Functional analysis of polyphenol oxidases by antisense/sense technology

Abstract

Polyphenol oxidases (PPOs) catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum) plants with modified PPO expression levels (suppressed PPO and overexpressing PPO). These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F.)), cotton bollworm (Helicoverpa armigera (Hübner)) and beet army worm (Spodoptera exigua (Hübner)), whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say)) feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants overexpressing PPO, suggesting that PPO may have a role in the development of plant water stress and potential for photoinhibition and photooxidative damage that may be unrelated to any effects on the Mehler reaction. These results substantiate the defensive role of PPO and suggest that manipulation of PPO activity in specific tissues has the potential to provide broad-spectrum resistance simultaneously to both disease and insect pests, however, effects of PPO on postharvest quality as well as water stress physiology should also be considered. In addition to the functional analysis of tomato PPO, the application of antisense/sense technology to decipher the functions of PPO in other plant species as well as for commercial uses are discussed.

Figure 1

In both compatible interactions (A, C) and incompatible interactions (B, D) between tomato and Pseudomonas syringae, PPO suppression results in enhanced susceptibility to infection. Antisense PPO transgenic plants have higher bacterial growth (A, B) and lesion numbers (C, D) than nontransformed controls. Results are presented as means ± SE. Leaf node 1 is closest to the apex [d].

Figure 2

Leaf area consumption (A) and simple growth rates (B) of beet armyworms (Spodoptera exigua) feeding on nodes 4 and 8 leaves of tomato vary in different genotypes differing in PPO activity levels. Relative growth rates (C) are not related to PPO activities. Durations of larval stages (D) of beet armyworms are similar among genotypes when fed on node 4 leaves, but are extended on OP plants and attenuated on SP plants compared to controls when fed on node 8 leaves. Results are presented as means ± SE. Leaf node 1 is closest to the apex.

Figure 3

Correlation between PPO activities and simple growth rates of cotton bollworms (Helicoverpa armigera) feeding on leaflets at node 4 of transgenic plants with suppressed and overexpressed PPO activity segregating for PPO activity levels (A), and between PPO activities and leaf areas consumed (B).

Figure 4

Leaf water potential (Ψ_L) during water stress declines more slowly in transgenic plants with suppressed PPO levels (A). During water stress these plants exhibits higher quantum yields of PSII electron transport (φ_PSII; B) but lower photoinhibition (C) compared to nontransformed controls. Suppression of PPO reduces chlorophyll loss (D) and anthocyanin accumulation (E) in leaflets at node 7. Results are presented as means ± SE [13].

Figure 5

Hypothetical mechanisms of action of tomato PPO in response to pathogen infection, insect infestation and water stress.