Ozone Induced Changes in Plant Secondary Metabolism

Marcello Iriti and Franco Faoro

11.1 Introduction

In their ecosystem, plants have to cope with a plethora of potentially unfavourable conditions. Stress factors affecting plant's fitness not only derive from natural sources, such as adverse temperature fluctuations (heating, chilling and freezing), high irradiance (photoinhibition, photooxidation), osmotic imbalance (salinity and drought), hypoxia/anoxia (flooding), mineral (macro- and micronutrient) deficiency, wounding, phytophagy and pathogen attack, but also from anthropogenic activities. The latter include xenobiotics employed in agriculture (herbicide, pesticides and fungicides), environmental (air, soil and water) pollutants and increased UV radiations. Particularly, many atmospheric pollutants, belonging to greenhouse gases, may increase the greenhouse effect, a natural warming process that prevents heat from diffusing to the outer atmosphere, thus balancing Earth cooling processes. Without the natural greenhouse effect, temperature on Earth would be much lower than it is now, and the existence of life would have not been possible. However, the rising emissions of greenhouse gases due to anthropogenic activities, namely carbon dioxide (CO2), chlorofluorocarbons (CFCs), nitrous oxide (N2O), tropospheric ozone (O3) and water vapour, may cause a short-term increase of the mean global temperature on the planet surface with consequent changes in precipitation patterns (Krupa and Kickert 1989). In this scenario, life on the earth depended from the co-evolution between atmosphere and biosphere, because the gradual and long-term climate changes enabled living organism adaptation to the new temperatures, precipitation patterns and other climate conditions (Voronin and Black 2007).

Regardless of natural or anthropogenic stress factors, plants have to cope with their stressors. From a pathophysiological point of view, a plant may avoid or adapt to a particular stress, with a dose-dependent mechanism (Fig. 11.1). Under a certain threshold, a mild stress may be compensated by the plant, whereas, at higher

Istituto di Patología Vegetale, Universita di Milano and Istituto di Virología Vegetale, CNR, Via Celoria 2, 20133 Milano, Italy e-mail: [email protected]

S.N. Singh (ed.), Climate Change and Crops, Environmental Science and Engineering, DOI 10.1007/978-3-540-88246-6.11. © Springer-Verlag Berlin Heidelberg 2009

concentration, the detrimental effects of a severe stress may cause irreversible damages, according to the stressor dose-stress effect relationship (Lichtenthaler 1998). Furthermore, the stress tolerance threshold depends not only on the type of stressor and exposure time, but also on the plant stress-coping capacity. Anyway, the shift between normal and stress metabolism represents a fundamental trait in plant acclimation (short-term) and adaptation (long-term) strategies, although it is almost impossible to define exactly the threshold between them (Heiser and Elstner 1998).

In this chapter, we deal with the phytotoxic potential of tropospheric ozone, due to partially reduced oxygen intermediates produced by the pollutant in biological systems. These intermediates are more reactive than molecular oxygen in its ground state and include both radical (superoxide anion, • O2- and hydroxyl radical, OH') and non-radical (hydrogen peroxide, H2O2) forms, collectively termed as reactive oxygen species (ROS) (Halliwell 2006). Plant tolerance mechanisms will also be discussed with emphasis on ozone-induced metabolic fluxes between primary (normal) and secondary (stress) metabolism, due to acute or chronic pollutant exposure. Besides, ozone is a greenhouse gas, although it plays a minor role in regulating the air temperature and in contributing to the warming effect (Wang et al. 1995).

11.2 The Ozone in the Atmosphere

Ozone is an important constituent of the atmosphere, although present in trace amounts. As a matter of fact, two different pools of O3 exists, the beneficial and the detrimental one (Fig. 11.2). In the stratosphere (the higher atmosphere, ranging approximately from 15 to 40 km in altitude), the ozone layer absorbs the harmful UV-B and UV-C radiations, thus saving the living organisms (Dutsch 1978; Kerr and McElroy 1993). In the past decades, emission of ozone-depleting chemicals led to the reduction of the ozone shield (commonly referred to as 'ozone hole') against UV radiation, worsening its harmful effects on animals and plants (Platt and Honninger 2003). Otherwise, in the troposphere (the lower part of the atmosphere, approximately from the earth surface to 10-12 km in altitude), that is to say the layer

Fig. 11.2 Beneficial and detrimental pools of ozone in the stratosphere and troposphere, respectively. In the stratosphere, this pool is depleted by fluorochlorocarbons (CCl2C2) while in the troposphere, it is raised by photochemical smog

where the climatic conditions originate, and temperature decreases with elevation, ozone is regarded as a pollutant (Logan 1985).

Tropospheric ozone is an oxidant constituent of the photochemical smog. It is a secondary pollutant produced through reactions among primary pollutants, emitted directly into the air (mainly nitric oxides, sulphur oxides, carbon oxides and hydrocarbons), catalyzed by sunlight (Crutzen and Lelieveld 2001) (Fig. 11.2). Hence, ozone is produced on bright sunny days over areas with intense primary pollution, mainly due to vehicle exhausts, fossil fuel burning and industrial processes, in the so-called photochemical cycle (Fowler et al. 1999; Kley et al. 1999). Meteorological conditions may exacerbate the rate of ozone formation too, particularly atmospheric inversion, a restricted air circulation associated to a warmer air layer above a cooler zone (Baumbach and Vogt 2003).

11.3 Chemistry of Ozone Injury

From the Greek ozein (to smell), ozone, the triatomic allotropic form of oxygen, is a colourless gas with a slightly sweet, water melon-like odour (odour threshold between 0.0076 and 0.036 ppm). Because of its strong oxidizing potential

(+ 2.07 eV), O3 is a powerful oxidizing agent capable of reacting with virtually any biomacromolecule, including lipids, proteins, nucleic acids and carbohydrates, although it is neither a radical species nor a ROS (Mustafa 1990; Kelly et al. 1995). Ozone is considered too reactive to penetrate far into tissues, so that only a minor amount of the pollutant is believed to pass not reacted through a membrane, and nothing through a cell (Pryor 1992). Furthermore, its toxicity can be greatly enhanced by the spontaneous hydroxyl radical (OH ) generation in the aqueous solution, strongly accelerated by traces of Fe2+ and favoured at alkaline pH, though occurring even at physiological pH (Pryor 1994).

In cell membrane, polyunsaturated fatty acids represent the primary target for ozone, stimulating lipid peroxidation and impairing membrane fluidity. The chemistry of O3-induced lipid peroxidation, known as Criegee ozonation pathway, involves ozonolysis of alkenes in polyunsaturated chains, i.e. the electrophilic O3 addition across the carbon-carbon double bonds, to give the Criegee ozonide (Criegee 1957). Afterwards, ozonide decomposes, under suitable conditions, to form organic radicals, aldehydes and peroxides. In further steps, H2O2 can react with transition metals (Cu or Fe), according to Fenton or Haber-Weiss reactions, to form other ROS (Pryor et al. 1991; Pryor 1993).

As a result of the ozone-induced oxidation, modification of proteins also occurs, both in their structure and activity. The pollutant directly or through highly reactive free radical mediated reactions, oxidizes the amino acidic residues, mainly of tyrosine, tryptophan, cysteine, methionine and histidine (Mudd et al. 1969). In particular, it reacts with the exposed sulphydryl groups to form disulphides bridges, and with tryptophan to give protein ozonides, in turn generating protein hydroperoxides and hydrogen peroxide (Freeman and Mudd 1981). Tyrosine residues can be cross-linked too, after the oxidation of their HO- groups, to give O,O'-dityrosine (Ignatenko et al. 1984). DNA damage can be produced as well, as shown by the increased activity of poly (ADP-ribose) synthetase, a chromatin-bound enzyme promoting damaged DNA repair (Hussain et al. 1985).

11.4 Plant Secondary Metabolism

In living organisms, secondary metabolites are not essential for growth and development unlike the products of primary metabolism. However, this does not mean they are not necessary. Infact, secondary metabolites relate plants with the components of their ecosystem, that is to say the physic environment (biotope) and the living community (biocenosis), thus resulting indispensable for the survival of the species. Additionally, stress metabolism could be regarded as a particular expression of secondary metabolism, when stressful conditions, of both biotic and abiotic nature, change the dynamic equilibrium of the ecosystem. As an instance, phytoalexins are compounds synthesized ex novo or whose synthesis increases after pathogen challenging, raising their concentration in the tissues (Frank 1993; Paiva 2000).

In plants, chemical diversity has determined their evolutionary success. Because of their sessile habit, plants are unable to avoid the worsening environmental conditions as well as they cannot escape the plethora of the laying before biotic stresses. Consequently, unlike animals, plants have evolved an enormous number of secondary metabolites to overcome any danger. The functional role of these phyto-chemicals ranges from the ecology to defence, improving protection against both biotic and abiotic stresses, besides being involved in ecological roles as attractants or repellents for pollinators and phytophagy, respectively, and colours and scents of reproductive organs (flowers and fruits).

Generally, precursors of secondary metabolic pathways are products of the primary metabolism. Therefore, a severe or long-lasting stress factor could induce an excessive shift between primary and secondary metabolism and consequently, a diversion of essential available resources from growth to defence. To a large extent, secondary metabolites derive from three biosynthetic routes, namely the phenyl-propanoid, isoprenoid and alkaloid pathways. Phytochemicals arising from these pathways include not only compounds with a broad-spectrum antibiotic activity, but also powerful antioxidants, able to efficiently scavenge ozone-induced ROS (Facchini 2001; Holstein and Hohl 2004; Iriti and Faoro 2004).

11.4.1 Phenylpropanoid Pathway

Phenylpropanoids are a class of phenylalanine derivatives with the basic skeletal C6-C3 (phenyl-propane) as depicted in Fig. 11.3.

Precursors of this pathway are phosphoenolpyruvate derived from glycolysis and erythrose 4-phosphate from pentose phosphate pathway, leading to two important intermediates, shikimic acid and chorismic acid. In further steps, after a branch point, phenylalanine and tyrosine are synthesized from prephenic and arogenic acid, whereas tryptophan comes from anthranilic acid (Weaver and Hermann 1997).

The removal of an amino group (deamination) from phenylalanine via the step catalyzed by the enzyme phenylalanine ammonia-lyase (PAL), leads to formation of cinnamic acid and, in turn, the precursor of hydroxycinnamates is produced after a series of hydroxylation of the benzene ring (Fig. 11.4). These compounds, including coumaric, ferulic and sinapic acids are reduced to the corresponding alcohol via aldehyde intermediates, namely coumaryl, coniferyl and sinapyl alcohol, collectively termed monolignols. Dimerization or polymerization of monolignols leads to formation of lignans and lignin, respectively (Fig. 11.4). Lignification is a complex reaction in which peroxidases catalyze the polymerization of lignin units consuming H2O2. Apposition of lignin in plant cell wall is a process occurring during the development of particular tissues, as well as in plant defence responses, in order to strengthen the cell wall and to protect the plasmalemma. Benzoic and hydroxybenzoic acids (C6-C1), such as salicylic acid (SA), a molecule involved in systemic acquired resistance (SAR), are another group of cinnamic acid derivatives,

Phosphoenolpyruvate (PEP) COO-

Pi-O H2

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