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BACKGROUND and AIMS Glucosinolates and isothiocyanates of young Camelina sativa (L.) Crantz can eliminate microbial species in the soil. It was aimed to demonstrate that only isothiocyanate insensitive microorganisms with phosphate solubilizing activity can be successfully used to improve the plant´s phosphate supply. METHODS We performed rhizotron growth studies and 33P–Imaging to study the uptake of phosphate solubilized from 33P-apatite by inoculated plants and determined the phosphate solubilization capacities of microorganisms by inductively coupled plasma mass spectrometry. Secondary metabolites of Camelina sativa and of the soil fungus Penicillium aurantiogriseum were analyzed by HPLC-MS/MS. Microorganisms and plant seedlings were cultured for growth inhibition studies. Phospholipid fatty acids in soil samples were investigated by gas chromatography and the data analyzed by clustering and principal component analysis. RESULTS 1. The inoculants Trichoderma viride and Pseudomonas laurentiana did not improve the phosphate uptake of Camelina sativa and Abutilon theophrasti. 2. The intrinsic soil microorganisms, including Penicillium aurantiogriseum, the inoculants and the assemblies of root colonizing microorganisms are able to solubilize phosphate from apatite and compete for phosphate. 3. Camelina´s glucosinolates and derived isothiocyanates and, as well secondary metabolites released by P. aurantiogriseum, particularly cyclo-(L-Leu-L-Pro), have the capacity to suppress plant and fungal growth and to destroy a phosphate solubilizing bacterial consortium that colonizes Camelina roots. 4. Loss of young Camelina´s key secondary metabolites, the glucosinolates, is of advantage for fungi, as verified by a green manure experiment. CONCLUSION The results underline functions of secondary metabolites when organisms are under competive conditions for phosphate uptake.

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUG Allelopathic activity of wheat (Triticum aestivum L.) has previously been associated with the production of phenolic acids and flavonoids (PAF), benzoxazinones (BXZs) and phenoxazinones (PXZs). The biosynthesis of BXZs is closely regulated during cereal growth, with accumulation highest in young tissues with variation associated with genotype and environmental conditions. This review is focused on BXZ metabolites and their impact on germination, seedling growth and physiological, biochemical, transcriptional and proteome traits of surrounding plants and weeds. The major pathways employed by plants for benzoxazinoid detoxification involve hydroxylation and glucosylation and polymerisation of intermediates in these pathways. Allelochemicals from various wheat genotypes have been shown to inhibit the growth of selected weed species, including Bromus japonicus, Chenopodium album, Portulaca oleracea, Avena fatua and Lolium rigidum. Wheat allelopathy is potentially exploited from the standpoint of crop mulches, incorporation of crop residues, tissue disruption, intercropping with allelopathic cultivars and application of aqueous wheat extracts. BXZs have been shown to suppress the growth and development of certain agricultural pests, including insects, fungal pathogens, and weeds. Many native plants, fungi and insect herbivores inherently possess varying tolerance levels towards BXZs. However, other BXZ- susceptible species are adversely impacted by elevated BXZ levels in crop plants. Thus, considerations for the selection and breeding of wheat genotypes possessing enhanced defensive ability via elevated BXZ contents are discussed. Here, these objectives are reconsidered with a focus on co-evolutionary aspects and their potential impacts on biodiversity in the agroecosystems under study. For future breeding efforts to be successful, it is important to take such potential adverse environmental impacts into account, in combination with an increased focus on enhancing beneficial allelopathic effects within agricultural systems.

Numerous allelopathic plant secondary metabolites impact plant–microorganism interactions by injuring plant-associated beneficial bacteria and fungi. Fungi belonging to the genus Trichoderma positively influence crops, including benzoxazinone-containing maize. However, benzoxazinones and their downstream metabolites such as benzoxazolinone and phenoxazinones are often fungitoxic. Specimen Trichoderma viride F-00612 was found to be insensitive to 100-µM phenoxazinone and 500-µM benzoxazolinone. Screening of 46 additional specimens of ascomycetes revealed insensitivity to phenoxazinones among fungi that cause disease in benzoxazinone-producing cereal crops, whereas many other ascomycetes were highly sensitive. In contrast, most of the screened fungi were insensitive to benzoxazolinone. T. viride F-00612 was associated with bacteria and, thus, existed as a consortium. By contrast, Enterobacter species and Acinetobacter calcoaceticus were prominent in the original specimen, and Bacillus species predominated after antibiotic application. Prolonged cultivation of T. viride F-00612 in liquid medium and on Czapek agar in the presence of T. viride F-00612 consortium may contribute to the ability to manage benzoxazinone downstream products and facilitate BOA-6-OH degradation via nitration.

For the characterization of BOA-OH insensitive plants, we studied the time-dependent effects of the benzoxazolinone-4/5/6/7-OH isomers on maize roots. Exposure of Zea mays seedlings to 0.5 mM BOA-OH elicits root zone-specific reactions by the formation of dark rings and spots in the zone of lateral roots, high catalase activity on root hairs, and no visible defense reaction at the root tip. We studied BOA-6-OH- short-term effects on membrane lipids and fatty acids in maize root tips in comparison to the benzoxazinone-free species Abutilon theophrasti Medik. Decreased contents of phosphatidylinositol in A. theophrasti and phosphatidylcholine in maize were found after 10–30 min. In the youngest tissue, α-linoleic acid (18:2), decreased considerably in both species and recovered within one hr. Disturbances in membrane phospholipid contents were balanced in both species within 30–60 min. Triacylglycerols (TAGs) were also affected, but levels of maize diacylglycerols (DAGs) were almost unchanged, suggesting a release of fatty acids for membrane lipid regeneration from TAGs while resulting DAGs are buildings blocks for phospholipid reconstitution, concomitant with BOA-6-OH glucosylation. Expression of superoxide dismutase (SOD2) and of ER-bound oleoyl desaturase (FAD2-2) genes were contemporaneously up regulated in contrast to the catalase CAT1, while CAT3 was arguably involved at a later stage of the detoxification process. Immuno-responses were not elicited in short-terms, since the expression of NPR1, POX12 were barely affected, PR4 after 6 h with BOA-4/7-OH and PR1 after 24 h with BOA-5/6-OH. The rapid membrane recovery, reactive oxygen species, and allelochemical detoxification may be characteristic for BOA-OH insensitive plants.

For the characterization of BOA-OH insensitive plants, we studied time-dependent effects of the benzoxazolinone-4/5/6/7-OH isomers on maize roots. Exposure of Zea mays seedlings to 0.5 mM BOA-OH elicits root zone specific reactions by formation of dark rings and spots in the zone of lateral roots, high catalase activity on root hairs and no visible defense reaction at the root tip. We studied BOA-6-OH- short term effects on membrane lipids and fatty acids in maize root tips in comparison to reference species Abutilon theophrasti Medik. Decreased contents of phosphatidylinositol in A. theophrasti and phosphatidylcholine in maize were found after 10-30 min. In the youngest tissue, α-linoleic acid (18:2), decreased considerably in both species and recovering within one hour. Disturbances in membrane phospholipid contents were balanced in both species within 30-60 min. Triacylglycerols were also affected, but levels of maize diacylglycerols were almost unchanged, suggesting a release of fatty acids for membrane lipid regeneration from TAGs while resulting DAGs are buildings blocks for phospholipid reconstitution, concomitant with BOA-6-OH glucosylation. Expression of superoxide dismutase (SOD2) and of ER-bound oleoyl desaturase (FAD2-2) genes were contemporaneously up regulated in contrast to the catalase CAT1, while CAT3 was arguably integrated at a later stage of the detoxification process. Immuno-responses were not elicited in short-terms, since expression of NPR1, POX12 were rather affected, PR4 after 6h with BOA-4/7-OH and PR1 after 24h with BOA-5/6-OH. The rapid membrane recovery, reactive oxygen species and allelochemical detoxification may be characteristic for BOA-OH insensitive plants.

Exposure of Zea maysseedlings to 0.5µM benzoxazolinone-4/5/6/7-OH allelochemicals elicits root zone specific reactions by formation of dark rings and spots in the zone of lateral roots,high catalase activity at root hairsand no visible defense reaction at the root tip. We studied short term effects on membrane lipids and fatty acids in maize root tips in comparison to those of Abutilontheophrastiafter BOA-6-OH incubations. We found a decrease mainly of phosphatidylinositol in Abutilon and phosphatidylcholinein maize after 10-30 min of exposure. In the youngest tissue, α-linoleic acid (18:2), decreased considerably in both species but amounts recovered within one hour.Disturbances in membrane phospholipid contents were balanced in both species within 30-60 min. The findings that triacylglycerols(TAGs) were also affected, but levels of maize diacylglycerols (DAG) were almost unchanged, suggestedthat fatty acids for membrane lipid regeneration are released from TAGs while resulting DAGs are buildings blocks for phospholipid reconstitution,concomitant with BOA-6-OH glucosylation. Theexpression of superoxide dismutase (SOD2)and of ER-bound oleoyldesaturase (FAD2-2) genes were contemporaneously up regulated in contrast to the catalase gene CAT1,while CAT3was arguably integrated at a later stage of the detoxification process. Major catalase activity seems to be attributed to root colonizing microorganisms. Immuno-responses were not elicited in short-terms, since expression of NPR1, POX12were rather affected,PR4 after 6h with BOA-4/7-OH and PR1 after 24hwith BOA-5/6-OH.The rapid membrane recovery, reactive oxygen species and allelochemical detoxification may be characteristic forBOA-OH insensitive plants.

Some sweet tasting plant secondary metabolites are non-caloric or low nutritive compounds that have traditional use in food formulations. This mini-review focuses on conventional and advanced cultivation regimes of plants that accumulate sweet tasting or sweet taste modulating secondary metabolites of potential economic importance, in particular mogrosides (Siraitia grosvenorii), phyllodulcin (Hydrangea macrophylla), glycyrrhizin (Glycyrrhiza glabra), steviol glycosides (Stevia grosvenorii), and rubusoside (Rubus suavissimus). Consequential obstacles during the cultivation of Hydrangea macrophylla cultivars outside their natural habitat in a protected cultivation environment are addressed. Culturing at non-habitat locations facilitates short transportation routes of plant material for processing, which can be a key to an economically and environmentally compatible usage. The biosynthetic pathways, as far as known, are shortly mentioned. The proved or hypothetical degradation pathways of the compounds to minimalize environmental contamination are another focal point.

Italian journal of agronomy = Rivista di agronomia 16(3), 1807 (2020). doi:10.4081/ija.2021.1807 special issue: "Special issue on "Integrated weed management" / Guest Editors: Vittoria Giannini; Donato Loddo;", Published by Forum Editrice Univ. Udinese, Udine

Benzoxazinoids, secondary metabolites of several Poaceae, and some benzoxazinoid downstream metabolites are bioactive compounds that act as allelochemicals and natural pesticides. Since a short lifetime of the substances is crucial to avoid long-term environmental effects, total degradation by microorganisms is of exceptional importance. We performed a screening with cultivable microorganisms (Species names and strain numbers: Mycobacterium fortuitum, 7; Bacillus aryabhattai, 34; Bacillus cereus, 59; Bacillus megaterium, 21, 48; Bacillus methylotrophicus, 58; Lysinibacillus xylanilyticus, 56; Paenibacillus polymyxa, 51; Aminobacter aminovorans, 49; the fungi Papulaspora sepedonioides, 12 and Trichoderma viride, 47) isolated from soil previously used for wheat and Persian clover mixed-culture systems to assess their behavior in the presence of the compounds. The microorganisms were exposed to glucosylated benzoxazinones, the benzoxazinones HBOA, DIBOA, and DIMBOA, the benzoxazolinones BOA, BOA-6-OH, and MBOA, and to several downstream products (AP, AAP, oHPMA, glucoside carbamate) in liquid culture to avoid interferences with soil minerals and other organisms. The microorganisms differed strongly in their metabolic activities in terms of growth, compound modification, and degradation. We observed degradation with DIBOA and GDIMBOA but rarely with DIMBOA, whereas BOA and MBOA showed almost no degradation when directly applied. Hydroxylation of BOA and demethylation of MBOA by the plant, resulting in BOA-6-OH, activated the benzoxazolinones for bacterial nitration. The resulting NBOA-6-OH was short-lived but could function temporarily as an allelochemical by inhibiting photosynthesis, e.g., in young seedlings of cress and kohlrabi. The BOA downstream products AP and oHPMA were converted to AAP, which can be nitrated to N-(2-OH-5-nitrophenyl)-acetamide and then degraded by A. aminovorans (49) and P. polymyxa (51). Only P. sepedonioides (12) and P. polymyxa (51) failed in the conversion of HBOA into AAP. While DIBOA, DIMBOA, MBOA, NBOA-6-OH, AP, AAP, and oHPMA reduced the growth of most microorganisms, glucoside carbamate promoted their growth. GDIMBOA had a stimulatory effect toward the fungi and three bacterial species. These findings lead to the hypothesis that in a natural habitat, such as the root surface, microorganisms may cooperate, perhaps by involving the plant, for the successful elimination of benzoxazinoids and their downstream metabolites.

Land plants are engaged in intricate communities with soil bacteria and fungi indispensable for plant survival and growth. The plant-microbial interactions are largely governed by specific metabolites. We employed a combination of lipid-fingerprinting, enzyme activity assays, high-throughput DNA sequencing and isolation of cultivable microorganisms to uncover the dynamics of the bacterial and fungal community structures in the soil after exposure to isothiocyanates (ITC) obtained from rapeseed glucosinolates. Rapeseed-derived ITCs, including the cyclic, stable goitrin, are secondary metabolites with strong allelopathic affects against other plants, fungi and nematodes, and in addition can represent a health risk for human and animals. However, the effects of ITC application on the different bacterial and fungal organisms in soil are not known in detail. ITCs diminished the diversity of bacteria and fungi. After exposure, only few bacterial taxa of the Gammaproteobacteria, Bacteriodetes and Acidobacteria proliferated while Trichosporon (Zygomycota) dominated the fungal soil community. Many surviving microorganisms in ITC-treated soil where previously shown to harbor plant growth promoting properties. Cultivable fungi and bacteria were isolated from treated soils. A large number of cultivable microbial strains was capable of mobilizing soluble phosphate from insoluble calcium phosphate, and their application to Arabidopsis plants resulted in increased biomass production, thus revealing growth promoting activities. Therefore, inclusion of rapeseed-derived glucosinolates during biofumigation causes losses of microbiota, but also results in enrichment with ITC-tolerant plant microorganisms, a number of which show growth promoting activities, suggesting that Brassicaceae plants can shape soil microbiota community structure favoring bacteria and fungi beneficial for Brassica plants.

The success of allelopathic plants is characterized by the releases of allelochemicals that suppress the growth of receiver plants directly and indirectly, due to concomitant effects on their microbiome. Therefore, negative effects of allelochemicals on microorganisms can enhance repercussions on plants. On the other hand, plants exposed to allelochemicals develop strategies to eliminate, detoxify or degrade the compounds, whereby plants can take advantage of metabolic properties of microorganisms associated with the root surface and the rhizosphere. Peroxidases and glucosyltransferases have important functions in those processes. In this chapter, methods are presented which allow an estimation of the extent of microbiome damage due to allelochemicals (signature lipid biomarker analysis combined with next generation sequencing). Assays for the determination of glucosyltransferases and peroxidases, important enzyme classes in detoxification processes, are presented with emphasis on benzoxazolinone detoxification as an example for the involvement of microorganisms in reaction sequences. Finally, methods to study alterations in the composition of cell wall polymers are presented as cell wall polymers can be modified during detoxification reactions.

Pantoea ananatis is a bacterium associated with other microorganisms on Abutilon theophrasti Medik. roots. It converts 6-hydroxybenzoxazolin-2(3H)-one (BOA-6-OH), a hydroxylated derivative of the allelochemical benzoxazolin-2(3H)-one, into 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one. The compound was identified by NMR and mass spectrometric methods. In vitro synthesis succeeded with Pantoea protein, with isolated proteins from the Abutilon root surface or with horseradish peroxidase in the presence of nitrite and H2O2. Nitro-BOA-6-OH is completely degraded further by Pantoea ananatis and Abutilon root surface proteins. Under laboratory conditions, 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one inhibits Lepidium sativum seedling growth whereas Abutilon theophrasti is much less affected. Although biodegradable, an agricultural use of 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one is undesirable because of the high toxicity of nitro aromatic compounds to mammals.

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.

6-Methoxy-benzoxazolin-2(3 H)-one (MBOA) is a degradation product derived from 2,4-Dihydroxy-7-methoxy-2 H-1,4-benzoxazin-3(4 H)-one (DIMBOA), one of the bioactive compounds found e.g., in maize. Here we present hitherto unknown 6-methoxy-4-nitro-benzoxazolin-2(3 H)-one (NMBOA) produced in Czapek medium by Pantoea ananatis (Enterobacteriaceae). P. ananatis is a member of a microbial consortium dominated by the zygomycete Actinomucor elegans, which was isolated from roots of Abutilon theophrasti. NMBOA was identified by NMR spectra and HR-ESI-MS analyses, revealing an unusual position of the nitro group at C-4. Nitration of MBOA initiates the degradation of the compound that is almost completed within three days by the entire consortium and isolated P. ananatis. The yeast Papiliotrema baii, another member of the consortium, is unable to degrade NMBOA but stored it at the surface of its polysacchararide capsule. NMBOA has negative effects on microbial growth in liquid medium whereas seedlings of Brassica oleracea var. gongylodes L. (kohlrabi) or Lepidium sativum (cress) are not impaired up to 500 μM. Degradation via nitration may be important to understand the behavior of microbial species and effects of microbiomes when exposed to MBOA.

Sulphur (S) has become a major limiting factor for plant production in industrial as well as in remote industrial rural areas. Limitation of S can reduce legume N2 fixation by affecting nodule development and function. In pot experiments with pea (Pisum sativum L.) and alfalfa (Medicago sativa L.), we investigated the influence of S on growth, ferredoxin, ATP and leghemoglobin concentrations. Addition of 200 mg S pot−1 increased yield of shoots, roots and nodules of both plant species significantly. However, the influence of S on nodule yield formation was most pronounced. Pea and alfalfa roots were found to have higher S concentrations than shoots and being up to 2.9 times the S concentration in the shoots of peas under S-sufficient conditions. Sulphur addition also increased N2 fixation significantly. The ferredoxin concentration in bacteroids of root nodules of pea was increased significantly by S only 10 weeks after planting and in bacteroids of root nodules of alfalfa 10 and 17 weeks after planting, while on per pot base the amounts of ferredoxin were higher throughout the experimental period of time. The ATP concentration of bacteroids of root nodules of both plant species as well as of mitochondria of root nodules of pea were significantly higher with optimum S supply. The effects of S deficiency on N2 fixation are likely to be caused by the shortage of ferredoxin and ATP. The amount of leghemoglobin was reduced in comparison to nodules of the S-sufficient plants.

Doratomyces stemonitis (Hyphomycetales, Dematiaceae) is a saprotrophic fungus belonging to the mycobiota of the cereal rhizosphere. The fungus is able to metabolize benzoxazolin-2-(3H)-one and a variety of its derivatives including higher plant detoxification products, microbial degradation products and the chemically rather stable 2-amino-(3H)-phenoxazin-3-one. D. stemonitis can use all of these compounds as sole C-sources but their utilization, especially that of microbial degradation products and 2-amino-(3H)-phenoxazin-3-one, seems to be highly energy consuming, resulting in slow mycelium growth and a change of colony morphology. Benzoxazolin-2-(3H)-one derived compounds induce the synthesis of different isoforms of a glycosylated protein with sequence homologies to the endo-1,3-β-glucanase Asp f2, an allergen from Aspergillus fumigatus and other Asp f2-like proteins e.g., from Verticillium dahliae or PRA1 antigen from Candida albicans. The induction of the protein is regarded as a stress response.

Benzoxazolinone detoxification is similar in plants grown under sulfur deficiency conditions and in control plants grown with an optimal S supply. However, when S-deficient plants were treated with metolachlor before benzoxazolin-2(3H)-one (BOA) incubation, detoxification was reduced, as indicated by a lower accumulation of the detoxification products BOA-6-O-glucoside and glucoside carbamate and by a loss of BOA-6-OH glucosyltransfease activity. Root colonizing microorganisms and the endophytic fungus Fusarium verticillioides participated in benzoxazolinone detoxification by converting the compound to 2-acetamidophenol (AAP) or O-hydroxyphenyl malonamic acid (OHPMA), a process accompanied by the appearance of phenoxazinone. Maize roots, however, absorbed AAP and OHPMA only in traces. Absorbed traces of OHPMA stimulated maize radicle growth, and traces of AAP stimulated cress. Phenoxazinone inhibited the growth of cress radicles at concentrations higher than 500 microM, whereas maize radicles were hardly affected. F. verticillioides did not convert benzoxazolinone to any known microbial degradation product when the medium of maize seedlings was inoculated with the fungus under sterile condition. Plant-fungus interactions seem to be important in plant survival of allelopathic attacks. This study points to a complicated network of allelopathic interactions that are influenced by biotic and abiotic factors, including herbicides.

In the present study, the influence of S supply on S concentrations, N2 fixation, available amounts of glucose and adenylates of Vicia faba minor L. were weekly investigated, starting 6 weeks after sowing. Glucose was determined photometrically in shoots, roots and nodules and in ATP, ADP and AMP by bioluminescence in roots, mitochondria and bacteroids. Sulphur deficiency resulted in significantly lower S concentrations of shoots, roots and nodules, in a reduced N2 fixation as well as in significantly lower amounts of glucose in shoots and nodules. In roots and bacteroids, S deficiency resulted in lower ATP concentrations, while the influence of S supply on ADP and AMP was less pronounced. With optimum S supply, the available amounts of glucose and ATP were strongly influenced by flower formation and seed development.

Protein extracts from Tobacco mosaic virus (TMV) infected young tobacco leaves exhibiting strong symptoms contain higher amounts of ubiquitin conjugates in comparison with non-infected control plants presumably due to stress reactions of host cells. Western blots with antibodies against TMV indicate as well an increase in coat protein or in ubiquitinated coat protein subunits. Immunogold labelling of infected leaf material revealed the accumulation of large amounts of coat protein in opaque inclusions which also reacted strongly with ubiquitin antibodies. These inclusions appear in close vicinity to chloroplasts in chlorotic areas of the leaf. It is concluded that most of the ubiquitin conjugates found in Western blots are due to ubiquitinated coat protein. As a consequence, a considerable amount of coat protein cannot be used for correct capsid construction as the stabilizing lysine residues are blocked by ubiquitination.

It is assumed that arylsulfatase contributes to the mineralization of organic S to SO42− for plant uptake. However, the impacts of agronomic measures (long-term organic manure) or crop species on enzyme activity are not well understood. Therefore, in the present investigations arylsulfatase activity was quantified within the rhizosphere of Sinapis album, Lolium perenne, Triticum aestivum and Brassica napus, grown on three different soils from a long-term field experiment. Highest activities were found within 0.25 mm from the root surface. Highest over all activities were found with B. napus and T. aestivum, lowest with S. album and L. perenne at a distance of 1.75 mm. As compared to soil supplemented with mineral fertilizer, compost and farmyard manure, respectively, arylsulfatase activity was higher in the soil with long-term compost application. In the protein extracts of sterile-grown roots a low arylsulfatase activity could be detected. The enzyme is induced under sulfate deficiency conditions. Preliminary biochemical data obtained from studies with T. aestivum arylsulfatase indicate conformity with biochemical properties of arylsulfatases originated from micro-organisms, vertebrates and invertebrata. From our results we assume that arylfulfatase activity determined in roots of sterile-grown plants is derived from endophytic bacteria and not by higher plants.

Abutilon theophrasti Medik., previously found to be rather insensitive to benzoxazinoid containing rye mulch and the allelochemical benzoxazolin-2(3H)-one (BOA), can be associated with the zygomycete Actinomucor elegans, whereby the fungus colonizes the root relatively superficially and mainly in the maturation zone. The fungus mitigates necrosis of the cotyledons when seedlings are incubated with 2 mM BOA, in contrast to those that lack the fungus. In liquid cultures of the fungus, tryptophan was identified. The accumulation of tryptophan is increased in presence of BOA. This amino acid seems to be important in protecting Abutilon against BOA and its derivatives since it suppressed the accumulation of BOA derived, highly toxic 2-aminophen-oxazin-3-one (APO) in the medium and on the root surface during BOA incubations of Abutilon seedlings. Although A. elegans is insensitive to BOA and APO, the fungus is not able to protect the plant against harmful effects of APO, when seedlings are treated with the compound. Abutilon can detoxify BOA via BOA-6-OH glucosylation probably by a cell wall associated glucosyltransferase, but only low amounts of the product accumulate. Low tryptophan concentrations can contribute to a degradation of the toxic intermediate BOA-6-OH by Fenton reactions, whereby the amino acid is oxidized. One of the oxidation products was identified as 4(1H)-quinolinone, which is the core substructure of the quorum sensing molecule 2-heptyl-3-hydroxy-4-quinolone. The mutualistic association of Abutilon theophrasti with Actinomucor elegans is considered as opportunistic and facultative. Such plant-fungus associations depend rather likely on environmental conditions, such as the mode of fertilization.

Western blots with anti-actin and anti-tubulin antibodies revealed an increase of both proteins upon 20–40 min of boron deprivation in roots of hydroponically grown Arabidopsis thaliana.Moreover,actin depolymerizing factor and myosin VIII showed a less pronounced but similiar response to boron deficiency. This is the first report of cytoskeletal responses of plants to short-term boron removal.

The capacity of benzoxazolinone metabolization of dicotyledonous species characteristic for the former vegetation classes Secalietea (grain field weed communities) and Chenopodietea (hoed vegetable communities) was estimated by the production of BOA-6-OH, BOA-6-β-O-glucoside, and BOA-N-glucoside. Except for Urtica urens, Galinsoga ciliata, and Polygonum aviculare (Chenopodietea), all species tested were able to synthesize BOA-6-OH, its glucoside, and BOA-N-glucoside, but effectiveness of BOA metabolism differed highly depending on species and plant organ. There was no correlation between bacterial phenoxazinone production and appearance of metabolites in the plants. Bioassays demonstrated that N-glucosylation is more efficient in BOA detoxification than O-glucosylation. The intermediate BOA-6-OH, however, is more harmful than BOA itself. It is therefore assumed that the ability to synthesize BOA-N-glucoside reduces the sensitivity to BOA strikingly. Since the detoxification capacity did not correlate with the taxonomic position, the affiliation of the species tested with the corresponding plant communities was taken into consideration. Evidently, the ecobiochemical potential of species to detoxify benzoxazolinone, regarded as an essential secondary compound in rye and wheat fields, reflects their occurrence in those plant associations. The ability to cope with the compound could be the result of co-evolutionary processes and presents a hidden aspect of allelopathic interaction.

Another detoxification product of Avena sativa roots incubated with the natural phytotoxin benzoxazolin-2(3 H )-one was isolated from their methanolic extracts and identified as 3-β- d -glucopyranosyl-benzoxazolin-2(3 H )-one. The structure was proven by synthesis. The ability of oat to produce the N-glucoside is supposed to be one reason for a less pronounced sensitivity to the phytotoxin. Methanolic extracts of Avena sativa roots previously incubated with the natural phytotoxin benzoxazolin-2(3 H )-one contain, in addition to two already described detoxification products, a third one of hitherto unknown structure. The compound has now been identified by NMR and mass spectral methods as 3-β- d -glucopyranosyl-benzoxazolin-2(3 H )-one. This structure was confirmed by an independent synthesis. The ability of oat to produce the N-glucoside is supposed to be one reason for a less pronounced sensitivity to the phytotoxin.

The action of 2,4-dihydroxy-1,4-benzoxazin-3-ones and their corresponding benzoxazolinone (BOA) derivatives on the activity of plasma membrane H + -ATPase from roots of Avena sativa and Vicia faba was investigated. Significant inhibitory effects were found with 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one in concentrations of 0.25 mM or higher. Benzoxazolinone compounds caused a weaker inhibition of the enzyme. At low effector concentrations stimulatory effects were observed. The H + -ATPase activity of A. sativa and V. faba was reduced by DIBOA to the same extent. The allelopathic effects of DIBOA and BOA on radicle elongation of A. sativa are correlated with the H + -ATPase activity.

Two dimensional gel electrophoresis of total cell protein extracts from not expanded, and primary leaves, petioles, and roots ofVicia faba resulted in four actin isoforms at 43 kDa with pI values from 5.9 to 6.05. In contrast to root extracts, in all leaf extracts an additional immunoreactive polypeptide with a molecular mass of 51 kDa and pI 5.75 was detected. This polypeptide was present in high amounts in protein extracts of purified chloroplasts, whereas no actin isoform at 43 kDa could be demonstrated. Compared to the tissue extracts, two actin isoforms at 43 kDa with pI values of 5.9 and 6.0 were enriched, when purified plasma membranes and the membranous fraction of vacuoles were analysed. In contrast, the soluble protein fraction of the plasma membrane preparation contained only two isoactins with pI values of 5.95 and 6.05 and a molecular mass of 43 kDa. These results indicate, that the four actin isoforms at 43 kDa detected in all examined tissues ofV. faba fulfill different functions at specific intracellular compartments, for example, the anchorage of actin microfilaments to membranes.

The small polypeptide ubiquitin participates in a variety of fundamental cellular events, such as cell differentiation, stress response, determination of steady state levels of regulatory proteins, cell cycle control, regulation of transcription, and programmed cell death. Although the complex mechanisms of these processes are not fully understood, ubiquitinylation of regulatory proteins involved in those events is obviously essential. Target proteins can be covalently coupled with one or a few ubiquitin molecules, which is supposed to present a (reversible) post-translational modification. Polyubiquitinylation, however, marks proteins selectively for degradation by the 26S proteasome. The ubiquitin system has been studied mostly with animal systems or yeast, but all basic reactions of ubiquitin appear in plants as well. The scope of this review is to summarize several implications of recent studies directed towards the plant ubiquitin system.

The allelopathic potency of rye (Secale cereale L.) is due mainly to the presence of phytotoxic benzoxazinones— compounds whose biosynthesis is developmentally regulated, with the highest accumulation in young tissue and a dependency on cultivar and environmental influences. Benzoxazinones can be released from residues of greenhouse-grown rye at levels between 12 and 20 kg/ha, with lower amounts exuded by living plants. In soil, benzoxazinones are subject to a cascade of transformation reactions, and levels in the range 0.5–5 kg/ha have been reported. Starting with the accumulation of less toxic benzoxazolinones, the transformation reactions in soil primarily lead to the production of phenoxazinones, acetamides, and malonamic acids. These reactions are associated with microbial activity in the soil. In addition to benzoxazinones, benzoxazolin-2(3H)-one (BOA) has been investigated for phytotoxic effects in weeds and crops. Exposure to BOA affects transcriptome, proteome, and metabolome patterns of the seedlings, inhibits germination and growth, and can induce death of sensitive species. Differences in the sensitivity of cultivars and ecotypes are due to different species-dependent strategies that have evolved to cope with BOA. These strategies include the rapid activation of detoxification reactions and extrusion of detoxified compounds. In contrast to sensitive ecotypes, tolerant ecotypes are less affected by exposure to BOA. Like the original compounds BOA and MBOA, all exuded detoxification products are converted to phenoxazinones, which can be degraded by several specialized fungi via the Fenton reaction. Because of their selectivity, specific activity, and presumably limited persistence in the soil, benzoxazinoids or rye residues are suitablemeans for weed control. In fact, rye is one of the best cool season cover crops and widely used because of its excellent weed suppressive potential. Breeding of benzoxazinoid resistant crops and of rye with high benzoxazinoid contents, as well as a better understanding of the soil persistence of phenoxazinones, of the weed resistance against benzoxazinoids, and of how allelopathic interactions are influenced by cultural practices, would provide the means to include allelopathic rye varieties in organic cropping systems for weed control.

Margot Schulz1, Dieter Sicker2, Frantisek Baluska3, Tina Sablofski1,3, Heinrich W. Scherer4 and Felix Martin Ritter1 1University of Bonn, Institute of Molecular Physiology and Biotechnology of Plants (IMBIO) 2University of Leipzig, Institute of Organic Chemistry 3Institute of Cellular and Molecular Botany (IZMB) 4Institut fur Nutzpflanzenwissenschaften und Ressourcenschutz(INRES) Plant Nutrition Germany

A recent greenhouse study revealed a significant reduction of germination and growth of redroot pigweed (Amaranthus retroflexus) and common purslane (Portulaca oleracea) by rye mulch, whereas velvetleaf (Abutilon theophrasti) and common lambsquarters (Chenopodium album) were not suppressed. Since BOA detoxification by metabolic alteration may influence the relation between the benzoxazinoid content of the soil mulch and weed suppression, we tested the dynamics in BOA detoxification in different plant organs of three and 10-day-old seedlings of four warm season weeds incubated with five BOA concentrations (4, 20, 40, 80, and 200 μmol g−1 fresh weight). In addition, germination and length of 3-day-old seedlings were measured after exposure to 0, 0.3, 1.5, 3, 6, and 15 μmol BOA. Finally, we tested the influence of the MDR translocator inhibitors verapamil, nifedipine, and the GST inhibitor ethycrynic acid on BOA accumulation and detoxification activity. Due to BOA-detoxification, all weeds were able to grow in environments with low BOA contents. At higher contents, Abutilon theophrasti and Chenopodium album had a better chance to survive because of highly active mechanisms that avoided the uptake of BOA (A. theophrasti) and of efficient detoxification activities in youngest seedlings (C. album). The interpretation of all of the data gave the following sequence of increasing sensitivity: A. theophrasti <<< C. album << P. oleracea ≤ A. retroflexus. The results were in agreement with recent findings of the suppression of these weeds by rye mulches and their benzoxazinoid contents. Our studies demonstrate for the first time that the detoxification of BOA influences the survival of certain weeds in environments enriched with this allelochemical. Therefore, detoxification processes affect the potential for weed suppression by soil allelochemicals in sustainable weed management.

Different tissues (leaf lamina and midveins, epidermis, primary roots, etiolated shoots, hypocotyl, petioles) and protoplasts from mesophyll, guard cells, and petioles of Vicia faba L., obtained from defined developmental stages were analyzed with polyclonal antibodies raised against mouse liver connexins 26 and 32. Immunostaining after treatment with CX 26 antibodies showed two polypeptides of 21 and 16 kD in all tissues examined except for hypocotyl and epidermis. Additionally, a stronger immunoresponse at 40 kD occurred in extracts from leaf material, mesophyll protoplasts, and roots. Incubation with the CX 32 antibodies resulted in an immunoreactive band at 29 kD in mesophyll protoplast samples, and, a very weak band in extracts from leaf material. A 36 kD polypeptide, present in samples prepared from root, etiolated shoots, petioles, and midveins, did not appear in leaf laminas and mesophyll protoplasts. Extracts from guard cells did not show any immunoreactive band. The tissue- and cell-specific distribution of CX 23- and CX 26-related proteins is supposed to reflect diversities in physiological functions together with alterations in plasmodesmatal ultrastructures during development.

Summary Membrane-bound enzyme activities in microsomal membrane fractions and the ubiquitin-protein-conjugate pattern of Vicia faba L. extracts were analyzed after clinostat treatment for 15 and 24 h and compared with a vertically rotated control. The activity of vanadate-sensitive ATPase was enhanced by clinostat treatment. The effect could be found in extracts of the youngest, not fully expanded leaves after 24 h of treatment, but as well as after 15 h of rotation and in primary leaf extracts. In addition, 24 h of clinostat rotation led to an increase in nitrate-sensitive ATPase activity and Antimycin A-insensitive NADH-cytochrome c reductase activity. No difference in latent IDPase activity was observed. The ubiquitin-protein-conjugate pattern of total cellular protein was changed by 24 h of clinostat treatment as revealed by SDS-PAGE and western-immunoblot analysis. The results indicate that the membrane system of leaf cells is influenced by omnilateral gravistimulation. Similar to the soluble and thylakoid membrane proteins an altered ubiquitination pattern points to a changed protein turnover.