ScBx gene based association analysis of hydroxamate content in rye (Secale cereale L.).
ABSTRACT: Hydroxamates (HX) are major secondary metabolites synthesized by rye and are responsible for some of the unique properties of this cereal, including good tolerance of biotic and abiotic stresses and allelopathy. Recently, five genes encoding enzymes taking part in HX biosynthesis have been sequenced and characterized, which was the starting point to undertake the present study. Association analysis of the content of six HX-HBOA (2-hydroxy-1,4-benzoxazin-3-one), GDIBOA (2,4-dihydroxy-1,4-benzoxazin-3(4H)-one glucoside), DIBOA (2,4-dihydroxy-1,4-benzoxazin-3(4H)-one), GDIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3(4H)-one glucoside), DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3(4H)-one) and MBOA (6-methoxy-benzoxazolin-2(3H)-one) in the above-ground parts of plants and roots was performed on a population consisting of 102 and 121 diverse inbred lines, in 2013 and 2014, respectively. Altogether, 48 single nucleotide polymorphisms (SNPs) were found to be associated with the content of at least one HX: 20 SNPs were associated with HX synthesized in the above-ground parts of rye plants (AG-SNP), and 28 were associated with HX synthesized in the roots (R-SNP). The highest number of SNPs was present in genes ScBx1 (9) and ScBx5 (11). The majority of SNPs were affected by environmental factors, except for two: ScBx4_1702 associated with GDIBOA and MBOA contents, and ScBx5_1105 associated with HBOA content in roots.
Project description:The preformed antimicrobial compounds produced by maize, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one and its desmethoxy derivative 2,4-dihydroxy-2H-1,4-benzoxazin-3-one, are highly reactive benzoxazinoids that quickly degrade to the antimicrobials 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA), respectively. Fusarium verticillioides (= F. moniliforme) is highly tolerant to MBOA and BOA and can actively transform these compounds to nontoxic metabolites. Eleven of 29 Fusarium species had some level of tolerance to MBOA and BOA; the most tolerant, in decreasing order, were F. verticillioides, F. subglutinans, F. cerealis (= F. crookwellense), and F. graminearum. The difference in tolerance among species was due to their ability to detoxify the antimicrobials. The limited number of species having tolerance suggested the potential utility of these compounds as biologically active agents for inclusion within a semiselective isolation medium. By replacing the pentachloronitrobenzene in Nash-Snyder medium with 1.0 mg of BOA per ml, we developed a medium that resulted in superior frequencies of isolation of F. verticillioides from corn while effectively suppressing competing fungi. Since the BOA medium provided consistent, quantitative results with reduced in vitro and taxonomic efforts, it should prove useful for surveys of F. verticillioides infection in field samples.
Project description:BACKGROUND:Intercropping systems could increase crop diversity and avoid vulnerability to biotic stresses. Most studies have shown that intercropping can provide relief to crops against wind-dispersed pathogens. However, there was limited data on how the practice of intercropping help crops against soil-borne Phytophthora disease. PRINCIPAL FINDINGS:Compared to pepper monoculture, a large scale intercropping study of maize grown between pepper rows reduced disease levels of the soil-borne pepper Phytophthora blight. These reduced disease levels of Phytophthora in the intercropping system were correlated with the ability of maize plants to form a "root wall" that restricted the movement of Phytophthora capsici across rows. Experimentally, it was found that maize roots attracted the zoospores of P. capsici and then inhibited their growth. When maize plants were grown in close proximity to each other, the roots produced and secreted larger quantities of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and 6-methoxy-2-benzoxazolinone (MBOA). Furthermore, MBOA, benzothiazole (BZO), and 2-(methylthio)-benzothiazole (MBZO) were identified in root exudates of maize and showed antimicrobial activity against P. capsici. CONCLUSIONS:Maize could form a "root wall" to restrict the spread of P. capsici across rows in maize and pepper intercropping systems. Antimicrobe compounds secreted by maize root were one of the factors that resulted in the inhibition of P. capsici. These results provide new insights into plant-plant-microbe mechanisms involved in intercropping systems.
Project description:Glycoside hydrolases catalyze the selective hydrolysis of glycosidic bonds in oligosaccharides, polysaccharides, and their conjugates. ?-glucosidases occur in all domains of living organisms and constitute a major group among glycoside hydrolases. On the other hand, the benzoxazinoids occur in living systems and act as stable ?-glucosides, such as 2-(2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one)-?-D-gluco-pyranose, which hydrolyse to an aglycone DIMBOA. Here, we synthesized the library of novel 1,3-benzoxazine scaffold based aglycones by using 2-aminobenzyl alcohols and aldehydes from one-pot reaction in a chloroacetic acid catalytic system via aerobic oxidative synthesis. Among the synthesized benzoxazines, 4-(7-chloro-2,4-dihydro-1H-benzo[d][1,3]oxazin-2-yl)phenol (compound 7) exhibit significant inhibition towards glucosidase compared to acarbose, with a IC50 value of 11.5 µM. Based upon results generated by in silico target prediction algorithms (Naïve Bayesian classifier), these aglycones potentially target the additional sodium/glucose cotransporter 1 (where a log likelihood score of 2.70 was observed). Furthermore, the in vitro glucosidase activity was correlated with the in silico docking results, with a high docking score for the aglycones towards the substrate binding site of glycosidase. Evidently, the in vitro and in vivo experiments clearly suggest an anti-hyperglycemic effect via glucose uptake inhibition by 4-(7-chloro-2,4-dihydro-1H-benzo[d][1,3]oxazin-2-yl)phenol in the starved rat model. These synthetic aglycones could constitute a novel pharmacological approach for the treatment, or re-enforcement of existing treatments, of type 2 diabetes and associated secondary complications.
Project description:Benzoxazinoids, such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), are secondary metabolites in grasses. In addition to their function in plant defence against pests and diseases above-ground, benzoxazinoids (BXs) have also been implicated in defence below-ground, where they can exert allelochemical or antimicrobial activities. We have studied the impact of BXs on the interaction between maize and Pseudomonas putida KT2440, a competitive coloniser of the maize rhizosphere with plant-beneficial traits. Chromatographic analyses revealed that DIMBOA is the main BX compound in root exudates of maize. In vitro analysis of DIMBOA stability indicated that KT2440 tolerance of DIMBOA is based on metabolism-dependent breakdown of this BX compound. Transcriptome analysis of DIMBOA-exposed P. putida identified increased transcription of genes controlling benzoate catabolism and chemotaxis. Chemotaxis assays confirmed motility of P. putida towards DIMBOA. Moreover, colonisation essays in soil with Green Fluorescent Protein (GFP)-expressing P. putida showed that DIMBOA-producing roots of wild-type maize attract significantly higher numbers of P. putida cells than roots of the DIMBOA-deficient bx1 mutant. Our results demonstrate a central role for DIMBOA as a below-ground semiochemical for recruitment of plant-beneficial rhizobacteria during the relatively young and vulnerable growth stages of maize.
Project description:BACKGROUND:The benzoxazinoids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA), are key defense compounds present in major agricultural crops such as maize and wheat. Their biosynthesis involves nine enzymes thought to form a linear pathway leading to the storage of DI(M)BOA as glucoside conjugates. Seven of the genes (Bx1-Bx6 and Bx8) form a cluster at the tip of the short arm of maize chromosome 4 that includes four P450 genes (Bx2-5) belonging to the same CYP71C subfamily. The origin of this cluster is unknown. RESULTS:We show that the pathway appeared following several duplications of the TSA gene (?-subunit of tryptophan synthase) and of a Bx2-like ancestral CYP71C gene and the recruitment of Bx8 before the radiation of Poaceae. The origins of Bx6 and Bx7 remain unclear. We demonstrate that the Bx2-like CYP71C ancestor was not committed to the benzoxazinoid pathway and that after duplications the Bx2-Bx5 genes were under positive selection on a few sites and underwent functional divergence, leading to the current specific biochemical properties of the enzymes. The absence of synteny between available Poaceae genomes involving the Bx gene regions is in contrast with the conserved synteny in the TSA gene region. CONCLUSIONS:These results demonstrate that rearrangements following duplications of an IGL/TSA gene and of a CYP71C gene probably resulted in the clustering of the new copies (Bx1 and Bx2) at the tip of a chromosome in an ancestor of grasses. Clustering favored cosegregation and tip chromosomal location favored gene rearrangements that allowed the further recruitment of genes to the pathway. These events, a founding event and elongation events, may have been the key to the subsequent evolution of the benzoxazinoid biosynthetic cluster.
Project description:Barnyardgrass (Echinochloa crus-galli) is a pernicious weed in agricultural fields worldwide. The molecular mechanisms underlying its success in the absence of human intervention are presently unknown. Here we report a draft genome sequence of the hexaploid species E. crus-galli, i.e., a 1.27 Gb assembly representing 90.7% of the predicted genome size. An extremely large repertoire of genes encoding cytochrome P450 monooxygenases and glutathione S-transferases associated with detoxification are found. Two gene clusters involved in the biosynthesis of an allelochemical 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and a phytoalexin momilactone A are found in the E. crus-galli genome, respectively. The allelochemical DIMBOA gene cluster is activated in response to co-cultivation with rice, while the phytoalexin momilactone A gene cluster specifically to infection by pathogenic Pyricularia oryzae. Our results provide a new understanding of the molecular mechanisms underlying the extreme adaptation of the weed.
Project description:Aphids are major pests in cereal crops that cause direct and indirect damage leading to yield reduction. Despite the fact that wheat provides 20% of the world's caloric and protein diet, its metabolic responses to aphid attack, in general, and specifically its production of benzoxazinoid defense compounds are poorly understood. The objective of this study was to compare the metabolic diversity of durum wheat seedlings (Triticum turgidum ssp. durum) under attack by three different cereal aphids: i) the English grain aphid (Sitobion avenae Fabricius), ii) the bird cherry-oat aphid (Rhopalosiphum padi L.), and iii) the greenbug aphid (Schizaphis graminum Rondani), which are some of the most destructive aphid species to wheat. Insect progeny bioassays and metabolic analyses using chromatography/Q-Exactive/mass spectrometry non-targeted metabolomics and a targeted benzoxazinoid profile were performed on infested leaves. The insect bioassays revealed that the plants were susceptible to S. graminum, resistant to S. avenae, and mildly resistant to R. padi. The metabolic analyses of benzoxazinoids suggested that the predominant metabolites DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin- 3-one) and its glycosylated form DIMBOA-glucoside (Glc) were significantly induced upon both S. avenae, and R. padi aphid feeding. However, the levels of the benzoxazinoid metabolite HDMBOA-Glc (2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside) were enhanced due to the feeding of S. avenae and S. graminum aphids, to which Svevo was the most resistant and the most susceptible, respectively. The results showed a partial correlation between the induction of benzoxazinoids and aphid reproduction. Overall, our observations revealed diverse metabolic responses of wheat seedlings to cereal aphid feeding.
Project description:Present study was undertaken to evaluate the analgesic activity of the ethanol extract of Chrysopogon aciculatus. In addition to bioassays in mice, chemical profiling was done by LC-MS and GC-MS to identify phytochemicals, which were further docked on the catalytic site of COX-2 enzymes with a view to suggest the possible role of such phytoconstituents in the observed analgesic activity. Analgesic activity of C. aciculatus was evaluated by acetic acid induced writhing reflex method and hot plate technique. Phytochemical profiling was conducted using liquid chromatography mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS). In docking studies, homology model of human COX-2 enzyme was prepared using Easy Modeler 4.0 and the identified phytoconstituents were docked using Autodock Vina. Preliminary acute toxicity test of the ethanol extract of C. aciculatus showed no sign of mortality at the highest dose of 4,000 mg/kg. The whole plant extract significantly (p < 0.05) inhibited acetic acid induced writhing in mice at the doses of 500 and 750 mg/kg. The extract delayed the response time in hot plate test in a dose dependent manner. LC-MS analysis of the plant extract revealed the presence of aciculatin, nudaphantin and 5?,8?-epidioxyergosta-6,22-diene-3?-ol. Three compounds namely citronellylisobutyrate; 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one and nudaphantin were identified in the n-hexane fraction by GC-MS. Among these compounds, six were found to be interacting with the binding site for arachidonic acid in COX-2 enzyme. Present study strongly supports the traditional use of C. aciculatus in the management of pain. In conclusion, compounds (tricin, campesterol, gamma oryzanol, and citronellyl isobutyrate) showing promising binding affinity in docking studies, along with previously known anti-inflammatory compound aciculatin can be held responsible for the observed activity.
Project description:The maize beta-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli, purified, crystallized and its structure solved at 2.5 Angstrom resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p-nitrophenyl beta-D-thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.