Förster resonance energy transfer (FRET) describes excitation energy exchange between two adjacent molecules typically in distances ranging from 2 to 10 nm. The process depends on dipole-dipole coupling of the molecules and its probability of occurrence cannot be proven directly. Mostly, fluorescence is employed for quantification as it represents a concurring process of relaxation of the excited singlet state S1 so that the probability of fluorescence decreases as the probability of FRET increases. This reflects closer proximity of the molecules or an orientation of donor and acceptor transition dipoles that facilitates FRET. Monitoring sensitized emission by 3-Filter-FRET allows for fast image acquisition and is suitable for quantifying FRET in dynamic systems such as living cells. In recent years, several calibration protocols were established to overcome to previous difficulties in measuring FRET-efficiencies. Thus, we can now obtain by 3-filter FRET FRET-efficiencies that are comparable to results from sophisticated fluorescence lifetime measurements. With the discovery of fluorescent proteins and their improvement toward spectral variants and usability in plant cells, the tool box for in vivo FRET-analyses in plant cells was provided and FRET became applicable for the in vivo detection of protein-protein interactions and for monitoring conformational dynamics. The latter opened the door toward a multitude of FRET-sensors such as the widely applied Ca2+-sensor Cameleon. Recently, FRET-couples of two fluorescent proteins were supplemented by additional fluorescent proteins toward FRET-cascades in order to monitor more complex arrangements. Novel FRET-couples involving switchable fluorescent proteins promise to increase the utility of FRET through combination with photoactivation-based super-resolution microscopy.

No abstract

The integration of redox- and reactive oxygen species-dependent signaling and metabolic activities is fundamental to plant acclimation to biotic and abiotic stresses. Previous data suggest the existence of a dynamically interacting module in the chloroplast stroma consisting of cyclophilin 20-3 (Cyp20-3), O-acetylserine(thiol)lyase B (OASTL-B), 2-cysteine peroxiredoxins A/B (2-CysPrx) and serine acetyltransferase 2;1 (SERAT2;1). The functionality of this COPS module is influenced by redox stimuli and oxophytodienoic acid (OPDA), which is the precursor for jasmonic acid. The concept of an integrating function of these proteins in stress signaling was challenged by combining transcriptome and biochemical analyses in Arabidopsis mutants devoid of oastlB, serat2;1, cyp20-3 and 2-cysprxA/B, and wild-type (WT). Leaf transcriptomes were analyzed 6 h after transfer to light intensity 10-fold in excess of growth light or under growth light. The survey of KEGG-based gene ontology groups showed common upregulation of translation- and protein homeostasis-associated transcripts under control conditions in all mutants compared with WT. The results revealed that the interference of the module was accompanied with disturbance of carbohydrate, sulfur and nitrogen metabolism, and also citric acid cycle intermediates. Apart from common regulation, specific responses at the transcriptome and metabolite level linked Cyp20-3 to cell wall-bound carbohydrates and oxylipin signaling, and 2-CysPrx to photosynthesis, sugar and amino acid metabolism. Deletion of either OASTL-B or SERAT2;1 frequently induced antagonistic changes in biochemical or molecular features. Enhanced sensitivity of mutant seedlings to OPDA and leaf discs to NaHS-administration confirmed the presumed functional interference of the COPS module in redox and oxylipin signaling.