Almost all terrestrial plants produce green leaf volatiles (GLVs), consisting of six-carbon (C6) aldehydes, alcohols and their esters, after mechanical wounding. C6 aldehydes deter enemies, but C6 alcohols and esters are rather inert. In this study, we address why the ability to produce various GLVs in wounded plant tissues has been conserved in the plant kingdom. The major product in completely disrupted Arabidopsis leaf tissues was (Z)-3-hexenal, while (Z)-3-hexenol and (Z)-3-hexenyl acetate were the main products formed in the intact parts of partially wounded leaves. ¹³C-labeled C6 aldehydes placed on the disrupted part of a wounded leaf diffused into neighboring intact tissues and were reduced to C6 alcohols. The reduction of the aldehydes to alcohols was catalyzed by an NADPH-dependent reductase. When NADPH was supplemented to disrupted tissues, C6 aldehydes were reduced to C6 alcohols, indicating that C6 aldehydes accumulated because of insufficient NADPH. When the leaves were exposed to higher doses of C6 aldehydes, however, a substantial fraction of C6 aldehydes persisted in the leaves and damaged them, indicating potential toxicity of C6 aldehydes to the leaf cells. Thus, the production of C6 aldehydes and their differential metabolisms in wounded leaves has dual benefits. In disrupted tissues, C6 aldehydes and their α,β-unsaturated aldehyde derivatives accumulate to deter invaders. In intact cells, the aldehydes are reduced to minimize self-toxicity and allow healthy cells to survive. The metabolism of GLVs is thus efficiently designed to meet ecophysiological requirements of the microenvironments within a wounded leaf.

Oxidative injury of the root elongation zone is a primary event in aluminum (Al) toxicity in plants, but the injuring species remain unidentified. We verified the hypothesis that lipid peroxide-derived aldehydes, especially highly electrophilic α,β-unsaturated aldehydes (2-alkenals), participate in Al toxicity. Transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis (Arabidopsis thaliana) 2-alkenal reductase (AER-OE plants), wild-type SR1, and an empty vector-transformed control line (SR-Vec) were exposed to AlCl₃ on their roots. Compared with the two controls, AER-OE plants suffered less retardation of root elongation under AlCl₃ treatment and showed more rapid regrowth of roots upon Al removal. Under AlCl₃ treatment, the roots of AER-OE plants accumulated Al and H₂O₂ to the same levels as did the sensitive controls, while they accumulated lower levels of aldehydes and suffered less cell death than SR1 and SR-Vec roots. In SR1 roots, AlCl₃ treatment markedly increased the contents of the highly reactive 2-alkenals acrolein, 4-hydroxy-(E)-2-hexenal, and 4-hydroxy-(E)-2-nonenal and other aldehydes such as malondialdehyde and formaldehyde. In AER-OE roots, accumulation of these aldehydes was significantly less. Growth of the roots exposed to 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal were retarded more in SR1 than in AER-OE plants. Thus, the lipid peroxide-derived aldehydes, formed downstream of reactive oxygen species, injured root cells directly. Their suppression by AER provides a new defense mechanism against Al toxicity.

Degradation of lipid peroxides leads to the formation of cytotoxic 2-alkenals and oxenes (collectively designated reactive carbonyls). The novel NADPH-dependent oxidoreductase 2-alkenal reductase (AER; EC 1.3.1.74) from Arabidopsis (Arabidopsis thaliana), which is encoded by the gene At5g16970, catalyzes the reduction of the α,β-unsaturated bond of reactive carbonyls, and hence is presumed to function in antioxidative defense in plants. Here we show that Arabidopsis AER (At-AER) has a broad substrate spectrum to biologically relevant reactive carbonyls. Besides 2-alkenals, the enzyme recognized as substrates the lipid peroxide-derived oxenes 9-oxo-octadeca-(10E),(12Z)-dienoic acid and 13-oxo-octadeca-(9E),(11Z)-dienoic acid, as well as the potent genotoxin 4-oxo-(2E)-nonenal, altogether suggesting AER has a key role in the detoxification of reactive carbonyls. To validate this conclusion by in vivo studies, transgenic tobacco (Nicotiana tabacum) plants that had 100- to 250-fold higher AER activity levels than control plants were generated. The engineered plants exhibited significantly less damage from either (1) the exogenously administered 4-hydroxy-(2E)-nonenal, (2) treatment with methyl viologen plus light, or (3) intense light. We further show that the At-AER protein fused with the Aequorea victoria green fluorescent protein localizes in cytosol and the nucleus in Bright-Yellow 2 cells. These results indicate that reactive carbonyls mediate photooxidative injury in leaf cells, and At-AER in the cytosol protects the cells by reducing the α,β-unsaturated bond of the photoproduced reactive carbonyls.

P1-ζ-crystallin (P1-ZCr) is an oxidative stress-induced NADPH:quinone oxidoreductase in Arabidopsis thaliana, but its physiological electron acceptors have not been identified. We found that recombinant P1-ZCr catalyzed the reduction of 2-alkenals of carbon chain C₃-C₉ with NADPH. Among these 2-alkenals, the highest specificity was observed for 4-hydroxy-(2E)-nonenal (HNE), one of the major toxic products generated from lipid peroxides. (3Z)-Hexenal and aldehydes without α,β-unsaturated bonds did not serve as electron acceptors. In the 2-alkenal molecules, P1-ZCr catalyzed the hydrogenation of α,β-unsaturated bonds, but not the reduction of the aldehyde moiety, to produce saturated aldehydes, as determined by gas chromatography/mass spectrometry. We propose the enzyme name NADPH:2-alkenal α,β-hydrogenase (ALH). A major portion of the NADPH-dependent HNE-reducing activity in A. thaliana leaves was inhibited by the specific antiserum against P1-ZCr, indicating that the endogenous P1-ZCr protein has ALH activity. Because expression of the P1-ZCr gene in A. thaliana is induced by oxidative stress treatments, we conclude that P1-ZCr functions as a defense against oxidative stress by scavenging the highly toxic, lipid peroxide-derived α,β-unsaturated aldehydes.