Callus and Differentiated Soybean Root Cultures

The metabolism of 11-14C12,4-dichlorophenoxyacetic acid (2,4-D) in soybean ( Glycine max IL.1 Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10-6 to 10-5 molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates. In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about I to 2 nanomoles per gram. Pea epicotyl and soybean cotyledon callus tissues metabolize 2,4-dichlorophenoxyacetic acid into metabolites soluble in water or diethyl ether. The diethyl ether-soluble fraction (pH 3) contains mainly unmetabolized 2,4-D and amino acid conjugates (2, 5, 8, 9). Several ring hydroxylated aglycones as well as 2,4-D can be recovered by treatment of the water-soluble fraction with 13-glucosidase. Thus, the water-soluble metabolites are mainly ring hydroxylated glycosides and the carboxylic glucoside ester of 2,4D (5, 8, 9). The 2,4-D metabolites qualitatively and quantitatively change with the length of incubation time (9). An increase in incubation time decreases the total amount of diethyl ether-soluble metabolites and increases the amount of water-soluble metabolites in soybean cotyledon callus tissues. The amount of each metabolite varies with incubation time which could reflect tissue age (9). The metabolism of 2,4-D in differentiated tissue may also differ from that of callus tissue inasmuch as it has been reported that excised roots do not metabolize 2,4-D readily (1, 17), but callus tissue does ' Authorized for publication as Paper No. 5399 in the Journal Series of the Pennsylvania Experiment Station. Supported in part by the Northeastern Regional Research Project NE-53 and Regional Research Funds. metabolize 2,4-D rapidly (5, 8, 9). The physiological significance of these 2,4-D metabolites is not completely understood. The hydroxylated aglycones have no auxin activity (10, II), but the amino acid conjugates have high activity (10). It has been suggested that auxin conjugates may contribute to the regulation of auxin levels in plants (3, 5, 13). Previous work (5) has shown that 2,4-D glutamic acid conjugate was metabolized to 2,4-D and other metabolites by soybean cotyledon callus tissues. Metabolism of 2,4-D was studied in undifferentiated soybean callus tissues and differentiated root cultures as a function of 2,4D concentration. The objective was to obtain evidence for regulation of internal 2,4-D levels by amino acid conjugate formation. MATERIALS AND METHODS Soybean root callus cultures were derived from 3-day-old roots of soybean (Glycine max [L.] Merrill var. Amsoy). Initial explants were cut 4 to 5 mm behind the root tip and cultured on an agarsolidified medium (14). Cultures were maintained at 25 C under cool-white fluorescent light (350 ergs cm-2 sec-'). After several weeks the cultures developed callus as well as differentiated roots. The roots were excised and grown in Miller's liquid medium (14), without auxin and kinetin, while the callus tissue was subcultured on solid Miller's medium with l0-5 M a-naphthalene acetic acid and 2.32 x 10-6 M kinetin. After subculturing the callus tissue lost its capacity to produce roots on this medium. Callus tissue (3-10 g) or differentiated root tissue (0.5-0.8 g) was aseptically transferred to 125-ml flasks containing 50 ml of the appropriate liquid medium (without added auxin) to which l0-5 to 10-6 M [-1_4C]2,4-D (52 mCi/mmol Amersham/Searle) was added. The tissue cultures were incubated on a shaker at 21 C for 48 hr. After incubation the tissue was collected on filter paper and surface-rinsed with cold-distilled H20 and weighed. The tissue was ground in a VirTis "45" tissue homogenizer with five times its weight of hot 95% ethanol. The homogenate was filtered and the residue washed with 80%1o ethanol. The filtrate was concentrated by evaporation, adjusted to pH 3, and extracted at room temperature with diethyl ether. This diethyl ether fraction was partitioned into sodium bicarbonate, and after acidification (pH 3), back into ether in the same manner as previously described (8, 9). The aqueous phase was extracted three times with equal volumes of l-butanol saturated with water. The l-butanol fraction was concentrated in a rotating evaporator in vacuo at 50 C to remove all of the 1-butanol and an aliquot dissolved in 20 ml of distilled H20 at pH 6 was incubated with ,B-glucosidase (14 mg of Emulsin, U.S. Biochemical Corp.) for 7 hr at 27 C. The solution was then acidified (pH 3) and extracted with diethyl ether. The diethyl ether and 1-butanol fractions were thin layer-chro80 www.plantphysiol.org on June 21, 2017 Published by Downloade from Copyright © 1978 American Society of Plant Biologists. All rights reserved. 2,4-D METABOLISM: SOYBEAN TISSUE CULTURE matographed on Supelcosil 12A thin layer plates containing a fluorescent indicator (zinc silicate). Metabolites were located by autoradiography and co-chromatography with nonlabeled standards. Two solvent systems were used: I: diethyl ether-petroleum ether (38-46 C)-formic acid (70:30:2, v/v/v); and II: chloroformmethanol-acetic acid (70:20:5, v/v/v). The radioactivity of each fraction was measured by liquid scintillation counting in Aquasol. The ethanol-insoluble residue was combusted by the 02 flask method (12) prior to counting. All counts were corrected for quenching using the channels ratio method. For mitotic index determinations, the tissue was fixed in Navishin's fluid, washed, treated with 10%7o glycerol in distilled H20 for 24 hr, and embedded in gelatin (15% gelatin, 2% glycerol by volume). Twelve-,m frozen sections were cut using a freezing microtome (International Cryostat) and the tissue was stained with gentian violet. The number of dividing nuclei/hundred (mitotic index) was determined by the examination of 2,000 nuclei/sample. RESULTS AND DISCUSSION The mitotic index of soybean root callus tissue decreased from a maximum of about 1.1 at 2 weeks after transfer to 0.6 at 3 weeks, and it was about 0.2 at 9 weeks after transfer. The mitotic index was used to evaluate the physiological state of the tissue at the time of each 2,4-D labeling experiment. At some unpredictable times, perhaps seasonally related, the tissue did not grow well and the mitotic index was lower than typical values given above. Such tissue was not used in the 2,4-D metabolism studies to be reported. The uptake of [14C]2,4-D by the tissue varied with concentration of 2,4-D (10-6 to l-5 M) as well as with the age and the amount of tissue. At 2 x 10-6 M the uptake was about 80%o of the label supplied in 3-week-old callus tissue and only about 20%o in 3week-old differentiated root cultures. The radioactivity in the ethanol-insoluble residue was about 10%o of the total taken up by the callus, but it was 209%o of the total taken up by the roots. The 2,4-D metabolites in this fraction remain unknown. The ethanol extract contained mainly amino acid conjugates, the hydroxylated 2,4-D glycosides, and free 2,4-D. The amount of ether-soluble, free 2,4-D was determined following TLC and is expressed separately. The remaining diethyl ether-soluble metabolites, as shown in Figures I to 3, represented primarily the amino acid conjugates. The aqueous metabolites (butanol extract) were primarily hydroxylated 2,4-D glycosides (5). Table I shows the composition of each fraction in a typical experiment in 3and 9week-old root callus and in 3-week-old excised roots. The glutamic TABLE I. The concentration of 2,4-D metabolites in various fractions after incubation of excised soybean roots or root callus with 2,4-D-1-14C for 48 hours. The weight of tissue per each 125-ml flask containing 50 ml of liquid medium was 5.5 and 5.9 for 3-week-old callus, 4.1 g for 9-week-old callus, and 0.7 g for 3-week-old excised roots. The tissues were incubated with the indicated concentration of 2,4-D-1-14C for 48 hr on a shaker at 21 C. Nanomoles of 14C label/g fresh wt