HORTSCIENCE 28(1):38-40. 1993. Localization of Peroxidase in Grapes using Nitrocellulose Blotting of Freezing/Thawing Fruits Antonio A. Calderón, Jose M. Zapata, Romualdo Muñoz, and A. Ros Barceló 1 Department of Plant Biology (Plant Physiology), University of Murcia, E-30100 Murcia, Spain Additional index words. isoenzymes, skin, tissue fractionation, Vitis vinifera Abstract. A technique has been developed to study the histochemical localization of peroxidase in Vitis vinifera by blotting freezing/thawing tissue sections on nitrocellulose membranes. After being stained with 4-methoxy- α α -naphthol and H 2 O 2 , peroxidase-mediated reaction products in mature ‘Gamay’ grapes were seen principally in the skin and, to a lesser extent, the pericarp, where discrete areas of reaction products were located in the vascular bundles. However, for immature ‘Gamay’ and ‘Grenache’ grapes, peroxidase activity in the skin was low and similar to that found in the pericarp. With this technique, fruit vascular bundle structure was preserved. The reliability of the technique in the histochemical localization of peroxidase in grapes was confirmed by fractionation and determining the peroxidase activity in the various tissues. Peroxidase (EC 1.11.1.7) is an enzyme studied widely in plants, and there is extensive literature describing changes in peroxidase activity and isoenzyme spectra as a function of ontogenic change and stress and as a specific characteristic of plant varieties and cultivars (Haard, 1977). Little effort, however, has been directed to understanding a physiological function for this enzyme. Recently, it has been proposed that peroxi- dase may participate in the hydrogen perox- ide-dependent metabolism of phenols (phe- nolic acids and flavonoids) in the vacuole (Takahama, 1991). In the grape cultivar Gamay, it has been shown that peroxidase, in conjunction with β- glucosidases, may be re- sponsible for anthocyanin turnover and deg- radation (Calderón et al., 1992a). This obser- vation is supported by the co-localization of peroxidase and anthocyanins in grapevine vacuoles (Calderón et al., 1992b). To clarify this possible role of peroxidases, histochemi- cal studies of peroxidase in grapes are neces- sary, since it is well known that anthocyanins are located mainly in the epidermal and the subepidermal cell layers that constitute the berry skin (Moskowitz and Hrazdina, 1981). Tissue printing has become an important tool invisualizing enzyme localization in plant tissues (Spruce et al., 1987). This technique Received for publication 13 Feb. 1992. Accepted for publication 14 Sept. 1992. This work was in part supported by a grant from the CICYT (Spain), project no. AGR 296/89. A.A. Calderón holds a fellowship (BPPI) from the MEC (Spain). We thank A. Martínez-Cutillas and J. Carreño-Espín (CRIA, Murcia, Spain) for providing the grape berries. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regula- tions, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1 To whom reprint requests should be addressed. 38 consists of blotting fresh tissue sections onto nitrocellulose membranes, using the proper- ties of nitrocellulose to absorb and retain pro- teins (Nibbering et al., 1986), and subsequently detecting enzyme activities on the blots using simple histochemical procedures. However, using this technique to localize enzymes in fleshy fruits is limited, since the tissue has to be hard or stretched so that cross sections can be obtained easily. Thus, it is difficult to blot grape berry sections on nitrocellulose mem- branes because of the near impossibility of obtaining thin sections without tearing the tissues. To avoid this problem, we have devel- oped a technique that consists of cutting fro- zen tissues. After thawing, the sections are blotted on nitrocellulose membranes. A histo- chemical peroxidase-staining technique on blotted tissues was developed using 4-methoxy- α- naphthol (4-MN) as a specific substrate, since it is not oxidizable by other hemoproteins (Ferrer et al., 1990). The results, confirmed by tissue fractionation, show that most peroxidase activity in mature grapes is located in the skin of the berry, as are the anthocyanins. Plant material and tissue printing. ‘Gamay’ rouge and ‘Grenache’ were grown in the field at the Hacienda Nueva Viticultural Experi- mental Station of the CRIA (Murcia, Spain), and sampled before (immature grapes) and after (mature grapes) veraison (pigmentation). Clusters were transported to the laboratory HORTSCIENCE , VOL. 28(1), JANUARY 1993