Reinventing metabolic pathways: Independent evolution of benzoxazinoids in flowering plants.

Benzoxazinoids (BXDs) form a class of indole-derived specialized plant metabolites with broad antimicrobial and antifeedant properties. Unlike most specialized metabolites, which are typically lineage-specific, BXDs occur sporadically in a number of distantly related plant orders. This observation suggests that BXD biosynthesis arose independently numerous times in the plant kingdom. However, although decades of research in the grasses have led to the elucidation of the BXD pathway in the monocots, the biosynthesis of BXDs in eudicots is unknown. Here, we used a metabolomic and transcriptomic-guided approach, in combination with pathway reconstitution in Nicotiana benthamiana, to identify and characterize the BXD biosynthetic pathways from both Aphelandra squarrosa and Lamium galeobdolon, two phylogenetically distant eudicot species. We show that BXD biosynthesis in A. squarrosa and L. galeobdolon utilize a dual-function flavin-containing monooxygenase in place of two distinct cytochrome P450s, as is the case in the grasses. In addition, we identified evolutionarily unrelated cytochrome P450s, a 2-oxoglutarate-dependent dioxygenase, a UDP-glucosyltransferase, and a methyltransferase that were also recruited into these BXD biosynthetic pathways. Our findings constitute the discovery of BXD pathways in eudicots. Moreover, the biosynthetic enzymes of these pathways clearly demonstrate that BXDs independently arose in the plant kingdom at least three times. The heterogeneous pool of identified BXD enzymes represents a remarkable example of metabolic plasticity, in which BXDs are synthesized according to a similar chemical logic, but with an entirely different set of metabolic enzymes.

Transcriptional up-regulation of host-specific terpene metabolism in aphid-induced galls of Pistacia palaestina.

Galling insects gain food and shelter by inducing specialized anatomical structures in their plant hosts. Such galls often accumulate plant defensive metabolites protecting the inhabiting insects from predation. We previously found that, despite a marked natural chemopolymorphism in natural populations of Pistacia palaestina, the monoterpene content in Baizongia pistaciae-induced galls is substantially higher than in leaves of their hosts. Here we show a general up-regulation of key structural genes in both the plastidial and cytosolic terpene biosynthetic pathways in galls as compared with non-colonized leaves. Novel prenyltransferases and terpene synthases were functionally expressed in Escherichia coli to reveal their biochemical function. Individual Pistacia trees exhibiting chemopolymorphism in terpene compositions displayed differential up-regulation of selected terpene synthase genes, and the metabolites generated by their gene products in vitro corresponded to the monoterpenes accumulated by each tree. Our results delineate molecular mechanisms responsible for the formation of enhanced monoterpene in galls and the observed intraspecific monoterpene chemodiversity displayed in P. palaestina. We demonstrate that gall-inhabiting aphids transcriptionally reprogram their host terpene pathways by up-regulating tree-specific genes, boosting the accumulation of plant defensive compounds for the protection of colonizing insects.

Identification and Characterization of Abiotic Stress Responsive CBL-CIPK Family Genes in Medicago.

The calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) play important roles in plant signal transduction and response to abiotic stress. Plants of Medicago genus contain many important forages, and their growth is often affected by a variety of abiotic stresses. However, studies on the CBL and CIPK family member and their function are rare in Medicago. In this study, a total of 23 CBL and 58 CIPK genes were identified from the genome of Medicago sativa as an important forage crop, and Medicaog truncatula as the model plant. Phylogenetic analysis suggested that these CBL and CIPK genes could be classified into five and seven groups, respectively. Moreover, these genes/proteins showed diverse exon-intron organizations, architectures of conserved protein motifs. Many stress-related cis-acting elements were found in their promoter region. In addition, transcriptional analyses showed that these CBL and CIPK genes exhibited distinct expression patterns in various tissues, and in response to drought, salt, and abscisic acid treatments. In particular, the expression levels of MtCIPK2 (MsCIPK3), MtCIPK17 (MsCIPK11), and MtCIPK18 (MsCIPK12) were significantly increased under PEG, NaCl, and ABA treatments. Collectively, our study suggested that CBL and CIPK genes play crucial roles in response to various abiotic stresses in Medicago.

Mechanism of Action of Endophytic Fungi Hypocrea lixii and Beauveria bassiana in Phaseolus vulgaris as Biopesticides against Pea Leafminer and Fall Armyworm.

Endophytic fungal isolates Hypocrea lixii F3ST1 and Beauveria bassiana G1LU3 were evaluated for their potential to endophytically colonize and induce active compounds in Phaseolus vulgaris, as a defense mechanism against pea leafminer (Liriomyza huidobrensis) and fall armyworm (Spodoptera frugiperda). Endophytic colonization was achieved through seed inoculation with the volatile emissions from P. vulgaris plants being analyzed using GC-MS. The crude extracts of P. vulgaris obtained using methanol and dichloromethane were assayed against leafminer and fall armyworm larvae using leaf dipping and topical application, respectively. The two isolates successfully colonized the entire host plant (roots, stems, and leaves) with significant variation (p < 0.001) between fungal isolates and the controls. The results showed qualitative differences in the volatile profiles between the control plants, endophytically colonized and insect-damaged plants attributed to fungal inoculation and leafminer damage. The crude methanol extracts significantly reduced the percentage pupation of 2nd instar leafminer larvae (p < 0.001) and adult-flies emergence (p < 0.05). The survival of the 1st instar fall armyworm larvae was also significantly reduced (p < 0.001) compared to the controls. This study demonstrated the high potential of endophytic fungi H. lixii and B. bassiana in inducing mainly specific defense compounds in the common bean P. vulgaris that can be used against pea leafminer and fall armyworm.

Production of Defense Phenolics in Tomato Leaves of Different Age.

Phenolics play an essential role in the defense reaction of crop plants against pathogens. However, the intensity of their production induced by infection may differ during the life of a plant. Here, we identified age-related differences in phenolic biosynthesis in the pathosystem Solanum lycopersicum cv. Amateur and Pseudomonas syringae pv. tomato DC3000. We analyzed concentrations of total phenolics, phenolic profiles, and concentrations of selected phenolic acids. The influence of bacterial infection, together with leaf and plant age, was assessed. The changes in concentrations of caffeic acid, 4-hydroxybenzoic acid, and salicylic acid glucoside caused by infection were found to be influenced by age. In concrete, the increases in the concentrations of these metabolites were all evident only in young plants.

Mistletoe Versus Host Pine: Does Increased Parasite Load Alter the Host Chemical Profile?

Stress caused by parasitic plants, e.g. mistletoes, alters certain host-plant traits as a response. While several physical implications of the parasite-host relation have been well studied, shifts in the host chemical profile remain poorly understood. Here we compare the chemical profiles of mistletoe (Viscum album subsp. austriacum) leaves and host pine (Pinus nigra subsp. salzmannii) needles and we investigate chemical changes in host needles of trees with different parasite loads (control, low, medium, and high). Our results reveal that despite the intimate contact between mistletoe and host pine, their chemical profiles differed significantly, revealing extremely low concentrations of defense compounds (including a complete lack of terpenes) and high levels of N concentrations in mistletoe leaves. On the other hand, parasitized pines showed unique chemical responses depending on parasite loads. Overall, the content in monoterpenes increased with parasitism. Higher parasitized pines produced higher amounts of defense compounds (phenols and condensed tannins) than less parasitized trees, but amounts in samples of the same year did not significantly differ between parasitized and unparasitized pines. Highly parasitized pines accumulated less N than pines with other parasite loads. The strongest response was found in sesqui- and diterpenes, which were at lower levels in pines under medium and high parasitism. Chemical responses of pines to mistletoe parasitism resembled reactions to other kinds of stress. Low levels induced reactions resembling those against drought stress, while medium and high parasitism elicited responses comparable to those against burning and defoliation.

The Application of Selenium and Copper Nanoparticles Modifies the Biochemical Responses of Tomato Plants under Stress by Alternaria solani.

Early blight is a disease that greatly affects Solanaceae, mainly damaging tomato plants, and causing significant economic losses. Although there are methods of biological control, these are very expensive and often their mode of action is slow. Due to this, there is a need to use new techniques that allow a more efficient control of pathogens. Nanotechnology is a new alternative to solve these problems, allowing the creation of new tools for the treatment of diseases in plants, as well as the control of pathogens. The aim of the present investigation was to evaluate the foliar application of selenium and copper in the form of nanoparticles in a tomato crop infested by Alternaria solani. The severity of Alternaria solani, agronomic variables of the tomato crop, and the changes in the enzymatic and non-enzymatic antioxidant compounds were evaluated. The joint application of Se and Cu nanoparticles decreases the severity of this pathogen in tomato plants. Moreover, high doses generated an induction of the activity of the enzymes superoxide dismutase, ascorbate peroxidase, glutathione peroxidase (GPX) and phenylalanine ammonia lyase in the leaves, and the enzyme GPX in the fruit. Regarding non-enzymatic compounds in the leaves, chlorophyll a, b, and totals were increased, whereas vitamin C, glutathione, phenols, and flavonoids were increased in fruits. The application of nanoparticles generated beneficial effects by increasing the enzymatic and non-enzymatic compounds and decreasing the severity of Alternaria solani in tomato plants.

A tandem array of UDP-glycosyltransferases from the UGT73C subfamily glycosylate sapogenins, forming a spectrum of mono- and bisdesmosidic saponins.

KEY MESSAGE: This study identifies six UGT73Cs all able to glucosylate sapogenins at positions 3 and/or 28 which demonstrates that B. vulgaris has a much richer arsenal of UGTs involved in saponin biosynthesis than initially anticipated. The wild cruciferous plant Barbarea vulgaris is resistant to some insects due to accumulation of two monodesmosidic triterpenoid saponins, oleanolic acid 3-O-ß-cellobioside and hederagenin 3-O-ß-cellobioside. Insect resistance depends on the structure of the sapogenin aglycone and the glycosylation pattern. The B. vulgaris saponin profile is complex with at least 49 saponin-like metabolites, derived from eight sapogenins and including up to five monosaccharide units. Two B. vulgaris UDP-glycosyltransferases, UGT73C11 and UGT73C13, O-glucosylate sapogenins at positions 3 and 28, forming mainly 3-O-ß-D-glucosides. The aim of this study was to identify UGTs responsible for the diverse saponin oligoglycoside moieties observed in B. vulgaris. Twenty UGT genes from the insect resistant genotype were selected and heterologously expressed in Nicotiana benthamiana and/or Escherichia coli. The extracts were screened for their ability to glycosylate sapogenins (oleanolic acid, hederagenin), the hormone 24-epibrassinolide and sapogenin monoglucosides (hederagenin and oleanolic acid 3-O-ß-D-glucosides). Six UGTs from the UGT73C subfamily were able to glucosylate both sapogenins and both monoglucosides at positions 3 and/or 28. Some UGTs formed bisdesmosidic saponins efficiently. At least four UGT73C genes were localized in a tandem array with UGT73C11 and possibly UGT73C13. This organization most likely reflects duplication events followed by sub- and neofunctionalization. Indeed, signs of positive selection on several amino acid sites were identified and modelled to be localized on the UGT protein surface. This tandem array is proposed to initiate higher order bisdesmosidic glycosylation of B. vulgaris saponins, leading to the recently discovered saponin structural diversity, however, not directly to known cellobiosidic saponins.

A tomatinase-like enzyme acts as a virulence factor in the wheat pathogen Fusarium graminearum.

During their interactions with plants, fungal pathogens employ large numbers of pathogenesis-associated molecules including secreted effectors and enzymes that can degrade various defence compounds. However, in many cases, in planta targets of pathogen-produced enzymes remain unknown. We identified a gene in the wheat pathogen Fusarium graminearum, encoding a putative enzyme that shows 84% sequence identity to FoTom1, a tomatinase produced by the tomato pathogen Fusarium oxysporum f. sp. lycopersici. In F. oxysporum f. sp. lycopersici, FoTom1 is a virulence factor involved in the degradation of tomato defence compound tomatine, a saponin compound. Given that wheat is unknown to produce tomatine, we tested the ability of F. graminearum to degrade tomatine and found that F. graminearum was unable to degrade tomatine in culture. However, FgTom1 degraded tomatine in vitro when heterologously expressed. To determine the possible function of FgTom1 in pathogen virulence, we generated FgTom1 knockout mutants (ΔTom1). ΔTom1 mutants were not different from wild type when grown in culture but showed significant reduction in pathogen virulence in root rot and head blight assays. In an attempt to identify possible in planta targets of FgTom1, the metabolomes of wheat heads infected with wildtype pathogen and ΔTom1 were compared and several peaks differentially abundant between treatments identified. Although the exact identity of these peaks is currently unknown, this result suggested that FgTom1 may have in planta targets in wheat, possibly tomatine-like saponin compounds. Overall, our results presented here show that FgTom1 is a new virulence factor in F. graminearum.

Impact of Argemone mexicana L. on tomato plants infected with Phytophthora infestans.

Background: Fungal diseases can cause significant losses in the tomato crop. Phytophthora infestans causes the late blight disease, which considerably affects tomato production worldwide. Weed-based plant extracts are a promising ecological alternative for disease control. Methods: In this study, we analyzed the plant extract of Argemone mexicana L. using chromatography-mass spectrometry analysis (GC-MS). We evaluated its impact on the severity of P. infestans, as well as its effect on the components of the antioxidant defense system in tomato plants. Results: The extract from A. mexicana contains twelve compounds most have antifungal and biostimulant properties. The findings of the study indicate that applying the A. mexicana extract can reduce the severity of P. infestans, increase tomato fruit yield, enhance the levels of photosynthetic pigments, ascorbic acid, phenols, and flavonoids, as well as decrease the biosynthesis of H2O2, malondialdehyde (MDA), and superoxide anion in the leaves of plants infected with this pathogen. These results suggest that using the extract from A. mexicana could be a viable solution to control the disease caused by P. infestans in tomato crop.

Unraveling the interactions of Esteya vermicola, pinewood nematode, and pine hosts: Insights into population dynamics and molecular responses.

Esteya vermicola has shown promise as an efficient biological control agent against pine wilt disease, a devastating disease in pine forests caused by the pinewood nematode (PWN, Bursaphelenchus xylophilus). However, the in vivo interactions among E. vermicola, PWN, and pine hosts are less understood, both at the population and molecular levels. In this study, we performed a series of bioassays to investigate E. vermicola colonization patterns in pine xylem and its population responses to PWN invasion in healthy and PWN-induced wilting trees. Our results demonstrated that although E. vermicola exhibits slow growth, its conidia germinate and grew along the pine tracheid, even producing lunate conidia capable of initiating PWN infections within the xylem. Interestingly, while fungal hyphae became undetectable in pine sapling xylem after inoculation, the E. vermicola population increased immediately in response to PWN invasion. Furthermore, we observed a "leap-frog" dispersal pattern of fungal colonization in PWN-induced wilting pines, facilitated by the migration of fungal-infected nematodes. Moreover, we explored the molecular mechanisms underlying fungal tolerance to pine defense systems using transcriptomic analysis. Comparative transcriptomics revealed that carbohydrate metabolism and abiotic stress-induced oxidoreductive activities are involved in the fungal tolerance to the pine defense compound ß-pinene. This study enhances our understanding of how E. vermicola colonizes and persists within pine xylem, its molecular responses to plant defense compounds, and its population dynamics upon PWN invasion, validating its efficacy as a biocontrol agent against pine wilt disease.

Sparking a sulfur war between plants and pathogens.

The biochemical versatility of sulfur (S) lends itself to myriad roles in plant-pathogen interactions. This review evaluates the current understanding of mechanisms by which pathogens acquire S from their plant hosts and highlights new evidence that plants can limit S availability during the immune responses. We discuss the discovery of host disease-susceptibility genes related to S that can be genetically manipulated to create new crop resistance. Finally, we summarize future research challenges and propose a research agenda that leverages systems biology approaches for a holistic understanding of this important element's diverse roles in plant disease resistance and susceptibility.

Current Understanding of Modes of Action of Multicomponent Bioactive Phytochemicals: Potential for Nutraceuticals and Antimicrobials.

Plants produce a diversity of plant secondary metabolites (PSMs), which function as defense chemicals against herbivores and microorganisms but also as signal compounds. An individual plant produces and accumulates mixtures of PSMs with different structural features using different biosynthetic pathways. Almost all PSMs exert one or several biological activities that can be useful for nutrition and health. This review discusses the modes of action of PSMs alone and in combinations. In a mixture, most individual PSMs can modulate different molecular targets; they are thus multitarget drugs. In an extract with many multitarget chemicals, additive and synergistic effects occur. Experiments with the model system Caenorhabditis elegans show that polyphenols and carotenoids can function as powerful antioxidative and longevity-promoting PSMs. PSMs of food plants and spices often exhibit antioxidant, anti-inflammatory, and antimicrobial properties, which can be beneficial for health and the prevention of diseases. Some extracts from food plants and spices with bioactive PSMs have potential for nutraceuticals and antimicrobials.

Comprehensive identification and characterization of abiotic stress and hormone responsive glycosyl hydrolase family 1 genes in Medicago truncatula.

ß-glucosidases (BGLUs) hydrolyze the ß-D-glycosidic bond with retention of anomeric configuration. BGLUs were associated with many aspects of plant physiological processes, in particular biotic and abiotic stresses through the activation of phytohormones and defense compounds. However, studies on systematic analysis of the stress- or hormone-responsive BGLU genes in plant are still rare. In this study, total 51 BGLU genes of the glycoside hydrolase family 1 were identified in the genome of the model legume plant Medicago truncatula, and they were classified into five distinct clusters. Sequence alignments revealed several conserved and characteristic motifs among these MtBGLU proteins. Analyses of their putative signal peptides and N-glycosylation site suggested that the majority of MtBGLU members have dual targeting to the vacuole and/or chloroplast. Many regulatory elements possibly related with abiotic stresses and phytohormones were identified in MtBGLU genes. Moreover, Microarray and qPCR analyses showed that these MtBGLU genes exhibited distinct expression patterns in various tissues, and in response to different abiotic stress and hormonal treatments. Notably, MtBGLU21, MtBGLU22, MtBGLU28, and MtBGLU30 in cluster I were dramatically activated by NaCl, PEG, IAA, ABA, SA and GA3 treatments. Collectively, our genome-wide characterization, evolutionary analysis, and expression pattern analysis of MtBGLU genes suggested that BGLU genes play crucial roles in response to various abiotic stresses and hormonal cues in M. truncatula.

Nanosilicon enhances maize resistance against oriental armyworm (Mythimna separata) by activating the biosynthesis of chemical defenses.

Silicon, in its nanoscale form, has shown plant-promoting and insecticidal properties. To date, however, we lack mechanistic evidence for how nanoscale silicon influences the regulation of plant chemical defenses against herbivore attacks. To address this gap, we compared the effect of Si nanodots (NDs) and sodium silicate, a conventional silicate fertilizer, on maize (Zea mays L.) chemical defenses against the oriental armyworm (Mythimna separata, Walker) caterpillars. We found that Si NDs and sodium silicate additions, at the dose of 50 mg/L, significantly inhibited the growth of caterpillars by 53.5% and 34.2%, respectively. This increased plant resistance was associated with a 44.2% increase in the production of chlorogenic acid, as well as the expression of PAL, C4H, 4CL, C3H and HCT, core genes involved in the biosynthesis of chlorogenic acid, by 1.7, 2.4, 1.9, 1.8 and 4.5 folds, respectively. Particularly, in the presence of M. separata, physiological changes in maize plants treated with 50 mg/L Si NDs, including changes in shoot biomass, leaf nutrients (e.g., K, P, Si), and chemical defense compounds (e.g., chlorogenic acid, total phenolics), were higher than those of plants added with equivalent concentrations of conventional silicate fertilizer. Taken together, our findings indicate that Si, in nanoscale form, could replace synthetic pesticides, and be implemented for a more effective and ecologically-sound management of insect pests in maize crop farming.

The Versatile Roles of Sulfur-Containing Biomolecules in Plant Defense-A Road to Disease Resistance.

Sulfur (S) is an essential plant macronutrient and the pivotal role of sulfur compounds in plant disease resistance has become obvious in recent decades. This review attempts to recapitulate results on the various functions of sulfur-containing defense compounds (SDCs) in plant defense responses to pathogens. These compounds include sulfur containing amino acids such as cysteine and methionine, the tripeptide glutathione, thionins and defensins, glucosinolates and phytoalexins and, last but not least, reactive sulfur species and hydrogen sulfide. SDCs play versatile roles both in pathogen perception and initiating signal transduction pathways that are interconnected with various defense processes regulated by plant hormones (salicylic acid, jasmonic acid and ethylene) and reactive oxygen species (ROS). Importantly, ROS-mediated reversible oxidation of cysteine residues on plant proteins have profound effects on protein functions like signal transduction of plant defense responses during pathogen infections. Indeed, the multifaceted plant defense responses initiated by SDCs should provide novel tools for plant breeding to endow crops with efficient defense responses to invading pathogens.

Transcriptional regulation of de novo biosynthesis of cyanogenic glucosides throughout the life-cycle of the burnet moth Zygaena filipendulae (Lepidoptera).

The six-spotted burnet moth Zygaena filipendulae (Lepidoptera) utilize the two cyanogenic glucosides (CNglcs) linamarin and lotaustralin as deterrents against predators throughout the entire life cycle. CNglcs can be hydrolyzed and bioactivated by ß-glucosidases, resulting in the release of toxic hydrogen cyanide. CNglcs are retained through metamorphosis, probably involved in mating communication, and transferred during mating from the male to the female as a nuptial gift. CNglcs can be biosynthesized de novo by Z. filipendulae larvae, but may also be sequestered from their food plant Lotus corniculatus (Fabaceae). These two strategies are tightly linked and adjusted according to the CNglc content and composition of the food plant in order to balance CNglc homeostasis in the larva. In this study, the amounts of CNglcs and transcript levels of the biosynthetic genes were monitored in all life-stages and tissues of Z. filipendulae. During pupation, transcription of the biosynthetic genes is turned off and the CNglc content slowly declines. In females but not males, transcription of the biosynthetic genes is re-activated at the end of pupation. Eggs and embryos do not biosynthesize CNglcs de novo, but are endowed with CNglcs following eclosion of the female. Similarly to larvae, de novo biosynthesis in female adults takes place in the integument from which CNglcs are then transported to other organs. This study demonstrates that Z. filipendulae has evolved the ability to adjust the production of CNglcs throughout its life-cycle for optimal utilization in defense and possibly other metabolic functions, while at the same time avoiding intoxication.

Sesquiterpenoid glycosides from glandular trichomes of the wild tomato relative Solanum habrochaites.

Profiles of terpenoid glycoside metabolites in glandular trichomes of Solanum habrochaites LA1777 leaves were generated using ultrahigh performance liquid chromatography/time-of-flight mass spectrometry with multiplexing of non-selective collision-induced dissociation (CID). Profiling data suggested a diverse group of 52 sesquiterpenoid glycosides, and fragment ions observed in both non-selective CID mass spectra and true tandem mass spectrometry (MS/MS) product ion spectra documented variation in extent of glycosylation and the presence of malonate or acetate esters. Up to 10 isomers were detected for some metabolites. Malonate and acetate esters of three sesquiterpene diol glucosides and one unmodified diglucoside were purified using reversed phase semipreparative HPLC and analyzed and identified using 1D and 2D NMR and mass spectrometry. All four of the isolated products were glucosides of campheranane-2,12-diol.