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Publikation

Bochnia, M.; Ziegler, J.; Glatter, M.; Zeyner, A.; Hypoglycin A in cow’s milk—A pilot study Toxins 13, 381, (2021) DOI: 10.3390/toxins13060381

Hypoglycin A (HGA) originating from soapberry fruits (litchi, and ackee) seeds or seedlings from the sycamore maple (SM) tree (related to Sapindaceae) may cause Jamaican vomiting sickness in humans and atypical myopathy in horses and ruminants. A possible transfer into dairy cow’s milk cannot be ruled out since the literature has revealed HGA in the milk of mares and in the offal of captured deer following HGA intoxication. From a study, carried out for another purpose, bulk raw milk samples from four randomly selected dairy farms were available. The cows were pastured in the daytime. A sycamore maple tree was found on the pasture of farm No. 1 only. Bulk milk from the individual tank or milk filling station was sampled in parallels and analyzed for HGA by LC-ESI-MS/MS. Measurable concentrations of HGA occurred only in milk from farm No. 1 and amounted to 120 and 489 nmol/L. Despite low and very variable HGA concentrations, the results indicate that the ingested toxin, once eaten, is transferred into the milk. However, it is unknown how much HGA the individual cow ingested during grazing and what amount was transferred into the bulk milk samples. As a prerequisite for a possible future safety assessment, carry-over studies are needed. Furthermore, the toxins’ stability during milk processing should also be investigated as well.
Publikation

Ziegler, J.; Bochnia, M.; Zeyner, A.; Aminosäurennachweis in geringsten ProbenmengenBestimmung von Hypoglycin A Wiley Analytical Science (2021)

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Publikation

Bochnia, M.; Sander, J.; Ziegler, J.; Terhardt, M.; Sander, S.; Janzen, N.; Cavalleri, J.-M. V.; Zuraw, A.; Wensch-Dorendorf, M.; Zeyner, A.; Detection of MCPG metabolites in horses with atypical myopathy PLOS ONE 14, e0211698, (2019) DOI: 10.1371/journal.pone.0211698

Atypical myopathy (AM) in horses is caused by ingestion of seeds of the Acer species (Sapindaceae family). Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A (HGA), is currently the only active toxin in Acer pseudoplatanus or Acer negundo seeds related to AM outbreaks. However, seeds or arils of various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also contain methylenecyclopropylglycine (MCPG), which is a structural analogue of HGA that can cause hypoglycaemic encephalopathy in humans. The active poison formed from MCPG is methylenecyclopropylformyl-CoA (MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that participate in β-oxidation and energy production from fat. The aim of our study was to investigate if MCPG is involved in Acer seed poisoning in horses. MCPG, as well as glycine and carnitine conjugates (MCPF-glycine, MCPF-carnitine), were quantified using high-performance liquid chromatography-tandem mass spectrometry of serum and urine from horses that had ingested Acer pseudoplatanus seeds and developed typical AM symptoms. The results were compared to those of healthy control horses. For comparison, HGA and its glycine and carnitine derivatives were also measured. Additionally, to assess the degree of enzyme inhibition of β-oxidation, several acyl glycines and acyl carnitines were included in the analysis. In addition to HGA and the specific toxic metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and MCPF-carnitine were detected in the serum and urine of affected horses. Strong inhibition of β-oxidation was demonstrated by elevated concentrations of all acyl glycines and carnitines, but the highest correlations were observed between MCPF-carnitine and isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-) glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here, for biochemical analysis of atypical myopathy of horses, it is necessary to take MCPG and the corresponding metabolites into consideration.
Publikation

Bochnia, M.; Scheidemann, W.; Ziegler, J.; Sander, J.; Vollstedt, S.; Glatter, M.; Janzen, N.; Terhardt, M.; Zeyner, A.; Predictive value of hypoglycin A and methylencyclopropylacetic acid conjugates in a horse with atypical myopathy in comparison to its cograzing partners Equine Vet. Educ. 30, 24-28, (2018) DOI: 10.1111/eve.12596

Hypoglycin A (HGA) was detected in blood and urine of a horse suffering from atypical myopathy (AM; Day 2, serum, 8290 μg/l; urine: Day 1, 574, Day 2, 742 μg/l) and in its cograzing partners with a high variability (46–1570 μg/l serum). Over the period of disease, the level of the toxic metabolites (methylencyclopropylacetic acid [MCPA]‐conjugates) increased in body fluids of the AM horse (MCPA‐carnitine: Day 2, 0.246, Day 3, 0.581 μmol/l serum; MCPA‐carnitine: Day 2, 0.621, Day 3, 0.884 μmol/mmol creatinine in urine) and HGA decreased rapidly (Day 3, 2430 μg/l serum). In cograzing horses MCPA‐conjugates were not detected. HGA in seeds ranged from 268 to 367 μg/g. Although HGA was present in body fluids of healthy cograzing horses, MCPA‐conjugates were not detectable, in contrast to the AM horse. Therefore, increasing concentrations of MCPA‐conjugates are supposed to be linked with the onset of AM and both parameters seem to indicate the clinical stage of disease. However, detection of HGA in body fluids of cograzing horses might be a promising step in preventing the disease.
Publikation

Bagchi, R.; Melnyk, C. W.; Christ, G.; Winkler, M.; Kirchsteiner, K.; Salehin, M.; Mergner, J.; Niemeyer, M.; Schwechheimer, C.; Calderón Villalobos, L. I. A.; Estelle, M.; The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases EMBO J. 37, 255-268, (2018) DOI: 10.15252/embj.201797159

The cullin‐RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin‐conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.
Publikation

Ziegler, J.; Schmidt, S.; Strehmel, N.; Scheel, D.; Abel, S.; Arabidopsis Transporter ABCG37/PDR9 contributes primarily highly oxygenated Coumarins to Root Exudation Sci. Rep. 7, 3704, (2017) DOI: 10.1038/s41598-017-03250-6

The chemical composition of root exudates strongly impacts the interactions of plants with microorganisms in the rhizosphere and the efficiency of nutrient acquisition. Exudation of metabolites is in part mediated by ATP-binding cassette (ABC) transporters. In order to assess the contribution of individual ABC transporters to root exudation, we performed an LC-MS based non-targeted metabolite profiling of semi-polar metabolites accumulating in root exudates of Arabidopsis thaliana plants and mutants deficient in the expression of ABCG36 (PDR8/PEN3), ABCG37 (PDR9) or both transporters. Comparison of the metabolite profiles indicated distinct roles for each ABC transporter in root exudation. Thymidine exudation could be attributed to ABCG36 function, whereas coumarin exudation was strongly reduced only in ABCG37 deficient plants. However, coumarin exudation was compromised in abcg37 mutants only with respect to certain metabolites of this substance class. The specificity of ABCG37 for individual coumarins was further verified by a targeted LC-MS based coumarin profiling method. The response to iron deficiency, which is known to strongly induce coumarin exudation, was also investigated. In either treatment, the distribution of individual coumarins between roots and exudates in the investigated genotypes suggested the involvement of ABCG37 in the exudation specifically of highly oxygenated rather than monohydroxylated coumarins.
Publikation

Ziegler, J.; Schmidt, S.; Chutia, R.; Müller, J.; Böttcher, C.; Strehmel, N.; Scheel, D.; Abel, S.; Non-targeted profiling of semi-polar metabolites in Arabidopsis root exudates uncovers a role for coumarin secretion and lignification during the local response to phosphate limitation J. Exp. Bot. 67, 1421-1432, (2016) DOI: 10.1093/jxb/erv539

Plants have evolved two major strategies to cope with phosphate (Pi) limitation. The systemic response, mainly comprising increased Pi uptake and metabolic adjustments for more efficient Pi use, and the local response, enabling plants to explore Pi-rich soil patches by reorganization of the root system architecture. Unlike previous reports, this study focused on root exudation controlled by the local response to Pi deficiency. To approach this, a hydroponic system separating the local and systemic responses was developed. Arabidopsis thaliana genotypes exhibiting distinct sensitivities to Pi deficiency could be clearly distinguished by their root exudate composition as determined by non-targeted reversed-phase ultraperformance liquid chromatography electrospray ionization quadrupole-time-of-flight mass spectrometry metabolite profiling. Compared with wild-type plants or insensitive low phosphate root 1 and 2 (lpr1 lpr2) double mutant plants, the hypersensitive phosphate deficiency response 2 (pdr2) mutant exhibited a reduced number of differential features in root exudates after Pi starvation, suggesting the involvement of PDR2-encoded P5-type ATPase in root exudation. Identification and analysis of coumarins revealed common and antagonistic regulatory pathways between Pi and Fe deficiency-induced coumarin secretion. The accumulation of oligolignols in root exudates after Pi deficiency was inversely correlated with Pi starvation-induced lignification at the root tips. The strongest oligolignol accumulation in root exudates was observed for the insensitive lpr1 lpr2 double mutant, which was accompanied by the absence of Pi deficiency-induced lignin deposition, suggesting a role of LPR ferroxidases in lignin polymerization during Pi starvation.
Publikation

Strehmel, N.; Mönchgesang, S.; Herklotz, S.; Krüger, S.; Ziegler, J.; Scheel, D.; Piriformospora indica Stimulates Root Metabolism of Arabidopsis thaliana Int. J. Mol. Sci. 17, 1091, (2016) DOI: 10.3390/ijms17071091

Piriformospora indica is a root-colonizing fungus, which interacts with a variety of plants including Arabidopsis thaliana. This interaction has been considered as mutualistic leading to growth promotion of the host. So far, only indolic glucosinolates and phytohormones have been identified as key players. In a comprehensive non-targeted metabolite profiling study, we analyzed Arabidopsis thaliana’s roots, root exudates, and leaves of inoculated and non-inoculated plants by ultra performance liquid chromatography/electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLC/(ESI)-QTOFMS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS), and identified further biomarkers. Among them, the concentration of nucleosides, dipeptides, oligolignols, and glucosinolate degradation products was affected in the exudates. In the root profiles, nearly all metabolite levels increased upon co-cultivation, like carbohydrates, organic acids, amino acids, glucosinolates, oligolignols, and flavonoids. In the leaf profiles, we detected by far less significant changes. We only observed an increased concentration of organic acids, carbohydrates, ascorbate, glucosinolates and hydroxycinnamic acids, and a decreased concentration of nitrogen-rich amino acids in inoculated plants. These findings contribute to the understanding of symbiotic interactions between plant roots and fungi of the order of Sebacinales and are a valid source for follow-up mechanistic studies, because these symbioses are particular and clearly different from interactions of roots with mycorrhizal fungi or dark septate endophytes
Publikation

Bochnia, M.; Ziegler, J.; Sander, J.; Uhlig, A.; Schaefer, S.; Vollstedt, S.; Glatter, M.; Abel, S.; Recknagel, S.; Schusser, G. F.; Wensch-Dorendorf, M.; Zeyner, A.; Hypoglycin A Content in Blood and Urine Discriminates Horses with Atypical Myopathy from Clinically Normal Horses Grazing on the Same Pasture PLOS ONE 10, e0136785, (2015) DOI: 10.1371/journal.pone.0136785

Hypoglycin A (HGA) in seeds of Acer spp. is suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy (AM) in Europe, fatal diseases in horses on pasture. In previous studies, this suspicion was substantiated by the correlation of seed HGA content with the concentrations of toxic metabolites in urine and serum (MCPA-conjugates) of affected horses. However, seed sampling was conducted after rather than during an outbreak of the disease. The aim of this study was to further confirm the causality between HGA occurrence and disease outbreak by seed sampling during an outbreak and the determination of i) HGA in seeds and of ii) HGA and MCPA-conjugates in urine and serum of diseased horses. Furthermore, cograzing healthy horses, which were present on AM affected pastures, were also investigated. AM-pastures in Germany were visited to identify seeds of Acer pseudoplatanus and serum (n = 8) as well as urine (n = 6) from a total of 16 diseased horses were analyzed for amino acid composition by LC-ESI-MS/MS, with a special focus on the content of HGA. Additionally, the content of its toxic metabolite was measured in its conjugated form in body fluids (UPLC-MS/MS). The seeds contained 1.7–319.8 μg HGA/g seed. The content of HGA in serum of affected horses ranged from 387.8–8493.8 μg/L (controls < 10 μg/L), and in urine from 143.8–926.4 μg/L (controls < 10 μg/L), respectively. Healthy cograzing horses on AM-pastures showed higher serum (108.8 ± 83.76 μg/L) and urine concentrations (26.9 ± 7.39 μg/L) compared to control horses, but lower concentrations compared to diseased horses. The range of MCPA-carnitine and creatinine concentrations found in diseased horses in serum and urine were 0.17–0.65 mmol/L (controls < 0.01), and 0.34–2.05 μmol/mmoL (controls < 0.001), respectively. MCPA-glycine levels in urine of cograzing horses were higher compared to controls. Thus, the causal link between HGA intoxication and disease outbreak could be further substantiated, and the early detection of HGA in cograzing horses, which are clinically normal, might be a promising step in prophylaxis.
Publikation

Buhtz, A.; Witzel, K.; Strehmel, N.; Ziegler, J.; Abel, S.; Grosch, R.; Perturbations in the Primary Metabolism of Tomato and Arabidopsis thaliana Plants Infected with the Soil-Borne Fungus Verticillium dahliae PLOS ONE 10, e0138242, (2015) DOI: 10.1371/journal.pone.0138242

The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant.
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