Oregon 97403-1229 (J.R.) Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidationsensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are 2318 mV for the cytoplasm and 2362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.Cellular redox status influences many processes in plants, including apoptosis (Cai and Jones, 1999), oxidative defense mechanisms (Foyer and Noctor, 2005), senescence (Groten et al., 2005), allosteric control of enzyme activities, transcription and translation (Apel and Hirt, 2004), and a variety of signal transduction pathways (Drö ge, 2002;Ermak and Davies, 2002;Neill et al., 2002). Yet, as central as is redox status to these processes, the redox potentials (oxidationreduction potential) of living plant cells have rarely been measured during the occurrence of these activities (Renew et al., 2005). Rather, most often plant tissues are homogenized and the homogenates subsequently assayed, either with redox-sensing electrodes, or, by measuring the ratios of the reduced and oxidized forms of glutathione and ascorbate, the two principal redox regulators in living systems (Foyer and Noctor, 2003). Recently the redox state of plant tissues has also been assessed using the dyes 5-(and 6-) carboxy-2#, 7#-dichlorodihydrofluorescein diacetate (C-400; Jiang et al., 2003) and dihydrofluorescein diacetate (N. Smirnoff, personal communication). While such approaches allow one to sum the oxidized and reduced species, and thereby to infer the overall redox status of a tissue, it is not possible with these approaches to obtain a measure of redox potential at the time the events of interest are occurring. Moreover, whole tissue homogenization does not allow one to more finely resolve redox status within the various compartments and organelles comprising a typical plant cell, nor does this approach allow for an assessment of the redox status of the cell wall. As well, homogenizing a tissue precludes the possibility of monitoring dynamic changes of redox status, including reversibility. As a consequence, plant biologists lack knowledge of the rapidity of redox changes in plant cells.Rece...
