbPYL1-like and NbPYL8-like receptors, which showed certain insensitivity to ABA for inhibition of seedling establishment, growth, and development of shoot and lateral roots as well as reduced sensitivity to the PYL1-agonist cyanabactin (CB). However, in these high-order mutants, regulation of transpiration was not affected and was responsive to ABA treatment. This reveals a robust and redundant control of transpiration in this allotetraploid plant that probably reflects its origin from the extreme habitat of central Australia.

Ubiquitylation of ABA Receptors and Protein Phosphatase 2C Coreceptors to Modulate ABA Signaling and Stress Response.

Post-translational modifications play a fundamental role in regulating protein function and stability. In particular, protein ubiquitylation is a multifaceted modification involved in numerous aspects of plant biology. Landmark studies connected the ATP-dependent ubiquitylation of substrates to their degradation by the 26S proteasome; however, nonproteolytic functions of the ubiquitin (Ub) code are also crucial to regulate protein interactions, activity, and localization. Regarding proteolytic functions of Ub, Lys-48-linked branched chains are the most common chain type for proteasomal degradation, whereas promotion of endocytosis and vacuolar degradation is triggered through monoubiquitylation or Lys63-linked chains introduced in integral or peripheral plasma membrane proteins. Hormone signaling relies on regulated protein turnover, and specifically the half-life of ABA signaling components is regulated both through the ubiquitin-26S proteasome system and the endocytic/vacuolar degradation pathway. E3 Ub ligases have been reported that target different ABA signaling core components, i.e., ABA receptors, PP2Cs, SnRK2s, and ABFs/ABI5 transcription factors. In this review, we focused specifically on the ubiquitylation of ABA receptors and PP2C coreceptors, as well as other post-translational modifications of ABA receptors (nitration and phosphorylation) that result in their ubiquitination and degradation.

An Update on Crop ABA Receptors.

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.

PYL8 ABA receptors of Phoenix dactylifera play a crucial role in response to abiotic stress and are stabilized by ABA.

The identification of those prevalent abscisic acid (ABA) receptors and molecular mechanisms that trigger drought adaptation in crops well adapted to harsh conditions such as date palm (Phoenix dactylifera, Pd) sheds light on plant-environment interactions. We reveal that PdPYL8-like receptors are predominantly expressed under abiotic stress, with Pd27 being the most expressed receptor in date palm. Therefore, subfamily I PdPYL8-like receptors have been selected for ABA signaling during abiotic stress response in this crop. Biochemical characterization of PdPYL8-like and PdPYL1-like receptors revealed receptor- and ABA-dependent inhibition of PP2Cs, which triggers activation of the pRD29B-LUC reporter in response to ABA. PdPYLs efficiently abolish PP2C-mediated repression of ABA signaling, but loss of the Trp lock in the seed-specific AHG1-like phosphatase PdPP2C79 markedly impairs its inhibition by ABA receptors. Characterization of Arabidopsis transgenic plants that express PdPYLs shows enhanced ABA signaling in seed, root, and guard cells. Specifically, Pd27-overexpressing plants showed lower ABA content and were more efficient than the wild type in lowering transpiration at negative soil water potential, leading to enhanced drought tolerance. Finally, PdPYL8-like receptors accumulate after ABA treatment, which suggests that ABA-induced stabilization of these receptors operates in date palm for efficient boosting of ABA signaling in response to abiotic stress.

Identification of ABA Receptor Agonists Using a Multiplexed High-Throughput Chemical Screening.

Small molecules that can activate abscisic acid (ABA) receptors represent valuable probes to study ABA perception and signaling. Additionally, these compounds have the potential to be used in the field to counteract the negative effect of drought stress on plant productivity. The PYR/PYL ABA receptors, in their ligand-bound conformation, inactivate protein phosphatases 2C (PP2Cs), triggering physiological responses that are essential for plant adaptation to environmental stresses, including drought. Based on this ligand-induced PP2C inactivation mechanism, we have developed an in vitro assay for the identification of ABA-receptor agonists by high-throughput screening of chemical libraries. The assay allows simultaneous use of different ABA receptors, increasing the chances to find new agonists and eliminates the need for parallel screening. In this chapter, we describe detailed procedures for the identification of ABA agonists using this multiplexed assay in a medium- (96-well plates) or a high-throughput (384-well plates) setup.

A Luciferase Reporter Assay to Identify Chemical Activators of ABA Signaling.

Plant stress tolerance relies on intricate signaling networks that are not fully understood. Several plant hormones are involved in the adaptation to different environmental conditions. Abscisic acid (ABA) has an essential role in stress tolerance, especially in the adaptation to drought. During the last years, chemical genomics has gained attention as an alternative approach to decipher complex traits. Additionally, chemical-based strategies have been very useful to untangle genetic redundancy, which is hard to address by other approaches such as classical genetics. Here, we describe the use of an ABA-inducible luciferase (LUC) reporter line for the high-throughput identification of chemical activators of the ABA signaling pathway. In this assay, seven-day-old pMAPKKK18-LUC+ seedlings are grown on 96-well plates and treated with test compounds. Next, the activity of the LUC reporter is quantified semiautomatically by image analysis. Candidate compounds able to activate the reporter are thus identified and subjected to a secondary screen by analyzing their effect on ABA-related phenotypes (e.g., inhibition of seed germination). This assay is fast, high-throughput, nondestructive, semiquantitative and can be applied to any other luciferase reporter lines, making it ideal for forward chemical genetic screenings.

Plant Osmotic Stress Signaling: MAPKKKs Meet SnRK2s.

Osmotic stress signaling in higher plants is crucial to cope with abiotic stress. RAF-like MAPKKKs are activated by hyperosmotic stress and activate downstream ABA-unresponsive and ABA-activated SnRK2s, integrating early osmotic stress and ABA signaling cascades. The connection of B2/B3/B4 RAF-like MAPKKKs with SnRK2s is a new paradigm in signal transduction.

Drug Discovery for Thirsty Crops.

Following virtual screening and structure-based ligand optimization, researchers have developed opabactin (OP), an abscisic acid (ABA)-receptor agonist with tenfold greater in vivo activity than ABA. This new ligand surpasses previous agonists for its potency and bioactivity on staple crops. OP leads a new class of agrochemicals designed to protect crops from drought.

RBR-Type E3 Ligases and the Ubiquitin-Conjugating Enzyme UBC26 Regulate Abscisic Acid Receptor Levels and Signaling.

The turnover of abscisic acid (ABA) signaling core components modulates the plant's response to ABA and is regulated by ubiquitination. We show that Arabidopsis (Arabidopsis thaliana) RING Finger ABA-Related1 (RFA1) and RFA4 E3 ubiquitin ligases, members of the RING between RING fingers (RBR)-type RSL1/RFA family, are key regulators of ABA receptor stability in root and leaf tissues, targeting ABA receptors for degradation in different subcellular locations. RFA1 is localized both in the nucleus and cytosol, whereas RFA4 shows specific nuclear localization and promotes nuclear degradation of ABA receptors. Therefore, members of the RSL1/RFA family interact with ABA receptors at plasma membrane, cytosol, and nucleus, targeting them for degradation via the endosomal/vacuolar RSL1-dependent pathway or 26S proteasome. Additionally, we provide insight into the physiological function of the relatively unexplored plant RBR-type E3 ligases, and through mutagenesis and biochemical assays we identified cysteine-361 in RFA4 as the putative active site cysteine, which is a distinctive feature of RBR-type E3 ligases. Endogenous levels of PYR1 and PYL4 ABA receptors were higher in the rfa1 rfa4 double mutant than in wild-type plants. UBC26 was identified as the cognate nuclear E2 enzyme that interacts with the RFA4 E3 ligase and forms UBC26-RFA4-receptor complexes in nuclear speckles. Loss-of-function ubc26 alleles and the rfa1 rfa4 double mutant showed enhanced sensitivity to ABA and accumulation of ABA receptors compared with the wild type. Together, our results reveal a sophisticated mechanism by which ABA receptors are targeted by ubiquitin at different subcellular locations, in which the complexity of the ABA receptor family is mirrored in the partner RBR-type E3 ligases.

The fungal sesquiterpenoid pyrenophoric acid B uses the plant ABA biosynthetic pathway to inhibit seed germination.

Pyrenophoric acid (P-Acid), P-Acid B, and P-Acid C are three phytotoxic sesquiterpenoids produced by the ascomycete seed pathogen Pyrenophora semeniperda, a fungus proposed as a mycoherbicide for biocontrol of cheatgrass, an extremely invasive weed. When tested in cheatgrass bioassays, these metabolites were able to delay seed germination, with P-Acid B being the most active compound. Here, we have investigated the cross-kingdom activity of P-Acid B and its mode of action, and found that it activates the abscisic acid (ABA) signaling pathway in order to inhibit seedling establishment. P-Acid B inhibits seedling establishment in wild-type Arabidopsis thaliana, while several mutants affected in the early perception as well as in downstream ABA signaling components were insensitive to the fungal compound. However, in spite of structural similarities between ABA and P-Acid B, the latter is not able to activate the PYR/PYL family of ABA receptors. Instead, we have found that P-Acid B uses the ABA biosynthesis pathway at the level of alcohol dehydrogenase ABA2 to reduce seedling establishment. We propose that the fungus P. semeniperda manipulates plant ABA biosynthesis as a strategy to reduce seed germination, increasing its ability to cause seed mortality and thereby increase its fitness through higher reproductive success.