Active oxygen species

Electrons usually exist as pairs in an atomic orbital and a free radical is defined as any species with one or more unpaired electrons (Halliwell and Gutteridge, 1984). By this definition oxygen itself is a free radical since it possesses two unpaired electrons, each located in a different n* anti-bonding orbital, with the same parallel spin quantum number (Figure 5.17) which renders the molecule relatively unreactive. This is because when another molecule is oxidised, by accepting a pair of electrons from it, both new electrons must be of parallel spin to fit into the vacant spaces in the n* orbitals.

However, such a pair would be expected to have anti-parallel spins (it) which slow and prevent many potential oxidations. Spin restriction can be overcome in biological systems by the presence of transition metals, especially iron, found at the active site of many oxygenases. The metals can do this by their ability to accept or donate single electrons. The reactivity of oxygen is also increased by moving one of the unpaired electrons to overcome the spin restriction and form singlet oxygen. Singlet oxygen has no unpaired electrons and is therefore not a radical, but can exist for long enough (2-4 ^s) to react with many biological molecules. It is commonly generated by excitation energy transfer from photosensitisers such as chlorophylls, porphyrins, flavins and retinal (Figure 5.18 ).

Figure 5.17 Bonding orbitals in diatomic oxygen ( reproduced with permission from Halliwell and Gutteridge, 1984, © The Biochemical Society).
Figure 5.18 Generation of singlet oxygen by photosensitisers.

The superoxide radical ( O2-) is formed when a single electron is accepted by ground state oxygen and the addition of a second electron, either enzymically by superoxide dismutase or non-enzymically, yields the peroxide ion ( O^-).

In addition, hydroxyl free-radicals (•OH) are formed in the presence of peroxide (H2O2) and ferrous salts in what is termed the Fenton Reaction (Figure 5.19). The reactivity of •OH is so great that they react immediately with whatever biological molecules are in the vicinity to produce secondary radicals.

H2O2

All four active oxygen species (1O2, O2- , .OH and H2O2) are naturally generated at the thylakoid and are normally quenched by a series of scavengers or antioxidants within the stroma or dissipated as heat.

Glutathione . (reduced)

rNADP+

Dehydroascorbate reductase

Glutathione reductase

Glutathione (oxidized)

Figure 5.19 Superoxide generation and detoxification at Photosystem I. PQ, paraquat; SOD, superoxide dismutase (after Shaaltiel and Gressel, 1986, http://www.biochemj.org).

cell death acclimation cell death acclimation

light

Figure 5.20 The fates of solar energy absorbed by the thylakoid light-harvesting complexes. *, denotes an excited state.

light

Figure 5.20 The fates of solar energy absorbed by the thylakoid light-harvesting complexes. *, denotes an excited state.

Chloroplast superoxide dismutase is a metalloprotein (apparently existing as Cu-Zn, Mn or Fe forms) partially bound to the thylakoid. Its product, hydrogen peroxide, can inhibit several stromal enzymes involved in photosynthetic carbon reduction, and is removed by the enzymes of the ascorbate-glutathione cycle (Figure 5. 19). Stromal ascor-bate and glutathione concentrations fluctuate seasonally and millimolar amounts are commonly measured in the summer months.

a-Tocopherol (vitamin E) is a most effective antioxidant and can react rapidly with singlet oxygen and lipid peroxide radicals. The carotenoid pigments, particularly P-carotene, are also important quenchers of triplet chlorophyll in addition to singlet oxygen, and lead to the dissipation of excess excitation as heat. More recent studies have suggested that polyamines and some flavonols may also act as natural photoprotectants.

Plant survival therefore depends on the balance between photo-oxidative stress and the effectiveness of natural antioxidant protection systems, which have the ability to 'mop up' oxygen free-radicals (Figure 5.20).

In the presence of PS II inhibitors, excitation energy generated by p680 cannot be dissipated by normal electron flow beyond QA, and so fluorescence yield is dramatically enhanced and activated oxygen species generated. Similarly, paraquat will divert electrons at higher energy from PS I and rapidly generate oxygen free-radicals, as previously described. Under these conditions the natural protective mechanisms are rapidly overloaded, especially at increased temperatures and photon flux densities, and lipid peroxidation is initiated in thylakoids by hydrogen abstraction (Figure 5.21).

Figure 5.21 The chain reaction of thylakoid lipid peroxidation.

Ethane Malondialdehyde

(breakdown products)

Figure 5.21 The chain reaction of thylakoid lipid peroxidation.

The free-radical attacks unsaturated membrane fatty acids and is quenched by hydrogen atom abstraction. Since a hydrogen atom has only one electron, it leaves behind an unpaired electron on a carbon atom. This carbon (lipid) radical rapidly reacts with oxygen to yield a hydroperoxy radical, which is itself able to abstract hydrogen atoms from other unsaturated lipid molecules, thus initiating a chain reaction of lipid peroxidation. Eventually, the unsaturated fatty acids of the thylakoid are totally degraded to

Figure 5.22 Ultrastructural symptoms following treatment with photosynthetic inhibitors. (A) Mesophyll cell chloroplast from an untreated leaf of Tripleurospermum maritimum subsp.inodora (scentless mayweed). Note appressed and non-appressed thylakoids (t), cell wall (cw), stroma (s) and chloroplast envelope (ce). (B) As (A), but seven days after treatment with ioxynil-sodium at a rate equivalent to 560 g active ingredient ha" 1. Note that the chloroplast is swollen and that vesicles (v) are present in both the stroma and the thylakoids. Such intergranal vacuolation is a typical symptom of photoperoxi-dative damage. (C, D) Mesophyll cell chloroplasts from Galium aparine leaves 3 hours after treatment with (C) water and (D) 100 jM of the diphenyl ether herbicide, acifluorfen. Note the invaginations (iv) and evaginations (ev) of the chloroplast envelope. Bar, 1 jiM (A and B from Sanders and Pallett, 1986, C and D from Derrick et al., 1988).

Figure 5.22 Ultrastructural symptoms following treatment with photosynthetic inhibitors. (A) Mesophyll cell chloroplast from an untreated leaf of Tripleurospermum maritimum subsp.inodora (scentless mayweed). Note appressed and non-appressed thylakoids (t), cell wall (cw), stroma (s) and chloroplast envelope (ce). (B) As (A), but seven days after treatment with ioxynil-sodium at a rate equivalent to 560 g active ingredient ha" 1. Note that the chloroplast is swollen and that vesicles (v) are present in both the stroma and the thylakoids. Such intergranal vacuolation is a typical symptom of photoperoxi-dative damage. (C, D) Mesophyll cell chloroplasts from Galium aparine leaves 3 hours after treatment with (C) water and (D) 100 jM of the diphenyl ether herbicide, acifluorfen. Note the invaginations (iv) and evaginations (ev) of the chloroplast envelope. Bar, 1 jiM (A and B from Sanders and Pallett, 1986, C and D from Derrick et al., 1988).

malondialdehyde and ethane, and the appressed thylakoid structure progressively opens up and disintegrates (Figure 5.22). Finally, cell membranes and tissues disintegrate from this chain reaction of free-radical attack.

The analysis of chlorophyll fluorescence has become a valuable experimental technique in recent years to investigate the effects of environmental stress, including herbicides, on

Figure 5.23 A characteristic fluorescent transient or Kautsky curve induced when a dark-adapted leaf is exposed to light in the presence and absence of DCMU (diuron).

PS II activity. Devices that are relatively simple to use and portable have become available, which enable rapid measurements in the laboratory, glasshouse and in the field. Indeed, chlorophyll fluorescence has become a useful screening technique for PS II inhibitors.

As shown in Figure 5.20 , chlorophyll in an excited state can be used in one of three ways: photochemistry to drive photosynthesis, dissipation as heat, or re-emission as fluorescence. The three processes are essentially competitive and environmentally sensitive. Under low light exposure about 97% of the absorbed photons are used in photochemistry, 2.5% are transformed to heat and 0.5% emitted as fluorescence. On the other hand, if all the PS II reaction centres are closed by high light or the presence of PS II inhibitors, 95-97% of the absorbed energy may be dissipated as heat and as much as 3-5% via fluorescence.

Kautsky and colleagues (1960) were the first to report the pattern of fluorescence induction when dark-adapted leaves are exposed to light. An example of this fluorescent transient or Kautsky curve is presented in Figure 5.23.

An initial rise from a dark-adapted low value (Fo) to a maximum value (Fm) is observed in less than a second (fast phase), followed by a slower phase, lasting minutes, before a constant value is reached. The fast phase is related to PS II photochemistry, while the slower phase is determined by thylakoid function such as electron transfer away from PS II and carbon metabolism in the stroma. Specifically, at Fo all the reaction centres at PS II are fully oxidised and ready to accept electrons. At Fm, all quinone carriers, especially QA, are reduced and unable to accept another electron until they have passed the first one on to the next carrier, QB . During this time the reaction centres are thought to be closed. Thus, when reaction centres are closed, photochemical efficiency is reduced and fluorescence yield is increased.

Note that in the presence of PS IHnhibiting herbicides that prevent electron flow between QA and QB, photochemistry is reduced to zero and Fm is sustained over a prolonged period.

Variable fluorescence, Fv, (Fm - Fo) is a valuable parameter that reflects the efficiency with which incident light energy is used in, and downstream of, the photosystems. It is also a valuable measure of how a plant may tolerate a herbicide, since metabolism in a crop plant will detoxify the herbicide and the Fm value will decline with time.

For a more detailed account of the underlying theory and interpretation of fluorescence data and photochemistry, the reader is referred to Maxwell and Johnson (2000) and Lawlor (2001), respectively.

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