Fluoroorganic Acids in Soybean Leaves Exposed to Fluoride Julie Y e - 0 Cheng, Ming-Ho Yu, Gene W. Miller, and George W. Welkie Botany Department. Utah State University, Logan, Utah 84321
Soybean plants were fumigated with H F or grown in nutrient solution containing sodium fluoride or fluoroacetate, and the organic acid fractions of the leaf extracts were compared. Organic acid extracts from these treatments contained fluoroorganic compounds that were chromatographically similar t o fluoroacetate and fluorocitrate. The fractions suspected of containing fluorocitrate were separated from HFand NaF-treated leaf extracts and found t o inhibit aconitase prepared from either pig heart or soybean leaves. The inhibition was similar to that exhibited by commercial fluorocitrate. Aconitase from soybean leaves was as sensitive to fluorocitrate as that isolated frompig heart. Fluorocitrate n’as identified in extracts of fluoride-treated plants by infrared spectroscopy.
F
luoride ions cause a number of physiological effects in plants and have been shown to affect several enzymic reactions (Yang and Miller, 1963). I n addition, a number of plants that are toxic to animals are known to contain fluoroorganic compounds. The toxic component fluoroacetate was isolated from the South African plant Dichapetalunz cyr?zosunz (Marais, 1944), a n Australian tree Acacia georginae (Oelrichs and McE\nan. 1961), and a Brazilian shrub Pdicourea rizurcgrcrrii (Oliveira. 1963). Liebecq and Peters (1949) found that fluoroacetate reduced the oxygen uptake of kidney homogenates and induced the accumulation of citrate. The accumulation of citrate was a result of a block in its metabolism by fluorocitrate rather than enhanced synthesis. The toxic substance fluorocitrate has been isolated and identified by Buffa, Peters, er ai.(1951). There is no e\,idence that the ability to synthesize fluoroacetate is B general property of plants. However, high levels of citrate are k n o u n to accumulate in soybean plants injured by H F (Yang and hliller, 1963). This might be the result of a block in the metabolism of citrate by fluorocitrate. The present study was initiated to determine whether soybean plants exposed to atmospheric fluoride were capable of forming fluororganic acids. Proceduws
Young so?bean plants (GZycine max, variety Hawkeye) growing in standard Hoagland’s nutrient solution were placed
in fluoride-fumigation chambers (Ross, Wiebe, er a/., 1962) and exposed t o 43 p.p.b. of HF or ambient air (0.06 p.p.b. of HF). Leaves of the HF-treated plants became necrotic about 10 days after initiation of treatment. Additional treatments of comparable plants consisted of a transfer of plants to one-tenth strength Hoagland’s nutrient solution which contained either 5 X 10-3 M N a F or sodium fluoroacetate. Control plants were grown in the same nutrient solution without added N a F or sodium fluoroacetate. After several days of culture necrotic lesions appeared on leaves of plants receiving either of the two treatments. The leaves were harvested when necrosis was evident and the organic and amino acids were extracted according to the procedures of Yang and Miller (1963). Fluoride content in the crude extracts, and in specific fluoroorganic acids separated by paper chromatography, was determined by the oxygen-combustion method of Wade, Ross, et a/. (1964). Separation and identification of fluoroorganic acids were accomplished by chromatography of organic acid extracts on Whatman No. 4 paper strips over a distance of 20 cm., using the methods that Ward and Peters (1961) used to detect fluoroacetate. Location and concentration of the fluoroorganic acids on chromatograms were determined by fluoride analysis of 10 successive segments of equal size. The short-strip chromatography method of Wade, Ross, et a/.(1964) was also employed in the identification and determination of the concentration of the fluoroorganic acids. Location of organic acids on 20-cm. chromatograms was detected by the color indicator method of Reid and Lederer (1951). Segments from the 20-cm. chromatograms which corresponded to the position of citrate and fluorocitrate were eluted with water, filtered, and concentrated. These extracts and similar ones prepared from organic acids of control plants tested positive for citric acid (Feigl, 1939). Aliquots of these extracts were tested for fluoride content and others were tested for inhibitory effects o n aconitase activity. The combined citrate and fluorocitrate fractions from fluoride-treated plants were further purified by the precipitation method of Peters and Wakelin (1953) with some minor modifications. These purified preparations and standards of citric acid and fluorocitric acid were mixed with solid KBr and made into thin KBr pellets. The pellets were analyzed with a Beckman infrared spectrophotometer and the spectra recorded. Aconitase was prepared from pig heart according to the method of Morrison (1954) and from soybean leaves according to the method of Hsu and Miller (1968). Aconitase activity was assayed as outlined by Hsu and Miller (1968). Volume 2, Number 5 , May 1968 367
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Rf Figure 3. Profile analysis of fluoride in chromatographic strips Organic acids extracted from plants treated aith fluoroacetate. Aliquots of extract used in chromatograph> corresponded to I gram of fresh leaves
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Rf Figure 1. Profile analysis of fluoride in organic acid extracts from HF-fumigated soybean plants Aliquots of extract used in chromatography corresponded to 1 gram of fresh leaves.
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Figure 2. Profile analysis of fluoride in organic acid extracts from NaF-treated soybean plants Aliquots of extract used in chromatography corresponded to 1 gram of fresh leaves
368 Environmental Science and Technology
Organic acid extracts from fumigated plants that received 43 p.p.b. of H F had a high fluoride content. The fluoride content of the amino acid fraction was also greater in the extracts from the fumigated plants than from the control plants. No further studies were conducted on this fraction. A trace of fluoride was occasionally detected in some chromatogram segments of the organic acid extracts from control plants. The profile analyses of fluoride in chromatograms of organic acid extracts from fluoride-treated plants were similar to each other, regardless of the source of fluoride. Fluoride was found in chromatogram segments 1 to 7 (Rfvalues 0.1 to 0.7), but little or none in segments 8 to 10 (Rf values 0.8 to 1.0). Segments corresponding to R f values 0.1,0.2, and 0.7 usually contained the highest fluoride concentrations (Figures 1, 2 , and and 3). When commercial fluoroacetate was chromatographed, fluoride was located in segments 6 and 7 (Figure 4).Commercial fluorocitrate, when chromatographed, showed fluoride primarily in segments 1 to 3, with some possible impurities at segment 5 (Figure 4). Chromatographed inorganic fluoride was found in the highest concentrations in segments 4 and 5 (Figure 4). Later studies indicate that commercial fluorocitrate contained significant quantities of inorganic fluoride. I n short-strip chromatography, fluoroacetate moved with the front, whereas inorganic fluoride and fluorocitrate remained immobile. The organic acid extracts from HF-fumigated plants yielded 3.5 pg. of fluoride per gram of fresh weight of leaves at the top of the front, which corresponded to fluoroacetate. Profile analysis of fluoride (20-cni. chromatogram) gave 4.0 pg. of fluoride per gram of fresh weight of leaves at R f values 0.6 and 0.7. These fluoroacetate values determined by two different methods were consistent with each other. The organic acid extracts from HF-fumigated plants contained a total of 30 pg. of fluoride per gram of fresh weight of leaves. Segments taken at Rf values 0.4 and 0.5 using profile analysis
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