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-in-doleacetic acid (IAA) has been sholwn to increase 100-fold in inoculated tobacco plants (7). The origin of IA'A in diseased 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 i's the determination of differences in synthetic pathways between both members, thus aldlowing 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. solanacearum (8) and one of its hosts, tobacco (6). The recovery of radioactive IAA from tobacco plants fed 14C-labeled tryptamine (TNHo) suggested that thi,s compound was a possible precursor of IAA synthesi,s in tobacco. Confirmation o!f 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-olid tobacco plants (Nicotiana tabacum var. Bottom Special) grown in sand culture at 280, under 1500 ft-c from a combination of General Blectric Cool \White, Sylvania Gro Lux and incandescent bulibs on a 12-houtr photoperiod. The apices were homogenized in a Sorvall Omni-mixer 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 0° and the supernatant fraction tu-sed without further treatmenit. If not homoge-nized immediately, apices were frozen in (Iry ice and stored at-200 for future use. Reaction mixtures containing tobacco cell-free extracts in 20 mm phosphate bluffer at pH 6.0 and 2.5 jumole/ml L-TTP were incubated for 1 hour at 300. The total voltume was 10 ml. Reactions were I terminated by heating in a boiling water bath for 5 minutes or by adding an equal volume of 10 % trichloroacetic acid. The protein precipitated 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 in-doles. Control mixtures containing heat-inactivated enzyme or no enzyme were treated similarly. In experiments utilizing labeled precursors, Df-TTP-1'-'4C (8.95 mc/mmole) or TNH2-1'-'AC bisuccinate (2.73 mc/mmo,le) were added to reaction mixtures at 0.10 c/ml. Indole compounds were separated into: A) ether-soltible acidic, B) ether-isoluble 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 HCl and acidic substances were extractedl with ether. The aqueous layer contained the ether-insoluble 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 plant was placed in a 250 ml beaker containing either 9.5 mg TNH2-HCl or 10.0 mg DL-TTP to which had been added 2 uc of the appropriate radioactive precursor. Each treatment involved 4 plants. Each plant absorbed the initial 50 ml of solution in about 3 hours. Disti,lled water was added as required over an incubation period of 44 hours, after which the plants were immediately frozen in dry ice. The frozen plants were finely grouind with a mortar and pestle at-78, and the powder was added slowly to 400 ml of hot ethyl acetate. The mixtuire was boiled for 15 mintutes, filtered through cheesecloth and the plant material was grouind in 300 ml distilled water in a Waring Blendor. The suispension was filtered through cheesecloth and the solids were discarded. The supernatant was adl-justed to pH 3.8 and extracted 3 times wilth 100 ml ethyl acetate. The acetate fraction was washedl once with distilled water and combined with the original acetate extract. The aqueous fractions were bulked, adjusted to pH 3.0 and extracted 3 times with 100 ml diethyl ether. This fraction was labeled 1. The aqu,eous layer was adjusted to pH 8.2 with 100 ml 5 % NaH,C03 and was extracted 1161