Analysing intracellular isoprenoid metabolites in diverse prokaryotic and eukaryotic microbes.

Isoprenoids, also known as terpenes or terpenoids, are a very large and diverse group of natural compounds. These compounds fulfil a myriad of critical roles in biology as well as having a wide range of industrial uses. Isoprenoids are produced via two chemically distinct metabolic pathways, the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. Downstream of these two pathways is the shared prenyl phosphate pathway. Because of their importance in both basic physiology and industrial biotechnology, extraction, identification, and quantification of isoprenoid pathway intermediates is an important protocol. Here we describe methods for extraction and analysis of intracellular metabolites from the MVA, MEP, and prenyl phosphate pathways for five key model microbes: the yeast Saccharomyces cerevisiae, the bacterium Escherichia coli, the diatom Phaeodactylum tricornutum, the green algae Chlamydomonas reinhardtii, and the cyanobacterium Synechocystis sp. PCC 6803. These methods also detect several central carbon intermediates. These protocols will likely work effectively, or be readily adaptable, to a variety of related microorganisms and metabolic pathways.

Analysis of glyphosate and aminomethylphosphonic acid in leaves from Coffea arabica using high performance liquid chromatography with quadrupole mass spectrometry detection.

Glyphosate is a commonly applied herbicide in coffee plantations. Because of its non-selective mode of action it can damage the crop exposed through spray drift. Therefore, it is of interest to study glyphosate fate in coffee plants. The aim of this study was to develop an analytical method for accurate and precise quantification of glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) at trace levels in coffee leaves using liquid chromatography with single-quadrupole mass spectrometry detection. The method is based on a two-step solid phase extraction (SPE) with an intermediate derivatization reaction using 9-fluorenylmethylchloroformate (FMOC). An isotope dilution method was used to account for matrix effects and to enhance the confidence in analyte identification. The limit of quantification (LOQ) for glyphosate and AMPA in coffee leaves was 41 and 111 µg kg(-1) dry weight, respectively. For the method optimization a design of experiments (DOE) approach was used. The sample clean-up procedure can be simplified for the analysis of less challenging matrices, for laboratories having a tandem mass spectrometry detector and for cases in which quantification limits above 0.1 mg kg(-1) are acceptable, which is often the case for glyphosate. The method is robust, possesses high identification confidence, while being suitable for most commercial and academic laboratories. All leaf samples from five coffee fields analyzed (n=21) contained glyphosate, while AMPA was absent. The simplified clean-up procedure was successfully validated for coffee leaves, rice, black beans and river water.

Glyphosate spray drift in Coffea arabica - sensitivity of coffee plants and possible use of shikimic acid as a biomarker for glyphosate exposure.

Glyphosate is widely used in coffee plantations to control weeds. Lacking selectivity, glyphosate spray drift is suspected to cause adverse effects in coffee plants. Symptoms caused by glyphosate can be similar to those produced by other stress factors. However, shikimic acid accumulation should be a useful biomarker for glyphosate exposure as shown for other crops. The aim of this study was to assess the sensitivity of coffee plants towards glyphosate on different biological response variables and to evaluate the use of shikimic acid as biomarker. Dose-response experiments yielded ED50 values (50% effect dose) in the range of 38-550 ga.e.ha(-1) depending on the quantitative or qualitative variable monitored. The frequency of plants showing symptoms was the most sensitive variable. The best sampling time for shikimic acid accumulation was 1-2 weeks after glyphosate application, depending on experimental conditions. The highest shikimic acid accumulation was observed in young leaves. Shikimic acid is a suitable biomarker for a glyphosate exposure in coffee, using only young leaves for the analysis. Young coffee plants are susceptible to glyphosate damage. If symptoms are absent the risk of severe crop damage or yield loss is low.