Plant Physiol. (1978) 61, 896-899

14C2H4 Metabolism in ' Received for publication November 21, 1977 and in revised form January 13, 1978

ELMO M. BEYER, JR. AND OLOF SUNDIN2 Central Research and Development Department, Experimental Station, E. I. du Pont de Nemours and Company, Wilmington, Delaware 19898

ABSTRACT beginning 3 days before and ending 2 days after full bloom. Data presented indicate that morning glory flowers, like carnation Flowers of Ipomoes tricolr Cav. (cv. Heavenly Blue) were cut at various flowers (9), have the metabolic competency at certain stages of stages ofdevelp_et and evaluated for their ability to metaboize ethylene. development to oxidize ethylene to CO2 and to incorporate Freshly cut buds or flowers were treated in glass containers for 8 hours ethylene into water-soluble tissue metabolites. This metabolism with 6 ,ul/lter of highbly purifled 14Cs21. Fofowing removal of dissolved correlates well with changes in ethylene sensitivity. 14C2H4, radioactivity was determined for the different flower tissues and trapped CO2. 14C2H4 oxidation to "4CO2 and tissue incorporation occurred at very low to nondetectable levels 2 to 3 days prior to flower opening. MATERIALS AND METHODS About I day prior to ful bom, just at the time when mature buds become Material. Seeds of I. tricolor Cav. (cv. Heavenly Blue) responsive to ethylene (Kende and Hanson, Plant Physiol 1976, 57: were purchased from Agway Inc. (Syracuse, N.Y.), were planted 523-527), there was a dramatic increase in the capacity of the buds to in a peat moss-Vermiculite sand mixture (3:1, v/v) and were oxidize 4C2H4 to 14CO2. This activity continued to increase until the watered daily with Hoagland nutrient solution. were grown flower was fuly opened reaching a peak activity of 2,500 dpm per three in an environmental growth room (1,500 ft-c; 12-hr photoperiod; flowers per 8 hours. It then declined as the flower closed and rapidly 26 C day; 21 C night; 60%1o RH) where flower opening and senesced. A simaar but smaller pak occurred in tissue Incorporation and senescence took place in a single day as previously described (14). it was folowed by a second peak during late flower senescence. This first Under these conditions, where the lights were turned on at 7:00 peak in tissue Incorporation and the dramatic peak in ethylene oxidation AM, the mature buds were fully opened by 9:00 AM and they slightiy preceded a large peak of natural ethylene production which accom- would begin to close between 4:00 and 5:00 PM of the same day panied flower secence. The ethyee metabolism observed was clearly (Fig. 1). By 6:00 PM all flowers were completely closed and petal dependent on celular metabolsm and did not involve microorganisms since drop was usually complete 24 to 48 hr later. heat kiling destroyed this activity and badly contaminated beat-killed Bud length (base of calyx to bud tip) served as a highly reliable flowers were unable to metabolize ethylene. predictor of ultimate flowering date (Fig. 1). Thus, it was possible to assign the following bud sizes to buds scheduled to open 24, 48, or 72 hr later: -72 hr = 2-2.2 cm; -48 hr = 3.2-3.6 cm; -24 hr = 4.6-5 cm. Prior to full bloom (9:00 AM), buds were given a minus designation while after full bloom they were given a plus designation as suggested by Kende and Hanson (15). Work on the metabolic fate of 1"C2H4 in pea seedlings (4, 5, 10) "C2H4 Treatment. Starting at 7:30 AM of the morning of an and more recently in cut carnation flowers (9) revealed a previ- experiment the buds on the plant were classified according to ously unrecognized active ethylene metabolic system in these length and tagged in the categories -72, -48, or -24. Buds tissues. This system oxidizes ethylene to CO2 and incorporates already opening were tagged 0 or full bloom, and flowers that had ethylene into water-soluble tissue metabolites. Ethylene purifica- opened the previous morning were tagged +24. These flowers tion by preparative gas chromatography (6) combined with aseptic were identified by tagging fully opened flowers at 9:00 AM the day techniques (4, 5) and specific ethylene trapping procedures (4) before the experiment. Three buds or flowers from each category have eliminated artifacts that might have been caused by labeled were then harvested and incubated with their stems in water for impurities or microorganisms. 8 hr in separate glass containers with 6 ,ll/l of highly purified Recently, several reports have appeared (3, 11, 14, 15) on the 14C2H4 (6) having a specific radioactivity of 119 mCi/mmol. This regulatory involvement ofethylene in the senescence ofthe morn- procedure was repeated with freshly cut buds or flowers at 5:00 ing glory flower, tricolor. Since cut carnations actively PM (8 hr after tagging) and again at 1:00 AM the next morning (16 metabolize ethylene during flower opening and senescence (9), it hr after tagging) to achieve complete 5-day coverage of the was of interest to see if flowers of . tricolor behave similarly. This synchronous development process starting 3 days before and metabolism would be essential in ethylene-sensitive flowers if ending 2 days after full bloom. ethylene metabolism were required for ethylene action as recently Flower stems were cut to a length of I cm for enclosure and suggested (4, 5). The metabolic rate might be expected to reflect fresh wt determined for each group of three buds or flowers. The changes in ethylene responsiveness which have been established glass containers were identical to those previously used for car- for I. tricolor during late bud development (15). nations (9) with the following exceptions: (a) there were three Flowers ofI. tricolor were periodically evaluated for their ability flowers/container; (b) the bottom of the vessel contained 6 ml of to metabolize ethylene. This evaluation covered a 5-day period water; and (c) a 5-ml beaker was placed inside the container to receive 2 ml of 1.5 N NaOH. All incubations took place in the dark at 23 C for 8 hr under mild agitation. ' Contribution No. 2539 from Central Research and Development Com- '4C2H4 concentrations in the container were checked by gas Department, Experimental Station, E. I. du Pont de Nemours and at the pany, Wilmington, Delaware 19898. chromatography 15 min after injection of 14C2H4 and again 2 Present address: Massachusetts Institute of Technology, Cambridge, end of the enclosure period. A flask prepared in the same manner Mass. 02139. with 6 ,ul/l of 14C2H4 but without buds or flowers served as the 896 Plant Physiol. Vol. 61, 1978 14C2H4METABOLISM 897

I. a' 24"|eg I I51:

Tip:m, -48 It-24

FIG. 1. Appearance ofbuds and flowers ofI. tricolor showing progession ofbud development, flower opening, and rapid senescence. Hr prior to full bloom are indicated by minuses; positive numbers indicate hr after full bloom. blank. '4C02 background radioactivity for a typical blank was 180 purged with ethylene-free air (air scrubbed through Purafil, H. E. dpm versus 2,500 dpm during peak activity for three open flowers. Burroughs and Associates, Inc., Chamblee, Ga.) at the beginning High humidity was maintained in each container by the 6 ml of of the 8-hr enclosure periods. Ethylene concentrations were deter- water in the bottom of the container. This was essential for mined by gas chromatography at the end of the 8-hr enclosure producing a low, stable background level of radioactivity in the period on an activated alumina column (6). For each enclosure blank flasks. period fresh buds or flowers were harvested as before for "4C2H4 14CO2 and 14C-Tissue. After incubation, 1.5 ml of NaOH from exposure. Respiratory CO2 production was determined in experi- inside the container was placed into a scintillation vial, brought to ments with and without 14C2H4. This was accomplished by deliv- dryness under N2 to remove dissolved 14C2H4, and after adding 4 ering 0.2 ml of the 1.5 N NaOH from each container to a 32-ml ml of water was counted in Aquasol (New England Nuclear). serum-capped flask, reducing the internal pressure to half an Following 14C2H4 treatment the three buds or opened flowers from atmosphere, and immediately injecting 0.4 ml of 1.5 N H2SO4 to each container were separated into the following parts: (a) petals liberate the trapped CO2. After 15 min on a shaker, the flasks were (removed by cuttingjust above the calyx); (b) "reproductive parts" vented with C02-free air to restore atmospheric pressure and the (pistil, stamens, receptacle, calyx, and the remaining basal few CO2 concentration determined by gas chromatography on a Po- mm of the petals enclosed by the calyx); and (c) the stem. The rapak Q column. stem and reproductive parts from the three flowers in each con- tainer were weighed, ground separately in mortars with I ml of RESULTS water, and transferred to scintillation vials with two additional rinses. The three corresponding petals were weighed, ground in 4 Like carnations (9), flowers of l. tricolor metabolize ethylene ml of water, and 2 ml were transferred to vials. The homogenized and this capacity varied depending on the stage of development. tissues were immediately frozen by placing the vials in dry ice, 14C2H4 oxidation to "4CO2 (Fig. 2, top) was not detected until the subsequently lyophilized to remove dissolved 14C2H4, and then buds were almost fully mature. While oxidation was not apparent rehydrated with I ml ofwater. The tissue was bleached to increase in buds harvested -72 to -16 hr before flowering buds harvested counting efficiency by adding 1 ml of 30% H202 for 1 hr. No loss during the -16 to 0 hr period prior to full bloom clearly possessed of 14C occurred by this addition. After discoloration, 2 ml of water this capability. During this period the rate of oxidation increased were added and the samples counted in Aquasol. All 14C data steadily reaching a peak of 2500 dpm three flowers-' 8 hr-' were corrected for counting efficiency and sample quenching by during the first enclosure period after full bloom. This activity the internal standard method using [14Cjhexadecane. then rapidly declined, stabilizing only briefly to produce a small C2HM Biosynthesis and Respiration. The pattern of endogenous shoulder on the oxidation peak. ethylene production during flower development was determined Tissue incorporation of 4C2H4 was also observed in some ofthe by tagging, harvesting, and enclosing flowers as described above flower parts of I. tricolor. Although tissue incorporation in the but no 14C2H4 was injected. Instead the vessels were carefully stem tissue was insignificant, easily detectable levels occurred in 898 BEYER AND SUNDIN Plant Physiol. Vol. 61, 1978 those data suggesting that ethylene is the natural accelerator of senescence in flowers of I. tricolor. Kende and Baumgartner (14) 2500 14C2H-o14C02 first studied the possible involvement of ethylene in the regulation of aging ofthese flowers. Evidence suggesting such a link included: i 2000 (a) a sharp increase in the rate of natural ethylene production - coincident with petal senescence; (b) the ability of added ethylene o ffi 1500 F 0S to induce ethylene production and senescence prematurely and (c) the ability of the ethylene antagonist, C02, and an ethylene o 1000 ,n trap to delay senescence. Additional support was obtained in CY 500 subsequent work with an excised corolla rib segment system (15). Using this system benzyladenine (15), the ethoxy analog of rhi- zobitoxine and Co2, or inhibited 0 (15), (3) markedly delayed ethylene production and spontaneous rolling up characteristic of 1000 A senescence in the intact flower. Inhibition with i14Y4_-oTW- 4C PEALS °ST - early rhizobitoxine - oz REPFUCM MKRS- suggested methionine as the ethylene precursor and this was 5001 verified by more definitive work with '4C-labeled methionine (12, 13). As with C02, the potent inhibitor of ethylene action Ag+ (7, e CCln 0 8) also delayed senescence as evidenced by an inhibition in the rolling up of the rib segments (3). 10 These and other data (15) indicated that the ethylene-producing C02 C2H4 system develops as an integral part of the aging process in Ipomoea 8 - PRODUCTION / \PRODUCTION and serves to accelerate aging but may not be the initial causative 6 agent. This accelerating effect of ethylene is apparently related to 1-* the autocatalytic nature of the ethylene biosynthetic system where 4 ethylene can induce ethylene biosynthesis. According to the model 2 proposed by Kende and Baumgartner (14) this autocatalysis occurs o because ethylene increases the permeability of the tonoplast facil- - 0 itating the mixing of ethylene precursor, presumably stored in the -72 -48 -24 0 +24 +48 vacuole, with the ethylene biosynthetic system located in the FULL BLOOM cytoplasm. Evidence (11) for an effect of ethylene on the perme- FLOWER STAGE RELATIVE TO FULL BLOOM (hr) ability of the tonoplast was obtained by the technique of com- FIG. 2. Top: oxidation of '4C2H4 to '4CO2 during bud development, partmental analysis with MRb+ and 3Cl- as markers. Whether or flowering, and senescence in morning glory expressed as radioactivity not this increase in membrane permeability is causally related to recovered in the NaOH tap during 15 separate 8-hr enclosure periods of an ethylene-induced loss in membrane phospholipid is unclear three buds or flowers with 6 1il/I of '4C2H4. Middle: radioactivity incor- (3). porated into various parts of the same three flowers. Bottom: CO2 produc- Because ethylene clearly influences natural flower aging in I. tion by '4C2H4-treated buds or flowers and ethylene production by similar, tricolor a study was undertaken to explore the ethylene metabolic but untreated, buds or flowers. Three separate experiments were conducted capabilities of such flowers during bud and flower development. with very similar results. At full bloom total fresh wt and dry wt for the Flowers of Ipomoea metabolize ethylene (Fig. 2, top and middle) three flowers were 1.750 and 0.176 g, respectively. and as with other tissues examined (4, 5, 9) this metabolic system results in the oxidation ofethylene to CO2 and in the incorporation the other parts of the buds and flowers (Fig. 2, middle). In the of ethylene into tissue metabolites. Moreover, these metabolites, reproductive parts the rate of 14C2H4 incorporation was essentially like those found in pea (10) and carnation (9) tissues, are also constant over the entire sampling period with reproductive parts water-soluble (data not shown). from three buds or flowers yielding 100 to 200 dpm. The petals As in other studies (4, 9) the capacity of the tissue to carry out incorporated 14C2H4 at a slightly higher rate with the rate remain- ethylene metabolism was not always the same. In the case of ing fairly constant until just about 16 hr before flower opening. ethylene oxidation, no activity was detected until about 16 hr After this time two peaks of activity were observed. The first before flower opening. At this time the buds appeared to develop occurred simultaneously with the 14C2H4 oxidation peak (Fig. 2, this capability and it rapidly increased with time reaching a peak middle) while a slightly smaller second peak occurred about 36 hr ust after flower opening. Taking into account differences in after full bloom and corresponded to the shoulder on the ethylene 4C2H4 specific radioactivities this peak activity in Ipomoea was oxidation peak. about 7% of that observed during peak activity in cut carnations As previously reported (14) natural ethylene production was (9). In carnations (9) the peak in ethylene oxidation was more difficult to detect until the time of flowering (Fig. 2, bottom). At associated with the later stages of senescence and developmentally this time ethylene production began to increase rapidly, reaching corresponded more with the second peak or shoulder of the oxidation in This shoulder a peak of 9 q g fresh wt'- hr-' several hr after visible senescence. ethylene peak Ipomoea (Fig. 2, top). This production, like oxidation, then rapidly declined. Surpris- could be related to petal drop which occurs during this period and ingly, CO2 evolution increased in the youngest buds during the which appears to be driven by turgor changes in the calyx causing -72 to -48 hr period prior to full bloom. However, after reaching the sepals to turn inward. oxidation and of the buds devel- a peak of 9 cc of CO2 10 g fresh wt-'- hr-' during the -56 to -48 Ethylene ethylene sensitivity hr sampling period the rate steadily declined reaching the low of oped in parallel. As reported by Kende and Hanson (15) segments 2 before 2.0 cc of CO2 10 g fresh wt-'. hr-' during the most advanced +40 taken from the corolla of buds days flowering (day -2) to +48 sampling period. do not roll up in response to added ethylene. However, segments taken from buds harvested the day before flower opening (day If oxidation to DISCUSSION -1) do respond to ethylene by rolling up. ethylene CO2 were a necessary aspect of ethylene action as suggested (4, 5) The visual, biochemical, and ultrastructural changes accompa- this inability to oxidize ethylene at day-2 (Fig. 2, top) could nying senescence in flowers of I. tricolor have been adequately account for the lack in ethylene sensitivity. The acquired sensitiv- described (2, 14-16). More pertinent to the data reported here are ity at day- could then be explained by the simultaneous appear- Plant Physiol. Vol. 61, 1978 14C2H4 METABOLISM 899 ance of the ethylene oxidation system. visualize how such a low level of removal would be significant. In With regard to tissue incorporation of ethylene the order of contrast, this amount of turnover could be significant from the activity was petals > reproductive parts > stems. This was in standpoint of ethylene action. contrast to cut carnation flowers (9) where the reproductive tissues Acknowledgments-Sincere appreciation is extended to A. Burr for her skillful technical assist- were exceptionally active as compared to the other flower parts. ance and to D. Bacon for her help in the preparation of the manuscript. However, in both Ipomoea (Fig. 2, middle) and carnation (9) peaks in ethylene oxidation occurred concomitantly with peaks in LITERATURE CITED tissue incorporation. 1. ABELEs FB 1973 Ethylene in Plant Biology. Academic Press, New York Although ethylene metabolism has been examined critically in 2. BAUMGARTNER B, H KENDE, P. MATILE 1975 Ribonuclease in senescing morning glory. Plant only a limited number of plant tissues (4, 5, 9, 10) it appears that Physiol 55: 734-737 this metabolism is an integral part ofthe plants' ethylene biochem- 3. BEUTELMANN P, H KENDE 1977 Membrane lipids in senescing flower tissue of Ipomoea istry. Like ethylene sensitivity and biosynthesis, the ability ofplant tricolor. Plant Physiol 59: 888-893. 4. BEYER EM JR 1975 "C-Ethylene incorporation and metabolism in pea seedlings. Nature 255: tissues to metabolize ethylene changes during development. Surely 144-147 this complex and delicate metabolic system (4, 5) serves some 5. BEYER EM JR 1975 4C21H4: its incorporation and metabolism by pea seedlings under aseptic functional role. It does not seem to serve as a detoxification or conditions. Plant Physiol 56: 273-278 degradative system since based solely on the amount of ethylene 6. BEYER EM JR 1975 14C2H4: its purification for biological studies. Plant Physiol 55: 845-848 7. BEYER EM JR 1976 A potent inhibitor of ethylene action in plants. Plant Physiol 58: 268-271 removed it would appear totally ineffective (4, 5). For example, in 8. BEYER EM JR 1976 Silver ion: a potent antiethylene agent in cucumber and tomato. Hort- the experiments with I. tricolor the peak of natural ethylene Science II: 195-1% production was I - g fresh wt-' hr-f and occurred during the 8- 9. BEYER EM JR 1977 "C2H14: its incorporation and oxidation to "CO2 by cut carnations. Plant to 16-hr enclosure perod after full bloom. During this period Physiol 60: 203-206 10. GIAQUINTA R, EM BEYER JR 1977 14C2H4: distribution of "4C-labeled tissue metabolites in pea ethylene was being metabolized at a rate (oxidation plus tissue seedlings. Plant Cel Physiol 18: 141-148 incorporation) ofapproximately 2,300 dpm three flowers-' 8 hr-' 11. HANSON AD, H KENDE 1975 Ethylene-enhanced ion and sucrose effiux in morning glory at an exposure level of 6 ,ul/l of '4C2H4. This rate of metabolism flower tissue. Plant Physiol 55: 663-669 clearly overestimates the rate of natural metabolism since, based 12. HANSON AD, H KENDE 1976 Biosynthesis of wound ethylene in morning-glory flower tissue. Plant Physiol 57: 538-541 on values for other similar tissues (1), the internal concentration 13. HANSON AD, H KENDE 1976 Methionine metabolism and ethylene biosynthesis in senescent would clearly be less than 0.7 tlI/I per qI1 g fresh wt-1 hr-1 (6 flower tissue of morning glory. Plant Physiol 57: 528-537 ,ul -1/9 uI g fresh wt-' hr-F). Nevertheless, even when this high 14. KENDE H, B BAUMGARTNER 1974 Regulation of aging in flowers of Ipomoea tricolor by metabolic rate is used and converted to Ijl g fresh wt-r hr-' based ethylene. Planta 1 16: 279-289 on a 119 15. KENDE H,AD HANSON 1976 Relationship between ethylene evolution and senescence in specific radioactivity of mCi/mmol, a value ofonly 0.017 morning glory flower tissue. Plant Physiol 57: 523-527 ,q g fresh wt-' hr-' is obtained. At this rate less than 0.2% of the 16. MATILE P, F WINKENBACH 1971 Function of lysosomes and lysosomal enzymes in the ethylene being produced would be metabolized. It is difficult to senescing corolla of the morning glory (). J Exp Bot 22: 759-771