Recent advances in the understanding of the molecular hasis of plant response to ozone attack are reviewed. Plants grown in elevated atmospheric ozone are known to undergo several hiochemical changes before any actual damage can be detected. These reactions include increases in the activities of enzymes associated with general plant defence mechanisms. Ozone exposure often causes a surge in the production of the plant hormone ethylene, as well as changes in polyamine metabolism and increases in the activities of several phenylpropanoid and flavonoid pathway enzymes. The activities of superoxide dismutase and peroxidases that protect cells from the oxidative damage caused by hydroxyl radicals, H2O2 and superoxides also increase. However, ozone-induced changes in plant cells at the gene level are almost unknown. The limited data available suggest close similarities between ozone-induced and pathogen-induced defence responses in plants. Several general defence genes that have been cloned in other studies will soon be applied to studies of gene expression in ozone-exposed plants. The use of molecular biological tools in ozone research should enable the development of highly specific and sensitive molecular markers for biomonitoring ozone-induced injuries in plants. Methionine (e) AdoMe sperm idine Spermine -^ Ethylene Figure 2. Biosynthesis of ethylene and polyamines in plants.Known inhibitory (-) interactions of the pathways are indicated by the dashed lines. Ozone is known to induce ethylene synthesis, transcription of the ACC synthase gene, changes in the polyamine concentrations and induction of ADC activity. Abbreviations used: AdoMet = S-adenosyl methionine; ACC= 1-aminocyclopropane-1-carbocylic acid. Enzymes catalysing the reactions are indicated by the following numbers: 1, ACC synthase; 2, ACC oxidase; 3, AdoMet decarboxylase; 4, ornithine decarboxylase; 5, arginine decarboxylase; 6, spermidine synthase; 7, spermine synthase; 8, AdoMet syntethase.Received 2 September 1993,

We show that above a certain threshold concentration, ozone leads to leaf injury in tomato (Lycopersicon esculentum). Ozone-induced leaf damage was preceded by a rapid increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, ACC content, and ethylene emission. Changes in mRNA levels of specific ACC synthase, ACC oxidase, and ethylene receptor genes occurred within 1 to 5 h. Expression of the genes encoding components of ethylene biosynthesis and perception, and biochemistry of ethylene synthesis suggested that ozone-induced ethylene synthesis in tomato is under biphasic control. In transgenic plants containing anLE-ACO1 promoter-β-glucuronidase fusion construct, β-glucuronidase activity increased rapidly at the beginning of the O3 exposure and had a spatial distribution resembling the pattern of extracellular H2O2 production at 7 h, which coincided with the cell death pattern after 24 h. Ethylene synthesis and perception were required for active H2O2 production and cell death resulting in visible tissue damage. The results demonstrate a selective ozone response of ethylene biosynthetic genes and suggest a role for ethylene, in combination with the burst of H2O2production, in regulating the spread of cell death.

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The response of a Cu-and Zn-tolerant birch (Betula pendula) clone to copper stress was investigated. The plants were exposed to control and EC50 concentrations of Cu (0·3 and 30 µM CuSO 4 , respectively) for 7 d in hydroponic culture. Total proteins were extracted from the roots and leaves and separated by two-dimensional polyacrylamide gel electrophoresis. The differences in protein patterns on silver-or Coomassie-stained gels were analysed. The most apparent quantitative difference was the increase in the amount of a 17 kDa polypeptide caused by Cu stress in both roots and leaves. The protein was identified as Bet v 1-Sc3 (according to the current nomenclature PR-10c) using N-terminal amino acid sequencing and on-line highperformance liquid chromatography/electrospray ionization/ion trap mass spectrometry. The present results indicate that PR-10 is not only activated by pathogens but also by excessive amounts of copper ions. PR-10 proteins in birch have been reported earlier not to be induced by Ag, Li or Cd in birch suspension culture, but Cu has not been previously tested.Key-words: birch pollen allergen; copper; Cu/Zn resistance; ESI-MS; heavy metals; pathogenesis-related protein; twodimensional electrophoresis.Abbreviations: CID, collision-induced dissociation; 2D, twodimensional; EC50, the concentration which inhibits the root growth by 50%; HPLC-ESI-MS, high-performance liquid chromatography/electrospray ionization/ion trap mass spectrometry; IEF, isoelectric focusing; PVDF, polyvinylidene difluoride. INTRODUCTIONPlants respond to copper stress through a variety of biochemical reactions (Van Assche & Clijsters 1990). Copper is a redox-active heavy metal which, when present in excessive amounts, causes uncontrolled redox reactions in the cells, resulting in the formation of damaging free radical species, peroxidative destruction of biomembranes and, finally, cell death (Hochstein et al. 1980; Sandman & Böger 1980a,b;Freedman et al. 1989). Toxic copper concentrations give rise to scavenging systems against oxidative stress, and stimulate catalase, peroxidase and ethylene production which, in turn, stimulate lipoxygenase capacity (Gora & Clijsters 1989;Weckx et al. 1993). Well-demonstrated responses to copper are potassium leakage due to destruction of biomembranes (McBrien & Hassall 1965) and induction of metal-binding polypeptides known as phytochelatins (Steffens 1990). The precise targets and primary toxic effects of copper within plant cells are not known for certain (Verkleij & Schat 1990). However, data on enzyme induction indicate that copper might act mainly at the root level (Van Assche & Clijsters 1990).We have screened several micropropagated birch clones in hydroponics for their tolerance to copper and zinc (Utriainen et al. 1997). The aim of the present study was to characterize Cu-responsive proteins in the roots of the most tolerant clone. In order to understand metal tolerance in plants we need to identify the molecular mechanisms involved in the biochemical adaptation. MATERIALS AN...