Stress-Related Compounds in Water-Stressed Carrots and Celery

Proc. Fla. State Hort. Soc. 99:100-102. 1986.

STRESS-RELATED COMPOUNDS IN WATER-STRESSED CARROTS AND CELERY

E. D. Lund Materials and Methods U. S. Citrus and Subtropical Products Laboratory Solutions were concentrated by evaporation in a rotary P. O. Box 1909 evaporator at 40-50°C/40 mmHg. A coarse sintered glass Winter Haven, Florida 33883-1909 filter was used for all filtrations. J. M. White Plant material. Two celery cultivars, 'Florida 2-14* and University of Florida 'Florimart' (a more pathogen-resistant cultivar), were Central Florida Research & Education Center grown at Zellwood, Florida (4 plots). Two plots of '2-14' P. O. Box909 were grown in waterlogged soil. One plot of Tlorida 2-14* Sanford, Florida 32771 and one of 'Florimart' were grown in soil exposed to the normal amount of water. One of the waterlogged plots was Additional index words. Daucus carota, Apium graveolens, sprayed with the fungicide Maneb (manganese drought, waterlogging, antifungal, Early Blight, Cercospora ethylenebisdithiocarbamate), and the other was not apii, polyacetylenes, falcarinol, falcarindiol, acetylfalcarin- sprayed. Plants grown in the waterlogged plots were sig diol. nificantly smaller in size than those grown in the control plots. All of the plants had become infected with Early Blight fungus (Cercospora apii Fres.). Evidence of fungal Abstract. Ten unidentified compounds were isolated by liquid attack included brown or yellow leaves and stalks, and chromatography from water (and fungus) stressed celery brown spots on normal leaves or stalks. Leaves or stalks and carrots. In addition, the three known stress-related polyacetylenes; falcarinol, falcarindiol, and acetylfalcarindiol were separated and the affected parts removed. The four were isolated from carrots. Seven of these unidentified fractions (affected and unaffected leaves and stalks) were compounds are structurally similar to these three known weighed, then extracted and analyzed separately. polyacetylenes. The remaining three compounds appear to be Carrots, cv 'Orlando Gold', were grown in Lauderhill new natural products. muck soil at Zellwood, Florida. Two plots were harvested. One plot was grown under normal conditions and har Celery and carrots are related vegetables (family Umbel- vested 14 Jan. 1985. The other plot was grown under liferae) grown in large quantities in Florida. They are fre drought conditions and harvested 13 June 1985. The quently exposed to stress conditions characteristic of this drought-stressed carrots were infected to a small extent by area, such as drought, waterlogging, freezing, fungus in unidentified microorganisms. The tops and root tips were fection, and unusual soil types. removed and any dark spots were excised prior to the In general, stressed plants produce chemicals that are weight determination. toxic to pests as part of a disease response mechanism. Extraction. Celery plants were harvested, then stored Three types of stress-related substances have been re for 1-5 days at 2°C. Samples were weighed, rinsed with ported for carrots: C17 polyacetylenes, CH2=CHC(R1) water, and allowed to drain. The sample was then cut into C=CC=CC(R2)CH=CH(CH2)6CH3, such as falcarinol (I*! pieces approximately 2 cm long and extracted with 95% - OH, R2 - H), falcarindiol (R, - OH, R2 - OH), and EtOH in a blendor (45 ml/100 g plant material). The resul acetylfalcarindiol (Rj - OAc, R2 - OH) (4,9); phenyl prop- tant slurry was extracted with an equal volume of distilled anoids (9); and isocoumarins (4,5). On the other hand, methylene chloride in a blendor. The mixture was filtered only the linear furanocoumarin group of stress metabolites and the methylene chloride layer concentrated to one-half has been reported for celery (1). the original volume. Extraction of a 100-g sample (wet Our purpose in this study was the identification of com weight)yielded 55 ml of concentrated methylene chloride pounds produced in celery {Apium graveolens L.) and car extract. The concentrated solution was fractionated by suc rots (Daucus carota L.) grown under various conditions of cessive extraction with IN HC1, 10% NaHCO3 and 10% water stress. These conditions included celery grown NaOH. The remaining (neutral) fraction was analyzed as under above normal rainfall (waterlogged soil) as well as described below. carrots grown under drought conditions. Carrot samples were washed by brushing lightly in a Samples of field-grown waterlogged celery and carrots water stream. They were cut into pieces (approximately exposed to drought in the field were extracted and 2.5-cm long) and extracted with methylene chloride con analyzed by liquid chromatography. Quantities of com taining BHT (0.2 mg/100 g plant material) in a blendor pounds related to stress in the stressed plants were com (200 ml methylene chloride/100 g plant material). The pared with amounts in the control samples. Physical data mixture was filtered and the solids washed with methylene were obtained on all of the stress-related compounds. chloride (1/2 the volume of the original extract). The fil trate was dried over Na2SO4, filtered, and the solution Mention of a trademark or proprietary product is for identification evaporated. The remaining solvent was removed at 40 only and does not imply an endorsement or warranty of the product by mmHg (25°C). The oily residue was mixed with hexane theU.S. Department of Agriculture over other products which may also (27 ml/100 g plant material). The resultant suspension was be suitable. centrifuged at 2000 G for 5 min. The supernatant hexane The authors wish to express appreciation to Dr. Joseph H. Bruemmer solution was removed by decantation. The residue was for advice and support, and Mr. J. M. Smoot for technical assistance. We thank Dr. Shelly G. Yates of the USDA, ARS, Northern Regional Re mixed with methyl-t-butyl ether (MTBE) (9 ml/100 g plant search Center, forcopies of the IR spectra of falcarinol and falcarindiol. material) and the resultant suspension centrifuged at 2000

100 Proc. Fla. State Hort. Soc. 99: 1986. G for 5 min. The supernatant MTBE solution was re visible and UV (254 and 366 nm) light. Bands were re moved by decantation. The supernatant hexane and moved and extracted with MTBE or methylene chloride. MTBE solutions were analyzed by chromatography as de Characterization. IR and UV-VIS spectra were obtained scribed in the following section. on all isolated compounds. HPLC and column chromatographic analysis. Solutions of the extracted sample were first purified on a column of 40 jim Cyanopropyl Sepralyte (Analytichem International, a Results and Discussion silica gel with covalently bonded cyanopropyl groups). Analysis of water-stressed celery and carrots showed a Celery solutions were purified by the following proce number of stress-related compounds. Waterlogged celery dure: A 50-ml sample of the filtered neutral fraction contained increased amounts of two unidentified com (methylene chloride solution) was filtered under pressure pounds. A third closely related substance was isolated from through a column (1 cm i.d. x 6 cm L) of the adsorbent stressed carrots. (packed in methylene chloride). The column was flushed Carrots grown under drought conditions contained a with 15 ml of EtOH. The combined eluates (methylene mixture of ten closely related polyacetylenes of the fal- chloride and EtOH solutions) were evaporated to dryness, carinol-falarindiol type. Concentrations of seven of the ten 30 ml of hexane added, and the mixture filtered. The fil compounds were increased compared with a sample grown trate was evaporated to 9 ml, filtered, and the filter washed under normal conditons. Most of these polyacetylenes have with 6 ml of hexane. The combined filtrates were finally not previously been reported as carrot constituents. evaporated to 3 ml. Celery. Celery (Apium graveolens L.) stressed by waterlog Carrot solutions were purified by a similar procedure. ging (and the associated fungus infection) was analyzed by The supernatants (150 ml hexane of 50 ml MTBE) were a general procedure. We did not attempt to determine passed through a 2.4 cm i.d. x 50 cm L column of Cyanop specific classes of compounds, such as the well-known ropyl Sepralyte (packed in the appropriate solvent, either stress metabolites of the psoralen group (1). hexane or MTBE). The hexane column was washed with Celery stressed by waterlogging (and the associated 40 ml hexane followed by 60 ml MTBE. The MTBE col fungus infection) contained two components (compounds umn was washed with 40 ml MTBE. The combined MTBE A and B) that were present at higher levels than in plants and hexane eluates were evaporated to 5-10 ml. grown under normal conditions. The increase in com Celery samples (500 jx 1) were separated on a 7.8 mm pound A was not correlated with fungal infection. The i.d. x 30 cm L Waters |x Bondapak CN (IOjji, reverse phase) increase in compound B was correlated with both waterlog semipreparative column (packed in hexane). The mobile ging and fungal infection, however, the increase due to phase was hexane and the flow rate was 2 ml/min. The fungal stress was much greater than that produced by eluting sample was detected by UV (211 nm). A series of waterlogging. The physical properties of the two com large (off-scale) peaks were observed, followed by a series pounds do not correspond to those reported for psoralens. of much smaller peaks. The unknowns were in the second A neutral, CH2C12 soluble fraction from the leaves and small peak (retention time 8-10 min). stalks of'Florida 2-14' was analyzed by HPLC (cyano polar column). A significant difference between control and Carrot samples were separated on a (20 mm i.d. x 37 stressed plants was observed in the HPLC trace of unaf cm L) column containing 40 |xm Cyanopropyl Sepralyte. fected leaf extract. This difference was observed in a reg For the hexane extract, the column was packed in hexane ion containing moderately polar compounds (i.e., esters). and the solvent composition was varied (non-linear) from One peak in the trace for stressed plants showed a particu 100% hexane to 20% hexane-80% MTBE by use of a sim larly large increase in size. TLC analysis of the fraction ple gradient former (a stirred reservoir and a large represented by this peak on a reverse-phase plate showed separatory funnel). For the MTBE extract, the solvent that it contained two stress-related compounds (A and B). varied from 90% hexane-10% MTBE to 20% hexane-80% Both compounds had two closely spaced low-intensity max MTBE. The flow rate for both columns was 45 ml/min. ima in the 250-260 nm region. The relative amounts of Eluting samples were detected by UV (211 nm). Prior to these two compounds, based on their UV absorption at injection, the hexane solution was concentrated to 0.8 ml 254 nm, are shown in Table 1. The concentration of these and the MTBE solution to 0.4 ml. The ppt was removed components in celery was 4.7 ppm (A) and 1.4 ppm (B) by filtration. Falcarinol and related compounds appeared (weight of isolated sample). The concentration of com only in the hexane soluble fraction. This group eluted pound A in stressed Tlorida 2-14' was approximately 50% from the column in those fractions containing 2% to 10% greater than that in the control. The concentration of B MTBE. The more polar compounds related to falcarindiol was doubled in the stressed-sprayed plants and increased eluted in fractions containing 10% to 40% MTBE. Two 5-6 fold in the stressed plants that were not sprayed. The esters (acetylfalcarindiol and an unidentified ester) eluted observation that unsprayed stressed plants were more se slightly before the diols (10% to 20% MTBE). Unidentified verely affected by fungal infection suggests that B may be compound C eluted in fractions containing 0% to 1% produced in response to fungal damage rather than water MTBE. logging. The level of both compounds was higher in TLC analysis. Mixtures collected as peaks from the col 'Florimart' than in the Tlorida 2-14' control leaves. umn were separated on Whatman LKC18F reverse phase Since we did not examine plants grown in the absence (C18) plates. The plates were prewashed with acetonitrile, of fungal infection, compound B could be either a fungal then with diethyl ether or methylene chloride. The de by-product or a product of the water stress. Compound A, veloping solvent was acetonitrile containing either 20% or on the other hand, appears to be a compound produced 50% MTBE. Bands were visualized by illumination with in increased amounts by the waterlogged plant.

Proc. Fla. State Hort. Soc. 99: 1986. 101