Induction of Rishitin-Metabolizing Activity in Potato Tuber Tissue Disks by Wounding and Identification of Rishitin Metabolites

Abstract

ISHIGURI, Y., K. TOMIYAMA, N. DOKE, A. MURAI, N. KA TSUI, F. Y AGIHASHI,and T. MASAMUNE. 1978. Inductionof rishitin-metabolizing activity in potato tuber tissue disks by wounding and identification of rishitin metabolites. Phytopathology 68: 720-725. Rishitin, the phytoalexin of potato, was metabolized by fresh and aged potato-tuber disks. In aged disks, radioactivity of rishitinC began to decrease almost simultaneously after incubation with rishitinC and continued to decrease at the rate of 3.6 μ.g rishitin/ g fresh wt/hr. The decrease in radioactivity of rishitinC was accompanied by an increase in radioactivity in the ethersoluble fraction (minus rishitin) and then followed by that in the water-soluble fraction. In fresh disks, on the contrary, there was a lag of a few hours before the initiation of decrease Additional key words: potato late blight. The sesquiterpene phytoalexins of potato [rishitin (7, 14, 26), rishitinol (8, 9), lubimin (10, 11, 13, 23), oxylubimin (10, 11), solavetivone (I) etc.] have been reported to play an important role in disease resistance ( 12, 21, 22). However, it is still not clear how induction of synthesis, accumulation, and disappearance of any of these phytoalexins occur. In a previous paper (3), it was reported that aged surface tissue of cut potato tuber noninfected or infected by an incompatible race of Phytophthora infestans could transform rishitin to other compounds. The metabolism of rishitin in these tissues may play a key role in accumulation and disappearance of rishitin. In this paper, we report: (i) the identification of metabolites of rishitin in potato tissue, (ii) the time course of rishitin conversion in potato tuber tissue, and (iii) the results of experiments carried out to determine whether fresh potato tuber disks could metabolize rishitin or not. MATERIALS AND METHODS Plant materials.-Tubers of the potato cultivar Rishiri carrying the R1 gene were used. They are highly resistant to race 0 of Phytophthora infestans which was used in the present experiments. Tubers were stored at 4 C, but were kept at 18 C for 24 hr before they were used for the 00032-949X/78/0001 21 $03.00/0 Copyright © 1978 The American Phytopathological Society, 3340 Pilot Knob Road, St. Paul, MN 55121. All rights reserved. in radioactivity of rishitinC. These results suggest that intact potato tissue has little activity to metabolize rishitin, but that the activity was induced by wounding. Two Clabeled compounds were isolated from the ether-soluble metabolites of rishitin in tuber tissue treated with C-rishitin. They were identified as rishitin-M-1 and rishitin-M-2 on the basis of R, values after thin-layer chromatography (TLC}, autoradiography, and color reactions after two-dimensional TLC. experiments. Zoospore suspensions of P. infestans were prepared as described previously (3). Preparation of rishitinC.-The 14C-labeled rishitin was prepared by the method of Horikawa et al. (3). A hole was made in the potato tuber and inoculum consisting of zoospores of race 0 was added. Then the hole was filled with 2 ml of acetate-214C solution (10 μCi/ml), which was prepared by dissolving acetate-214 C (56 mCi/ mmole, Radiochemical Center, Amersham, England) in distilled water. Rishitin14C was isolated from the fluid in the holes and from the tissue surrounding the holes according to the method of Horikawa et al. (3). Fifty mg of nonlabeled rishitin was added to the extract and final purification of the crude rishitin obtained was effected by formation of the crystalline bis(3,5-dinitrobenzoate) followed by hydrolysis and distillation in vacuo (26). When the rishitin14 C obtained (336 dpm/ μg) was chromatographed on silica gel by thin-layer chromatography (TLC), a single peak of radioactivity was obtained at the proper R1. Incubation of tuber tissue with rishitinC.-Tissue disks ( 16 mm in diam, I 0-mm thick), were cut out with a cork borer from the central tissue of potato tubers. The disks were washed with running water for 30 min and then used immediately or after incubation at about 18 C for 24 hr. The upper surfaces of the disks were sectioned into slices 0.7 mm thick. Fifteen slices (about 2.2 g) were placed in 1 ml of sterile water containing pure rishitin14C