Inhibition of Ethylene Biosynthesis by Aminoethoxyvinylglycine and by Polyamines Shunts Label from 3,4-[14CjMethionine into Spermidine in Aged Orange Peel Discs1

The flux of radioactivity from 3,4-114Clmethionine into S-adenosyl_L- methionine (SAM), 1-aminocyclopropane-l-carboxylic acid (ACC), spermine, and spermidine while inhibiting conversion of ACC to ethylne by 100 millimolar phosphate and 2 millimolar Co2' was studied in aged peel discs of orange (Citrus sixensis L. Osbeck) fruit. Inhibition up to 80% of ethylene production by phosphate and cobalt was accompanied by a 3.3 times increase of label in ACC while the radioactivity in SAM was only slightiy reduced. Aminoethoxyvinylglycine (AVG) increased the label in SAM by 61% and reduced it in ACC by 47%. Different combinations of standard solution, in which putrescine or spermidine were administered alone or with AVG, demonstrated clearly that inhibition of ethylene biosynthesis-at the conversion of SAM to ACC-by AVG, exogenous putrescine or exogenous spermidine, stimulated the incorporation of 3,4-114CImethionine into spermine.

Biosynthesis of ethylene in higher plants has been extensively studied and reviewed (14). Following the discovery in 1966 by Lieberman et al. (15) of methionine as a precursor of ethylene in higher plants, Adams and Yang (2) and Lurssen et al. (17) have shown independently that ACC, a metabolite of SAM4 (5,27), is efficiently converted to ethylene. These observations have now been extended to other plant tissues and confirmed elsewhere (7,9,18,26). It seems likely that ethylene is synthesized from methionine via the following metabolic sequence: methionine -. SAM ACC -* ethylene (Fig. 1). Thus, SAM metabolism in plants assumes a particular significance, inasmuch as it is also a well established precursor of polyamines (19,23) (Fig. 1). SAM may be utilized by plants for the biosynthesis of either polyamines or ethylene, or both, depending upon the conditions that regulate its pathway. However, the conditions appropriate for channeling SAM towards either of the two are not yet determined. Interestingly, the functions of polyamines and ethylene in higher ' Supported by a grant from the Israeli Ministry of Absorption to Z. E. C. and by a grant from the Israeli Ministry of Agriculture to R. G. plant metabolism differ diametrically. Although ethylene is a plant-aging hormone leading to retardation of growth and promotion of senescence (1, 6), polyamines delay senescence in excised leaves and protoplasts (3,13,20). Polyamines also inhibit ethylene biosynthesis in several plant tissues (4,25), but the mechanism of this inhibition was not yet determined. The possibility that the utilization of SAM in the biosynthesis of either ethylene or polyamine is regulated in the plant cell may have important physiological implications and therefore needs further investigation. Ethylene production by albedo tissue of mandarin fruit (11), and by wounded tissue of citrus fruit (12,29) has been previously reported. The induction of ethylene formation by ABA and the inhibitory effect of AVG on its production in citrus bud culture and leaf explants were also studied (10,21). In the present investigation, we have used orange peel tissue to study relative incorporation of radioactivity from 3,4-[14CJmethionine into SAM, ACC, spermine, and spermidine while inhibiting biosynthesis of ethylene from ACC by cobalt and phosphate ions. By specifically inhibiting the terminal step of ethylene biosynthesis (Fig. 1), unwanted effects of excess ethylene on tissue metabolism were kept to a minimum. Under these conditions, we show that inhibition of ethylene biosynthesis at the step of conversion of SAM to ACC by AVG and the polyamines, putrescine and spermidine, stimulates 3,4-['4Clmethionine incorporation into spermidine, while lowering the concentration of ACC.

MATERIALS AND METHODS
Plant Material. Mature Shamouti orange fruits (Citrus sinensis L. Osbeck) were picked, washed with water, surface-sterilized with 70%o ethanol, and peeled. The peelings were then put in a dark humid chamber for 72 h at 25°C to age, and used to prepare discs of 5 mm in diameter and 5 mm thickness. The discs were incubated in test tubes (four per tube, tissue weighing approximately 0.8 g) with 0.75 ml of a medium (pH 5.5) containing 0.25 M sucrose and 0.75 ,uCi of 3,4-['4CJmethionine (49 mCi/mmol) in H20; when indicated, 2 mM Co2+, 100 mm Na-phosphate, 2.6 mm AVG, 10 mM putrescine, 10 mm spermidine, or 5 mm Ca2+ were added either alone or in various combinations. Each medium was infiltrated into the tissue under vacuum, and the tubes were then placed in a vial with two compartments (one of the compartments contained 2 ml saturated KOH for trapping total CO2 including I'4C1O2 and the other contained 1 ml of 0.25 M mercuric perchlorate to absorb ethylene). After 20 h incubation, radioactive CO2 and ethylene were determined and the tissue was frozen at -20°C until analyzed. The experiments were repeated twice and averages of the data are presented. Standard errors between experiments were generally in the range of 5 to 10%1o of the means. Plant Physiol. Vol. 69, 1982 (16), after extracting the tissue for ACC by the method of Yu and Yang (28). Analysis of Polyamines. Polyamines were analyzed by the procedure described by Seiler and Wiechmann (22). Orange peel discs were extracted with 4% HC104, dansylated, and separated by TLC, using the solvent system ethylacetate:cyclohexane (2:3, v/v). The zones on the TLC co-migrating with authentic dansylated samples of spermidine and spermine were scraped off the TLC plates and radioactivity was determined in a scintillation spectrometer.

RESULTS AND DISCUSSION
Carbons 3 and 4 of methionine are specifically converted into the carbon skeleton of ethylene in most of the higher plants tested (14). Under the experimental conditions, aged orange peel discs (Table I), and a good proportion of the label was seen in SAM and ACC, the intermediates of ethylene biosynthesis (Fig. 1). The amount of radioactive CO2 produced was relatively very low. Addition of 100 mm phosphate to the incubation medium reduced the incorporation of methionine into ethylene by 30%, while 50% more ACC accumulated in the tissue. However, an addition of Co2+ together with phosphate caused 80% inhibition of ethylene synthesis and a 3.3-fold increase in [14C]ACC (Table I). Also, incorporation into radioactive SAM was not changed by these treatments. The findings suggest that inhibitory effects of phosphate and CO2' are more pronounced at the conversion of ACC to ethylene. Indeed, the ratios of both ACC:SAM and ACC:ethylene increased from 0.62 and 0.45 in the control to 1.12 and 1.00 in the presence of phosphate, and to 2.15 and 7.35 in that of phosphate and Co2+ (Table I). These results complement previous reports in which phosphate (8,9) was shown to inhibit ethylene synthesis in tomato fruit plugs, pea seedling segments, and carrot slices, and Co2+ (28) was found to inhibit the conversion of ACC to ethylene in auxin-induced ethylene production. When incorporation of labeled methionine into [14Clethylene was inhibited by 80% in the presence of Co2' and phosphate, some label from methionine was recovered in spermidine and spermine; however, the label incorporated into spermine was twice as much as that incorporated into spermidine (Table II). AVG, which is a potent inhibitor of ethylene biosynthesis in higher plants (14) including citrus (10,21), was shown recently to inhibit the conversion of SAM to ACC (5, 27). In orange peel tissue also (Table   II) the presence of AVG resulted in 60%o more [14CISAM than the control, apparently at the cost of label in [14CJACC which was reduced by 50%7o as compared with control. Concomitant with the inhibition of ethylene formation at the stage of SAM conversion to ACC by AVG, a dramatic increase (475%) in label from 3,4- ["4CImethionine was seen in ["4C]spermidine, whereas in spermine radioactivity was slightly reduced. In fact, the radioactivity in spermine remained very nearly the same under different treatments, an interesting finding which may mean that its level is under strict metabolic regulation; this, however, was not explored further. When the incubation medium contained both unlabeled putrescine and AVG, the incorporation of 3,4-['4Cjmethionine into radioactive spermidine increased 2-fold as compared with AVG alone, and about 10-fold as compared with control (Table   II). Moreover, putrescine prevented the AVG-mediated accumulation of label in SAM and caused further decrease of label in ACC. Incubation medium containing unlabeled spermidine and AVG also prevented the AVG-mediated increase in the transfer of label from methionine into SAM; however, unlabeled spermidine in the presence of AVG could not cause further increase in radioactive spermidine as was seen with putrescine (Table II). It seems that although AVG   SAM, and that this process is amplified when the conversion of SAM to ACC is decreased, for instance in the presence of AVG, spermidine, or putrescine (see below). However, it seems that below a certain threshold, putrescine may limit the conversion rate of SAM to s ermidine, inasmuch as when it was supplied exogenously, 3,4-[ 4Cjmethionine incorporation into spermidine was further increased in tbe presence of AVG. This conclusion is supported by other data (Table II), showing that when no AVG was present, putrescine increased the incorporation of label from 3,4-[ 4C]methionine into spermidine 5-fold, while inhibiting the conversion of SAM to ACC. AVG is known also to inhibit endogenous synthesis of methionine by affecting ,8-cystathionase activity (14). Therefore, under these conditions, when methionine is limiting, any exogenously provided substrate, for example labeled methionine, would be expected to get utilized for either the formation of ethylene or/ and polyamines via SAM. Since AVG also prevents the conversion of SAM to ACC, the flux of label from 3,4-[14CJmethionine would be expected to shunt away into spermidine/spermine. This is precisely what was observed (Table II). However, inasmuch as only the flux of radioactivity was determined in the present investigation, proof that spermidine/spermine synthesis is promoted is not complete. How far AVG or polyamines affect the specific activity of various intermediates and biosynthetic end products remains to be investigated. Spermidine alone also inhibited the conversion of SAM to ACC while increasing the formation of labeled spermidine (Table II), indicating that it may be acting as a feed-forward activator (autocatalyst) of its own synthesis from methionine. It might therefore be that one metabolic site of polyamine-mediated inhibition observed previously (4) is at the conversion of SAM to ACC. Because these experiments were not designed to study the stage at which the conversion of ACC to ethylene occurs, we do not rule out the possibility that polyamines inhibit not only the conversion of SAM to ACC but perhaps also the conversion of ACC to ethylene.
Although AVG, putrescine, and spermidine all increased the incorporation of labeled methionine into spermidine, it is possible that the mechanism of action for AVG may be different from that for putrescine and spermidine. AVG decreased the incorporation of SAM into ACC and caused SAM to accumulate. This accumulation of SAM may also enhance the conversion of methionine into spermidine through a mass-action effect. However, spermidine appeared to inhibit slightly the conversion of 3,4-['4C]methionine into SAM while putrescine was slightly stimulatory (Table  II). Therefore, the methionine to SAM step does not appear to be a metabolic site of polyamine action. The stimulation of methionine incorporation into spermidine by polyamines seems to be a result of their effect at a later step in the biosynthetic pathway.
An earlier work showed that Ca2+ reversed the inhibitory effect of polyamines on ethylene production partly or in full (4), and that Ca2+, supplied together with polyamines, diminished their action, indicating probable involvement of an initial ionic attach-ment mechanism (13). We have found ( The data presented may suggest that there is a close physiological link between ethylene and polyamine biosynthesis. This indicates that hormonal interactions with one of the two biosynthetic pathways may affect the other and be reflected in a physiological response. For example, it was found (10,21) that ABA-induced ethylene formation is inhibited by AVG. Does this mean that polyamine biosynthesis is increased concomitantly? Or, is the reduction of both the activity of arginine decarboxylase and putrescine level by ABA (24) accompanied by increased ethylene formation? Further work with various plant systems at different stages of development will provide the answers to these intriguing questions. In fact, putrescine and AVG, supplied independently, were found recently to increase the incorporation of label from 3,4-["C]methionine into spermidine and spermine in mature green tomato fruit tissue (Adams, Wang, and Lieberman, personal communication). In the tomato experiments cited above (Adams et al, personal communication) the last step from ACC to ethylene was not inhibited as was done in the present study, thus showing that under conditions in which ethylene production from ACC is not blocked, the observed effects of AVG and polyamine can be as well obtained. That polyamines are capable of directly inhibiting ethylene formation (4) further emphasizes the importance of the proposed interactions of plant growth regulators with biosynthetic pathways of polyamines and ethylene (Fig. 1).