Synthesis of Indoleacetic Acid via Tryptamine by a Cell-free System from Tobacco Terminal Buds

Abstract

Plants infected with the vascular pathogen, Pseudomonas solanacearum, exhibit symptoms associated with auxin imbalance. The content of 3-indoleacetic acid (IAA) has been shown to increase 100-fold in inoculated tobacco plants (7). The origin of IAIA in di;seased tissues has remained relatively obscure, although preliminary data suggest that the host contributes most of the auxin during early stages of pathogenesis (6). Progress in this area has been impeded by the similarity in pathways of biosynthesis of IAA from tryptophan (TTP) in higher plants and microorganisms. A possible approach to the problem of IAA synthesis in the host-parasite complex is the determination of differences in synthetic pathways between both mernbers, thus allowing the use of radioactive precursors which only one member can convert to IAA. Alternate pathways in the conversion of TTP to IAA have been reported for P. solanacearutm (8) and one of its hosts, tobacco (6). The recovery of radioactive IAA from tobaccoo plants fed 14C-labeled tryptamine (TNH2) suggesIted that this compound was a possible precursor of IAA synthesiis in tobacco. Confirmation of these results was sought by studying the synthesis of auxin in a cell-free system from tobacco terminal buds. Apical stem sections, 3 cm in length, were removed from 4-week-old tobacco plants (Nicotiana tabacum var. Bottom Special) grown in sand culture at 280, under 1500 ft-c from a combination of General Blectric Co-ol White, Sylvania Gro Lux and incandescent bullbs on a 12-hour photoperiod. The apices were homogenized in a Sorvall Omnimixer for 4 minutes in cold 20 mM potassium phosphate (pH 6.0) at 2 ml per g fresh weight of tissue. The homogenate was centrifuged at 5000 g for 10 minutes at 00 and the supernatant fraction used without further treatment. If not homogenized immediately, apices were frozen in dry ice and stored at -20? for future use. Reaction mixtures containing tobacco celil-free extracts in 20 mm phosphate buffer at pH 6.0 and 2.5 ptmole/ml L-TTP were incubated for 1 hour at 300. The total volume was 10 ml. Reactions were terminated by heating in a boiling water bath for 5 minutes or by adding an equal volume of 10 % trichloroacetic acid. The protein preciipittated by heat or trichloroacetic acid treatment was removed by centrifugation at 10,000 g for 10 minutes and the supernatant was retained for extraction of indoles. Control mixtures containing heat-inactivated enzyme or no enzyme were treated similarly. In experiments utilizing labeled precursors, DL-TTPl'-*C (8.95 mrc/mmnole) or TNH2-1'-'*C bi,succinate (2.73 m'c/mmole) were added to reaction mixtures at 0.10 Ac/Aml. Indole compounds were separated into: A) Other-solu'ble acidic, B) ether-soluble basic plus neutral, and C) ether-insoluble components. For this purpose, the reaction mixture was adjusted to pH 8.2 with solid NaHCO3 and basic and neutral substances were removed by extraction with diethyl ether. The aqueous layer was adjusted to pH 3.0 with 1 N HOC and acidic substances were extracted with ether. The aqueous layer contained the etherinsoluble components. The 3 fractions were evaporated to 1 ml under reduced pressure at 250. Three-week-old tobacco plants, grown in the manner already described, were removed from the pots and the sand was washed off the roolts. Each pliant was placed in a 250 ml beaker containing either 9.5 mg TNH2-HCI or 10.0 mg DL-TTP to which had been added 2 pc of the appropriate radioactive precursor. Each treatment involved 4 plants. Each plant absorbed the initial 50 ml of solution in about 3 hours. Distifled water was added as required over an incubation peried of 44 hours, after which the plants were immediately frozen in dry ice. The frozen plants were finely ground with a m,ortar and pestle at -78? and the powder was added slowly to 400 ml of hot ethyl acetate. The mixture was boiled for 15 minutes, filtered through cheesecloth and the plant material was ground in 300 ml distilled water in a Waring Blendor. The suspension was filtered through cheesecloth and the solids were discarded. The supernatant was adjusted to pH 3.8 and extracted 3 times with 100 ml ethyl acetate. The acetate fraiction was washed once with distilled water and combined with the original ac-etate extract. Tihe aqueous fractions were bulked, adjusted to pH 3.0 and extracted 3 times with 100 ml diethyl ether. This fraction was labeled 1. The aqueous layer was adjusted to pH 8.2 with 100 ml 5 % NaH003 and was extracted 1 Supported by Research Grants -Nos. GB125 and GB4209 from the National Science Foundation. 2 Published with the approval of the Director, Wisconsin Agricttltural Experiment Station. Project 1209.