J. AMER. SOC. HORT. SCI. 117(6):1012-1016. 1992. Expression of a Chimeric Tobacco Peroxidase Gene in Transgenic Tomato Plants L. Mark Lagrimini 1 , Jill Vaughn 2 , John Finer 3 , Karen Klotz 4 , and Patrick Rubaihayo 5 The Ohio State University, Columbus, Ohio 43210 Additional index words. soluble solids concentration, tissue blots, isoelectric focusing, Lycopersicon esculentum Abstract. Tomato plants (Lycopersicon esculentum cv. OH 7814) were transformed via Agrobacterium tumefaciens with a chimeric tobacco anionic peroxidase (EC 1.11.1.7) gene joined to the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants obtained by selection on kanamycin were found to have more than five times the total leaf peroxidase activity of control plants. Transformed tomato plants chronically wilted upon reaching sexual maturity. Two independently selected transformants were self-fertilized, and progeny were obtained that were homozygous for the foreign gene. Isoelectric focusing gels stained for peroxidase activity revealed a new tomato leaf peroxidase isoenzyme with a pI of 3.75, which is similar to that seen in Nicotiana sylvestris L. Mature tomato fruit were found to have up to 1600-fold higher peroxidase activity in transformants expressing the tobacco anionic peroxidase (TobAnPOD) than control plants. Tissue blots showed the tobacco enzyme evenly distributed throughout the tomato fruit tissue. Progeny plants possessing the tobacco peroxidase gene (now homozygous) showed stunting, and fruit size was reduced by >80%. However, fruit set was normal and the rate of ripening was not altered from control plants. Fruit from transformed plants were found to have normal pigmentation, but the soluble solids concentration was 400% higher than in control tomato fruit. This result was predicted from the peroxidase-induced water stress. Possible roles for the tobacco anionic peroxidase in growth, development, and stress resistance are discussed. Plant peroxidases (donor : hydrogen-peroxide oxidoreduc- tase) carry out single-electron oxidations of a wide variety of compounds in the presence of H 2 O 2 or O 2 (Gaspar et al., 1982). Cinnamyl alcohols, phenolic acids, aromatic amines, and in- doles are some of the natural compounds that serve as electron donors in peroxidase-catalyzed reactions. In addition to the large variety of potential substrates found in plants, there are also a multitude of peroxidase isoenzymes. Tobacco and tomato each have > 12 peroxidase isoenzymes (Lagrimini and Rothstein, 1987; Marangoni et al., 1989). This makes it difficult to assign spe- cific functions to individual peroxidases. Some of the physio- logical processes in which peroxidases have been implicated include the regulation of cell elongation (Goldberg et al., 1986), cross-linking of cell wall polysaccharides (Fry, 1986), lignifi- cation (Grisebach, 1981), wound-healing (Espelie et al., 1986), pathogen defense (Hammerschmidt et al., 1982), and phenol oxidation (Strivastava and Huystee, 1977). Several reviews have been written on the numerous biochemical and physiological functions of peroxidase (Everse and Grisham, 1991; Greppin et al., 1986). Although peroxidases are ubiquitous to vascular plants and are certainly crucial to growth and development, many of the proposed functions for peroxidases are based solely on in vitro data. It has been difficult to determine the actual in vivo role for individual peroxidase isoenzymes using standard biochem- ical techniques, due in part to the many substrates and isoen- zyme forms. Based on prior biochemical and cytological data, the TobAnPOD is considered to participate in the formation of Received for publication 10 Mar. 1992. Accepted for publication 8 June 1992. Supported in part by a grant from the United States Department of Energy [DE- FG02-89ER14004) and by state and federal funds to the Ohio Agricultural Research and Development Center. The Ohio State Univ. J. Article no. 12- 92. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1 Dept. of Horticulture, to whom reprint requests should be addressed. 2 Dept. of Food Science and Technology. 3 Dept. of Agronomy. 4 Dept. of Horticulture. 5 Visiting Scholar. Department of Crop Science, Makerere University, Kampala, Uganda. lignin (Lagrimini, 1991; Mäder et al., 1977). Recently, trans- genic tobacco plants have been generated with altered peroxi- dase activity (Lagrimini et al., 1990). These transgenic tobacco plants had a chimeric TobAnPOD gene under control of the CaMV 35S promoter introduced to direct the overexpression of the TobAnPOD. The plants were characterized by chronic wilt- ing, which began at the time of flowering (Lagrimini et al., 1990). This result suggested a connection between peroxidase activity, root growth, and water relations. Also, a wound-in- duced browning reaction occurred in pith tissue excised from tobacco plants that overproduced the TobAnPOD isoenzyme (Lagrimini, 1991). This browning reaction in damaged plant tissue was caused by deposition of polyphenolic acids in cell walls. Tomato plants may have as many as 12 peroxidase isoen- zymes, however, tomato fruit expresses only one isoenzyme with an isoelectric point of 3.5 (Evans, 1970). The tomato en- zyme has an approximate molecular weight of 40-42 kD. Unlike the TobAnPOD, this enzyme has a strict requirement of Ca for activity (Marangoni et al., 1989), and the tomato fruit enzyme has been implicated in the oxidation of indole3-acetic acid (Brooks, 1986). These characteristics differ from those of TobAnPOD (Lagrimini and Rothstein, 1987), and these en- zymes may perform different functions in their respective tissues and organisms. In this study, tomato plants were transformed with this same chimeric TobAnPOD gene to determine if the dramatic pheno- types seen in tobacco could also be obtained in tomato. The effect of TobAnPOD on the tomato fruit with respect to food quality was also investigated, since peroxidase has been impli- cated in excessive browning and fiber formation in harvested fruits and vegetables (Haard, 1977). Also, the precipitation of proteins by polyphenols can lead to an astringent flavor and a decrease in palatability (Ozawa et al., 1987). Polyphenols can also contribute to decreased digestibility through the denatura- tion of protein and cross-links to carbohydrates (e.g., lignocel- lulose) (Jung and Fahey, 1983). For these reasons, we were Abbreviations: CaMV, cauliflower mosaic virus; POD, peroxidase; Tob- AnPOD, tobacco anionic peroxidase. 1012 J. Amer. Soc. Hort. Sci. 117(6):1012-1016. 1992.