Reactive Oxygen Species

SEB Bulletin Otober 2006 - ROS: A radical paradox

The evolution of photosynthetic organisms resulted in a gradual, but massive, increase in the level of atmospheric molecular oxygen (O2) that enabled aerobic life to blossom on earth. All life on earth is based on redox reactions (reduction; the gain of an electron and oxidation; the loss of an electron - ref 1), using reductive processes to store energy and oxidative processes to release it (refs 2, 3, 4). As a result of its unusual chemistry, it was possible to integrate highly reactive oxygen in life-giving redox metabolism. The total reduction of oxygen produces water, however partial reduction produces REACTIVE OXYGEN SPECIES (ROS) including superoxide anion (O2 -), hydrogen peroxide (H2O2) and the hydroxyl radical (OH·).

ROS are produced as a consequence of electron transport processes in photosynthesis and aerobic respiration. ROS, as their name suggests, are reactive and potentially harmful to cells, causing oxidation of lipids, proteins and DNA. High levels of ROS production lead to a process that is often referred to as 'oxidative stress'. In animals, the deleterious effects of ROS have been linked with aging, carcinogenesis and atherosclerosis in humans (ref 5,6).
As aerobic life evolved, so did mechanisms to scavenge the ROS and it has been a long-held view that a major purpose of such scavengers is to protect the organism from the damaging effects of ROS. These scavengers known as antioxidants (ref 7), have received much media attention in recent years as potential anti-aging compounds and agents that promote good health (ref 8).

Whilst uncontrolled ROS accumulation is generally accepted as deleterious particularly for animals, the idea that ROS are the unavoidable, unwelcome, by-products of aerobic respiration is a misconception. As Christine Foyer (ref 9) of Rothamsted Research, UK points out “there is a considerable body of evidence to show that ROS have a positive influence on plant growth and survival” (ref 10). Similarly, our bodies could not fight invading organisms or destroy foreign, unneeded or damaged cells without ROS production, which plays a key role in disease-resistance, cell-mediated immunity and microbiocidal activity.

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ROS are generated at very high rates in plants. They are produced by organelles with a high oxidising metabolic activity or intense rate of electron flow, e.g. chloroplasts, mitochondria and microbodies, or by the oxidases (ref 11), a large class of enzymes. Examples of ROS-producing oxidases include the plasma membrane NADPH oxidases (ref 12, 13), peroxidises (ref 14, 15), oxalate oxidases (ref 16) and amine oxidases (ref 17). From the plethora of mechanisms that can generate ROS it is clear how plants are able to produce them in large quantities and that this generation can be flexible. This flexibility has enabled plants to utilise ROS in a wide variety of physiological processes such as the regulation of photosynthesis, cell wall metabolism and for defence against pathogens (ref 18, 19, 20, 21, 22) and more recently they have been shown to play important roles in the control of gene expression (ref 23, 24) and plant development (ref 25). Moreover, ROS are part of the repertoire of signals that facilitates interactions between plants and other organisms such as bacteria and fungi in order to form beneficial structures such as mychorriza and N-fixing nodules. For ROS to be effective in these roles Graham Noctor of the University of Paris, notes that “the production and concentration of ROS requires effective regulation by a powerful antioxidant system”.

The plant antioxidative system is continuously processing ROS, by acting as electron donors the antioxidants are themselves oxidised in the process of neutralising the ROS. This regulates the accumulation of ROS by determining their lifespan and spatial distribution and by controlling signal specificity. Just as a vast number of mechanisms for the generation of ROS exist, as do antioxidative compounds Contributors to the plant antioxidative system include superoxide dismutase (ref 26) (catalyses the generation of oxygen and H2O2 from superoxide), catalase (ref 27) (decomposition of H2O2 to water and oxygen), ascorbate (vitamin C, water soluble providing protection in the cytosol and appoplast), tocopherols (ref 28) (vitamin E, lipid soluble antioxidants that provide protection to lipid membranes), thioredoxin (ref 29) (promotes active metabolism and detoxifies ROS by reducing disulphide bonds), and glutathione (ref 30) (GSH, maintains ascorbate and thiol/disulphide balance).

Redox homeostasis21 is maintained in plants by pools of antioxidants, such as ascorbate and glutathione. The antioxidants are generally maintained in their 'active' reduced state by specific enzymes, like glutathione reductase. The balance of reduced and oxidised antioxidants can be utilised by plants to detect changes in the environment via changes in ROS concentrations. For example, ROS generation by the chloroplast, the major source of ROS, can be increased by excess light, drought, salt stress and in some cases conditions of limiting carbon dioxide by activation of the photorespiratory pathway resulting in H2O2 production in the peroxisomes. The ROS will oxidise the antioxidants and the redox balance will be disrupted. The collaborative research interests of Graham Noctor and Christine Foyer lie in trying to understand how the interplay between ROS and antioxidants is used by plants to perceive and respond to environmental and developmental cues.

There is a clear need for plants to perceive and react to their environment, light energy for example can vary and is often in vast excess of that required for metabolism. As mentioned above, excess light results in increased production of ROS, which can trigger changes in many parts of the cell including the photosynthetic machinery. Plants therefore need to detect such environmental fluctuations and implement appropriate changes to balance the need to maximise the efficiency of harvesting light energy whilst limiting the level of inactivation and inhibition. Redox components appear to be important in the sensing of such changes. Plastoquinone (PQ) (ref 31) is an electron acceptor that forms part of photosynthetic electron transport chain and is implicated in the adaptive response of plants to changes in light. In the short-term (seconds to minutes) as the PQ pool becomes reduced protein kinases are activated, which in turn mediates re-organisation of the photosynthetic machinery. Recent evidence has shown a long-term role for the PQ pool, mediating changes in transcription and translation of numerous genes including antioxidants and the genes that encode the photosynthetic machinery itself.

Redox signalling events are also involved in plant responses to temperature stresses (ref 32). Here the ROS appear to play a more direct role in the induction of heat shock proteins (HSPs), but this does not rule out other indirect mechanisms. It would appear that stress sensors in the photosynthetic and respiratory electron transport chains activate redox-sensitive transcription factors that in turn up-regulate the expression of genes that encode HSPs and related proteins, ROS-scavenging enzymes and factors involved in the amplification of the ROS signal by activation of NADPH oxidases.

In addition to sensing the environment and abiotic stresses, ROS also play an important role in plant defence responses to pathogens. The hypersensitive response (HR), is an example of programmed cell death (PCD) characterised by cell death at the site of infection. One of the earliest events in HR is the rapid accumulation of ROS, which can be directly toxic to the pathogen, but recent evidence suggests that the ROS, in particular H2O2, are the signal molecules that trigger HR and other defence mechanisms such as systemic acquired resistance (SAR) and activation of defence genes. Much of the ROS generated during pathogen attack are produced by the plant NADPH oxidases.

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ROS are produced in response to many hormones such as auxin, abscisic acid and salicylic acid. It is no surprise therefore that ROS are important not only in sensing and responding to environmental changes, but also in orchestrating plant movement (stomatal closure and tropism) responses and in development.
Liam Dolan (ref 33) of the John Innes Centre, UK and his colleagues have demonstrated a role for ROS in the development of root hairs (ref 34, 35). Root hairs are cellular protuberances and are essential for healthy plant growth as they enable access to immobile inorganic ions e.g. phosphate. The production of the ROS signal for root growth is highly organised and tightly regulated to ensure the production of a single root hair.

It is still often taught that ROS are the unwanted by-products of photosynthesis and aerobic respiration and that they need to be 'mopped up' from biological systems to limit oxidative damage. While the jury is still out with regard to effects in animals, it has become increasingly apparent that, far from being hugely deleterious, ROS production in plants is absolutely required for healthy growth and development. Without such a system plants would not have the ability to develop organs such as root hairs or communicate with other organisms in the soil and therefore obtain essential nutrients. Recent evidence suggests that the developmental regulation described above for roots also operates in many aspects of plant development. In addition, without a mechanism to detect changes in the environment and an ability to signal these changes to elicit a response, the plants survival would be severely jeopardised. The observation that plant themselves actively generate ROS, through the action of oxidases and other enzymes, as part of signal cascades also strongly suggests that they are not the unwelcome companions of aerobic life, they were once believed to be.

Rebecca Poole
University of Bristol

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SEB Bulletin Otober 2006 - ROS: A radical paradox

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