Thomas Etzerodt

Transformation kinetics of 6-methoxybenzoxazolin-2-one in soil

Wheat (Triticum aestivum L.) and other cereals produce allelochemicals as natural defense compounds against weeds, fungi, insects and soil-borne diseases. The main benzoxazinoid allelochemical of wheat is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), bound as β-glucoside and released upon plant injury. When leached from wheat to soil, DIMBOA is microbially transformed to 6-methoxy-benzoxazolin-2-one (MBOA). Exploiting benzoxazinoids and their degradation products as substitutes for synthetic pesticides depends on knowledge of transformation pathways and kinetics. In an MBOA degradation experiment at a concentration of 2400 nmol g–1 soil, the previously identified transformation products 2-amino-7-methoxy-phenoxazin-3-one (AMPO) and 2-acetylamino-7-methoxy-phenoxazin-3-one (AAMPO) were quantified. Three different kinetic models were applied to MBOA transformation kinetics; single first-order (SFO), first-order multi-compartment, and double first-order in parallel. SFO proved to be adequate and was subsequently applied to the transformations of MBOA, AMPO and AAMPO. Degradation endpoints, expressed as degradation time (DT), were calculated for MBOA, AMPO and AAMPO to test whether the maximum values for synthetic pesticides set by the European Commission and the Danish Environmental Protection Agency were exceeded. DT50 values for MBOA and AMPO were 5.4 d and 321.5 d, respectively, and DT90 values were 18.1 d and 1068 d, respectively. The DT50 value for AMPO exceeded the maximum value. The persistence, concentrations and toxicity of metabolites such as AMPO should be considered when breeding cereal crops with increased levels of benzoxazinoids.

2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) inhibits trichothecene production by Fusarium graminearum through suppression of Tri6 expression.

Fusarium head blight (FHB) is a devastating disease of wheat (Triticum aestivum L.) caused by a mycotoxigenic fungus Fusarium graminearum resulting in significantly decreased yields and accumulation of toxic trichothecenes in grains. We tested 7 major secondary metabolites from wheat for their effect on trichothecene production in liquid cultures of F. graminearum producing trichothecene 15-acetyldeoxynivalenol (15-ADON). 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) benzoxazinoid completely abolished toxin production without any apparent effect on fungal growth. DIMBOA strongly affected the expression of Tri6, encoding a major transcriptional regulator of several genes of the trichothecene biosynthesis pathway. DIMBOA also repressed expression of Tri5, encoding trichodiene synthase, the first enzyme in the trichothecene biosynthesis pathway. Thus, DIMBOA could play an important role against the accumulation of trichothecenes in wheat grain. Breeding or engineering of wheat with increased levels of benzoxazinoids could provide varieties with increased resistance against trichothecene contamination of grain and lower susceptibility to FHB.

Fructan metabolism and changes in fructan composition during cold acclimation in perennial ryegrass

Perennial ryegrass (Lolium perenne L.) produces high levels of fructans as a mixture of oligosaccharides and polysaccharides with different degrees of polymerization (DP). The present study describes the analysis of the compositional changes in the full spectrum of fructans, fructan distribution between above ground biomass (top) and the roots, and the transcription of candidate genes involved in fructan metabolism during cold acclimation in perennial ryegrass variety “Veyo” and ecotype “Falster” from distinct geographical origins. We observed changes in fructan composition and induction of low-DP fructans, especially DP = 4, in both the top and the roots of “Veyo” and “Falster” in response to low-temperature stress. The accumulation of DP > 50 fructans was only apparent in the top tissues where the Lp1-FFT expression is higher compared to the roots in both “Veyo” and “Falster.” Our results also show the accumulation and depolymerization of fructans with different DP, together with the induction of genes encoding fructosyltransferases and fructan exohydrolases in both “Veyo” and “Falster” during cold acclimation, supporting the hypothesis that fructan synthesis and depolymerization occurring simultaneously. The ecotype “Falster,” adapted to cold climates, increased total fructan content and produced more DP > 7 fructans in the roots than the variety “Veyo,” adapted to warmer climates. This indicates that high-DP fructan accumulation in roots may be an adaptive trait for plant recovery after abiotic stresses.

Changes in Lolium perenne transcriptome during cold acclimation in two genotypes adapted to different climatic conditions

BackgroundActivation of numerous protective mechanisms during cold acclimation is important for the acquisition of freezing tolerance in perennial ryegrass (Lolium perenne L.). To elucidate the molecular mechanisms of cold acclimation in two genotypes (‘Veyo’ and ‘Falster’) of perennial ryegrass from distinct geographical origins, we performed transcriptome profiling during cold acclimation using RNA-Seq.MethodsWe cold-acclimated plants from both genotypes in controlled conditions for a period of 17 days and isolated Total RNA at various time points for high throughput sequencing using Illumina technology. RNA-seq reads were aligned to genotype specific references to identify transcripts with significant changes in expression during cold acclimation.ResultsThe genes induced were involved in protective mechanisms such as cell response to abiotic stimulus, signal transduction, redox homeostasis, plasma membrane and cell wall modifications, and carbohydrate metabolism in both genotypes. ‘Falster’ genotype, adapted to cold climates, showed a stronger transcriptional differentiation during cold acclimation, and more differentially expressed transcripts related to stress, signal transduction, response to abiotic stimulus, and metabolic processes compared to ‘Veyo’. ‘Falster’ genotype also showed an induction of more transcripts with sequence homology to fructosyltransferase genes (FTs) and a higher fold induction of fructan in response to low-temperature stress. The circadian rhythm network was perturbed in the ‘Veyo’ genotype adapted to warmer climates.ConclusionIn this study, the differentially expressed genes during cold acclimation, potentially involved in numerous protective mechanisms, were identified in two genotypes of perennial ryegrass from distinct geographical origins. The observation that the circadian rhythm network was perturbed in ‘Veyo’ during cold acclimation may point to a low adaptability of ‘Veyo’ to low temperature stresses. This study also revealed the transcriptional mechanisms underlying carbon allocation towards fructan biosynthesis in perennial ryegrass.

Quantification of azoxystrobin and identification of two novel metabolites in lettuce via liquid chromatography–quadrupole-linear ion trap (QTRAP) mass spectrometry

ABSTRACT Pesticide metabolite identification is gaining increased attention because of the interest in potential metabolite toxicity. Azoxystrobin is one of the most prevalent pesticide residues in foods in Europe. The majority of azoxystrobin metabolites have been identified using radiolabelled standards, which are either expensive or not readily available. Thus, alternative approaches for metabolite identification are desirable. Here, an LC-MS/MS method for quantifying azoxystrobin and identifying its metabolites using quadrupole-linear ion trap mass spectrometry is reported. Seven metabolites of azoxystrobin were identified 2 and 4 weeks after spraying lettuce with azoxystrobin. Among them, two metabolites are reported for the first time. The hydrolysis, reduction, hydroxylation, photoisomerisation and hydrolytic cleavage of ether bonds are identified as biotransformation processes involved in azoxystrobin metabolism in lettuce.

Correlation of Deoxynivalenol Accumulation in Fusarium-Infected Winter and Spring Wheat Cultivars with Secondary Metabolites at Different Growth Stages.

Fusarium infection in wheat causes Fusarium head blight, resulting in yield losses and contamination of grains with trichothecenes. Some plant secondary metabolites inhibit accumulation of trichothecenes. Eighteen Fusarium infected wheat cultivars were harvested at five time points and analyzed for the trichothecene deoxynivalenol (DON) and 38 wheat secondary metabolites (benzoxazinoids, phenolic acids, carotenoids, and flavonoids). Multivariate analysis showed that harvest time strongly impacted the content of secondary metabolites, more distinctly for winter wheat than spring wheat. The benzoxazinoid 2-β-glucopyranoside-2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), α-tocopherol, and the flavonoids homoorientin and orientin were identified as potential inhibitors of DON accumulation. Several phenolic acids, lutein and β-carotene also affected DON accumulation, but the effect varied for the two wheat types. The results could form a basis for choosing wheat cultivars using metabolite profiling as a marker for selecting wheat cultivars with improved resistance against Fusarium head blight and accumulation of trichothecene toxins in wheat heads.

Additional file 3: of Changes in Lolium perenne transcriptome during cold acclimation in two genotypes adapted to different climatic conditions

Major molecular functions of differentially expressed transcripts in ‘Veyo’ genotype of perennial ryegrass ( Lolium perenne L.) during cold acclimation identified by the DAVID tool. (XLSX 12 kb)