Tomato pomace food waste from different variants as a high antioxidant potential resource.

Pomaces obtained from three San Marzano tomato genotypes including the wild type (WT), Sun Black (SB), and colorless fruit epidermis (CL) were dried at 50 °C and analyzed for nutritional composition, total polyphenol (TPC), flavonoid (TFC) content, polyphenol qualitative profile, total antioxidant capacity (TAC), and antimicrobial activity. Commercial dried tomato powder (CTRP) was included as a control. No differences were detected nutritionally, in TPC and antimicrobial activity, but significant changes were observed for TFC and TAC, underlying variation in the phenolic profile. SB pomace (SBP) had the highest TFC and TAC. LC-HRMS analysis showed a flavonoid-enriched profile in SBP besides the exclusive presence of anthocyanins, with petanin and negretein as the most abundant. Among flavonoids, quercetin-hexose-deoxyhexose-pentose, naringenin, and rutin were the major. Overall, we showed the potential of dried tomato pomace, especially SBP, as an extremely valuable waste product to be transformed into a functional ingredient, reducing the food industry waste.

Oligogalacturonide application increases resistance to Fusarium head blight in durum wheat.

Fusariosis causes substantial yield losses in the wheat crop worldwide and compromises food safety because of the presence of toxins associated with the fungal disease. Among the current approaches to crop protection, the use of elicitors able to activate natural defense mechanisms in plants is a strategy gaining increasing attention. Several studies indicate that applications of plant cell-wall-derived elicitors, such as oligogalacturonides (OGs) derived from partial degradation of pectin, induce local and systemic resistance against plant pathogens. The aim of this study was to establish the efficacy of OGs in protecting durum wheat (Triticum turgidum subsp. durum), which is characterized by an extreme susceptibility to Fusarium graminearum. To evaluate the functionality of OGs, spikes and seedlings of cv. Svevo were inoculated with OGs, F. graminearum spores, and a co-treatment of both. Results demonstrated that OGs are active elicitors of wheat defenses, triggering typical immune marker genes and determining regulation of fungal genes. Moreover, bioassays on spikes and transcriptomic analyses on seedlings showed that OGs can regulate relevant physiological processes in Svevo with dose-dependent specificity. Thus, the OG sensing system plays an important role in fine tuning immune signaling pathways in durum wheat.

ROS and RNS production, subcellular localization and signaling triggered by immunogenic danger signals.

Plants continuously monitor the environment to detect changing conditions and to properly respond, avoiding deleterious effects on their fitness and survival. An enormous number of cell-surface and intracellular immune receptors are deployed to perceive danger signals associated with microbial infections. Ligand binding by cognate receptors represents the first essential event in triggering plant immunity and determining the tissue invasion attempt outcome. Reactive oxygen and nitrogen species (ROS/RNS) are secondary messengers rapidly produced in different subcellular localizations upon the perception of immunogenic signals. Danger signal transduction inside the plant cells involves cytoskeletal rearrangements as well as several organelles and interactions between them to activate key immune signaling modules. Such immune processes depend on ROS and RNS accumulation, highlighting their role as key regulators in the execution of the immune cellular programme. In fact, ROS and RNS are synergic and inter-dependent intracellular signals required for decoding danger signals and for the modulation of defense-related responses. Here we summarize the current knowledge on ROS/RNS production, compartmentalization and signaling in plant cells that have perceived immunogenic danger signals.

Differential modulation of Target of Rapamycin activity under single and combined iron and sulfur deficiency in tomato plants.

Over the past few decades, a close relationship between sulfur (S) and iron (Fe) in terms of functionality and nutrition was demonstrated in the tomato. However, very little is known about the regulatory mechanisms underlying S/Fe interactions. Recently, the potential role of citrate in plant adaptation to Fe deficiency and combined S and Fe deficiency has been described. It is known that an impaired organic acid metabolism may stimulate a retrograde signal, which has been proven to be linked to the Target of Rapamycin (TOR) signaling in yeast and animal cells. Recent reports provided evidence of TOR involvement in S nutrient sensing in plants. This suggestion prompted us to investigate whether TOR may play a role in the cross-talk of signaling pathway occurring during plant adaptation to combined nutrient deficiency of Fe and S. Our results revealed that Fe deficiency elicited an increase of TOR activity associated with enhanced accumulation of citrate. In contrast, S deficiency resulted in decreased TOR activity and citrate accumulation. Interestingly, citrate accumulated in shoots of plants exposed to combined S/Fe deficiency to values between those found in Fe- and S-deficient plants, again correlated with TOR activity level. Our results suggest that citrate might be involved in establishing a link between plant response to combined S/Fe deficiency and the TOR network.

Biodiversity and Bioprospecting of Fungal Endophytes from the Antarctic Plant Colobanthus quitensis.

Microorganisms from extreme environments are considered as a new and valuable reservoir of bioactive molecules of biotechnological interest and are also utilized as tools for enhancing tolerance to (a)biotic stresses in crops. In this study, the fungal endophytic community associated with the leaves of the Antarctic angiosperm Colobanthus quitensis was investigated as a new source of bioactive molecules. We isolated 132 fungal strains and taxonomically annotated 26 representative isolates, which mainly belonged to the Basidiomycota division. Selected isolates of Trametes sp., Lenzites sp., Sistotrema sp., and Peniophora sp. displayed broad extracellular enzymatic profiles; fungal extracts from some of them showed dose-dependent antitumor activity and inhibited the formation of amyloid fibrils of α-synuclein and its pathological mutant E46K. Selected fungal isolates were also able to promote secondary root development and fresh weight increase in Arabidopsis and tomato and antagonize the growth of pathogenic fungi harmful to crops. This study emphasizes the ecological and biotechnological relevance of fungi from the Antarctic ecosystem and provides clues to the bioprospecting of Antarctic Basidiomycetes fungi for industrial, agricultural, and medical applications.

The Triple Jags of Dietary Fibers in Cereals: How Biotechnology Is Longing for High Fiber Grains.

Cereals represent an important source of beneficial compounds for human health, such as macro- and micronutrients, vitamins, and bioactive molecules. Generally, the consumption of whole-grain products is associated with significant health benefits, due to the elevated amount of dietary fiber (DF). However, the consumption of whole-grain foods is still modest compared to more refined products. In this sense, it is worth focusing on the increase of DF fractions inside the inner compartment of the seed, the endosperm, which represents the main part of the derived flour. The main components of the grain fiber are arabinoxylan (AX), ß-glucan (ßG), and resistant starch (RS). These three components are differently distributed in grains, however, all of them are represented in the endosperm. AX and ßG, classified as non-starch polysaccharides (NSP), are in cell walls, whereas, RS is in the endosperm, being a starch fraction. As the chemical structure of DFs influences their digestibility, the identification of key actors involved in their metabolism can pave the way to improve their function in human health. Here, we reviewed the main achievements of plant biotechnologies in DFs manipulation in cereals, highlighting new genetic targets to be exploited, and main issues to face to increase the potential of cereals in fighting malnutrition.

The Fusarium graminearum FGSG_03624 Xylanase Enhances Plant Immunity and Increases Resistance against Bacterial and Fungal Pathogens.

Fungal enzymes degrading the plant cell wall, such as xylanases, can activate plant immune responses. The Fusarium graminearum FGSG_03624 xylanase, previously shown to elicit necrosis and hydrogen peroxide accumulation in wheat, was investigated for its ability to induce disease resistance. To this aim, we transiently and constitutively expressed an enzymatically inactive form of FGSG_03624 in tobacco and Arabidopsis, respectively. The plants were challenged with Pseudomonas syringae pv. tabaci or pv. maculicola and Botrytis cinerea. Symptom reduction by the bacterium was evident, while no reduction was observed after B. cinerea inoculation. Compared to the control, the presence of the xylanase gene in transgenic Arabidopsis plants did not alter the basal expression of a set of defense-related genes, and, after the P. syringae inoculation, a prolonged PR1 expression was detected. F. graminearum inoculation experiments of durum wheat spikes exogenously treated with the FGSG_03624 xylanase highlighted a reduction of symptoms in the early phases of infection and a lower fungal biomass accumulation than in the control. Besides, callose deposition was detected in infected spikes previously treated with the xylanase and not in infected control plants. In conclusion, our results highlight the ability of FGSG_03624 to enhance plant immunity, thus decreasing disease severity.

Biotechnological Resources to Increase Disease-Resistance by Improving Plant Immunity: A Sustainable Approach to Save Cereal Crop Production.

Plant diseases are globally causing substantial losses in staple crop production, undermining the urgent goal of a 60% increase needed to meet the food demand, a task made more challenging by the climate changes. Main consequences concern the reduction of food amount and quality. Crop diseases also compromise food safety due to the presence of pesticides and/or toxins. Nowadays, biotechnology represents our best resource both for protecting crop yield and for a science-based increased sustainability in agriculture. Over the last decades, agricultural biotechnologies have made important progress based on the diffusion of new, fast and efficient technologies, offering a broad spectrum of options for understanding plant molecular mechanisms and breeding. This knowledge is accelerating the identification of key resistance traits to be rapidly and efficiently transferred and applied in crop breeding programs. This review gathers examples of how disease resistance may be implemented in cereals by exploiting a combination of basic research derived knowledge with fast and precise genetic engineering techniques. Priming and/or boosting the immune system in crops represent a sustainable, rapid and effective way to save part of the global harvest currently lost to diseases and to prevent food contamination.

The intracellular ROS accumulation in elicitor-induced immunity requires the multiple organelle-targeted Arabidopsis NPK1-related protein kinases.

Recognition at the plasma membrane of danger signals (elicitors) belonging to the classes of the microbe/pathogen- and damage-associated molecular patterns is a key event in pathogen sensing by plants and is associated with a rapid activation of immune responses. Different cellular compartments, including plasma membrane, chloroplasts, nuclei and mitochondria, are involved in the immune cellular program. However, how pathogen sensing is transmitted throughout the cell remains largely to be uncovered. Arabidopsis NPK1-related Proteins (ANPs) are mitogen-activated protein kinase kinase kinases previously shown to have a role in immunity. In this article, we studied the in vivo intracellular dynamics of ANP1- and ANP3-GFP fusions and found that under basal physiological conditions both proteins are present in the cytosol, while ANP3 is also localized in mitochondria. After elicitor perception, both proteins are present also in the plastids and nuclei, revealing a localization pattern that is so far unique. The N-terminal region of the protein kinases is responsible for their localization in mitochondria and plastids. Moreover, we found that the localization of ANPs coincides with the sites of elicitor-induced ROS accumulation and that plants lacking ANP function do not accumulate intracellular ROS.

Ligand-induced monoubiquitination of BIK1 regulates plant immunity.

Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens1-3. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants4. The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.

Provitamin A Biofortification of Durum Wheat through a TILLING Approach.

Macro- and micronutrients, essential for the maintenance of human metabolism, are assimilated daily through the diet. Wheat and other major cereals are a good source of nutrients, such as carbohydrates and proteins, but cannot supply a sufficient amount of essential micronutrients, including provitamin A. As vitamin A deficiency (VAD) leads to several serious diseases throughout the world, the biofortification of a major staple crop, such as wheat, represents an effective way to preserve human health in developing countries. In the present work, a key enzyme involved in the branch of carotenoids pathway producing ß-carotene, lycopene epsilon cyclase, has been targeted by a Targeting Induced Local Lesions in Genomes (TILLING) approach in a "block strategy" perspective. The null mutant genotype showed a strong reduction in the expression of the lcyE gene and also interesting pleiotropic effects on an enzyme (ß-ring hydroxylase) acting downstream in the pathway. Biochemical profiling of carotenoids in the wheat mutant lines showed an increase of roughly 75% in ß-carotene in the grains of the complete mutant line compared with the control. In conclusion, we describe here the production and characterization of a new wheat line biofortified with provitamin A obtained through a nontransgenic approach, which also sheds new light on the molecular mechanism governing carotenoid biosynthesis in durum wheat.

Disruption of endocytosis through chemical inhibition of clathrin heavy chain function.

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.

Proteolytic Processing of SERK3/BAK1 Regulates Plant Immunity, Development, and Cell Death.

Plants have evolved many receptor-like kinases (RLKs) to sense extrinsic and intrinsic cues. The signaling pathways mediated by multiple Leucine-rich repeat (LRR) RLK (LRR-RLK) receptors require ligand-induced receptor-coreceptor heterodimerization and transphosphorylation with BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1)/SOMATIC EMBRYOGENESIS RECEPTOR KINASES family LRR-RLKs. Here we reveal an additional layer of regulation of BAK1 via a Ca2+-dependent proteolytic cleavage process that is conserved in Arabidopsis (Arabidopsis thaliana), Nicotiana benthamiana, and Saccharomyces cerevisiae The proteolytic cleavage of BAK1 is intrinsically regulated in response to developmental cues and immune stimulation. The surface-exposed Asp (D287) residue of BAK1 is critical for its proteolytic cleavage and plays an essential role in BAK1-regulated plant immunity, growth hormone brassinosteroid-mediated responses, and cell death containment. BAK1D287A mutation impairs BAK1 phosphorylation on its substrate BOTRYTIS-INDUCED KINASE1 (BIK1), and its plasma membrane localization. Intriguingly, it aggravates BAK1 overexpression-triggered cell death independent of BIK1, suggesting that maintaining homeostasis of BAK1 through a proteolytic process is crucial to control plant growth and immunity. Our data reveal that in addition to layered transphosphorylation in the receptor complexes, the proteolytic cleavage is an important regulatory process for the proper functions of the shared coreceptor BAK1 in diverse cellular signaling pathways.

POLAR-guided signalling complex assembly and localization drive asymmetric cell division.

Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD), which is necessary for plant survival1-4. In Arabidopsis thaliana, the GLYCOGEN SYNTHASE KINASE3 (GSK3)/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) phosphorylates both the mitogen-activated protein kinase (MAPK) signalling module5,6 and its downstream target, the transcription factor SPEECHLESS (SPCH)7, to promote and restrict ACDs, respectively, in the same stomatal lineage cell. However, the mechanisms that balance these mutually exclusive activities remain unclear. Here we identify the plant-specific protein POLAR as a stomatal lineage scaffold for a subset of GSK3-like kinases that confines them to the cytosol and subsequently transiently polarizes them within the cell, together with BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), before ACD. As a result, MAPK signalling is attenuated, enabling SPCH to drive ACD in the nucleus. Moreover, POLAR turnover requires phosphorylation on specific residues, mediated by GSK3. Our study reveals a mechanism by which the scaffolding protein POLAR ensures GSK3 substrate specificity, and could serve as a paradigm for understanding regulation of GSK3 in plants.

Corrigendum: Loss of the Arabidopsis Protein Kinases ANPs Affects Root Cell Wall Composition, and Triggers the Cell Wall Damage Syndrome.

[This corrects the article DOI: 10.3389/fpls.2017.02234.].

The crossroads of receptor-mediated signaling and endocytosis in plants.

Plants deploy numerous plasma membrane receptors to sense and rapidly react to environmental changes. Correct localization and adequate protein levels of the cell-surface receptors are critical for signaling activation and modulation of plant development and defense against pathogens. After ligand binding, receptors are internalized for degradation and signaling attenuation. However, one emerging notion is that the ligand-induced endocytosis of receptor complexes is important for the signal duration, amplitude, and specificity. Recently, mutants of major endocytosis players, including clathrin and dynamin, have been shown to display defects in activation of a subset of signal transduction pathways, implying that signaling in plants might not be solely restricted to the plasma membrane. Here, we summarize the up-to-date knowledge of receptor complex endocytosis and its effect on the signaling outcome, in the context of plant development and immunity.

Methods of Isolation and Characterization of Oligogalacturonide Elicitors.

Oligogalacturonides (OGs) are pectic fragments derived from the partial degradation of homogalacturonan in the plant cell wall and able to elicit plant defence responses. Recent methodological advances in the isolation of OGs from plant tissues and their characterization have confirmed their role as bona fide plant Damage-Associated Molecular Patterns. Here, we describe the methods for the isolation of OGs from Arabidopsis leaf tissues and for the characterization of OG structure and biological activity.

Immune responses induced by oligogalacturonides are differentially affected by AvrPto and loss of BAK1/BKK1 and PEPR1/PEPR2.

Plants possess an innate immune system capable of restricting invasion by most potential pathogens. At the cell surface, the recognition of microbe-associated molecular patterns (MAMPs) and/or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) represents the first event for the prompt mounting of an effective immune response. Pathogens have evolved effectors that block MAMP-triggered immunity. The Pseudomonas syringae effector AvrPto abolishes immunity triggered by the peptide MAMPs flg22 and elf18, derived from the bacterial flagellin and elongation factor Tu, respectively, by inhibiting the kinase function of the corresponding receptors FLS2 and EFR, as well as their co-receptors BAK1 and BKK1. Oligogalacturonides (OGs), a well-known class of DAMPs, are oligomers of α-1,4-linked galacturonosyl residues, released on partial degradation of the plant cell wall homogalacturonan. We show here that AvrPto affects only a subset of the OG-triggered immune responses and that, among these responses, only a subset is affected by the concomitant loss of BAK1 and BKK1. However, the antagonistic effect on auxin-related responses is not affected by either AvrPto or the loss of BAK1/BKK1. These observations reveal an unprecedented complexity among the MAMP/DAMP response cascades. We also show that the signalling system mediated by Peps, another class of DAMPs, and their receptors PEPRs, contributes to OG-activated immunity. We hypothesize that OGs are sensed through multiple and partially redundant perception/transduction complexes, some targeted by AvrPto, but not necessarily comprising BAK1 and BKK1.

Loss of the Arabidopsis Protein Kinases ANPs Affects Root Cell Wall Composition, and Triggers the Cell Wall Damage Syndrome.

The Arabidopsis NPK1-related Protein kinases ANP1, ANP2 and ANP3 belong to the MAP kinase kinase kinase (MAPKKK) superfamily and were previously described to be crucial for cytokinesis, elicitor-induced immunity and development. Here we investigate the basis of their role in development by using conditional ß-estradiol-inducible triple mutants to overcome lethality. In seedlings, lack of ANPs causes root cell bulging, with the transition zone being the most sensitive region. We uncover a role of ANPs in the regulation of cell wall composition and suggest that developmental defects of the triple mutants, observed at the cellular level, might be a consequence of the alterations of the pectic and cellulosic cell wall components. Lack of ANPs also induced a typical cell wall damage syndrome (CWDS) similar to that observed in plants treated with the cellulose biosynthesis inhibitor isoxaben (ISX). Moreover, anp double mutants and plants overexpressing single ANPs (ANP1 or ANP3) respectively showed increased and reduced accumulation of jasmonic acid and PDF1.2 transcripts upon ISX treatment, suggesting that ANPs are part of the pathway targeted by this inhibitor and play a role in cell wall integrity surveillance. Highlights: The loss of ANP function affects cell wall composition and leads to typical cell wall damage-induced phenotypes, such as ectopic lignification and jasmonic acid accumulation.

Danger-associated peptide signaling in Arabidopsis requires clathrin.

The Arabidopsis thaliana endogenous elicitor peptides (AtPeps) are released into the apoplast after cellular damage caused by pathogens or wounding to induce innate immunity by direct binding to the membrane-localized leucine-rich repeat receptor kinases, PEP RECEPTOR1 (PEPR1) and PEPR2. Although the PEPR-mediated signaling components and responses have been studied extensively, the contributions of the subcellular localization and dynamics of the active PEPRs remain largely unknown. We used live-cell imaging of the fluorescently labeled and bioactive pep1 to visualize the intracellular behavior of the PEPRs in the Arabidopsis root meristem. We found that AtPep1 decorated the plasma membrane (PM) in a receptor-dependent manner and cointernalized with PEPRs. Trafficking of the AtPep1-PEPR1 complexes to the vacuole required neither the trans-Golgi network/early endosome (TGN/EE)-localized vacuolar H(+)-ATPase activity nor the function of the brefeldin A-sensitive ADP-ribosylation factor-guanine exchange factors (ARF-GEFs). In addition, AtPep1 and different TGN/EE markers colocalized only rarely, implying that the intracellular route of this receptor-ligand pair is largely independent of the TGN/EE. Inducible overexpression of the Arabidopsis clathrin coat disassembly factor, Auxilin2, which inhibits clathrin-mediated endocytosis (CME), impaired the AtPep1-PEPR1 internalization and compromised AtPep1-mediated responses. Our results show that clathrin function at the PM is required to induce plant defense responses, likely through CME of cell surface-located signaling components.