Interspecies-cooperations of abutilon theophrasti with root colonizing microorganisms disarm BOA-OH allelochemicals.

A facultative, microbial micro-community colonizing roots of Abutilon theophrasti Medik. supports the plant in detoxifying hydroxylated benzoxazolinones. The root micro-community is composed of several fungi and bacteria with Actinomucor elegans as a dominant species. The yeast Papiliotrema baii and the bacterium Pantoea ananatis are actively involved in the detoxification of hydroxylated benzoxazolinones by generating H2O2. At the root surface, laccases, peroxidases and polyphenol oxidases cooperate for initiating polymerization reactions, whereby enzyme combinations seem to differ depending on the hydroxylation position of BOA-OHs. A glucosyltransferase, able to glucosylate the natural benzoxazolinone detoxification intermediates BOA-5- and BOA-6-OH, is thought to reduce oxidative overshoots by damping BOA-OH induced H2O2 generation. Due to this detoxification network, growth of Abutilon theophrasti seedlings is not suppressed by BOA-OHs. Polymer coats have no negative influence. Alternatively, quickly degradable 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one can be produced by the micro-community member Pantoea ananatis at the root surfaces. The results indicate that Abutilon theophrasti has evolved an efficient strategy by recruiting soil microorganisms with special abilities for different detoxification reactions which are variable and may be triggered by the allelochemical´s structure and by environmental conditions.

Bottom-up approach for the reaction of xenobiotics and their metabolites with model substances for natural organic matter by electrochemistry-mass spectrometry (EC-MS).

Risk assessment of xenobiotics requires a comprehensive understanding of their transformation in the environment. As most of the transformation processes usually involve a redox reaction or a hydrolysis as the first steps of the transformation, we applied an approach that uses an electrochemical cell to investigate model "redox" reactions in aqueous solutions for environmental processes. We investigated the degradation of a variety of xenobiotics from polar to nonpolar and analyzed their degradation products by on-line coupling of electrochemistry with mass spectrometry (EC-MS). Furthermore, we evaluated possible binding reactions with regard to the generation of non-extractable residues with some model substances (catechol, phthalic acid, γ-L-Glutamyl-L-cysteinyl-glycine (GSH) and L-histidine) deduced from a natural organic matter (NOM) structure model and identified possible binding-sites. Whereas typically investigations in soil/water-systems have been applied, we used to our knowledge for the first time a bottom-up approach, starting from the chemicals of interest and different model substances for natural organic matter to evaluate chemical binding mechanisms (or processes) in the EC-MS under redox conditions. Under oxidative conditions, bindings of the xenobiotics with catechol, GSH and histidine were found, but no reactions with the model compound phthalic acid were observed. In general, no chemical binding has yet been found under reductive conditions. In some cases (i.e. benzo[a]anthracene) the oxidation product only underwent a binding reaction, whereas the xenobiotic itself did not undergo any reactions. EC-MS is a promising fast and simple screening method to investigate the environmental behavior of xenobiotics and to evaluate the potential risks of newly synthesized substances.

Long-term persistence of various 14C-labeled pesticides in soils.

The fate of the 14C-labeled herbicides ethidimuron (ETD), methabenzthiazuron (MBT), and the fungicide anilazine (ANI) in soils was evaluated after long-term aging (9-17 years) in field based lysimeters subject to crop rotation. Analysis of residual 14C activity in the soils revealed 19% (ETD soil; 0-10 cm depth), 35% (MBT soil; 0-30), and 43% (ANI soil; 0-30) of the total initially applied. Accelerated solvent extraction yielded 90% (ETD soil), 26% (MBT soil), and 41% (ANI soil) of residual pesticide 14C activity in the samples. LC-MS/MS analysis revealed the parent compounds ETD and MBT, accounting for 3% and 2% of applied active ingredient in the soil layer, as well as dihydroxy-anilazine as the primary ANI metabolite. The results for ETD and MBT were matching with values obtained from samples of a 12 year old field plot experiment. The data demonstrate the long-term persistence of these pesticides in soils based on outdoor trials.

Elucidation of the final reactions of DIMBOA-glucoside biosynthesis in maize: characterization of Bx6 and Bx7.

Benzoxazinoids were identified in the early 1960s as secondary metabolites of the grasses that function as natural pesticides and exhibit allelopathic properties. Benzoxazinoids are synthesized in seedlings and stored as glucosides (glcs); the main aglucone moieties are 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) and 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA). The genes of DIBOA-glc biosynthesis have previously been isolated and the enzymatic functions characterized. Here, the enzymes for conversion of DIBOA-glc to DIMBOA-glc are identified. DIBOA-glc is the substrate of the dioxygenase BENZOXAZINLESS6 (BX6) and the produced 2,4,7-trihydroxy-2H-1,4-benzoxazin-3-(4H)-one-glc is metabolized by the methyltransferase BX7 to yield DIMBOA-glc. Both enzymes exhibit moderate K(m) values (below 0.4 mm) and k(cat) values of 2.10 s(-1) and 0.25 s(-1), respectively. Although BX6 uses a glucosylated substrate, our localization studies indicate a cytoplasmic localization of the dioxygenase. Bx6 and Bx7 are highest expressed in seedling tissue, a feature shared with the other Bx genes. At present, Bx6 and Bx7 have no close relatives among the members of their respective gene families. Bx6 and Bx7 map to the cluster of Bx genes on the short arm of chromosome 4.

A conserved motif is prerequisite for the interaction of NAC with ribosomal protein L23 and nascent chains.

In eukaryotes, newly synthesized proteins interact co-translationally with a multitude of different ribosome-bound factors and chaperones including the conserved heterodimeric nascent polypeptide-associated complex (NAC) and a Hsp40/70-based chaperone system. These factors are thought to play an important role in protein folding and targeting, yet their specific ribosomal localizations, which are prerequisite for their functions, remain elusive. This study describes the ribosomal localization of NAC and the molecular details by which NAC is able to contact the ribosome and gain access to nascent polypeptides. We identified a conserved RRK(X)nKK ribosome binding motif within the beta-subunit of NAC that is essential for the entire NAC complex to attach to ribosomes and allow for its interaction with nascent polypeptide chains. The motif localizes within a potential loop region between two predicted alpha-helices in the N terminus of betaNAC. This N-terminal betaNAC ribosome-binding domain was completely portable and sufficient to target an otherwise cytosolic protein to the ribosome. NAC modified with a UV-activatable cross-linker within its ribosome binding motif specifically cross-linked to L23 ribosomal protein family members at the exit site of the ribosome, providing the first evidence of NAC-L23 interaction in the context of the ribosome. Mutations of L23 reduced NAC ribosome binding in vivo and in vitro, whereas other eukaryotic ribosome-associated factors such as the Hsp70/40 chaperones Ssb or Zuotin were unaffected. We conclude that NAC employs a conserved ribosome binding domain to position itself on the L23 ribosomal protein adjacent to the nascent polypeptide exit site.