Polyphenol Oxidases- Expression, Assembly and Function

Polyphenol oxidases (PPOs) participate in the preparation of many plant products on the one hand and cause considerable losses during processing of plant products on the other hand. However, the physiological functions of plant PPO were still a subject of controversy at the onset of the project. Preliminary observations that suggested involvement of PPOs in resistance to herbivores and pathogens held great promise for application in agriculture but required elucidation of PPO's function if modulation of PPO expression is to be considered for improving plant protection or storage and processing of plant products. Suggestions on a possible role of PPO in various aspects of chloroplast metabolism were also relevant in this context. The characterization of plant PPO genes opened a way for achieving these goals. We reasoned that "understanding PPO targeting and routing, designing ways to manipulate its expression and assessing the effects of such modifications will enable determination of the true properties of the enzyme and open the way for controlling its activity". The objective of the project was to "obtain an insight into the function and biological significance of PPOs" by examining possible function(s) of PPO in photosynthesis and plant-pest interactions using transgenic tomato plants; extending our understanding of PPO routing and assembly and the mechanism of its thylakoid translocation; preparing recombinant PPOs for use in import studies, determination of the genuine properties of PPOs and understanding its assembly and determining the effect of PPO's absence on chloroplast performance. Results obtained during work on the project made it necessary to abandon some minor objectives and devote the effort to more promising topics. Such changes are mentioned in the 'Body of the report' which is arranged according to the objectives of the original proposal. The complex expression pattern of tomato PPO gene family was determined. Individual members of the family are differentially expressed in various parts of the plant and subjected to developmentally regulated turnover. Some members are differentially regulated also by pathogens, wounding and chemical wound signals. Wounding systemically induces PPO activity and level in potato. Only tissues that are developmentally competent to express PPO are capable of responding to the systemic wounding signal by increased accumulation of PPO mRNA. Down regulation of PPO genes causes hyper susceptibility to leaf pathogens in tomato while over expression regulation of PPO expression in tomato plants is their apparent increased tolerance to drought. Both the enhanced disease resistance conferred by PPO over expression and the increased stress tolerance due to down regulation can be used in the engineering of improved crop plants. Photosynthesis rate and variable fluorescence measurements in wild type, and PPO-null and over expressing transgenic tomato lines suggest that PPO does not enable plants to cope better with stressful high light intensities or reactive oxygen species. Rather high levels of the enzyme aggravate the damage caused under such conditions. Our work suggests that PPO's primary role is in defending plants against pathogens and herbivores. Jasmonate and ethylene, and apparently also salicylate, signals involved in responses to wounding and defense against herbivores and pathogens, enhance markedly and specifically the competence of chloroplasts to import and process pPPO. The interaction of the precursor with thylakoid membranes is primarily affected. The routing of PPO shows other unusual properties: stromal processing occurs in two sites, resulting in intermediates that are translocated across thylakoids by two different mechanisms - a DpH- and a Sec-dependent one. It is suggested that the dual pattern of processing and routing constitutes a'fail safe' mechanism, reflecting the need for a rapid and flexible response to defense challenges. Many of the observations described above should be taken into consideration when manipulation of PPO expression is contemplated for use in crop improvement.