Scrutinizing the Impact of Alternating Electromagnetic Fields on Molecular Features of the Model Plant Arabidopsis thaliana.

Natural and anthropogenic electromagnetic fields (EMFs) are ubiquitous in the environment and interfere with all biological organisms including plants. Particularly the quality and quantity of alternating EMFs from anthropogenic sources are increasing due to the implementation of novel technologies. There is a significant interest in exploring the impact of EMFs (similar to those emitted from battery chargers of electric cars) on plants. The model plant Arabidopsis thaliana was exposed to a composite alternating EMF program for 48 h and scrutinized for molecular alterations using photosynthetic performance, metabolite profiling, and RNA sequencing followed by qRT-PCR validation. Clear differences in the photosynthetic parameters between the treated and control plants indicated either lower nonphotochemical quenching or higher reduction of the plastoquinone pool or both. Transcriptome analysis by RNA sequencing revealed alterations in transcript amounts upon EMF exposure; however, the gene ontology groups of, e.g., chloroplast stroma, thylakoids, and envelope were underrepresented. Quantitative real-time PCR validated deregulation of some selected transcripts. More profound were the readjustments in metabolite pool sizes with variations in photosynthetic and central energy metabolism. These findings together with the invariable phenotype indicate efficient adjustment of the physiological state of the EMF-treated plants, suggesting testing for more challenging growth conditions in future experiments.

Biochemical Characterization of 13-Lipoxygenases of Arabidopsis thaliana.

13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (Vmax app) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s-1·mg protein-1), with a Km value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg2+, Ca2+, Cu2+ and Cd2+, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.

Förster Resonance Energy Transfer Measurements in Living Plant Cells.

Sensitized emission-based Förster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.