PerspectivesBotany

Sending ROS on a Bullet Train

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Science Signaling  29 Sep 2009:
Vol. 2, Issue 90, pp. pe60
DOI: 10.1126/scisignal.290pe60

Abstract

Plants have to contend with biotic stress, such as disease, mechanical wounding, and herbivory, as well as abiotic stress, such as heat, cold, and salinity. An early warning system for these threats would prevent or reduce the damage suffered by plants. Such a warning system should allow the signal to be rapidly generated and sent over long distances. The study of systemic signaling in plants has been a major scientific challenge. Reactive oxygen species (ROS) are among the systemic signals that have been proposed. Now, the exciting discovery that systemic ROS signaling is mediated by an NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase opens the door to understanding the molecular mechanisms that initiate and propagate a rapid systemic signal.

Plants possess complex signaling systems to survive and thrive under biotic and abiotic stresses. These signaling systems include generating and receiving intracellular signaling to trigger local responses and long-distance cell-to-cell signaling in response to diverse stimuli. Thus, rapid systemic signaling is important to alert undamaged cells about incoming danger and give the undamaged cells time to invoke suitable defense responses (1, 2). In plants, two types of signals are propagated over long distances: electrical and chemical signals. The electrical signals can be divided into action potentials (APs) and variation potentials (VPs) or slow-wave potentials (SWPs). APs are “all-or-nothing” transient membrane depolarizations and are usually considered to be a general stress signal (3, 4). VPs are non–self-perpetuating transient depolarizations of variable amplitudes that are released during wounding (3, 4). Both electrical signals may precede the slower chemical signals. The quest for plant systemic chemical signals has been intense, particularly in the field of plant immunity. Methyl jasmonate (MeJA), methyl salicylate (MeSA), and azelaic acid have been reported to be signals for systemic acquired resistance (57). Nevertheless, as is the case with other plant mobile signals, their roles still need to be verified. Previously, reactive oxygen species (ROS), particularly H2O2, have been proposed to be a systemic signal that is produced in response to various stimuli (8, 9). Now, Miller et al. have discovered that in Arabidopsis thaliana (thale cress), initiation and propagation of a signal requires the function of a respiratory burst oxidase homolog D (RbohD) gene, which encodes an NADPH oxidase (10).

ROS were once considered to be the unwanted byproduct of aerobic metabolism. Now, despite their toxicity, they are regarded as key signal molecules for regulating cell function and development (11). In plants, ROS play diverse roles from cell signaling to organ development, in processes such as plant immune responses, cell death, abiotic stress, stomatal closure, and root hair development (12, 13). Plant NADPH oxidase, also known as RBOH, is an important generator of ROS, particularly superoxide. Superoxide is highly reactive and is rapidly converted to H2O2 by the superoxide dismutase or chemically converted to other ROS. Hydrogen peroxide is less reactive than superoxide but is more water-soluble and, thus, better suited as a signaling molecule (14). Nevertheless, the cytotoxicity of ROS at high concentrations means that a delicate balance between ROS production and removal must be achieved for cell survival and signaling (15).

Previously, Mittler’s group developed an elegant system to monitor ROS concentrations in vivo by fusing the luciferase (Luc) reporter to the promoter of the gene Zat12, the expression of which is rapidly increased by wounding and oxidative stress in Arabidopsis (9, 16). In the present study, Miller et al. expressed Zat12::Luc in rbohD mutant and showed that RbohD is required for wounding-induced systemic ROS accumulation in the extracellular space at a site distal from the initial wounding (10). Rboh genes encode homologs of the catalytic subunit of phagocyte NADPH oxidase complex and have been isolated from many plant species, including rice, Arabidopsis, tobacco, tomato, and potato (12). Ten Rboh genes have been identified in the Arabidopsis genome (12). One might expect that functional redundancy among the Rboh genes would obscure the effect of rbohD deficiency on the wounding-induced systemic ROS accumulation. Surprisingly, microarray analysis indicates that ROS, particularly H2O2, did not induce systemic increases in Zat12 transcript in other rboh mutants (rbohF or rbohC), unlike in rbohD (10). Incidentally, the study also reaffirmed the notion that H2O2 is the key signal molecule in ROS signaling.

Although H2O2 is involved in many signaling pathways (17), one may ask, “How can a highly reactive molecule act as a long-distance signal?” Indeed, H2O2 would soon react with other molecules and the original signal would be lost, but not if the signal has an “auto-propagating” ability, as proposed by Miller et al. (10). A systemic signal does not need to be transported from the origin to a distal site if the signal from the originating cell can self-perpetuate after it diffuses or is transported into the neighboring cell. Therefore, after it is formed, the water-soluble H2O2 may enter a neighboring cell and rapidly find its target, which in turn activates RBOH to regenerate the H2O2 signal (Fig. 1). For such a signal relay to work well, the signal needs to be regenerated very rapidly. The authors also showed that ectopic application of H2O2 initiates systemic signaling, further supporting the notion that RBOHD activation is required for signal propagation. Surprisingly, this systemic signaling is independent of JA, SA, or ethylene, hormones that are known to be involved in stress and wounding responses and ROS production. However, this is possible if ROS act downstream of these hormones and if systemic signal propagation does not require these hormones. Miller et al. (10) also showed that RbohD-mediated systemic signaling is required for resistance to aphid infection. Therefore, resistance to aphid feeding may involve another signaling pathway that activates RBOHD.

Fig. 1

RBOHD-dependent ROS-mediated rapid systemic signaling. RBOH is activated by local stimuli to produce O2, which is rapidly dismutated to H2O2 by apoplast-localized superoxide dismutase. H2O2 diffuses to the neighboring cells and reacts with its target, the identity of which is yet unknown. The oxidized target may activate the RBOH to regenerate a new signal molecule to propagate the signal to distal sites.

Finally, now that we know RBOH mediates the rapid systemic signaling, we may begin to tackle the question of RBOH activation. Several clues to this riddle have been reported. RBOHs are regulated by cytosolic Ca2+ elevation, phosphorylation by calcium-dependent protein kinases, Rop guanosine triphosphatase, and phospholipase Dα1 and its product phosphatidic acid (1823). Perhaps the key to understand the regulation of RBOHD in this rapid systemic signaling is simply to ask how RBOHD can be activated so quickly. In most studies, ROS production is observed in minutes or even hours. We need to look at a mechanism that is much faster.

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