The secreted redox sensor roGFP2-Orp1 reveals oxidative dynamics in the plant apoplast.

Specific generation of reactive oxygen species (ROS) is important for signalling and defence in many organisms. In plants, different types of ROS serve useful biological functions in the extracellular space (apoplast), influencing polymer structures as well as signalling during immune responses. The current knowledge of apoplastic ROS dynamics is limited, as dynamic monitoring of extracellular redox processes in vivo remains difficult. We employed evolutionary distant land plant model species from bryophytes and flowering plants to test whether the genetically encoded redox biosensor roGFP2-Orp1 can be used to assess extracellular redox dynamics. Secreted roGFP2-Orp1 can provide information about local diffusion barriers and protein cysteinyl oxidation rate in the apoplast, after pre-reduction. Observed re-oxidation rates were slow - within the range of hours. Compared to Physcomitrium patens, re-oxidation in Arabidopsis thaliana was faster and increased after triggering an immune response. Comparing roGFP2-Orp1 signals in tip-growing P. patens protonema and Nicotiana tabacum pollen tubes, we consistently find no intracellular redox gradient, but a partially reduced extracellular sensor in pollen tubes. Our data indicate differences in extracellular oxidative processes between species and within a species, depending on cell type and immune signalling.