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I

n the 1950s, a new type of air pollution damage was observed on crop plants and on Pinus ponderosaneedles in the vicinity of Los Angeles, as well as on tobacco in the Eastern USA. Damage was attributed to photochemical smog containing ozone as a major component. Today, ozone is recognized as the most phytotoxic of the common air pollutants. National and interna-tional limits for air ozone concentrations are regularly exceeded in North America and Europe. Nevertheless, the characteristic, vis-ible symptoms of ozone damage are with few exceptions [e.g. black cherry tree (Prunus serotina); grapevine (Vitis vinifera)] not detected on field sites, and forest decline phenomena have only been clearly attributed to ozone in Southern California1

. Chronic stress effects of ozone seem to be much more important.

This review focuses on reaction types known to be triggered by pathogens in the hypersensitive response (HR) or in systemic acquired resistance (SAR). The HR reactions result in plant cell death at the site of infection so that further spread of pathogens is inhibited2Ð4

. SAR leads to induced resistance and ÔimmunizationÕ5 throughout the plant so that fewer or no lesions develop following a second pathogen infection2Ð6

. Fungal elicitors2,5,7

and certain sig-nal molecules such as salicylic acid6

and jasmonic acid are pro-posed to be involved in both HR and SAR. The major induced reaction types considered here with regard to ozone are summa-rized in Fig. 1.

Phytoalexins

The isoflavonoid phytoalexins of soybean had already been found to be induced by ozone in 19758

and, later on, pine needles were observed to accumulate stilbene phytoalexins and their biosyn-thetic enzymes upon ozone treatment9

. This process is initiated at the level of transcription10

, as was also shown in grapevine11 . Cat-echin, an allelochemical and antioxidant, is strongly induced by ozone in spruce (Picea abies) and pine. The elevated levels of cat-echin and stilbenes persist over several months and may be related to the ÔmemoryÕ effect of ozone in conifers12

. In addition to the furanocoumarin phytoalexins of parsley plants, ozone can also induce the genes, enzymes and metabolites of the UV-B-induced flavone malonyl-glycoside pathway13

, which is known not to respond to fungal elicitor. The ability of ozone to mimic other stresses has been termed cross-induction13

. This process can lead to cross-tolerance against pathogens as well as numerous abiotic stresses that are proposed to act via activated oxygen species9,14

.

Cellular barriers

Ozone induces the lignin biosynthesis enzyme, cinnamyl alcohol dehydrogenase, at the protein and transcript levels9,10

, but the derived lignan- or lignin-like product has so far not been identified.

Elicitor-induced lignin from cell cultures of spruce was shown to have an unusual juvenile structure, and to be tightly associated with extensins15

. The latter are induced at the transcript level by ozone in several tree species16

and in parsley13

, and transcripts for a glycine-rich wall protein increase in Atriplex17

. Callose is induced by ozone in parsley13

and tobacco9 .

Pathogenesis-related proteins

Basic b-1,3-glucanase, basic chitinase, acidic chitinase and patho-genesis-related (PR) protein 1b were found to be induced by ozone, in this temporal sequence, at the transcript level in tobacco18

. Basic b-1,3-glucanase mRNA accumulation is restricted to the ozone-treated leaf area and no systemic induction occurs19

. The PR proteins 1a and 1b were subsequently also shown to be induced by ozone in another tobacco cultivar20

. The ozone induction of tobacco b-1,3-glucanases and chitinases could furthermore be demonstrated at the levels of enzyme activity9

and immuno-reactive protein21

. An ozone-induced apoplastic chiti-nase, PR-3b, was identified by protein microsequencing21

. In pars-ley plants, transcripts for PR1-1, PR1-3, PR1-4 and the elicitor-induced protein Eli-16 are increased upon ozone treat-ment22

. In Arabidopsis, transcripts for PR-protein 1 were induced along with transcripts for glutathione-S-transferase-1 (GST-1) and phenylalanine-ammonia lyase23Ð25

. In spruce, ozone-respon-sive PR-proteins can be detected by immunoblotting and b -1,3-glucanase activity26

. An ozone-induced 8.6 kDa protein from

Arabidopsis was identified as a novel type of stress-related protein also induced by pathogens27

.

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reviews

Ozone: an abiotic elicitor of plant

defence reactions

Heinrich Sandermann, Jr, Dieter Ernst, Werner Heller and Christian Langebartels

The air pollutant ozone has recently been found to resemble fungal elicitors – it can induce plant signal molecules such as ethylene and salicylic acid, as well as certain genes and biosynthetic pathways associated with pathogen and oxidative defence. The action of ambi-ent ozone on the plant defence system may predispose the plant to enhanced attack by pathogens, but may also lead to induced resistance. The results mean that ozone can be regarded as a new experimental tool for analyzing stress responses.

Fig. 1. General scheme for the action of fungal elicitors2,3,5,7

. In the case of ozone, the receptor(s) and the initial signal (possibly acti-vated oxygen species3,4,9,14,29,30

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Signal substances

Studies on signal pathways are important because most of the ozone taken up through stomata is decomposed in the apoplast9,28

. A specific ozone receptor or redox sensor has not been identified to date. The hypothetical oxidative burst caused by ozone9,14,29,30

might act as a second messenger for ozone and might be respon-sible for the induction of GST-1 (Refs 3 and 23Ð25), PR-1-type proteins18,23Ð25

, glutathione peroxidase and polyubiquitin3

, as well as further (third) messengers for ozone. For example, ethylene biosynthesis is an early and well-known response to ozone9,29

. Ethylene may act as a second or third messenger depending on whether its induction is caused by ozone itself or by the hypo-thetical secondary oxidative burst. Ethylene induction is also detectable at the levels of the ethylene biosynthesis enzyme activ-ities, 1-aminocyclopropyl-1-carboxylic acid (ACC) synthase and ACC oxidase9,29,31,32

. More recently, ozone has been shown to acti-vate the transcription of specific members of the S -adenosyl-methionine synthase (sam3) and ACC synthase (LE-acs2) gene families in tomato33

and potato29,34

. LE-ACS2 (Ref. 33) in tomato is 96% identical at the amino acid level to the ozone-responsive ACC synthase isoform in potato29

, and in both cases fungal pathogens or elicitors were also inducers29,34

. ACC oxidase tran-script induction has been demonstrated as one of the fastest ozone responses (<30 min)29,33

. Some of the transcript levels increased by ozone may be a result of the ethylene induced by stress. This indirect mechanism has been proposed for the basic PR-pro-teins18,19

, stilbene synthase (STS)11

, accelerated senescence gener-ally29

, and more specifically the loss of Rubisco activity and the transcript of its small subunit32

. Recently, the STS promoter has been shown to respond to ethylene (D. Ernst et al., unpublished). Ethylene is also considered to be a modulator of programmed plant cell death3,4

. In a similar manner to the fungal induction of the oxidative burst and ethylene evolution3

, ethylene induction by ozone was blocked by the protein kinase inhibitor K252A and promoted by a protein phosphatase inhibitor33

.

Salicylic acid Ð a proposed signal of both HR and SAR (Refs 3Ð6) Ð and its b-D-glucoside, are induced in tobacco by ozone20. This response is accompanied by increased tolerance towards tobacco mosaic virus20

. In Arabidopsis, salicylic acid and its con-jugates are also induced along with induced resistance to the pathogen Pseudomonas syringae25

. There is a controversy on transgenic plants that contain a bacterial salicylate hydroxylase and therefore cannot accumulate salicylic acid. This enzyme dis-rupts SAR (Ref. 6), and both higher ozone sensitivity in Arabid-opsis25

and tolerance in tobacco35

have been reported. As noted

for ethylene, salicylic acid may also act as a second (or third) messenger for ozone and may mediate some typical SAR-like ozone responses such as induction of the acidic PR-proteins in tobacco, parsley and

Arabidopsis18,21,22,25

. Lipoxygenases poss-ibly involved in jasmonic acid biosynthesis were increased by ozone in lentil (Lens culinaris)36

, but not in Arabidopsis23

. Applied methyl-jasmonate or wounding prevented the visible symptoms caused by high ozone concentrations on tobacco leaves35

.

Antioxidative systems

The oxidative stress caused by ozone appears to have two opposing effects: it is generally held responsible for the detri-mental effects of ozone14,28

and may serve as the initial signal for programmed cell death and HR (Ref. 4). This signalling may involve ethylene4

, which was specifically induced in the ozone-hypersensitive biomonitoring plant tobacco Bel W3 (Refs 9 and 14). The detrimental effect of activated oxy-gen species is well illustrated by an ascorbic acid-deficient

Arabidopsismutant that is hypersensitive to ozone, as well as to sulphur dioxide (SO2) and UV-B (Ref. 37). However, active

oxy-gen species are involved in the protective HR- or SAR-type sig-nalling pathways and the defence reactions shown in Fig. 19,14,29,30

. Hydrogen peroxide and the superoxide anion radical are potential primary signalling species, only hydrogen peroxide being able to permeate over longer distances3,14,28

.

Ozone, and to a lesser extent SO2and UV-B, cause an increase

in transcript levels of the antioxidative enzymes catalase 2 and glutathione peroxidase. There are small and delayed effects on tran-script levels of several superoxide dismutase isoforms and ascor-bate peroxidase38

. In another study with tobacco, the cytosolic ascorbate peroxidase was shown to be up-regulated by high-level ozone exposure as well as by methyljasmonate35

. Somewhat similar results were obtained for ozone in Arabidopsis39

, where transcripts for the antioxidative enzymes Cu/Zn-superoxide dismutase, ascor-bate peroxidase and GST-1, but not catalase, were induced by ozone23Ð25

. GST-1 induction in Arabidopsiswas only in part medi-ated by salicylic acid25

. GST-1 is also induced by many other oxi-dative stresses, including pathogens24,25

, ethylene and salicylic acid3,24,25

. The particular antioxidative response to ozone generally depends on the developmental stage of the plant and other param-eters9,28

. Only certain members of the antioxidative gene families seem to respond to ozone. Furthermore, compartmentation of antioxidative systems (e.g. apoplast, cytosol and chloroplast) has been shown to be of key importance for ozone sensitivity and for generating ozone tolerance by gene transfer9,14,29

.

Additional ozone-responsive genes

Ozone has been shown to increase several transcripts whose poss-ible response to fungal elicitor, ethylene or salicylic acid has not been well studied. This concerns transcripts for a pine terpene-biosynthesis enzyme (HMG-CoA-synthase40

, a small heat-shock protein from parsley22

, peroxidases in Arabidopsis23,39,41

and pars-ley13

, and thiol protease and protease inhibitors in Atriplex17

. Poly-ubiquitin transcripts with ten repeats have been shown to be induced by ozone in pine seedlings42

. Activated oxygen species appeared to act as inducing agents, and the ubiquitin system was proposed to be involved in the degradation of proteins that are damaged by ozone42

by analogy with its role in plant senescence and stress responses.

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February 1998, Vol. 3, No. 2

Fig. 2. Structure of the stilbene synthase promoter from grapevine (Vitis vinifera)11 . The depicted minimal promoter regions responding to ozone and fungal pathogen, respectively, were determined by studying the effect of promoter deletions on b-GUS expression. The fol-lowing regulatory elements obtained from the DNA sequence are marked: A, TATA-box; B, ethylene-responsive like element; C, elicitor-responsive element. Several further regulatory elements were also identified11

.

2430 Minimal promoter, ozone

2280 _{Minimal promoter,}

pathogen

Coding region A

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trends in plant science

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Ozone-responsive promoters

The biosynthesis of stilbene phytoalexins constitutes one of the most sensitive ozone responses in pine9

and grapevine11

. Tobacco plants transformed with the

grapevine STS gene were previously

found to possess fungal tolerance. The grapevine promoter used to transform these plants is now known to be induced by ozone11

. When segments of the grapevine STSpromoter were coupled to

the b-GUS reporter gene, an

ozone-responsive region could be defined by use of transgenic tobacco plants11

. Minimal upstream promoter sequences to positions

2430 and 2280 were required for induc-tion by ozone and fungal pathogen, respectively11

. The promoter sequence shown schematically in Fig. 2 contained an inverse elicitor-responsive element (ERE) at positions 2312 to 2317 of the ozone-responsive region. ERE-like se-quences occur in promoters of different defence-related genes, including those for

PR1 protein isoforms of parsley that are induced by ozone as well as fungal elicitor22

. The presence of this ERE in the STSpromoter may indicate that the signalling pathway for ozone and pathogens results in the activation of the same cis-element by trans-factors. Interestingly, ERE-like motifs have also been found in the chal-cone synthase (CHS) promoter of maize11

. Ozone treatment of parsley has been shown to lead to the induction of CHS tran-scripts13

. As EREs are present in various stress-related promoters, one may speculate that the ERE is frequently involved in the induction of plant defence genes. A GCC-element resembling ethylene regulatory sequences was also present in the STS promoter11

.

The ozone induction of the STS-promoter/b-GUS construct led to a spotted pattern (Fig. 3)11

. This pattern is reminiscent of early visible ozone symptoms in another tobacco line (Fig. 3a). A sys-tematic comparison has yet to be performed, but a similar spotted pattern was also detected when ozone-treated tobacco leaves were stained for activated oxygen species with diaminobenzidine14

. All these recent observations are consistent with early ozone lesions having an HR-like nature, comparable with HR-like viral lesions of tobacco leaves6

.

Ambient ozone as an elicitor

Ozone can easily be generated and applied, so that it is a conveni-ent model elicitor that supplemconveni-ents existing preparations (intact pathogens, fungal elicitors or signalling substances). Among these, ozone and ethylene are the only model elicitors that can easily be removed at any time after application.

In most of the studies described here, the ozone concentrations applied in the lab were several-fold higher than ambient, but the applied ozone dose (product of concentration and time) was always lower than the ambient ozone dose per growing season9

. In several examples, effects were demonstrated at non-elevated ozone concentrations. For example, the ozone-sensitive line tobacco Bel W3 showed a fourfold-increased b-1,3-glucanase activity after exposure to only 2 d of ambient ozone43

. In the pres-ence of the frequently occurring synergist, NO2, near-ambient

ozone induced b-1,3-glucanase activity 22-fold even in the ozone-tolerant tobacco line Bel B (Ref. 43). Spruce is usually quite ozone tolerant, but the needle catechin content responded strongly

to treatment with 80 nl l21

(i.e. ppb) ozone12

. Visible damage or decreases of photosynthesis have frequently been observed at near-ambient ozone concentrations9,28

.

Present mean ambient ozone concentrations are about two- to fivefold higher than in the last century. Therefore, only the anthro-pogenic portion appears to make present-day tropospheric ozone a significant environmental elicitor. The plant responses to ozone are, paradoxically, twofold: induced resistance to viral or mi-crobial pathogens, or to insects; or predisposition to higher sus-ceptibility to these various pathogens9

. Ozone thus influences the likelihood of the disease by acting on the plant defence status.

Biotechnological implications

It has been reported that pathogen resistance can be induced in plants by certain chemicals, such as elicitor-like compounds (e.g. oligosaccharides and chitosan5Ð7

), or salicylic acid, 2,6-dichloro-isonicotinic acid and benzothiadiazole6

. The extensive research on resistance-inducing chemicals has so far not taken into account the possible interference by ambient ozone. Furthermore, trans-genes for defence-related trans-genes under the control of elicitor-inducible promoters may respond to ozone, independently of fungal attack, as has been shown to be the case for the STSgene11

. Deletion of the STSpromoter sequences down to position 2280 resulted in loss of ozone inducibility and yielded a minimal pro-moter that was only responsive to fungal pathogens11

. This exam-ple indicates that it may be possible to eliminate cross-induction of antifungal gene constructs by ozone. Many ozone-responsive plant genes have been discovered, but the full ecological signifi-cance of ambient ozone as an unregulated elicitor and disease agent remains to be elucidated.

Acknowledgements

We thank Drs K. Davis, J. KangasjŠrvi, R. Last and Eva Pell for preprints and information. Special thanks are due to our co-work-ers for their valuable contributions and to Prof. JŸrgen Ebel for critical comments. Financial support by the Bundesministerium fŸr Bildung, Wissenschaft, Forschung und Technologie, Bayer-isches Staatsministerium fŸr Landesentwicklung und Umwelt-fragen, EUROSILVA, Limagrain and Fonds der Chemischen Industrie is gratefully acknowledged.

Fig. 3. Visual appearance of early ozone symptoms in tobacco Bel W3 (a) and of b-GUS staining in transgenic tobacco (var. Petit Havanna SR1) containing the stilbene synthase promoter coupled to a b-GUS reporter gene11

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Heinrich Sandermann, Jr*, Dieter Ernst, Werner Heller and Christian Langebartels are at the GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, Institut für Biochemische Pflanzenpathologie, D-85764 Oberschleißheim, Germany.