Interplay between secondary metabolites and plant hormones in silver nitrate-elicited Arabidopsis thaliana plants.

Plants produce a myriad of specialized compounds in response to threats such as pathogens or pests and different abiotic factors. The stress-related induction of specialized metabolites can be mimicked using silver nitrate (AgNO3) as an elicitor, which application in conservation agriculture has gained interest. In Arabidopsis thaliana, AgNO3 triggers the accumulation of indole glucosinolates (IGs) and the phytoalexin camalexin as well as pheylpropanoid-derived defensive metabolites such as coumaroylagmatins and scopoletin through a yet unknown mechanism. In this work, the role of jasmonic (JA) and salicylic acid (SA) signaling in the AgNO3-triggered specialized metabolite production was investigated. To attain this objective, AgNO3, MeJA and SA were applied to A. thaliana lines impaired in JA or SA signaling, or affected in the endogenous levels of IGs and AGs. Metabolomics data indicated that AgNO3 elicitation required an intact JA and SA signaling to elicit the metabolic response, although mutants impaired in hormone signaling retained certain capacity to induce specialized metabolites. In turn, plants overproducing or abolishing IGs production had also an altered hormonal signaling response, both in the accumulation of signaling molecules and the molecular response mechanisms (ORA59, PDF1.2, VSP2 and PR1 gene expression), which pointed out to a crosstalk between defense hormones and specialized metabolites. The present work provides evidence of a crosstalk mechanism between JA and SA underlying AgNO3 defense metabolite elicitation in A. thaliana. In this mechanism, IGs would act as retrograde feedback signals dampening the hormonal response; hence, expanding the signaling molecule concept.

From Classical to Modern Computational Approaches to Identify Key Genetic Regulatory Components in Plant Biology.

The selection of plant genotypes with improved productivity and tolerance to environmental constraints has always been a major concern in plant breeding. Classical approaches based on the generation of variability and selection of better phenotypes from large variant collections have improved their efficacy and processivity due to the implementation of molecular biology techniques, particularly genomics, Next Generation Sequencing and other omics such as proteomics and metabolomics. In this regard, the identification of interesting variants before they develop the phenotype trait of interest with molecular markers has advanced the breeding process of new varieties. Moreover, the correlation of phenotype or biochemical traits with gene expression or protein abundance has boosted the identification of potential new regulators of the traits of interest, using a relatively low number of variants. These important breakthrough technologies, built on top of classical approaches, will be improved in the future by including the spatial variable, allowing the identification of gene(s) involved in key processes at the tissue and cell levels.

New approaches to improve crop tolerance to biotic and abiotic stresses.

During the last years, a great effort has been dedicated at the development and employment of diverse approaches for achieving more stress-tolerant and climate-flexible crops and sustainable yield increases to meet the food and energy demands of the future. The ongoing climate change is in fact leading to more frequent extreme events with a negative impact on food production, such as increased temperatures, drought, and soil salinization as well as invasive arthropod pests and diseases. In this review, diverse "green strategies" (e.g., chemical priming, root-associated microorganisms), and advanced technologies (e.g., genome editing, high-throughput phenotyping) are described on the basis of the most recent research evidence. Particularly, attention has been focused on the potential use in a context of sustainable and climate-smart agriculture (the so called "next agriculture generation") to improve plant tolerance and resilience to abiotic and biotic stresses. In addition, the gap between the results obtained in controlled experiments and those from application of these technologies in real field conditions (lab to field step) is also discussed.

Abscisic Acid as an Emerging Modulator of the Responses of Plants to Low Oxygen Conditions.

Different environmental and developmental cues involve low oxygen conditions, particularly those associated to abiotic stress conditions. It is widely accepted that plant responses to low oxygen conditions are mainly regulated by ethylene (ET). However, interaction with other hormonal signaling pathways as gibberellins (GAs), auxin (IAA), or nitric oxide (NO) has been well-documented. In this network of interactions, abscisic acid (ABA) has always been present and regarded to as a negative regulator of the development of morphological adaptations to soil flooding: hyponastic growth, adventitious root emergence, or formation of secondary aerenchyma in different plant species. However, recent evidence points toward a positive role of this plant hormone on the modulation of plant responses to hypoxia and, more importantly, on the ability to recover during the post-hypoxic period. In this work, the involvement of ABA as an emerging regulator of plant responses to low oxygen conditions alone or in interaction with other hormones is reviewed and discussed.

Identification of ABA-Mediated Genetic and Metabolic Responses to Soil Flooding in Tomato (Solanum lycopersicum L. Mill).

Soil flooding is a compound abiotic stress that alters soil properties and limits atmospheric gas diffusion (O2 and CO2) to the roots. The involvement of abscisic acid (ABA) in the regulation of soil flooding-specific genetic and metabolic responses has been scarcely studied despite its key importance as regulator in other abiotic stress conditions. To attain this objective, wild type and ABA-deficient tomatoes were subjected to short-term (24 h) soil waterlogging. After this period, gas exchange parameters were reduced in the wild type but not in ABA-deficient plants that always had higher E and g s . Transcript and metabolite alterations were more intense in waterlogged tissues, with genotype-specific variations. Waterlogging reduced the ABA levels in the roots while inducing PYR/PYL/RCAR ABA receptors and ABA-dependent transcription factor transcripts, of which induction was less pronounced in the ABA-deficient genotype. Ethylene/O2-dependent genetic responses (ERFVIIs, plant anoxia survival responses, and genes involved in the N-degron pathway) were induced in hypoxic tissues independently of the genotype. Interestingly, genes encoding a nitrate reductase and a phytoglobin involved in NO biosynthesis and scavenging and ERFVII stability were induced in waterlogged tissues, but to a lower extent in ABA-deficient tomato. At the metabolic level, flooding-induced accumulation of Ala was enhanced in ABA-deficient lines following a differential accumulation of Glu and Asp in both hypoxic and aerated tissues, supporting their involvement as sources of oxalacetate to feed the tricarboxylic acid cycle in waterlogged tissues and constituting a potential advantage upon long periods of soil waterlogging. The promoter analysis of upregulated genes indicated that the production of oxalacetate from Asp via Asp oxidase, energy processes such as acetyl-CoA, ATP, and starch biosynthesis, and the lignification process were likely subjected to ABA regulation. Taken together, these data indicate that ABA depletion in waterlogged tissues acts as a positive signal, inducing several specific genetic and metabolic responses to soil flooding.

Early Molecular Responses of Tomato to Combined Moderate Water Stress and Tomato Red Spider Mite Tetranychus evansi Attack.

Interaction between plants and their environment is changing as a consequence of the climate change and global warming, increasing the performance and dispersal of some pest species which become invasive species. Tetranychus evansi also known as the tomato red spider mite, is an invasive species which has been reported to increase its performance when feeding in the tomato cultivar Moneymaker (MM) under water deficit conditions. In order to clarify the underlying molecular events involved, we examined early plant molecular changes occurring on MM during T. evansi infestation alone or in combination with moderate drought stress. Hormonal profiling of MM plants showed an increase in abscisic acid (ABA) levels in drought-stressed plants while salicylic acid (SA) levels were higher in drought-stressed plants infested with T. evansi, indicating that SA is involved in the regulation of plant responses to this stress combination. Changes in the expression of ABA-dependent DREB2, NCED1, and RAB18 genes confirmed the presence of drought-dependent molecular responses in tomato plants and indicated that these responses could be modulated by the tomato red spider mite. Tomato metabolic profiling identified 42 differentially altered compounds produced by T. evansi attack, moderate drought stress, and/or their combination, reinforcing the idea of putative manipulation of tomato plant responses by tomato red spider mite. Altogether, these results indicate that the tomato red spider mite acts modulating plant responses to moderate drought stress by interfering with the ABA and SA hormonal responses, providing new insights into the early events occurring on plant biotic and abiotic stress interaction.

Inheritance, Fitness Cost, and Management of Lambda-Cyhalothrin Resistance in a Laboratory-Selected Strain of Ceratitis capitata (Wiedemann).

The management of the medfly, Ceratitis capitata, in Spanish citrus crops relies mainly on the use of insecticides and the release of sterile males. However, the development of resistance to different insecticides in field populations, including lambda-cyhalothrin, implies a threat for the sustainable control of this pest. The inheritance, fitness cost, and management of lambda-cyhalothrin resistance were examined in the laboratory-selected W-1Kλ strain. We have demonstrated that lambda-cyhalothrin resistance in W-1Kλ is autosomic, completely dominant, and polygenic. In addition, individuals from W-1Kλ showed a lower embryo to pupal viability, a slower developmental time from egg to pupae, and an increase in adults' weight and longevity. We did not find significant trade-offs in the activity of digestive hydrolytic enzymes, with the exception of higher α-amylase activity in W-1Kλ females. A comparative study with different insecticide treatment strategies showed that lambda-cyhalothrin resistance increased when several consecutive treatments with this insecticide were applied. However, the alternation of this insecticide with spinosad was enough to delay the development of resistance. Our results indicate that the rotation of lambda-cyhalothrin with spinosad-a practice already used in some fields-may contribute to prevent the development of resistance.

The Role of ABA in Plant Immunity is Mediated through the PYR1 Receptor.

ABA is involved in plant responses to a broad range of pathogens and exhibits complex antagonistic and synergistic relationships with salicylic acid (SA) and ethylene (ET) signaling pathways, respectively. However, the specific receptor of ABA that triggers the positive and negative responses of ABA during immune responses remains unknown. Through a reverse genetic analysis, we identified that PYR1, a member of the family of PYR/PYL/RCAR ABA receptors, is transcriptionally upregulated and specifically perceives ABA during biotic stress, initiating downstream signaling mediated by ABA-activated SnRK2 protein kinases. This exerts a damping effect on SA-mediated signaling, required for resistance to biotrophic pathogens, and simultaneously a positive control over the resistance to necrotrophic pathogens controlled by ET. We demonstrated that PYR1-mediated signaling exerted control on a priori established hormonal cross-talk between SA and ET, thereby redirecting defense outputs. Defects in ABA/PYR1 signaling activated SA biosynthesis and sensitized plants for immune priming by poising SA-responsive genes for enhanced expression. As a trade-off effect, pyr1-mediated activation of the SA pathway blunted ET perception, which is pivotal for the activation of resistance towards fungal necrotrophs. The specific perception of ABA by PYR1 represented a regulatory node, modulating different outcomes in disease resistance.

A customizable method to characterize Arabidopsis thaliana transpiration under drought conditions.

BACKGROUND: Characterization of the dynamic response of plant transpiration to decreasing soil water content in a reproducible way is required for the correct phenotyping of traits related to water saving strategies. Nowadays, an increasing number of automated high throughput platforms are available, but their development requires a great economic investment and it is not always desirable/feasible to outsource these analyses. We propose a medium-throughput protocol to characterize transpiration responses to decreasing soil moisture in a quantitative and highly reproducible way with a minimum investment of resources. RESULTS: The quantitative characterization of plant responses to a decreasing soil water content using our phenotyping platform has showed high reproducibility between different experiments. The proposed irrigation strategy allowed us to harvest plants ranging from a well-watered condition to the loss-of-turgor point in a predictable and controlled way. Coupling this protocol with hormone profiling allows investigation of hormonal responses (metabolite accumulation as well as plant sensitivity) to water stress. As a proof-of-concept, we have characterized the dynamic responses of leaf transpiration to decreasing soil water contents in an abscisic acid (ABA) deficient genotype (aba1-1) as well as in genotypes with altered sensitivity to ABA (abi1-1 and hab1-1abi1-1), which are insensitive and hypersensitive to ABA, respectively. CONCLUSIONS: This protocol allows for assessment of quantitative differences in rosette transpiration responses to water depletion in both ABA biosynthesis mutants and genotypes with altered sensitivity to the hormone. Data indicate a correlation between ABA levels and/or hormone perception and growth rate and/or water content. The protocol guarantees the correct application of water stress to adult plants, which is essential to understand responses of mutants and/or natural accessions.

Plant-Mediated Effects of Water Deficit on the Performance of Tetranychus evansi on Tomato Drought-Adapted Accessions.

Climate change is expected to increase drought periods and the performance and dispersal of some invasive species such as Tetranychus evansi, which has been reported to take advantage of the nutritional changes induced by water-shortage on the tomato cultivar Moneymaker (MM). We have examined the implications for mite's biology of four accessions of the drought-adapted tomatoes, "Tomàtiga de Ramellet" (TR), under moderate drought stress. Mite performance was enhanced by drought in two accessions (TR61 and TR154), but not in the other two accessions (TR58 and TR126). We selected one accession of each outcome (i.e., TR154 and TR126) to further analyze plant nutritional parameters. We found that free sugars and most essential amino acids for mites were induced by drought and/or mite infestation on MM and TR154 plants, whereas sugars were not altered and a reduced number of essential amino acids were induced by drought in TR126. Remarkably, mite performance was enhanced by leaf infiltration of free sugars, essential amino acids mixture, and L-proline on well-watered MM and by free sugars on drought-stressed TR126 plants. These results indicate a positive link between the induction of soluble carbohydrates and amino acids used by the plant for osmotic adjustment and mite performance. The effects of drought and/or mite infestation on the defense response of plants was analyzed at three levels: phytohormone accumulation, the transcript levels of marker genes linked to jasmonates (JAs), salicylic acid (SA), and abscisic acid (ABA) pathways, and the activity of defense proteins. The ability of T. evansi to downregulate the accumulation of defense-related phytohormones was noted on MM and the two TR accessions analyzed (TR126 and TR154), though differences in the induction of protein defense genes and activities by drought and/or mite infestation were observed among them. These results emphasize the importance of studying plant biotic and abiotic stress factors in combination and provides an experimental framework for screening drought-tolerant tomato accessions that will be also resistant to herbivore mites.

Depletion of abscisic acid levels in roots of flooded Carrizo citrange (Poncirus trifoliata L. Raf. × Citrus sinensis L. Osb.) plants is a stress-specific response associated to the differential expression of PYR/PYL/RCAR receptors.

Soil flooding reduces root abscisic acid (ABA) levels in citrus, conversely to what happens under drought. Despite this reduction, microarray analyses suggested the existence of a residual ABA signaling in roots of flooded Carrizo citrange seedlings. The comparison of ABA metabolism and signaling in roots of flooded and water stressed plants of Carrizo citrange revealed that the hormone depletion was linked to the upregulation of CsAOG, involved in ABA glycosyl ester (ABAGE) synthesis, and to a moderate induction of catabolism (CsCYP707A, an ABA 8'-hydroxylase) and buildup of dehydrophaseic acid (DPA). Drought strongly induced both ABA biosynthesis and catabolism (CsNCED1, 9-cis-neoxanthin epoxycarotenoid dioxygenase 1, and CsCYP707A) rendering a significant hormone accumulation. In roots of flooded plants, restoration of control ABA levels after stress release was associated to the upregulation of CsBGLU18 (an ABA ß-glycosidase) that cleaves ABAGE. Transcriptional profile of ABA receptor genes revealed a different induction in response to soil flooding (CsPYL5) or drought (CsPYL8). These two receptor genes along with CsPYL1 were cloned and expressed in a heterologous system. Recombinant CsPYL5 inhibited ΔNHAB1 activity in vitro at lower ABA concentrations than CsPYL8 or CsPYL1, suggesting its better performance under soil flooding conditions. Both stress conditions induced ABA-responsive genes CsABI5 and CsDREB2A similarly, suggesting the occurrence of ABA signaling in roots of flooded citrus seedlings. The impact of reduced ABA levels in flooded roots on CsPYL5 expression along with its higher hormone affinity reinforce the role of this ABA receptor under soil-flooding conditions and explain the expression of certain ABA-responsive genes.

The whole genome sequence of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), reveals insights into the biology and adaptive evolution of a highly invasive pest species.

BACKGROUND: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a major destructive insect pest due to its broad host range, which includes hundreds of fruits and vegetables. It exhibits a unique ability to invade and adapt to ecological niches throughout tropical and subtropical regions of the world, though medfly infestations have been prevented and controlled by the sterile insect technique (SIT) as part of integrated pest management programs (IPMs). The genetic analysis and manipulation of medfly has been subject to intensive study in an effort to improve SIT efficacy and other aspects of IPM control. RESULTS: The 479 Mb medfly genome is sequenced from adult flies from lines inbred for 20 generations. A high-quality assembly is achieved having a contig N50 of 45.7 kb and scaffold N50 of 4.06 Mb. In-depth curation of more than 1800 messenger RNAs shows specific gene expansions that can be related to invasiveness and host adaptation, including gene families for chemoreception, toxin and insecticide metabolism, cuticle proteins, opsins, and aquaporins. We identify genes relevant to IPM control, including those required to improve SIT. CONCLUSIONS: The medfly genome sequence provides critical insights into the biology of one of the most serious and widespread agricultural pests. This knowledge should significantly advance the means of controlling the size and invasive potential of medfly populations. Its close relationship to Drosophila, and other insect species important to agriculture and human health, will further comparative functional and structural studies of insect genomes that should broaden our understanding of gene family evolution.

FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates Its Delivery to the Vacuolar Degradation Pathway.

Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of Arabidopsis thaliana This suggested that ubiquitylated abscisic acid (ABA) receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knockdown fyve1 alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, fyve1 mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling.

Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling.

Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca(2+) are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca(2+) signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca(2+)-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca(2+) sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca(2+)-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.

A Direct Link between Abscisic Acid Sensing and the Chromatin-Remodeling ATPase BRAHMA via Core ABA Signaling Pathway Components.

Optimal response to drought is critical for plant survival and will affect biodiversity and crop performance during climate change. Mitotically heritable epigenetic or dynamic chromatin state changes have been implicated in the plant response to the drought stress hormone abscisic acid (ABA). The Arabidopsis SWI/SNF chromatin-remodeling ATPase BRAHMA (BRM) modulates response to ABA by preventing premature activation of stress response pathways during germination. We show that core ABA signaling pathway components physically interact with BRM and post-translationally modify BRM by phosphorylation/dephosphorylation. Genetic evidence suggests that BRM acts downstream of SnRK2.2/2.3 kinases, and biochemical studies identified phosphorylation sites in the C-terminal region of BRM at SnRK2 target sites that are evolutionarily conserved. Finally, the phosphomimetic BRM(S1760D S1762D) mutant displays ABA hypersensitivity. Prior studies showed that BRM resides at target loci in the ABA pathway in the presence and absence of the stimulus, but is only active in the absence of ABA. Our data suggest that SnRK2-dependent phosphorylation of BRM leads to its inhibition, and PP2CA-mediated dephosphorylation of BRM restores the ability of BRM to repress ABA response. These findings point to the presence of a rapid phosphorylation-based switch to control BRM activity; this property could be potentially harnessed to improve drought tolerance in plants.

Inactivation of PYR/PYL/RCAR ABA receptors by tyrosine nitration may enable rapid inhibition of ABA signaling by nitric oxide in plants.

Abscisic acid (ABA) is a phytohormone that inhibits growth and enhances adaptation to stress in plants. ABA perception and signaling rely on its binding to receptors of the pyrabactin resistance1/PYR1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) family, the subsequent inhibition of clade A type 2C protein phosphatases (PP2Cs), and the phosphorylation of ion channels and transcription factors by protein kinases of the SnRK2 family. Nitric oxide (NO) may inhibit ABA signaling because NO-deficient plants are hypersensitive to ABA. Regulation by NO often involves posttranslational modification of proteins. Mass spectrometry analysis of ABA receptors expressed in plants and recombinant receptors modified in vitro revealed that the receptors were nitrated at tyrosine residues and S-nitrosylated at cysteine residues. In an in vitro ABA-induced, PP2C inhibition assay, tyrosine nitration reduced receptor activity, whereas S-nitrosylated receptors were fully capable of ABA-induced inhibition of the phosphatase. PYR/PYL/RCAR proteins with nitrated tyrosine, which is an irreversible covalent modification, were polyubiquitylated and underwent proteasome-mediated degradation. We propose that tyrosine nitration, which requires NO and superoxide anions, is a rapid mechanism by which NO limits ABA signaling under conditions in which NO and reactive oxygen species are both produced.

The IBO germination quantitative trait locus encodes a phosphatase 2C-related variant with a nonsynonymous amino acid change that interferes with abscisic acid signaling.

Natural genetic variation is crucial for adaptability of plants to different environments. Seed dormancy prevents precocious germination in unsuitable conditions and is an adaptation to a major macro-environmental parameter, the seasonal variation in temperature and day length. Here we report the isolation of IBO, a quantitative trait locus (QTL) that governs c. 30% of germination rate variance in an Arabidopsis recombinant inbred line (RIL) population derived from the parental accessions Eilenburg-0 (Eil-0) and Loch Ness-0 (Lc-0). IBO encodes an uncharacterized phosphatase 2C-related protein, but neither the Eil-0 nor the Lc-0 variant, which differ in a single amino acid, have any appreciable phosphatase activity in in vitro assays. However, we found that the amino acid change in the Lc-0 variant of the IBO protein confers reduced germination rate. Moreover, unlike the Eil-0 variant of the protein, the Lc-0 variant can interfere with the activity of the phosphatase 2C ABSCISIC ACID INSENSITIVE 1 in vitro. This suggests that the Lc-0 variant possibly interferes with abscisic acid signaling, a notion that is supported by physiological assays. Thus, we isolated an example of a QTL allele with a nonsynonymous amino acid change that might mediate local adaptation of seed germination timing.