Naturally-Occurring and Ethylene-Induced Phenolic Compounds in the Carrot Root

526

S. K. SARKARI* and C. T. PHAN2 Department ofFood Science, University ojAlberta, Edmonton, Alberta T6G 2N2, Canada and Department des Sciences Biologiqus, Universite de Montreal, Cas Postale 6128, Montreal, Quebec H3C 317, Canada

(Received for publication August 14, 1978) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021

ABSTRACT periods of storage (3). The carrots processed soon after Carrots accumulate phenols and often develop off-flavor and color harvest were free from this bitter flavor. In an attempt to after long periods of storage. To investigate probable causes for such find a solution to this bitterness problem, investigations physiological disorder, the effect of ethylene on various aspects of metabolism of carrot roots was studied. Ethylene, when applied at into various aspects of carrot production, including moderate level (100 ppm), caused an increase in total phenol content of cultural and post-harvest handling practices, were the roots. It caused an increased accumulation of the phenols normally initiated by various workers at the Agricultural present in the tissue, especially isochlorogenic acid. Moreover, Experiment Station in Geneva, New York. Atkin (3) relatively longer exposure to a moderate level (100 ppm) and short reported the results of a survey of various cultural exposure to high levels (2000 and 50,000 ppm) of ethylene induced formation of new compounds, viz, isocoumarin, eugenin, and two practices of carrot growers. Application of oils (to control others yet unidentified. Studies with [l-14C]acetate, [2- 14C]malonate and weeds), insecticides, fertilizers, and crop rotation were [3-14C]acetoacetate indicated that the newly synthesized compounds the major management procedures. The results of his are probably synthesized via the acetate pathway. Ethylene stimulated survey failed to provide any correlation between above the rate of 0 2 uptake and C02 evolution by carrot slices, indicating cultural practices and the development of bitterness in probable relationship of glucose metabolism with de novo synthesis of "stress-metabolites". Studies with specifically labelled glucose showed carrots. However, Atkin always observed that whenever that both the Embden-Meyerhof-Parnas (EMP) and the Pentose bitterness occurred, it did so in refrigerated storage. Phosphate (PP) pathways operate in carrots, and that ethylene The nature of the compound(s) responsible for preferentially stimulated the EMP pathway. Like ethylene, dinitro bitterness in carrots was investigated by Sondheimer (24). phenol (DNP) induced isocoumarin synthesis in carrots. Methylene blue, an electron acceptor often used for stimulating glucose He suggested that bitter flavor in carrots was caused by catabolism via the PP pathway, also induced isocoumarin synthesis in the presence of several compounds and that 8-hydroxy-3- carrots. The effect of cycloheximide. an inhibitor of protein synthesis, methyl-6-methoxy-3,4-dihydroisoioumarin was one of suggested that the de novo synthesis of enzyme protein(s) might be them. He named this compound as 'isocoumarin', and required for ethylene-induced isocoumarin synthesis in carrots. In con this name has been used ever since. clusion, it appears that ethylene triggers changes in the metabolism of carrots during storage, which result in, among other things, synthesis What causes induction of isocoumarin synthesis in of so-called "stress-metabolites," namely isocoumarin and eugenin and carrots was the subject of further investigation, and related compounds. various workers suggested that ethylene might be a "triggering factor" (5,6, 7). Carrot root is an important component of the vegetable portion of our diet. It is a good source of Although ethylene was suggested as a causative agent vitamins, minerals and fiber. And it adds rich color and for synthesis of isocoumarin in carrots on the basis of aroma to our food. On the basis of the United States isocoumarin's ability to fluoresce under U .V .light (5,6, 7), Department of Agriculture statistics for 1970 (1), carrot no attempt was made by these workers to isolate and root has been ranked tenth in terms of nutritional value characterize isocoumarin from ethylene-treated carrots among 38 other fruits and vegetables, and seventh for its to ascertain whether the observed fluorescence was contribution to nutrition. caused only by isocoumarin and not any other Current advancement in agriculture has enabled us to compound(s). Further, the question whether isocoumarin produce more carrots than can be marketed as fresh is the only compound synthesized in carrots in the produce, which leaves a large amount of produce to be presence of ethylene was unanswered until we started our processed in some form for later use. The time lag investigation some 12 years later. Our results show that between harvest and processing, or future marketing as not only isocoumarin but three additional related fresh produce, appears to be crucial in relation to compounds are synthesized de novo in carrots after undesirable color and flavor development in carrots (8). exposure to ethylene. Further, the levels of existing During the early 1950s, processing industries in the phenols, including hydroxy-cinnamic acid derivatives, eastern U.S. and Canada encountered a problem of increased considerably in ethylene-treated carrots. bitterness in some carrots that were processed after some The cinnamic acid derivatives, namely various 'University ofAlberta. phenolic compounds encompassing flavonoids, lignins, 'Universite de Montreal. and tannins, although classified by plant physiologists as PHENOLIC COMPOUNDS IN CARROTS 527 secondary metabolites, occupy an important place in ether to obtain two fractions: (a) ether-soluble and (b) food color and flavor. Therefore, it must be recognized ether-insoluble. The presence of phenolic compounds in by people dealing with foods of plant origin that unless the ether-soluble fraction was not detectable in carrots adequate care in handling and storage of fresh produce is stored at 3 ± 1 C and ca. 90% relative humidity, and in taken, undesirable color and/or flavor development may the absence of ethylene. No isocoumarin or any other result from various phenolic compounds that are present related compounds were present in the extract. in these tissues. The ether-insoluble portion revealed the presence of a We, in this paper, will describe the results of our number of phenolic compounds on the paper chromato studies on the effects of ethylene on various phenolic gram (Table 2). Three of these spots were identified by compounds in carrot roots. co-chromatography with known compounds; caffeic acid (I), isochlorogenic acid (II) and chlorogenic acid (III). EXPERIMENTAL The rest of the spots were not identified as such but

All the carrots used for experiments were grown at the Horticultural Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021 Research Station, Brooks, Alberta. These were stored at 3 ± 1 C and 98% relative humidity, in the absence of light. The methods of extraction, separation and identification of various phenolic compounds have been published previously (13.17-21), and a detailed description will not be given here. However, it should be noted that we have used intact carrots as well as slices for our studies. The slices were used either for feeding experiments with labelled compounds or to augment the effects of ethylene on carrots over a short time, resulting from an increased surface for exposure to ethylene. The entire phenol extract of carrots was classified, according to (I) solubility, into ether-soluble and methanol-soluble (or ether-insoluble) fractions. The composition of extracts was analyzed by paper, thin-layer and gas chromatography.

(II) RESULTS AND DISCUSSION E]Ject ofstoroge on phenol content ofcarrots The total phenol content of carrots stored at 3 ± 1 C HOO~COH ~ increased steadily with time. Figure 1 shows that the HO o-t-CH=CH-o amount of phenols in 100 g of carrots increased from 35 ::,...1 to 210 mg during a period of 9 months. Chubey and I ' OH Nylund (8) observed a similar increase in phenol content OH of carrot roots during storage at various temperatures. They found that the amount of phenol accumulated was (III) (IV) the highest at 10 C. Our laboratory is currently looking at the effect of storage on phenol content of potatoes.

Probable physiological basis for phenol accumulation ncH=CH-COOH in carrots during storage will be described later, but from Ho-V a practical standpoint increased phenol content poses I OCH additional problems in post-harvest handling. Carrots 3 richer in phenols are more susceptible to surface ( V) browning and other concomitant problems (8). The concern over increased phenol content in carrots in relation to surface browning is heightened because of p-coumaric acid (IV), ferulic acid (V) and caffeic acid the existence of a concentration gradient from core to the (I) were identified by thin-layer and gas-liquid surface of the carrot root (fable 1). About 85o/oof phenol chromatography after acid and alkaline hydrolysis of the is present in the 1-mm thick surface layer of the root. original extract (19). We found that under the above This would easily explain why carrots with bruises on the storage conditions the levels of hydroxycinnamic acid surface tend to develop brown color (8). Although an derivatives increased with storage time. increase in phenols in carrots augments the potential for Effect of ethylene on phenol content ofcarrots surface browning, no bitterness, as described by The situation is quite different, however, if carrots are Sondheimer (24), was detected. stored even for a short time in a refrigerated storage in Composition ofphenolic compounds in carrots the presence of ethylene. In carrots treated with ethylene The next question that arose was what was the (100 ,_d!l), the amount of phenol (Fig. 2) increased 5-fold composition of phenolic compounds in carrots, and how after a 3-day exposure (I) and about 7-fold after a 7-day is it affected by storage? To answer this question, the exposure (II). We studied the effects of various ethylene aqueous extract of phenols was further extracted with concentrations (1-2000 ppm) on phenol content of carrot 528 SARKAR AND PHAN

I I I ---,- 2101- 100

80 1901- ;: / 60 .~· 1701- I - "'~ ~· • n. 40 ~ ·y· 150- - ,.. 20 0 I r~:--·----'--'-·------· c: • ~--·..J__·-· Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021 ..! 0. 130- 0 2 3 6 7 0 I - Ooy~ ot mcpo\uu~ 0 Figure 2. E.tfect of continued exposure to 100 !llil ethylene on the .... total phenol content of carrots. expressed as mg of chlorogenic acid per 1101- g of dry weight. Total phenol in ethylene-treated carrots (.6. ); total - phenol in air-treated carrots { • ). I and II refer to two different experiments. // exposure to ethylene (Fig. 3). When the concentrations of isochlorogenic acid and chlorogenic acid were monitored ./ 90~ - in ethylene-treated carrots over a few days, the former showed a steady increase whereas the latter remained ~ unchanged (Fig. 4). Although such observation was not I I l I made previously and therefore cannot be related to any 0 2 6 8 10 Months of storage Figure 1. Variation of total phenol content of carrots during stnrage at 3 ± 1 C. Total phenol is expressed as mg chlorogenic acid per 100 g olfresh weight.

TABLE 1. Ether-insoluble phenols separated by paper chromato graphy.ab

Identity 0.82 CatTeic acid 0.76 lsochlorogenic acid 0.64 Chlorogenic acid 0.57 Not identified 0.52 Not identified 0.39 Not identified v c 0.31 Not identified 0 :-2 "'0 v c 0 Sarkar (In. ;:, system was the organic phase of the mixture: n-butanol:acetic ..0 v 0 :-2 ·c: acid:water (4: 1:5). u 0 Ol 0 v TABLE 2. Centripetal distribution qftotal phenol content in carrots. ·;:: ..Q exposed to air or 100 ppm ethylene for 2 days. a ..r. c c c: c: v 3: 3: 3: 3: Ol 0 0 0 0 0 _g "'0 Total phenol content: c: c c: ·g ..-" ~ ~ ~ 0 mg cblorogenic acid per g dry wt. c c c c: :::c :::) :::) :::) :::) v u r- :! Peel 21.87 43.21 0 Phloem 2.26 4.46 r- v 1.35 2.88 Sarkar and Ph an (l9). roots, and found that the higher the concentration the '-'---- n faster the rate of synthesis of all phenols, including ll - 0.31 0.39 0.53 0.56 0.63 0.76 0.82 isocoumarin and related compounds. Rf values Effect of ethylene on ether-insoluble phenols. When Figure 3. Rf values and relative abundances qf ether-insoluble the ether-insoluble phenols were analyzed by paper phenols separated by paper chromatography. Relative abundances chromatography, isochlorogenic acid concentration re were estimated visually on the basis of area qf'jluorescence of different compounds under UV light. Solid bar represents phenols in air-treated, gistered a marked increase, whereas chlorogenic acid and blank bar represents phenols in 100 ppm of ethylene-treated and caffeic acid concentration declined slightly on carrots. PHENOLIC COMPOUNDS IN CARROTS 529

IO,r------r------~r------~

E 08 c 0 <"") <"")

>- X Q> -o -o u c c 0 0 c Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021 d:l d:l -o0 c • ::> ...0 0 0 ? 1 Q> Day\ of P.xpowrP. > Figure 4. Time course ofisochlorogenic acid accumulation in carrots -0 upon exposure to ethylene (1 00 ppm). Isochlorogenic acid in Q.j ethylene-treated (.A. ), and ( e ) isochlorogenic acid in air-treated IX carrots. Broken lines: Chlorogenic acid in ethylene-treated carrots ( 4 ), chlorogenic acid in air-treated carrots ( e ). c c: particular physiological event, we have proposed ~ ~ N 0 0 elsewhere that the accumulation of a diester such as c: c -o ...:>1: ...:>1: c isochlorogenic acid probably occurs as a result of c c: 0 :::> :::> d:l ethylene-induced stimulation of the shikimic acid pathway. Effect of ethylene on ether-soluble phenols. The effect of ethylene on the ether-soluble phenols in carrots was 0.27 0.39 OA4 0.48 0.53 0.58 0.69 0.83 0.95 particularly striking. Four more phenolic compounds were synthesized in carrots on exposure to ethylene Rf values (Fig. 5). Compounds of band X ffif0.69) and bandY (Rf Figure S. Rf values and relative abundances of ether-soluble 0.58) were the two most abundant phenols, and were compounds separated by thin-layer chromatography. Solid bars isolated in crystalline form by column chromatography represent air-treated. and blank bars represent ethylene-treated (17), These compounds were characterized unequivocally carrots. as 8-hydroxy-3-methyl-6-methoxy-3,4-dihydroisocouma rin, the so-called 'isocoumarin' (band X) and 5-hydroxy- isocoumarin in carrot disc, and isocoumarin exhibited 7-methoxy-2-methylchromone or eugenin (band Y) by fungitoxic properties. Further investigation into the elemental analysis as well as U.V., LR., P.M.R. spectral fungitoxic properties of isocoumarin led to its in· and mass spectrometric data (19). The compounds of elusion as a phytoalexin (10). Subsequent workers in band Z and M were not identified but their U.V. spectra this area (7,9) found that C. .fimbriata also produces and color reactions to phenol detecting reagents were ethylene and Chalutz et al. (7) correlated isocoumarin very similar to those of isocoumarin. This would suggest synthesis in carrots with ethylene production by C. the presence of a similar chromophore in these two fi.mbriata. Later, Condon et al. (9) tested four different compounds. fungi for their ability to induce isocoumarin synthesis in Why and how more isochlorogenic acid is synthesized carrots. and to produce ethylene. They found only one in carrot roots in the presence of ethylene is a question fungus capable of producing ethylene, whereas all four that should intrigue plant physiologists and biochemists. induced isocoumarin formation in carrot roots. There To people dealing with post-harvest problems of carrots, fore, a strict correlation between ethylene production and increased isochlorogenic acid level makes these roots isocoumarin synthesis was not found. Things became more susceptible to browning. even more complicated when isocoumarin as well as The other phenols namely isocoumarin and eugenin, other closely related compounds were isolated from which are synthesized de novo in carrots on exposure to various fungi, including Ceratocystis .fimbriata (4, 11, 12, ethylene, pose an altogether different problem. Isocou 25). Thus the time was right to redefine the role of marin has been associated with bitter-tasting carrots ethylene in isocoumarin synthesis in carrot roots. We have (24), and there has been some controversy as to the real not studied the effects of microorganisms on synthesis of role of ethylene in the induction of isocoumarin isocoumarin in carrots, but our results clearly show that formation in carrots. Condon et al. (9) found that carrot ethylene is a causative agent for the synthesis of discs inoculated with Ceratocystis .fimbriata produced isocoumarin. 530 SARKAR AND PHAN

Eugenin has not been reported as a normal constituent of carrot root. Its occurrence after ethylene treatment indicates that the gas brings about a profound change in the metabolism of carrot root tissues. Time course studies on ethylene-induced isocoumarin synthesis The rate of isocoumarin formation in carrots can be seen in Fig. 6; up to day 2 accumulation of isocoumarin continued rapidly, from day 2 to day 3 it increased at a 0') slower rate and after day 3 it stayed nearly unchanged. _,E c: A time-course study of ethylene-induced isocoumarin .... formation in carrots indicated the presence of a lag 0 E Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021 period of about 16 h before the presence of isocoumarin ::;) 0 could be detected (Fig. 7). Increasing the concentration v 0 0 16 32 48 ..:!!. of ethylene shortened the lag time to 4 h, and increased Hours of exposure the initial rate of isocoumarin synthesis but not the final amount accumulated (Fig. 8). The amount of isocou 8. Rate of ethylene-induced isocoumarin synthesis in carrot with 0.2% and 5% ethylene. ( e ) · 0.2"7o ethylene, and ( .i.) · 5% marin accumulated in 2 days with 100 ppm of ethylene ethylene. could be attained after a day with 0.2 o/oor 5%ethylene. Ejj'ects of ethylene on the biosynthetic pathways of phenols From the above data, it is clear that ethylene exerted a pronounced effect on the phenol content of carrot root. This effect is manifested both as an accumulation of existing phenols such as isochlorogenic acid, and as a de novo synthesis of at least four phenolic compounds. 0> It is important to note that the two moities in 0 isochlorogenic acid (caffeic acid and quinic acid) are Q 15 ~ synthesized via the shikimic acid pathway. Thus an 0> increased synthesis of isochlorogenic acid must occur as E a result of ethylene-induced stimulation of the shikimic - acid pathway. One of the key enzymes of this pathway is .... L-phenylalanine ammonialyase (PAL). The effect of 0 E ethylene on the activity ofthis enzyme in carrot roots was :::> investigated. The results (Fig. 9) show an initial increase 0 u in PAL activity, which declined to a value less than that 0 til of control after 3 days of exposure to ethylene. Ethylene also failed to produce any effect on a cell-free extract from carrot root (20). This would indicate that a direct 0 2 3 4 5 correlation does not exist between enhanced isochloro Days of exposure genic acid level and in vitro PAL activity. What happens Figure 6. Rate of ethylene-induced isocoumarin synthesis in carrot in vivo is a matter that needs further investigation. slices at 25 ± 0.5 C. Ethylene-treated ( .i. ), and air· treated ( • ) Structural analysis of 8-hydroxy-6-methoxy-3-methyl- carrots. 3,4-dihydro-isocoumarin (so-called isocoumarin) and I 5-hydroxy-7 -methoxy-2-methylchromone (commonly • •i known as eugenin) suggests that these are most likely --·I synthesized via the acetate pathway. Incorporation ·- studies with r4C]acetate, [14C]malonate, and 14aceto acetate were carried out on carrot slices in presence of ethylene (21). The results lTables 3 and 4) clearly indicated that isocoumarin is synthesized from acetate as c: 0 5 suggested earlier by Condon et al. (9). E Similarly, as expected, acetate was readily incorpor 0 1 "u ated into eugenin (Table 5). In this instance, it was _g 40 56 72 further shown that addition of the methyl group on Hours of exposure position 7 was probably the last step in the sequence of Figure 7. Time course of isocoumarin formation in carrot slices on eugenin synthesis because 5,7-dihydroxy-2-methyl-chro ethylene treatment. ( .i. ) and (e) represent two different batches of carrots. mone (DHMC) was characterized as the immediate PHENOLIC COMPOUNDS IN CARROTS 531

0-methyl groups (15,16). Thus the biosynthetic route for eugenin may be as follows:

"~ lQjj_Q~HJWHI I "'' H, ) H, ~ ~ I")_, ~ II H I OH 0 L Our results from all the above sections bring to light two important observations regarding the effect of ethylene on phenol synthesis in carrots: (a) ethylene stimulates the synthesis of phenols derived via the shikimic acid pathway, and (b) ethylene induces synthesis of four more phenolic compounds that are synthesized Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021 via the acetate pathway of origin of aromatic compounds. Therefore, we conclude that ethylene stimulates both the pathways of biosynthesis of aromatic compounds in carrots. Ethylene-induced phenol synthesis in carrots and its relation to other metabolic activities 0 Effect of ethylene on respiration and glycolytic Exposure pathways. For syntheses of aromatic compounds via Figure 9. Effect of eJ:posure time on the development of PAL activity either pathway, the required substrates and energy must in carrots. Ethylene· treated (A ), and air-treated (e) carrots. land ll represent two different experiments. come from a process or processes that is/are induced or modified by the action of ethylene. One of the main precursor of eugenin by trapping 14C-labeled DHMC biological functions affected by ethylene is respiration. from carrots fed [1 4C]acetate. This is in agreement with Although the mode of action of ethylene on the the data obtained with other phenolic compounds having respiratory processes is not yet clear, it is well-established

TABLE 3. Incorporation of [1· 14C]acetate and [2· 14C]malonate into isocoumarin. a Substrate: one mm carrot slices were shaken with aqueous solutions (5 m{J of the labelled compounds for 2 days in presence of a continuous stream of 0.2% ethylene. Initial spec!fic activity qf acetate and malonate was 10.4 mCilmmol.

---·-~ ·--~·····------

Activity added to Activity taken up Activity of isocoumarin Specific activity Weight oft he theJissue by the tissue in th~ tissue of isocoumarin Substrate tissue (g) (10' dpm) no-6dpm) (10· dpm) Conversion(%) ( ~Ci/mmol) [1-14 C]acetate 3.00 12.7 9.4 0.15 1.6 135 [2-14C]malonate 3.10 12.7 8.8 0.12 1.3 104 a From Sarkar and Ph an {21).

TABLE 4. lncorporation qfl3-14C]acetoacetate alone or together with unlabelled acetate into isocoumarin. a !1·14C]acetate together with unlabelled acetate was .fed by infiltration into the tissue after8 h ofprior exposure to 0.2o/oethylene done for another40 hat room tempe ration.

[3-14c]aeetoacetate Weight of Activity added L' nlabelled acetate Activity taken up Activity of iS<>eoumarin (in6.7ml) the tissue to thg tissue (in 6.7 ml) by the tissue in th§ tissue (~MJ (g) no- dpm) (eMJ no-6dpm) (10' dpm) Conversion(%) 21 3.10 3.5 none added 1.00 17.1 !.70 21 3.20 3.5 210 0.96 6.4 0.67 21. 3.05 3.5 420 1.03 7.7 0.75 21 3.00 3.5 630 0.99 18.3 1.86 Sarkar and Ph an 1.;21).

TABLE 5. l ncorporation of [1· 14C]acetate into eugenin and DHM C in the presence or absence of tmla belled D HM C. a The solution q( acetate (5 m{J alone or along with unlabelled DHMC was shaken with l·mm thick tissue slices days of prior exposure to 2000 iJ.llethylene in the presence of the same concentration

Activity Activity Activity of eugenin Weight of Metabolic added to taken up by and DHMC in the Specific activity the tissue period the tissue the tissue Conversion % tissue, w-6 dpm ( ~ Ci/mmol) Substrate (g) (hl (10'6dpm) no-6dpm) Eugenin Eugenin DHMCb

8.15 60 39.0 37.4 0.79 153

6.90 J2 39.0 0.22c 0.8oc 0.1 0.3 18 5 Sarkar and Phan {21). specific activity ofDHMC is after dilution with the carrier. CEased on the activity added. 532 SARKAR AND PHAN that ethylene markedly enhances respiration in plant The pattern of conversion of specifically labelled tissues. It has been suggested recently that ethylene glucose into isocoumarin is given in Table 7. It will be induced rise in respiration might result from a greater seen that the yield as well as the specific activity of participation of cyanide-resistant electron path (23). Our isocoumarin was much higher when obtained from data (Fig. 10) clearly showed that ethylene enhanced the [6- 14C]glucose than those obtained from [I-1 4C]glucose or respiratory activity of carrot tissues. Peel (1-mm thick) 3,4- 14C]glucose. This would indicate that the EMP tissues were more active and responded more noticeably pathway contributes mere to isocoumarin synthesis in to ethylene treatment than internal tissues (17). Both 0 2 ethylene-treated carrots. This may have happened uptake and C02 output increased, and the R.Q. because of ethylene-induced shift from the PP to the remained unchanged and was close to one. Thus, EMP pathway of carbohydrate dissimilation as discussed carbohydrate appears to be the source of carbon dioxide above. produced. Effect of metabolic inhibitors on isocoumarin synthe To ascertain the distribution of glucose breakdown sis in carrots. The effects of DNP and methylene blue on Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021 between the Embden-Meyerhof-Parnas (EMP) and the carrot slices are presented in Table 8. At pH 7.1, DNP concentrations of 5 M and 10- 4 M were inactive, and 250 w- only at 10- 3 M was isocoumarin synthesis induced. The effect ofDNP cannot be considered as mediated through ethylene as it depressed ethylene production (14). DNP is ~200:;... known to stimulate respiration and glycolysis, and acts as an uncoupler of oxidative phosphorylation, but ethylene 150 :> Q. has not been shown to uncouple oxidative phosphoryla :; 0 tion. 0100 100 0 This indicated that both the PP and the EMP pathways u were operative in carrots, as has been reported earlier (2). In ethylene-treated samples (lower half of Fig. 11), the 14 0 6 12 18 24 rate of C02 production from [6- C]glucose increased Hours of exposure 17So/oover that what was obtained in air-treated carrots, Figure !0. Ejji•ct oferhylent? on the respiratory activity of carrot root 14 while the rate of CO 2 production from [1- C]glucose was slices. Dotted lines: ethylene-treated. Plain lines: air controls. slightly depressed. These changes in rates of C02 Pentose Phosphate (PP) pathways of glucose utilization, production resulted in lower C-1/C-6 ratios (Table 6), specifically labelled glucose was fed to air-treated and which meant a preferential stimulation of the EMP ethylene-treated carrot slices. The curves in the upper pathway in presence of ethylene. Such a shift from the PP half of Fig. 11 show that in air-treated samples the rate to the EMP has been reported by Tager (26) in ripening 4 of C02 production from [P C]glucose was higher than bananas and its concomitant ethylene production. that from either [6- 14C] or [3,4- 14C]glucose, and the In view of the studies with DNP, it is reasonable to C-1 /C-6 ratios were always greater than one (fable 6). suggest that acetate, the recognized precursor of isocoumarin and eugenin (21), is formed through an 151-' '.J increased activity of the glycolytic pathway, and the (I) .--· .--c,--·, energy required for the synthetic processes comes from 10 the operation of the sequence EMP-Krebs cycle and ./~ 0---- .. --·' electron transport chain, probably the cyanide resistant //' .. path (23). p 51- ~~0---- - Methylene blue, too, induced the production of .. :--::::~.:::::::::: ..~ isocoumarin in carrots (Table 8). Although methylene ..,~I blue is known to stimulate respiration, and enhance the operation of the PP pathway (2), it might be suggested, in this case, that synthesis of isocoumarin followed higher ethylene production; production of ethylene by carrot 0 slices was stimulated by methylene blue (21). Never :!u 10 theless, the role of increased respiratory activity (in the presence of methylene blue) in isocoumarin synthesis 5 cannot be ruled out. The data in Table 9 show that a 10-millimolar solution of arsenite completely inhibited isocoumarin synthesis; a 2 3 s 6 7 8 millimolar solution partially inhibited it, while a Hours 0.1-millimolar solution stimulated isocoumarin produc Fi;:nrc I l . Y iclds q(1 4C0zfrom specifically labelled glucose in air (/) .md ethylene (100 ppm) (2). From [l· 14 C]glucose (.A. J: from tion. Its stimulatory action at low concentration is 14 14 !3.-l· ( 'l_,;lucosc ( 0 );from [6· C]g!ucose ( e l. intriguing. Could this be caused by a stimulation of PHENOLIC COMPOUNDS IN CARROTS 533 ethylene production by low concentrations of arsenite, as a decrease in pyruvate oxidase mediated acetyl CoA shown by Shimokawa and Kasai (,22)? At higher production. concentration, arsenite is known to function as an In light of the above three inhibitor studies, we are inhibitor of keto acid synthesis (27), and its role in proposing that ethylene enhances the production of inhibition of isocoumarin synthesis may be described by pyruvate by stimulating glycolysis. The pyruvate thus TABLE 6.

Control Ethylene (2000 ppm) treated %ofC-1 0roofC-6 "kofC-1 %ofC-6 Experiment Time converted to converted to Ratio converted to converted to Ratio number (h) C02 C02 C-1/C-6 C02 C02 C-1/C-6 0-3 5.23 2.47 2.11 4.80 3.41 1.40 3-6 6.65 3.40 1.95 6.00 4.80 1.25

2 0-2 3.75 l.56 2.40 2.81 1.61 1.61 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021 2-4 5.46 2.65 2.06 4.97 3.73 1.33 3 0-2&1/2 2.79 1.28 2.18 2.23 1.40 1.59 2&112-5 3.98 2.13 1.87 3.55 2.54 1.39

*C-1 release was determined using [1-14C]glucose. **C-6 release was determined using l6-14C]glucose.

TABLE 7.

Substrate Activity taken up ( x1Q·6 dpm) Isocoumarin (dpm) 0;0 ccnversion

1 18.10 15.33 77.284 0.504 activity 2 15.32 13.50 75,104 0.556 10.7 mCi/mole [6-14 C]glucose. specific 1 18.10 17.29 119,800 0.693 activity 2 15.32 13.88 107,496 0.774 10.7 mCi/mole [3,4- 14C]glucose, specific 18.10 16.85 17,025 0.101 activity 2 15.32 12.99 15,055 0.115 10.7 mCilmole Ph an and Sarkar (21). conversion was determined on the basis of labeled glucose taken up by the tissue. Average of duplicate determinations.

TABLE 8. Ejfect qf'dinitrophenol and methylene blue on isocoumarina carrot slices. b

lsocoumarin (mg/100 g fresh weight) Inhibitor Days after treatment Molarity of solutions

1 10 4 M Not detectable Not detectable 1 10 3M Not detectable 10.76e 2 10- 3M Not detectable 22.17 3 10" 3M Not detectable 31.85 Methylene blue 1 1Q"3M Not detectable 10.08 2 10" 3M Not detectable 31.35 eugenin was detected. Phan and Sarkar (21). ccontrols were infiltrated with water. dLimitofdetection: 0.1 11g. e Average of duplicate determinations.

TABLE 9. Effect of sodium arsenite on the synthesis ofisocoumarin in carrots. a

Isocoumarinb (mg/100 g carrots) Expt. No. Ethylene treatment (100 ppm) Molarity of arsenite solution Control Arsenite-treated 1 day None added 11.65 1 day 1 X 10" 2M None 1 day 1 X 10" 3M 7.19 1 day 1 X 104M 18.19 2 2 days None added 19.85 2days 1 X 10" 2M None 2 days 1 X JO"lM 16.89 2 days 1 X 10~4M 25.67 Phan and Sarkar (21). of duplicate determinations. 534 SARKAR AND PHAN formed produces more acetyl CoA than can be used by Agricultural Experiment Station, Geneva, N.Y. TCA. The excess acetyl CoA is then diverted to the 4. Aue, R., R. Mauli, and H. P. Sigg. 1966. Production of 6-methoxy synthesis of acetogenins, namely isocoumarin, eugenin mellein by Sporormia bipartis Cain. Experientia 22:575. 5. Bessey, P.M., R. L. Carolus, and M. H. Sell. 1956. Fluorescence and related compounds. In a similar fashion an and ethylene as related to bitterness in carrots. Abstr. Annu. Mtg., increased production of ethanol and acetaldehyde in Amer. Soc. Hort. Sci. p. 2a. carrots on DNP treatment has been explained. 6. Carlton, B. C., C. E. Peterson, and N. E. Tolbert. 1961. Effects of The data in Table 10 show that cycloheximide ethylene and oxygen on prQduction of a bitter compound by carrot completely inhibited isocoumarin formation only when it roots. Plant Physiol. 36:550-552. 7. Chalutz, E., J. E. Devay, and E. C. Maxie. 1969. Ethylene-induced was added to tissues that did not receive prior ethylene isocoumarin formation in carrot root tissue. Plant Physiol. 44: treatment. Partial inhibition was observed when cyclo- 235-241. 8. Chubey, B. B., and R. E. Nylund. 1969. Surface browning in TABLE 10. Influence of the time of in.filtration of cycloheximide carrots. Can. J. Plant Sci. 49:421-426. U x 10- 4M) on the isocoumarin content of carrots after 24 hrs of

9. Condon, P., J. Kuc, and H. N. Draut. 1963. Production of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/6/526/1650161/0362-028x-42_6_526.pdf by guest on 27 September 2021 ethylene treatment. a 3-methyl-6-methoxy-3,4-dihydroisocoumarin by carrot root tissue. Phytopathology 53:1244-1250. 10. Cruikshank, I. A. M. 1963. Phytoalexins. Ann. Rev. Phytopathol. Time of infiltration of Expt. cycloheximide (h after Isocoumarinb 1:351-374. No. ethylene application) (mg/100 g carrots) 11. Curtis, R. F. 1968. 6-methoxymellein as a phytoalexin. Experientia 0 Not detectable 24:1187-1188. 4 7.91 12. McGahren, W. J., and L.A. Mitscher. 1968. Dihydroisocoumarins No cycloheximide 11.60 from a Sporormia fungus. J. Org. Chern. 33:1577-1580. 2 0 Not detectable 13. Phan, C. T., H. Hsu, and S. K. Sarkar. 1973. Physical and chemical 4 6.15 changes occurring in the carrot root during storage. Can. J. Plant No cycloheximide 9.80 Sci. 53:635-641. a From Phan and Sar kar (I 4). 14. Phan, C. T., and S. K. Sarkar. 1975. Studies on the biosynthesis of b Average of duplicate determinations. isocoumarins in carrot root tissues: induction by substances other than ethylene and effects of metabolic inhibitors. Rev. Can. Bioi. heximide was added to carrots after 4 h of ethylene 34:23-32. treatment. The results indicate that upon ethylene 15. Rhodes, A., B. Boothroyd, M. P. McGonagle, and G. A. treatment there is an active , de novo synthesis of Sommerfield. 1961. Biosynthesis of griseofulvin: the methylated enzyme(s) necessary for isocoumarin synthesis. This benzophenol intermediates. Biochem. J. 81:28-37. 16. Rickards, R. W. 1961. In: W. D. Ollis (ed.) Chemistry of natural would explain the existence of a lag phase preceding the phenolic compounds. Pergaman Press, London. appearance of isocoumarin synthesis in ethylene-treated 17. Sarkar, S. K. 1972. Ethylene and phenol metabolism in stored carrots. ·carrots. Ph. D. Thesis, University of Alberta, Edmonton, Alberta, Canada. CONCLUSIONS 18. Sarkar, S. K., and C. T. Ph an. 1974. Some hydroxy-cinnamic acids of the carrot root. Plant Sci. Letters 2:41-44. On the basis of foregoing findings it seems certain that 19. Sarkar, S. K., and C. T. Phan. 1974. Effect of ethylene on the ethylene causes an increased accumulation of existing qualitative and quantitative composition of the phenol content of phenolic compounds in carrots. Ethylene has also been carrot roots. Physiol. Plant. 30:72-76. 20. Sarkar, S. K., and C. T. Phan. 1974. Effect of ethylene on the established as a cause for inducing the synthesis of phenylalanine ammonia-lyase activity of carrot tissues. Physiol. isocoumarin and eugenin in carrots. Although the Plant. 32:318-321. concentrations of ethylene (100 and 2000 ppm) used in 21. Sarkar, S. K., and C. T. Phan.1975. The biosynthesis of8-hydroxy- these studies are much higher than the physiological 6-methoxy-3-methyl-3,4-dihydroisocoumarin and 5-hydroxy-7- levels, the above fmdings may be extrapolated to the methoxy-2-methylchromone in carrot root tissues treated with ethylene. Physiol. Plant. 33:108-112. effects of lower concentrations of ethylene.lt is suggested 22. Shimokawa, K., and Z. Kasai. 1966. Biogenesis of ethylene in apple that low levels of ethylene produced by carrots may tissue. I. Formation of ethylene from glucose, acetate, pyruvate and induce slowly the production of isocoumarin in carrots acetaldehyde in apple tissue. Plant Cell Physiol. 7:1-9. during storage and render them bitter. What effect(s) 23. Solomos, T., and G. Laties. 1975. The mechanism of ethylene eugenin and other phenols (synthesized upon ethylene and cyanide action in triggering the rise in respiration in potato treatment) may have on the taste and other qualities of tubers. Plant Physiol. 55:73-78. 24. Sondheimer, E. 1957. Bitter flavor in carrots. III. The isolation of a carrots will be worth studying. compound with spectral characteristics similar to hydrocarbon extracts of bitter carrots. Food Res. 22:296-299. REFERENCES 25. Stoessl, A. 1969. 8-hydroxy-6-methoxy-3-methylisocoumarin and 1. Agricultural Statistics. 1971. U.S. Department of Agriculture, other metabolites of Ceratocystis fimbriata. Biochem. Biophys. Washington, D.C. Res. Commun. 35: 186-192. 2. Ap. Rees, T., and H. Beevers. 1960. Pathways of glucose 26. Tager, J. M. 1956. The role of the pentose cycle in the ripening dissimilation in carrots. Plant Physiol. 35:830-838. banana. South Aft. J. Sci. 53:167-170. 3. Atkin, J. D. 1956. Bitter flavor in carrots. II. Progress Report on 27. Webb, J. L. 1966. In: Enzymes and metabolic inhibitors, Vol. 3. field and storage experiments. Bulletin No. 774, New York State Academic Press, N.Y.