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...

Embedded within the meristem of all Angiosperm roots is a population of slowly dividing cells designated the quiescent center (QC). In maize roots the QC can constitute upwards of 800-1200 cells, most of which spend an extended period of time (180-200 hours) in the G 1 phase of the cell cycle. How the QC forms and is maintained is not known. Here we report that cells of the QC are characterized by their highly oxidized status. Glutathione and ascorbic acid occur predominately in the oxidized forms in the QC. This is contrasted with the status of these redox intermediates in adjacent, rapidly dividing cells in the root meristem, in which the reduced forms of these two species are favored. Using a redox sensitive fluorescent dye we were able to visualize an overall oxidizing environment in the QC, and we also made comparisons with the adjacent, rapidly dividing cells in the root meristem. Altering the distribution of auxin and the location of the auxin maximum in the root tip activates the QC, and cells leave G1 and enter mitosis. Commencement of relatively more rapid cell division in the QC is preceded by changes in the overall redox status of the QC, which becomes less oxidizing. We discuss how the position of the auxin maximum may influence the redox status of the QC and thereby modulate the cell cycle.

The establishment of the Angiosperm root apical meristem is dependent on the specification of a stem cell niche and the subsequent development of the quiescent center at the presumptive root pole. Distribution of auxin and the establishment of auxin maxima are early formative steps in niche specification that depend on the expression and distribution of auxin carriers. Auxin specifies stem cell niche formation by directly and indirectly affecting gene activities. Part of the indirect regulation by auxin may involve changes in redox, favoring local, oxidized microenvironments. Formation of a QC is required for root meristem development and elaboration. Many signals likely pass between the QC and the adjacent root meristem tissues. Disappearance of the QC is associated with roots becoming determinate. Given the many auxin feedback loops, we hypothesize that roots evolved as part of an auxin homeostasis mechanism.

Recent studies suggest that intercellular transport via plasmodesmata (PD) is regulated by cellular redox state. Until now, this relationship has been unclear, as increased production of reactive oxygen species (ROS) has been associated with both increased and decreased intercellular transport via PD. Here, we show that silencing two genes that both increase transport via PD, INCREASED SIZE EXCLUSION LIMIT1 (ISE1) and ISE2, alters organelle redox state. Using redox-sensitive green fluorescent proteins targeted to the mitochondria or plastids, we show that, relative to wild-type leaves, plastids are more reduced in both ISE1-and ISE2-silenced leaves, whereas mitochondria are more oxidized in ISE1-silenced leaves. We further show that PD transport is positively regulated by ROS production in mitochondria following treatment with salicylhydroxamic acid but negatively regulated by an oxidative shift in both chloroplasts and mitochondria following treatment with paraquat. Thus, oxidative shifts in the mitochondrial redox state positively regulate intercellular transport in leaves, but oxidative shifts in the plastid redox state counteract this effect and negatively regulate intercellular transport. This proposed model reconciles previous contradictory evidence relating ROS production to PD transport and supports accumulating evidence that mitochondria and plastids are crucial regulators of PD function.