Earlier studies with Arabidopsis thaliana exposed to ultraviolet 6 (UV-6) and ozone (O,) have indicated the differential responses of superoxide dismutase and glutathione reductase. In this study, we have investigated whether A. thaliana genotype Landsberg erecfa and its flavonoid-deficient mutant fransparent testa ( t f 5 ) is capable of metabolizing UV-B-and O,-induced activated oxygen species by invoking similar antioxidant enzymes. UV-6 exposure preferentially enhanced guaiacol-peroxidases, ascorbate peroxidase, and peroxidases specific to coniferyl alcohol and modified the substrate affinity of ascorbate peroxidase. O, exposure enhanced superoxide dismutase, peroxidases, glutathione reductase, and ascorbate peroxidase to a similar degree and modified the substrate affinity of both glutathione reductase and ascorbate peroxidase. Both UV-B and O, exposure enhanced similar Cu,Zn-superoxide dismutase isoforms. New isoforms of peroxidases and ascorbate peroxidase were synthesized in tt5 plants irradiated with UV-B. UV-B radiation, in contrast to O,, enhanced the activated oxygen species by increasing membrane-localized NADPH-oxidase activity and decreasing catalase activities. These results collectively suggest that (a) UV-B exposure preferentially induces peroxidase-related enzymes, whereas O, exposure invokes the enzymes of superoxide dismutase/ ascorbate-glutathione cycle, and (b) in contrast to O,, UV-B exposure generated activated oxygen species by increasing NADPHoxidase activity.

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Recent studies suggest that cross-talk between salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O(3)) exposure activates a hypersensitive response (HR)-like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O(3)-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O(3)-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O(3)-induced H(2)O(2) content and SA concentrations and completely abolished O(3)-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O(3) exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O(3) of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O(3)-induced HR-like cell death.

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INTRODUCTIONPlants continuously produce active oxygen species (AOS) such as superoxide radical ( • O 2 Ϫ ), hydrogen peroxide (H 2 O 2 ), and singlet oxygen ( 1 O 2 ) as a consequence of normal cellular metabolism. Under normal conditions, plants rapidly metabolize these AOS with the help of constitutive antioxidant enzymes or metabolites (Scandalios, 1997). However, when subjected to environmental stresses such as cold, high light, ozone (O 3 ), pathogens, and UV irradiation, excess AOS is generated. This increase in AOS production necessitates the activation of additional defenses (Doke, 1997;Scandalios, 1997). Unless these AOS are efficiently metabolized, they rapidly oxidize and damage membrane lipids, proteins, and other cellular components. This leads to cellular dysfunction and can ultimately cause cell death, which is manifested by the appearance of necrotic lesions (Mudd, 1997). Based on the assumption that AOS are highly reactive and lead to cellular dysfunction, AOS generation is frequently considered deleterious and harmful. However, recent studies have demonstrated that AOS are important components of signaling pathways that influence plant defense responses, including cell death (Jabs et al., 1996;Alvarez et al., 1998;Karpinski et al., 1999;Solomon et al., 1999).H 2 O 2 and • O 2 Ϫ have been implicated as signal molecules that can activate defense responses. Treating cell cultures with H 2 O 2 alone induces defense responses and cell death (Levine et al., 1994;Solomon et al., 1999). However, although sublethal H 2 O 2 concentrations induce expression of defense genes, complete induction of defense genes and cell death requires additional signaling molecules such as salicylic acid (SA) at the whole-plant level (Chamnongpol et al., 1998;Rao and Davis, 1999). These studies have established that AOS probably require additional downstream components to transduce or amplify the signal Bolwell, 1999). Among several molecules proposed to act downstream of AOS, SA, jasmonic acid (JA), and ethylene are considered major regulators of plant defense responses (Dong, 1998;Glazebrook, 1999). SA is one of the most widely studied stress-signaling molecules; its role in influencing plant resistance to pathogens and other stress factors is well documented (Draper, 1997;Shirasu et al., 1997;Surplus et al., 1998;Rao and Davis, 1999). Similar to SA, JA also is believed to play an important role in influencing plant resistance to pathogens and other stress factors (Creelman and Mullet, 1997;Penninckx et al., 1998;Vijayan et al., 1998). The recent cloning of COI1 has increased our understanding of the involvement of protein degradation in JA-induced responses (Xie et al., 1998); however, little is 1 Current address: Paradigm Genetics, Inc., 104 Alexander Drive, Research Triangle Park, NC 27709. 2 Current address: Mendel Biotechnology, Inc., 21375 Cabot Boulevard, Hayward, CA 94545. 3 To whom correspondence should be addressed. E-mail kdavis @paragen.com; fax 919-572-6764. 1634The Plant Cell known about other potential m...

SummaryEthylene is known to influence plant defense responses including cell death in response to both biotic and abiotic stress factors. However, whether ethylene acts alone or in conjunction with other signaling pathways is not clearly understood. Ethylene overproducer mutants, eto1 and eto3, produced high levels of ethylene and developed necrotic lesions in response to an acute O 3 exposure that does not induce lesions in O 3 -tolerant wild-type Col-0 plants. Treatment of plants with ethylene inhibitors completely blocked O 3 -induced ethylene production and partially attenuated O 3 -induced cell death. Analyses of the responses of molecular markers of specific signaling pathways indicated a relationship between salicylic acid (SA)-and ethylene-signaling pathways and O 3 sensitivity. Both eto1 and eto3 plants constitutively accumulated threefold higher levels of total SA and exhibited a rapid increase in free SA and ethylene levels prior to lesion formation in response to O 3 exposure. SA pre-treatments increased O 3 sensitivity of Col-0, suggesting that constitutive high SA levels prime leaf tissue to exhibit increased magnitude of O 3 -induced cell death. NahG and npr1 plants compromised in SA signaling failed to produce ethylene in response to O 3 and other stress factors suggesting that SA is required for stress-induced ethylene production. Furthermore, NahG expression in the dominant eto3 mutant attenuated ethylene-dependent PR4 expression and rescued the O 3 -induced HR (hypersensitive response) cell death phenotype exhibited by eto3 plants. Our results suggest that both SA and ethylene act in concert to influence cell death in O 3 -sensitive genotypes, and that O 3 -induced ethylene production is dependent on SA.

The potential role of antioxidant enzymes in protecting maize (Zea mays 1.) seedlings from chilling injury was examined by analyzing enzyme activities and isozyme profiles of chilling-susceptible (CO 316) and chilling-tolerant (CO 328) inbreds. Leaf superoxide dismutase (SOD) activity in CO 316 was nearly one-half that of CO 328, in which the high activity was maintained during the chilling and postchilling periods. Activity of glutathione reductase (CR) was much higher in roots than in leaves. CO 328 also possessed a new CR isozyme that was absent in roots of CO 316. Ascorbate peroxidase (APX) activity was considerably lower in leaves of CO 328 than in CO 316, and nearly similar in roots. Paclobutrazol treatment of CO 316 induced several changes in the antioxidant enzyme profiles and enhanced their activities, especially those of SOD and APX, along with the induction of chilling tolerance. These results suggest that increased activities of SOD in leaves and CR in roots of CO 328, as well as SOD and APX in leaves and roots of paclobutrazol-treated CO 31 6, contribute to their enhanced chilling tolerance.