Giuseppe Forlani

Degradation of the phosphonate herbicide glyphosate in soil: evidence for a possible involvement of unculturable microorganisms

Abstract The properties of microbial strains responsible for the rapid mineralization of the herbicide glyphosate in soil were investigated in soil–water mixtures supplemented with 10 mmol l −1 active ingredient. Over 2 weeks degradation kinetics were linear, as expected in the case of non-growth-linked metabolization, and the rate of utilization was not enhanced following repeated treatment of the soil with increasing herbicide doses. The availability of exceeding phosphorus, nitrogen and carbon sources did not affect the rate of glyphosate utilization, that was maximal under conditions of neutral pH, high oxygen and low osmolarity. The screening of 1200 bacterial strains isolated on a rich medium in the absence of the herbicide failed to identify any strain able to cleave the glyphosate molecule. When antibiotics with different mode of action were added to the mixtures, while some inhibitors of protein synthesis exerted considerable effects, those that are active only against actively-proliferating cells were scarcely effective. An MPN analysis was performed to enumerate degrading microorganisms, but in no dilution the same extent of utilization measured in the original mixture could be found. Results suggest that at least the first steps in herbicide degradation could be accomplished by some microbial species unable to grow in vitro and form visible colonies on plates.

Biochemical Bases for a Widespread Tolerance of Cyanobacteria to the Phosphonate Herbicide Glyphosate

Possible non-target effects of the widely used, non-selective herbicide glyphosate were examined in six cyanobacterial strains, and the basis of their resistance was investigated. All cyanobacteria showed a remarkable tolerance to the herbicide up to millimolar levels. Two of them were found to possess an insensitive form of glyphosate target, the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase. Four strains were able to use the phosphonate as the only phosphorus source. Low uptake rates were measured only under phosphorus deprivation. Experimental evidence for glyphosate metabolism was also obtained in strains apparently unable to use the phosphonate. Results suggest that various mechanisms may concur in providing cyanobacterial strains with herbicide tolerance. The data also account for their widespread ability to metabolize the phosphonate. However, such a capability seems limited by low cell permeability to glyphosate, and is rapidly repressed when inorganic phosphate is available.

Physiological implications of arginine metabolism in plants

Nitrogen is a limiting resource for plant growth in most terrestrial habitats since large amounts of nitrogen are needed to synthesize nucleic acids and proteins. Among the 21 proteinogenic amino acids, arginine has the highest nitrogen to carbon ratio, which makes it especially suitable as a storage form of organic nitrogen. Synthesis in chloroplasts via ornithine is apparently the only operational pathway to provide arginine in plants, and the rate of arginine synthesis is tightly regulated by various feedback mechanisms in accordance with the overall nutritional status. While several steps of arginine biosynthesis still remain poorly characterized in plants, much wider attention has been paid to inter- and intracellular arginine transport as well as arginine-derived metabolites. A role of arginine as alternative source besides glutamate for proline biosynthesis is still discussed controversially and may be prevented by differential subcellular localization of enzymes. Apparently, arginine is a precursor for nitric oxide (NO), although the molecular mechanism of NO production from arginine remains unclear in higher plants. In contrast, conversion of arginine to polyamines is well documented, and in several plant species also ornithine can serve as a precursor for polyamines. Both NO and polyamines play crucial roles in regulating developmental processes as well as responses to biotic and abiotic stress. It is thus conceivable that arginine catabolism serves on the one hand to mobilize nitrogen storages, while on the other hand it may be used to fine-tune development and defense mechanisms against stress. This review summarizes the recent advances in our knowledge about arginine metabolism, with a special focus on the model plant Arabidopsis thaliana, and pinpoints still unresolved critical questions.

Requirement of proline synthesis during Arabidopsis reproductive development

BackgroundGamete and embryo development are crucial for successful reproduction and seed set in plants, which is often the determining factor for crop yield. Proline accumulation was largely viewed as a specific reaction to overcome stress conditions, while recent studies suggested important functions of proline metabolism also in reproductive development. Both the level of free proline and proline metabolism were proposed to influence the transition to flowering, as well as pollen and embryo development.ResultsIn this study, we performed a detailed analysis of the contribution of individual proline biosynthetic enzymes to vegetative development and reproductive success in Arabidopsis. In contrast to previous reports, we found that pyrroline-5-carboxylate (P5C) synthetase 2 (P5CS2) is not essential for sexual reproduction although p5cs2 mutant plants were retarded in vegetative development and displayed reduced fertility under long-day conditions. Single mutant plants devoid of P5CS1 did not show any developmental defects. Simultaneous absence of both P5CS isoforms resulted in pollen sterility, while fertile egg cells could still be produced. Expression of P5C reductase (P5CR) was indispensable for embryo development but surprisingly not needed for pollen or egg cell fertility. The latter observation could be explained by an extreme stability of P5CR activity, which had a half-life time of greater than 3 weeks in vitro. Expression of P5CR-GFP under the control of the endogenous P5CR promoter was able to restore growth of homozygous p5cr mutant embryos. The analysis of P5CR-GFP-fluorescence in planta supported an exclusively cytoplasmatic localisation of P5CR.ConclusionsOur results demonstrate that potential alternative pathways for proline synthesis or inter-generation transfer of proline are not sufficient to overcome a defect in proline biosynthesis from glutamate during pollen development. Proline biosynthesis through P5CS2 and P5CR is limiting for vegetative and reproductive development in Arabidopsis, whereas disruption of P5CS1 alone does not affect development of non-stressed plants.

Differential sensitivity of plant-associated bacteria to sulfonylurea and imidazolinone herbicides

The side effects of sulfonylurea and imidazolinone herbicides on plant-associated bacteria were investigated under pure culture conditions. Eighteen isolates, belonging to the genera Azotobacter, Azospirillum, Bacillus, Enterobacter Pseudomonas and Serratia, were exposed to four active compounds at concentration ranges similar to those in field soil. The sulfonylureas chlorsulfuron and rimsulfuron inhibited the growth of one of two Azospirillum and one of four Pseudomonas strains, while the imidazolinones imazapyr and imazethapyr were effective on two out of five Bacillus isolates. Surfactants in commercial formulation significantly enhanced rimsulfuron toxicity. With the exception of one Azospirillum strain, the differential tolerance of rhizobacteria to these herbicides was related to a differential sensitivity of their target, the activity of the first enzyme in branched-chain amino acid biosynthesis, acetohydroxyacid synthase (AHAS).Greenhouse pot studies were performed to assess the occurrence of inhibitory effects on bacterial growth in field conditions. Maize seedlings were bacterized with the two strains which had shown in vitro sensitivity to sulfonylureas. Following the application to the soil of a commercial formulation of rimsulfuron at rates of 0, 0.2 and 0.5 μmol a.i. kg−1, significative differences in the resulting degree of bacterial root colonization were found. Moreover, upon co-inoculation with two strains, one tolerant and one sensitive to the herbicide, the presence of rimsulfuron significantly enhanced root occupancy by resistant bacteria, suggesting that shifts in the microbial community structure of crop rhizosphere could indeed result as a consequence of weed control by AHAS inhibitors.

Tailoring the Structure of Aminobisphosphonates To Target Plant P5C Reductase

Using the structure of (3,5-dichlorophenyl)aminomethylenebisphosphonic acid as a lead compound, 25 new phosphonates were synthesized and evaluated as possible inhibitors of Arabidopsis thaliana delta1-pyrroline-5-carboxylate (P5C) reductase. Derivatives substituted in the phenyl ring retained the inhibitory potential, though to a different extent. On the contrary any variation in the scaffold, i.e., the replacement of the second phosphonate moiety with a hydroxyl or an amino residue, resulted in a significant loss of biological activity. The availability of several structures capable of interfering with the catalytic mechanism in the micromolar to millimolar range allowed a proper structure-activity relationship analysis, leading us to hypothesize about the steric and electronic requirements for maintenance or enhancement of the inhibitory properties. Reversal experiments with suspension cultured cells provided evidence for the occurrence of enzyme inhibition in vivo. Because in higher plants the step catalyzed by P5C reductase is shared by all pathways leading to proline synthesis, these compounds may be exploited for the design of new substances endowed with herbicidal activity.

Synthesis of Photosynthesis-Inhibiting Nostoclide Analogues

A series of 34 3-benzyl-5-(arylmethylene)furan-2(5H)-ones, designed using the naturally occurring toxins nostoclides as a lead structure, was synthesized as potential inhibitors of the photosynthetic electron transport. All compounds were fully characterized by IR, NMR (1H and 13C), and MS spectrometry. HMBC and HSQC bidimensional experiments allowed 13C and 1H assignments. Their biological activities were evaluated in vitro as the ability to interfere with light-driven reduction of ferricyanide by isolated spinach chloroplasts. About two-thirds of the compounds exhibited inhibitory properties in the micromolar range against the basal electron flow from water to K3[Fe(CN)6]. The inhibitory potential of these 3-benzyl-5-(arylmethylene)furan-2(5H)-one lactones is higher than that of other nostoclide analogues previously synthesized in the same laboratories.

Amino acid content and nectar choice by forager honeybees (Apis mellifera L.)

Dual choice feeding tests were performed to determine a preference of forager honeybees for specific amino acids. Artificial nectar containing proline was preferred over those containing only sugars. Nectar containing alanine was preferred on the first day, but preference was no longer significant thereafter. On the contrary, a negative response was found for serine. When the bees were given the choice between two nectars enriched with different compounds, proline was preferred above both alanine and serine, and alanine above serine.

A glyphosate-resistant 5-enol-pyruvyl-shikimate-3-phosphate synthase confers tolerance to a maize cell line

Abstract Among a few cell lines of maize ( Zea mays L., cv. Black Mexican Sweet (MS)) tested, one showed a natural, remarkable tolerance to glyphosate at concentrations as high as 10 mM. Two activities of 5- enol -pyruvyl-shikimate-3-phosphate (EPSP) synthase, the target enzyme of the herbicide, were separated in the tolerant culture by anion-exchange chromatography. One peak of activity was not significantly inhibited by glyphosate even at millimolar concentrations. While the glyphosate-sensitive isoform persisted throughout the growth cycle of the culture, the glyphosate-resistant EPSP synthase increased only after the onset of exponential growth and declined in the stationary phase. Tolerance to the herbicide was accompanied by a reduced affinity of the enzyme for the substrate phospho- enol -pyruvic acid.

Biochemical evidence for multiple acetoin-forming enzymes in cultured plant cells

Abstract Acetoin (3-hydroxy-2-butanon) production was investigated in extracts from suspension cultured cells of carrot, tobacco, maize and rice. Crude extracts were able to catalyze acetoin synthesis from pyruvate and/or acetaldehyde at rates ranging from 0.02 to 0.1 mkat kg −1 protein, while no evidence was found for acetolactate-deriving acetoin production. Three acetoin-forming enzymes were resolved upon adsorption chromatography. A minor peak of activity was deduced as due to a partial, non-enzymatic decarboxylation of the acetolactate produced by acetolactate synthase under the same experimental conditions, being completely abolished by the addition of an acetolactate synthase inhibitor. The other two activities were characterized following further purification by gel filtration chromatography. A low molar ratio between acetoin production and pyruvate utilization, the capability of producing acetaldehyde from pyruvate at higher rate, an optimal activity at acidic pH values and its increase in extracts from cells grown under hypoxic condition strongly suggested the former as a side reaction of pyruvate decarboxylase. The latter activity, which showed maximal rate at neutral pH values, was on the contrary found to quantitatively convert acetaldehyde and pyruvate to acetoin. This pyruvate carboligase, which increased in actively proliferating cells and declined in a late logarithmic phase and was not induced under anaerobiosis, was present at similar levels in all four plant species.