The endophytic fungus Serendipita indica affects auxin distribution in Arabidopsis thaliana roots through alteration of auxin transport and conjugation to promote plant growth.

Plants share their habitats with a multitude of different microbes. This close vicinity promoted the evolution of interorganismic interactions between plants and many different microorganisms that provide mutual growth benefits both to the plant and the microbial partner. The symbiosis of Arabidopsis thaliana with the beneficial root colonizing endophyte Serendipita indica represents a well-studied system. Colonization of Arabidopsis roots with S. indica promotes plant growth and stress tolerance of the host plant. However, until now, the molecular mechanism by which S. indica reprograms plant growth remains largely unknown. This study used comprehensive transcriptomics, metabolomics, reverse genetics, and life cell imaging to reveal the intricacies of auxin-related processes that affect root growth in the symbiosis between A. thaliana and S. indica. Our experiments revealed the sustained stimulation of auxin signalling in fungus infected Arabidopsis roots and disclosed the essential role of tightly controlled auxin conjugation in the plant-fungus interaction. It particularly highlighted the importance of two GRETCHEN HAGEN 3 (GH3) genes, GH3.5 and GH3.17, for the fungus infection-triggered stimulation of biomass production, thus broadening our knowledge about the function of GH3s in plants. Furthermore, we provide evidence for the transcriptional alteration of the PIN2 auxin transporter gene in roots of Arabidopsis seedlings infected with S. indica and demonstrate that this transcriptional adjustment affects auxin signalling in roots, which results in increased plant growth.

The Beneficial Fungus Mortierella hyalina Modulates Amino Acid Homeostasis in Arabidopsis under Nitrogen Starvation.

Non-mycorrhizal but beneficial fungi often mitigate (a)biotic stress-related traits in host plants. The underlying molecular mechanisms are mostly still unknown, as in the interaction between the endophytic growth-promoting soil fungus Mortierella hyalina and Arabidopsis thaliana. Here, abiotic stress in the form of nitrogen (N) deficiency was used to investigate the effects of the fungus on colonized plants. In particular, the hypothesis was investigated that fungal infection could influence N deficiency via an interaction with the high-affinity nitrate transporter NRT2.4, which is induced by N deficiency. For this purpose, Arabidopsis wild-type nrt2.4 knock-out and NRT2.4 reporter lines were grown on media with different nitrate concentrations with or without M. hyalina colonization. We used chemical analysis methods to determine the amino acids and phytohormones. Experimental evidence suggests that the fungus does not modulate NRT2.4 expression under N starvation. Instead, M. hyalina alleviates N starvation in other ways: The fungus supplies nitrogen (15N) to the N-starved plant. The presence of the fungus restores the plants' amino acid homeostasis, which was out of balance due to N deficiency, and causes a strong accumulation of branched-chain amino acids. We conclude that the plant does not need to invest in defense and resources for growth are maintained, which in turn benefits the fungus, suggesting that this interaction should be considered a mutualistic symbiosis.

The Root-Colonizing Endophyte Piriformospora indica Supports Nitrogen-Starved Arabidopsis thaliana Seedlings with Nitrogen Metabolites.

The root-colonizing endophytic fungus Piriformospora indica promotes the root and shoot growth of its host plants. We show that the growth promotion of Arabidopsis thaliana leaves is abolished when the seedlings are grown on media with nitrogen (N) limitation. The fungus neither stimulated the total N content nor did it promote 15NO3- uptake from agar plates to the leaves of the host under N-sufficient or N-limiting conditions. However, when the roots were co-cultivated with 15N-labelled P. indica, more labels were detected in the leaves of N-starved host plants but not in plants supplied with sufficient N. Amino acid and primary metabolite profiles, as well as the expression analyses of N metabolite transporter genes suggest that the fungus alleviates the adaptation of its host from the N limitation condition. P. indica alters the expression of transporter genes, which participate in the relocation of NO3-, NH4+ and N metabolites from the roots to the leaves under N limitation. We propose that P. indica participates in the plant's metabolomic adaptation against N limitation by delivering reduced N metabolites to the host, thus alleviating metabolic N starvation responses and reprogramming the expression of N metabolism-related genes.

Two types of GLR channels cooperate differently in light and dark growth of Arabidopsis seedlings.

BACKGROUND: GLutamate Receptor-like (GLR) channels are multimeric, ionotropic, ligand-gated plant transmembrane receptors. They are homologous to mammalian glutamate receptors, iGLuRs, which are critical to neuronal function. GLRs have been reported several times to play a role in photomorphogenesis. However, to date, no study has looked at the mechanism of their involvement in this process. Here we focused on examining the impact of GLRs on the regulation of early seedling growth in blue light, red light, and in the dark. RESULTS: Wild type and six photoreceptor mutant seedlings were grown on media supplemented with known iGLuR/GLR channel antagonists: MK-801, which non-competitively blocks NMDA channels in mammalian cells, and CNQX, known for competitive blocking of AMPA channels in mammalian cells. The lengths of hypocotyls and roots were measured in seedlings of phyA, phyB, phot1, phot2, cry1, and cry2 mutants after 7 days of in vitro culture. Changes in growth parameters, both in light and in darkness upon application of chemical antagonists, show that both types of GLR channels, NMDA-like and AMPA-like, are involved in the regulation of seedling growth irrespective of light conditions. Analysis of seedling growth of photoreceptor mutants indicates that the channels are influenced by signaling from phot1, phot2, and cry1. To extend our analysis, we also evaluated the elicitation of a calcium wave, which is likely to be partially driven by GLRs, in Arabidopsis seedlings. The changes in cellobiose-induced calcium waves observed after applying GLR inhibitors suggest that both types of channels likely cooperate in shaping Arabidopsis seedling growth and development. CONCLUSIONS: Our work provides the first experimental evidence that two types of GLR channels function in plants: NMDA-like and AMPA-like. We also demonstrate that the channels are involved in seedling growth and development, at least partially through modulation of calcium signaling, but they are unlikely to play a major role in photomorphogenesis.

CORK1, A LRR-Malectin Receptor Kinase, Is Required for Cellooligomer-Induced Responses in Arabidopsis thaliana.

Cell wall integrity (CWI) maintenance is central for plant cells. Mechanical and chemical distortions, pH changes, and breakdown products of cell wall polysaccharides activate plasma membrane-localized receptors and induce appropriate downstream responses. Microbial interactions alter or destroy the structure of the plant cell wall, connecting CWI maintenance to immune responses. Cellulose is the major polysaccharide in the primary and secondary cell wall. Its breakdown generates short-chain cellooligomers that induce Ca2+-dependent CWI responses. We show that these responses require the malectin domain-containing CELLOOLIGOMER-RECEPTOR KINASE 1 (CORK1) in Arabidopsis and are preferentially activated by cellotriose (CT). CORK1 is required for cellooligomer-induced cytoplasmic Ca2+ elevation, reactive oxygen species (ROS) production, mitogen-associated protein kinase (MAPK) activation, cellulose synthase phosphorylation, and the regulation of CWI-related genes, including those involved in biosynthesis of cell wall material, secondary metabolites and tryptophan. Phosphoproteome analyses identified early targets involved in signaling, cellulose synthesis, the endoplasmic reticulum/Golgi secretory pathway, cell wall repair and immune responses. Two conserved phenylalanine residues in the malectin domain are crucial for CORK1 function. We propose that CORK1 is required for CWI and immune responses activated by cellulose breakdown products.

The calcium sensor CBL7 is required for Serendipita indica-induced growth stimulation in Arabidopsis thaliana, controlling defense against the endophyte and K+ homoeostasis in the symbiosis.

Calcium is an important second messenger in plants. The activation of Ca2+ signalling cascades is critical in the activation of adaptive processes in response to environmental stimuli. Root colonization by the growth promoting endophyte Serendipita indica involves the increase of cytosolic Ca2+ levels in Arabidopsis thaliana. Here, we investigated transcriptional changes in Arabidopsis roots during symbiosis with S. indica. RNA-seq profiling disclosed the induction of Calcineurin B-like 7 (CBL7) during early and later phases of the interaction. Consistently, reverse genetic evidence highlighted the functional relevance of CBL7 and tested the involvement of a CBL7-CBL-interacting protein kinase 13 signalling pathway. The loss-of-function of CBL7 abolished the growth promoting effect and affected root colonization. The transcriptomics analysis of cbl7 revealed the involvement of this Ca2+ sensor in activating plant defense responses. Furthermore, we report on the contribution of CBL7 to potassium transport in Arabidopsis. We analysed K+ contents in wild-type and cbl7 plants and observed a significant increase of K+ in roots of cbl7 plants, while shoot tissues demonstrated K+ depletion. Taken together, our work associates CBL7 with an important role in the mutual interaction between Arabidopsis and S. indica and links CBL7 to K+ transport.

Comprehensive Genome-Wide Identification and Expression Profiling of Eceriferum (CER) Gene Family in Passion Fruit (Passiflora edulis) Under Fusarium kyushuense and Drought Stress Conditions.

Plant surfaces are covered with cuticle wax and are the first barrier between a plant and environmental stresses. Eceriferum (CER) is an important gene family involved in wax biosynthesis and stress resistance. In this study, for the first time, 34 CER genes were identified in the passion fruit (Passiflora edulis) genome, and PeCER proteins varied in physicochemical properties. A phylogenetic tree was constructed and divided into seven clades to identify the evolutionary relationship with other plant species. Gene structure analyses revealed that conserved motifs ranged from 1 to 24, and that exons ranged from 1 to 29. The cis-element analysis provides insight into possible roles of PeCER genes in plant growth, development and stress responses. The syntenic analysis revealed that segmental (six gene pairs) and tandem (six gene pairs) gene duplication played an important role in the expansion of PeCER genes and underwent a strong purifying selection. In addition, 12 putative ped-miRNAs were identified to be targeting 16 PeCER genes, and PeCER6 was the most targeted by four miRNAs including ped-miR157a-5p, ped-miR164b-5p, ped-miR319b, and ped-miR319l. Potential transcription factors (TFs) such as ERF, AP2, MYB, and bZIP were predicted and visualized in a TF regulatory network interacting with PeCER genes. GO and KEGG annotation analysis revealed that PeCER genes were highly related to fatty acid, cutin, and wax biosynthesis, plant-pathogen interactions, and stress response pathways. The hypothesis that most PeCER proteins were predicted to localize to the plasma membrane was validated by transient expression assays of PeCER32 protein in onion epidermal cells. qRT-PCR expression results showed that most of the PeCER genes including PeCER1, PeCER11, PeCER15, PeCER17, and PeCER32 were upregulated under drought and Fusarium kyushuense stress conditions compared to controls. These findings provide a foundation for further studies on functions of PeCER genes to further facilitate the genetic modification of passion fruit wax biosynthesis and stress resistance.

The Ca2+ sensor proteins CML37 and CML42 antagonistically regulate plant stress responses by altering phytohormone signals.

KEY MESSAGE: Calmodulin-like-proteins (CML) belong to a family of calcium-sensing proteins that are unique for plants and involved in many different developmental and stress-related reactions. In defense against herbivory, some pathogens and drought, CML37 acts as a positive and CML42 as a negative regulator, respectively. We provide evidence that both CMLs act antagonistically in the regulation of induced defense responses. A double knock-out line, cml37 x cml42, thus shows wild-type phenotypes upon all kind of stresses we used. A transient increase in the cytosolic calcium concentration is one of the first reactions that can be measured in plant cells upon abiotic as well as biotic stress treatments. These calcium signals are sensed by calcium binding proteins such as calmodulin-like proteins (CMLs), which transduce the sensed information into appropriate stress responses by interacting with downstream target proteins. In previous studies, CML37 has been shown to positively regulate the plants' defense against both the insect herbivore Spodoptera littoralis and the response to drought stress. In contrast, CML42 is known to negatively regulate those two stress responses. Here, we provide evidence that these two CMLs act antagonistically in the regulation of induced responses directed against drought and herbivory stress as well as in the defense against the necrotrophic pathogen Alternaria brassicicola. Both CMLs shape the plant reactions by altering the phytohormone signaling. Consequently, the phytohormone-regulated production of defensive compounds like glucosinolates is also antagonistically mediated by both CMLs. The finding that CML37 and CML42 have antagonistic roles in diverse stress-related responses suggests that these calcium sensor proteins represent important tools for the plant to balance and fine-tune the signaling and downstream reactions upon environmental stress.

Identification of Fungal Pathogens to Control Postharvest Passion Fruit (Passiflora edulis) Decays and Multi-Omics Comparative Pathway Analysis Reveals Purple Is More Resistant to Pathogens than a Yellow Cultivar.

Production of passion fruit (Passiflora edulis) is restricted by postharvest decay, which limits the storage period. We isolated, identified, and characterized fungal pathogens causing decay in two passion fruit cultivars during two fruit seasons in China. Morphological characteristics and nucleotide sequences of ITS-rDNA regions identified eighteen isolates, which were pathogenic on yellow and purple fruit. Fusarium kyushuense, Fusarium concentricum, Colletotrichum truncatum, and Alternaria alternata were the most aggressive species. Visible inspections and comparative analysis of the disease incidences demonstrated that wounded and non-wounded yellow fruit were more susceptible to the pathogens than the purple fruit. Purple cultivar showed higher expression levels of defense-related genes through expression and metabolic profiling, as well as significantly higher levels of their biosynthesis pathways. We also found fungi with potential beneficial features for the quality of fruits. Our transcriptomic and metabolomics data provide a basis to identify potential targets to improve the pathogen resistance of the susceptible yellow cultivar. The identified fungi and affected features of the fruit of both cultivars provide important information for the control of pathogens in passion fruit industry and postharvest storage.

The effect of the root-colonizing Piriformospora indica on passion fruit (Passiflora edulis) development: Initial defense shifts to fitness benefits and higher fruit quality.

Passion fruit (Passiflora edulis) has an important economic value as exotic ingredient in juice blends. We inoculated the passion fruit cultivar Passiflora edulis Sims f. edulis's roots with the beneficial root-colonizing fungus Piriformospora indica under greenhouse conditions. The experiments were performed at three different locations and times (between 2017 and 2019). After transient initial growth retardation associated with a mild salicylic-acid (SA)-dependent defense activation and reduced sucrose metabolism, plant performance and growth are promoted during later stages. The elevated SA level in the aerial parts stimulates the plant immune system and promotes pathogen resistance in the adult plants and the fruit peels. P. indica stimulates the fruit size and fruit quality, and the higher amounts of defense-related secondary metabolites in the peels restrict growth of herbivorous insect larvae fed with peel extracts. We conclude that application of P. indica to passion fruits stimulates the plants' immune system and improves the fruits' quality.

ANNEXIN1 mediates calcium-dependent systemic defense in Arabidopsis plants upon herbivory and wounding.

Cellular calcium (Ca) transients are endogenous signals involved in local and systemic signaling and defense activation upon environmental stress, including wounding and herbivory. Still, not all Ca2+ channels contributing to the signaling have been identified, nor are their modes of action fully known. Plant annexins are proteins capable of binding to anionic phospholipids and can exhibit Ca channel-like activity. Arabidopsis ANNEXIN1 (ANN1) is suggested to contribute to Ca transport. Here, we report that wounding and simulated-herbivory-induced cytosolic free Ca elevation was impaired in systemic leaves in ann1 loss-of-function plants. We provide evidence for a role of ANN1 in local and systemic defense of plants attacked by herbivorous Spodoptera littoralis larvae. Bioassays identified ANN1 as a positive defense regulator. Spodoptera littoralis feeding on ann1 gained significantly more weight than larvae feeding on wild-type, whereas those feeding on ANN1-overexpressing lines gained less weight. Herbivory and wounding both induced defense-related responses on treated leaves, such as jasmonate accumulation and defense gene expression. These responses remained local and were strongly reduced in systemic leaves in ann1 plants. Our results indicate that ANN1 plays an important role in activation of systemic rather than local defense in plants attacked by herbivorous insects.

Harnessing symbiotic plant-fungus interactions to unleash hidden forces from extreme plant ecosystems.

Global climate change is arguably one of the biggest threats of modern times and has already led to a wide range of impacts on the environment, economy, and society. Owing to past emissions and climate system inertia, global climate change is predicted to continue for decades even if anthropogenic greenhouse gas emissions were to stop immediately. In many regions, such as central Europe and the Mediterranean region, the temperature is likely to rise by 2-5 °C and annual precipitation is predicted to decrease. Expected heat and drought periods followed by floods, and unpredictable growing seasons, are predicted to have detrimental effects on agricultural production systems, causing immense economic losses and food supply problems. To mitigate the risks of climate change, agricultural innovations counteracting these effects need to be embraced and accelerated. To achieve maximum improvement, the required agricultural innovations should not focus only on crops but rather pursue a holistic approach including the entire ecosystem. Over millions of years, plants have evolved in close association with other organisms, particularly soil microbes that have shaped their evolution and contemporary ecology. Many studies have already highlighted beneficial interactions among plants and the communities of microorganisms with which they coexist. Questions arising from these discoveries are whether it will be possible to decipher a common molecular pattern and the underlying biochemical framework of interspecies communication, and whether such knowledge can be used to improve agricultural performance under environmental stress conditions. In this review, we summarize the current knowledge of plant interactions with fungal endosymbionts found in extreme ecosystems. Special attention will be paid to the interaction of plants with the symbiotic root-colonizing endophytic fungus Serendipita indica, which has been developed as a model system for beneficial plant-fungus interactions.

Beneficial and Pathogenic Arabidopsis Root-Interacting Fungi Differently Affect Auxin Levels and Responsive Genes During Early Infection.

Auxin (indole-3-acetic acid, IAA) is an important phytohormone involved in root growth and development. Root-interacting beneficial and pathogenic fungi utilize auxin and its target genes to manipulate the performance of their hosts for their own needs. In order to follow and visualize auxin effects in fungi-colonized Arabidopsis roots, we used the dual auxin reporter construct DR5::EGFP-DR5v2::tdTomato and fluorescence microscopy as well as LC-MS-based phytohormone analyses. We demonstrate that the beneficial endophytic fungi Piriformospora indica and Mortierella hyalina produce and accumulate IAA in their mycelia, in contrast to the phytopathogenic biotrophic fungus Verticillium dahliae and the necrotrophic fungus Alternaria brassicicola. Within 3 h after exposure of Arabidopsis roots to the pathogens, the signals of the auxin-responsive reporter genes disappeared. When exposed to P. indica, significantly higher auxin levels and stimulated expression of auxin-responsive reporter genes were detected both in lateral root primordia and the root elongation zone within 1 day. Elevated auxin levels were also present in the M. hyalina/Arabidopsis root interaction, but no downstream effects on auxin-responsive reporter genes were observed. However, the jasmonate level was strongly increased in the colonized roots. We propose that the lack of stimulated root growth upon infection with M. hyalina is not caused by the absence of auxin, but an inhibitory effect mediated by high jasmonate content.

Alternaria Brassicae Induces Systemic Jasmonate Responses in Arabidopsis Which Travel to Neighboring Plants via a Piriformsopora Indica Hyphal Network and Activate Abscisic Acid Responses.

Stress information received by a particular local plant tissue is transferred to other tissues and neighboring plants, but how the information travels is not well understood. Application of Alternaria Brassicae spores to Arabidopsis leaves or roots stimulates local accumulation of jasmonic acid (JA), the expression of JA-responsive genes, as well as of NITRATE TRANSPORTER (NRT)2.5 and REDOX RESPONSIVE TRANSCRIPTION FACTOR1 (RRTF1). Infection information is systemically spread over the entire seedling and propagates radially from infected to non-infected leaves, axially from leaves to roots, and vice versa. The local and systemic NRT2.5 responses are reduced in the jar1 mutant, and the RRTF1 response in the rbohD mutant. Information about A. brassicae infection travels slowly to uninfected neighboring plants via a Piriformospora Indica hyphal network, where NRT2.5 and RRTF1 are up-regulated. The systemic A. brassicae-induced JA response in infected plants is converted to an abscisic acid (ABA) response in the neighboring plant where ABA and ABA-responsive genes are induced. We propose that the local threat information induced by A. brassicae infection is spread over the entire plant and transferred to neighboring plants via a P. indica hyphal network. The JA-specific response is converted to a general ABA-mediated stress response in the neighboring plant.

Herbivory-responsive calmodulin-like protein CML9 does not guide jasmonate-mediated defenses in Arabidopsis thaliana.

Calcium is an important second messenger in plants that is released into the cytosol early after recognition of various environmental stimuli. Decoding of such calcium signals by calcium sensors is the key for the plant to react appropriately to each stimulus. Several members of Calmodulin-like proteins (CMLs) act as calcium sensors and some are known to mediate both abiotic and biotic stress responses. Here, we study the role of the Arabidopsis thaliana CML9 in different stress responses. CML9 was reported earlier as defense regulator against Pseudomonas syringae. In contrast to salicylic acid-mediated defense against biotrophic pathogens such as P. syringae, defenses against herbivores and necrotrophic fungi are mediated by jasmonates. We demonstrate that CML9 is induced upon wounding and feeding of the insect herbivore Spodoptera littoralis. However, neither different CML9 loss-of-function mutant lines nor overexpression lines were impaired upon insect feeding. No difference in herbivore-induced phytohormone elevation was detected in cml9 lines. The defense against the spider mite Tetranychus urticae was also unaffected. In addition, cml9 mutant lines showed a wild type-like reaction to the necrotrophic fungus Alternaria brassicicola. Thus, our data suggest that CML9 might be a regulator involved only in the defense against biotrophic pathogens, independent of jasmonates. In addition, our data challenge the involvement of CML9 in plant drought stress response. Taken together, we suggest that CML9 is a specialized rather than a general regulator of stress responses in Arabidopsis.

Verticillium dahliae-Arabidopsis Interaction Causes Changes in Gene Expression Profiles and Jasmonate Levels on Different Time Scales.

Verticillium dahliae is a soil-borne vascular pathogen that causes severe wilt symptoms in a wide range of plants. Co-culture of the fungus with Arabidopsis roots for 24 h induces many changes in the gene expression profiles of both partners, even before defense-related phytohormone levels are induced in the plant. Both partners reprogram sugar and amino acid metabolism, activate genes for signal perception and transduction, and induce defense- and stress-responsive genes. Furthermore, analysis of Arabidopsis expression profiles suggests a redirection from growth to defense. After 3 weeks, severe disease symptoms can be detected for wild-type plants while mutants impaired in jasmonate synthesis and perception perform much better. Thus, plant jasmonates have an important influence on the interaction, which is already visible at the mRNA level before hormone changes occur. The plant and fungal genes that rapidly respond to the presence of the partner might be crucial for early recognition steps and the future development of the interaction. Thus they are potential targets for the control of V. dahliae-induced wilt diseases.

A Poly(A) Ribonuclease Controls the Cellotriose-Based Interaction between Piriformospora indica and Its Host Arabidopsis.

Piriformospora indica, an endophytic root-colonizing fungus, efficiently promotes plant growth and induces resistance to abiotic stress and biotic diseases. P. indica fungal cell wall extract induces cytoplasmic calcium elevation in host plant roots. Here, we show that cellotriose (CT) is an elicitor-active cell wall moiety released by P. indica into the medium. CT induces a mild defense-like response, including the production of reactive oxygen species, changes in membrane potential, and the expression of genes involved in growth regulation and root development. CT-based cytoplasmic calcium elevation in Arabidopsis (Arabidopsis thaliana) roots does not require the BAK1 coreceptor or the putative Ca2+ channels TPC1, GLR3.3, GLR2.4, and GLR2.5 and operates synergistically with the elicitor chitin. We identified an ethyl methanesulfonate-induced mutant (cytoplasmiccalcium elevation mutant) impaired in the response to CT and various other cellooligomers (n = 2-7), but not to chitooligomers (n = 4-8), in roots. The mutant contains a single nucleotide exchange in the gene encoding a poly(A) ribonuclease (AtPARN; At1g55870) that degrades the poly(A) tails of specific mRNAs. The wild-type PARN cDNA, expressed under the control of a 35S promoter, complements the mutant phenotype. Our identification of cellotriose as a novel chemical mediator casts light on the complex P. indica-plant mutualistic relationship.

Evidence for GABA-Induced Systemic GABA Accumulation in Arabidopsis upon Wounding.

The non-proteinogenic amino acid γ-aminobutyric acid (GABA) is present in all plant species analyzed so far. Its synthesis is stimulated by either acidic conditions occurring after tissue disruption or higher cytosolic calcium level. In mammals, GABA acts as inhibitory neurotransmitter but its function in plants is still not well understood. Besides its involvement in abiotic stress resistance, GABA has a role in the jasmonate-independent defense against invertebrate pests. While the biochemical basis for GABA accumulation in wounded leaves is obvious, the underlying mechanisms for wounding-induced GABA accumulation in systemic leaves remained unclear. Here, the Arabidopsis thaliana knock-out mutant lines pop2-5, unable to degrade GABA, and tpc1-2, lacking a wounding-induced systemic cytosolic calcium elevation, were employed for a comprehensive investigation of systemic GABA accumulation. A wounding-induced systemic GABA accumulation was detected in tpc1-2 plants demonstrating that an increased calcium level was not involved. Similarly, after both mechanical wounding and Spodoptera littoralis feeding, GABA accumulation in pop2-5 plants was significantly higher in local and systemic leaves, compared to wild-type plants. Consequently, larvae feeding on these GABA-enriched mutant plants grew significantly less. Upon exogenous application of a D2-labeled GABA to wounded leaves of pop2-5 plants, its uptake but no translocation to unwounded leaves was detected. In contrast, an accumulation of endogenous GABA was observed in vascular connected systemic leaves. These results suggest that the systemic accumulation of GABA upon wounding does not depend on the translocation of GABA or on an increase in cytosolic calcium.

Synthesis, metabolism and systemic transport of a fluorinated mimic of the endogenous jasmonate precursor OPC-8:0.

Jasmonates (JAs) are fatty acid derivatives that mediate many developmental processes and stress responses in plants. Synthetic jasmonate derivatives (commonly isotopically labeled), which mimic the action of the endogenous compounds are often employed as internal standards or probes to study metabolic processes. However, stable-isotope labeling of jasmonates does not allow the study of spatial and temporal distribution of these compounds in real time by positron emission tomography (PET). In this study, we explore whether a fluorinated jasmonate could mimic the action of the endogenous compound and therefore, be later employed as a tracer to study metabolic processes by PET. We describe the synthesis and the metabolism of (Z)-7-fluoro-8-(3-oxo-2-(pent-2-en-1-yl)cyclopentyl)octanoic acid (7F-OPC-8:0), a fluorinated analog of the JA precursor OPC-8:0. Like endogenous jasmonates, 7F-OPC-8:0 induces the transcription of marker jasmonate responsive genes (JRG) and the accumulation of jasmonates after its application to Arabidopsis thaliana plants. By using UHPLC-MS/MS, we could show that 7F-OPC-8:0 is metabolized in vivo similarly to the endogenous OPC-8:0. Furthermore, the fluorinated analog was successfully employed as a probe to show its translocation to undamaged systemic leaves when it was applied to wounded leaves. This result suggests that OPC-8:0 - and maybe other oxylipins - may contribute to the mobile signal which triggers systemic defense responses in plants. We highlight the potential of fluorinated oxylipins to study the mode of action of lipid-derived molecules in planta, either by conventional analytical methods or fluorine-based detection techniques.

Additional evidence against jasmonate-induced jasmonate induction hypothesis.

Jasmonates are phytohormones involved in development and stress reactions. The most prominent jasmonate is jasmonic acid, however, the bioactive jasmonate is (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile). Biosynthesis of jasmonates is long time known; compartmentalization, enzymes and corresponding genes are well studied. Because all genes encoding these biosynthetic enzymes are jasmonate inducible, a hypothesis of jasmonate-induced-jasmonate-biosynthesis is widely accepted. Here, this hypothesis was revisited by employing the synthetic JA-Ile mimic coronalon to intact and wounded leaves, which excludes structural cross-contamination with endogenous jasmonates. At an effective concentration that induced various jasmonate-responsive genes in Arabidopsis, neither accumulation of endogenous jasmonic acid, JA-Ile, nor of their hydroxylated metabolites was detected. Results indicate that in spite of jasmonate-induced biosynthetic gene expression, no jasmonate biosynthesis/accumulation takes place supporting a post-translational regulation.