Comparison of Chorismate Mutase Isozyme Patterns in Selected

A wide variety of plants have been assayed to determine if they contain three isozymes of chorismate mutase (EC 5.4.99.5) as does alfalfa (Medicago sadva L.) or two isozymes, as does mung bean (Phaseolus aureus). The isozymes were separated by disc electrophoresis. All anthophyta with the exception of some closely related Leguminosae contained three isozymes of chorismate mutase. The one coniferophyta (a pine), and pterophyta (a fern) and one microphyUophyta (a Selaginetll) assayed contained two isozymes of chorismate mutase. All plants assayed contained measurable chorismate mutase levels and at least two isozymes of chorismate mutase. The shikimic acid pathway (13) appears to be the biosynthetic pathway for the aromatic amino acids in all organisms studied so far, with the possible exception of Trichophytron rubrum (29) (Fig. 1). More detailed investigations, however, reveal significant diversity in the enzyme aggregations, isozymic patterns, and the processes regulating the shikimic acid pathway (Tables I and II). Investigations of photosynthetic organisms, those cited in Table II, as well as those of Zurawski and Brown (28) with Chlamydomonas reinhardi, Kaney and Jhabvala (20) with Synechoccus cedrorum, and Chu and Widholm (3) with a variety of higher plant tissue cultures, indicate that in these organisms, enzymes of the shikimic acid pathway are not subject to control by repression and/or derepression. However, some of the shikimic acid pathway enzymes in photosynthetic organisms do occur in isozymic form and are sensitive to the presence of metabolites (Table II). Most algae, both blue-green (18, 19) and green (26), contain one DAHP3 synthase (EC 4.1.2.15) and one chorismate mutase (EC 5.4.99.5). Work with the higher plants is somewhat scattered as there is no one plant whose complete shikimic acid pathway enzymes have been investigated. Husman and Kosuge (16) found one isozyme ofDAHP synthase in cauliflower, and Gilchrist et al. (8) found two isozymes of chorismate mutase in etiolated mung bean. We (27) found three chorismate mutase isozymes (CM,, CM2, CM3) in alfalfa. Both CM1 and CM3 are inhibited by phenylalanine and tyrosine and activated by tryptophan. The CM2 is unaffected by the aromatic amino acids but is inhibited by the lignin precursor caffeic acid as is CM1. The CM3 is the only isozyme affected by 3,4-dimethoxycinnamic acid (an activator) and the lignin precursor ferulic acid (an inhibitor). ' This work was supported by National Institutes of Health Grant AM13737:04. 2Presefit address: Sea and Ski Corporation, Reno, Nevada. 3Abbreviations: DAHP: deoxyarabino-heptulosonic-acid-phosphate; SK: shikimate kinase. In an effort to determine if most plants contain two or three isozymes of chorismate mutase, we surveyed a wide variety of anthophyta and a more limited variety of more primitive plants. MATERIALS AND METHODS Plant Material. Unless otherwise indicated all plant material was 6to 7-day-old seedlings grown on cheesecloth wicks. Green plants were grown in a greenhouse and exposed to normal spring day lengths. Etiolated plants were grown in sealed drawers. Unless otherwise indicated the entire seedling was used for making extracts. Pine needles were gathered on April 25 from a pine growing in the wooded area near the laboratory. All other mature plant material was taken from greenhouse-grown plants just before or after flowering. Extracts. Nitrogen powders were prepared by dropping pieces of plant material into a Waring Blendor containing liquid N2 and grinding until well powdered. Acetone powders were prepared by grinding plant material in a Waring Blendor containing dry ice and acetone, collecting the powders on a Buchner funnel, and washing them with cold acetone until the washings were colorless. Powders were extracted twice with 0.10 M K-phosphate (pH 6.5) at a ratio of 2 g of nitrogen powder to 1 ml of buffer or I g of acetone powder to I ml of buffer. The extracts were clarified by centrifugation for 20 min at 30,000g in the SS34 head of a Sorvall model RC2-B refrigerated centrifuge. All operations including electrophoresis and elution were performed at 4 C. Electrophoresis. Electrophoresis was performed on 6% (w/v) polyacrylamide gels prepared according to Davis and Ornstein (5) with the exception that no spacer or sample gels were employed. Bromphenol blue was employed as the tracking dye. Fifty,l of the first mycelial extraction made 10% (w/v) in sucrose was applied gently under buffer to each of six gels, each approximately 9 cm long. The gels were subjected to a current of 3 to 4 mamp/tube. Immediately at the end of each run five gels of each set were frozen in liquid N2 and stored overnight at 20 C. One gel of each set was stained for protein by immersion for 12 hr in 2.5% (w/v) Amido Schwarz. These were destained by repeated flushing with 7% (w/v) acetic acid. Gel Extraction and Assay. The frozen gels were lined up with dye fronts matching and cut into sections 3 mm long. Corresponding sections from each gel were pooled and put into a test tube (1.2 x 7.5 cm) to which was added I ml of 0.10 M K-phosphate (pH 6.5). Gels and buffer were incubated for 4 to 6 hr with occasional hand shaking. When the dye band was evenly distributed between gel and buffer, we assumed that protein elution was completed. Elution of chorismate mutase activity ranged from 80 to 100%o of that originally applied, with the exception of that from beans, 67% (Table I-II). Appropriate aliquots were used for assays of chorismate mutase, using a slight variation of a method developed by Nishioka and Woodin (24). Assay mixtures consisting of 0.60 ml of 0.10 M K-phosphate (pH 6.5), containing I ,umol of 949 www.plantphysiol.org on November 11, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. WOODIN, NISHIOKA, AND HSU Plant Physiol. Vol. 61, 1978