Assessing redox state and reactive oxygen species in circadian rhythmicity.

Redox homeostasis is an important parameter of cell function and cell signaling. Spatial and temporal alterations of redox state control metabolism, developmental processes, as well as acute responses to environmental stresses and stress acclimation. Redox homeostasis is also linked to the circadian clock. This chapter introduces methods to assess important redox parameters such as the low molecular weight redox metabolites glutathione and ascorbate, their amount and redox state, and H2O2 as reactive oxygen species. In vivo redox cell imaging is described by use of the reduction-oxidation sensitive green fluorescent protein (roGFP). Finally, on the level of posttranslational redox modifications of proteins, methods are shown to assess hyperoxidation of 2-cysteine peroxiredoxin and glutathionylation of peroxiredoxin IIE. The redox state of 2-cysteine peroxiredoxin has been identified as a transcription-independent marker of circadian rhythmicity.

Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells.

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 Ca(2+)-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.

The conformational bases for the two functionalities of 2-cysteine peroxiredoxins as peroxidase and chaperone.

2-Cysteine peroxiredoxins (2-CysPrxs) are ubiquitous and highly abundant proteins that serve multiple functions as peroxidases, chaperones, and thiol oxidases and in redox-dependent cell signalling. The chloroplast protein plays a role in seedling development and protection of the photosynthetic apparatus. This study aimed to unequivocally link conformation and function. To this end, a set of non-tagged site-directed mutagenized At2-CysPrx variants was engineered, which mimicked the conformational states and their specific functions: hyperoxidized form (C54D), reduced form (C54S, C176S), oxidized form (C54DC176K), phosphorylated form (T92D), reduced ability for oligomerization by interfering with the dimer-dimer interface (F84R) and a C-terminally truncated form [ΔC (-20 aa)]. These variants were fully or partly fixed in their quaternary structure and function, respectively, and were analysed for their conformational state and peroxidase and chaperone activity, as well as for their sensitivity to hyperoxidation. The presence of a His6-tag strongly influenced the properties of the protein. The ΔC variant became insensitive to hyperoxidation, while T92D and F84R became more sensitive. The C54D variant revealed the highest chaperone activity. The highest peroxidase activity was observed for the F84R and ΔC variants. Efficient interaction with NADP-dependent thioredoxin reductase C depended on the presence of Cys residues and the C-terminal tail. The results suggest that the structural flexibility is important for the switch between peroxidase and chaperone function and that evolution has conserved the functional switch instead of maximizing a single function. These variants are ideal tools for future conformation-specific studies in vivo and in vitro.

Elements of transcriptional machinery are compatible among plants and mammals.

In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.