Plant Physiol. (1977) 59, 888-893

Membrane Lipids in Senescing Flower Tissue ofIpomoea tricolor'Received for publication October 19, 1976 and in revised form January 18, 1977

PETER BEUTELMANN2 AND HANS KENDEMichigan State University/Energy Research and Development Administration, Plant Research Laboratory,Michigan State University, East Lansing, Michigan 48824

ABSTRACT

Rib segments excised from flower buds ofIpomoea tricolor Cav. passthrough the same phases of senescence as the respective tissue on theintact plant. Such segments were used to correlate changes in lipidcontent with known symptoms of aging, such as rolling up of the ribs andethylene formation. It was found that the level of phospholipid hadalready started to decline before visible signs of senescence were evident.As the segments began to roll up and to produce ethylene, the rate ofphospholipid loss accelerated sharply. During the same period, the levelof fatty acids esterified to phospholipids also fell by 40%. No qualitativechanges in any lipid component could be detected during senescence.Labelng experiments using 33P as marker showed that the rate at whichradioactivity was lost from phospholipids during aging was parallel to therate at which the level of total phospholipids declined. Exogenouslyapplied ethylene accelerated the loss of phospholipid and the senescenceof rib segments while benzyladenine retarded both of these processes.

Ag+, which counteracts the effect of ethylene in many plants, in-hibited rolling up of the rib segments but did not affect either sponta-neous and ethylene-induced ethylene generation, or phospholpid loss.In contrast, Co2+, a purported inhibitor of ethylene synthesis, reducedethylene production, rolling up, and phosphoUpid loss. The inhibition ofethylene-induced rolling up by Co2+ could not be overcome with exoge-nous ethylene, however.Our results indicate that phospholipid loss is a marker for membrane

degradation in rib segments. Changes in membrane integrity and incellular compartmentation may be the basis for ethylene synthesis duringaging of flower tissue.

Flowers of the morning glory Ipomoea tricolor Cav. produceethylene during aging, and their senescence can be advanced bytreatment with exogenous ethylene (8). Treating the flowers for60 min with ethylene induces premature ethylene biosynthesis(8), a phenomenon which has been observed previously withother flower and fruit tissue and which has often been referred toas "autocatalysis." We suggested that the burst in ethyleneproduction, either during natural aging or following ethylenetreatment, was due to loss of cellular compartmentation whichcaused intermixing of previously sequestered components of theethylene-generating system (8, 9). In a first attempt to test thishypothesis, we investigated cellular compartmentation duringaging of segments excised from the ribs of the morning glorycorolla (5). Such rib segments exhibit the same symptoms ofsenescence as the intact tissue. When aging and ethylene produc-

1 This research was supported by a fellowship of the Max KadeFoundation to P. B. and by the United States Energy Research andDevelopment Administration under Contract E(11-1)-1338.

2 Present address: Institut fur Allgemeine Botanik, Universitat Mainz,D-6500 Mainz, Federal Republic of Germany.

tion commence, they start to roll up, just as the ribs of the intactcorolla, and this process can be quantitated to assess the progressof senescence (5). The use of segments also permits measuringthe release of cellular components into the bathing solution.Employing the technique of compartmental analysis with 36Cl-and 86Rb+ (12, 13) as markers and measuring the rates of effluxof sucrose and organic acids, we were able to demonstrate anabrupt change in cellular compartmentation during natural andethylene-induced aging (5).

Since a change in compartmentation most likely involves analteration of membrane properties, we initiated a study on thecomposition of membranes of juvenile and senescing flowertissue. In addition, we also investigated the effects of cobalt andsilver ions on phospholipid metabolism and senescence, sinceboth of these metal ions have been reported to affect ethylene-induced processes (1, 10).

MATERIALS AND METHODS

Plant Material. Seeds of I. tricolor Cav. (cv. Heavenly Blue)were purchased from Agway Inc. (Syracuse, N.Y.), and plantswere grown in an environmental chamber as described previ-ously (5). Rib tissue of the corolla was prepared according toKende and Hanson (9), except that in most experiments two 1-cm pieces were cut from each rib of the corolla. This permitted amore economic utilization of plant material and shortened thetime needed to cut rib segments. The aging response of ribsegments originating from the edge and from the inner part ofthe corolla was the same.The following designations are used to describe the age of the

flower tissue: day 0, day of opening and fading of the flower; day-1, 1 day before opening of the flower.Incubation of Rib Segments. Usually, the rib segments were

excised from flower buds between 3:00 and 6:00 PM on day -1.The segments were placed in Petri dishes containing three layersof Whatman No. 1 filter paper soaked with 5 mm KCl solution.The dishes were kept overnight in the same growth chamber inwhich the plants were grown. Between 7:30 and 8:30 AM, thesegments were blotted on filter paper and transferred into 25-mlErlenmeyer flasks, containing 1 ml 1% (w/v) agar in 5 mm KCl.Usually, 10 rib segments were used per flask. The flasks weresealed with serum vial caps, flushed for 2 min with ethylene-freeair, and placed in a darkroom at 27 C.

Tissue treated this way passes through the same develop-mental stages as the respective tissue on the intact flower. Onday -1, the segments are purple and slightly twisted, followingthe original shape of the bud. At one of the two longitudinalsides of the segment, the corolla tissue is folded over the rib.During overnight incubation the segments turn blue, unfold, andflatten. Around noon, the typical symptoms of senescence be-come evident; the rib segments become purple, they roll up, andbegin to produce ethylene at a sharply increased rate. Treatmentof the segments with various chemicals was usually carried outduring the overnight incubation. The respective substances were

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MEMBRANE LIPIDS AND AGING

dissolved in 5 mm KCI which was used to wet the filter paper.Only AgNO3 was applied in water.For exogenous ethylene treatment, the appropriate amount of

0.1% (v/v) ethylene in air was injected into sealed Erlenmeyerflasks through serum vial caps.Measurement of Rolling Up of Rib Segments. Angular meas-

urements were taken during the rolling up process by aligningthe sides of the segments in the flask alongside a protractor (5).When the segments were flat and unrolled, the angle was scoredas 1800; as rolling up occurred, the angle increased to >3600with the segment ends overlapping.

Ethylene Determination. Ethylene was measured by gas chro-matography as described earlier (9).

Determination of Total Phospholipid. Ten rib segments wereground in a 2-ml tissue grinder in 1 ml of boiling isopropylalcohol. The extract was allowed to cool down, and 1 ml metha-nol and 4 ml of chloroform were added to it. This mixture wasshaken for 90 min in the cold room at 4 C. After filtration andwashing of the residue with a mixture of chloroform-methanol(2:1), the combined organic phase was extracted with 0.2 vol-umes of an aqueous 0.73% (w/v) NaCl solution and dried overanhydrous Na2SO4 (4). Aliquots were evaporated to dryness andsubjected to phosphorus determination according to Rouser etal. (14).

Chromatography of Lipids. For extraction of total lipids, ribsegments (usually 50) were ground in 5 ml boiling isopropylalcohol; to this homogenate 5 ml methanol and 20 ml chloro-form were added, and the mixture was shaken for 90 min at 4 C.After filtration and extraction with 0.2 volumes of 0.73% (w/v)NaCl solution, the organic phase was taken to dryness, redis-solved in 3 ml chloroform, and applied to a silicic acid column.The column was prepared according to Rouser et al. (15) withminor modifications by suspending 1 g of silicic acid in 5 mlchloroform and pouring the slurry into a glass column of 1-cmdiameter. After settling, the silicic acid was washed with 10 ml ofchloroform before application of the sample. Fraction 1, con-taining mainly mono-, di-, and triglycerides, sterols and freefatty acids, was obtained by eluting the column with 13 ml ofchloroform. Fraction 2, eluted with 13 ml acetone, consistedmainly of the glycolipids. A final wash with 13 ml of methanol(fraction 3) eluted the phospholipids. Fraction 3 was concen-trated in vacuo to 1 ml, of which 50 to 100-,ul aliquots were usedfor TLC analysis on silica gel plates (Brinkmann, precoatedplastic sheets, Sil Gel, without binder). The plates were devel-oped in two dimensions with chloroform-methanol-concentratedNH4OH (18:14:3, v/v) in the first direction, and chloroform-acetone-methanol-acetic acid-water (6:8:2:2:1, v/v) in the sec-ond (14). The phospholipids were identified by co-chromatogra-phy with authentic standards and by group-specific reagents suchas molybdenum, ninhydrin, Dragendorf and periodate-Schiffreagents (17). For quantitative determination, the phospholipidspots were localized with iodine vapor, scraped from the plate,and analyzed for phosphorus by the method of Rouser et al.(14).

Fatty Acid Analysis. Free fatty acids were extracted from thefirst column fraction with 1% Na2CO3 solution, following themethod of Draper (2). Methyl esters of free and previouslyesterified fatty acids were produced according to Luddy et al.(11), using a methanolic base reagent, which was prepared bydissolving 1.45 g metallic potassium in 100 ml anhydrous metha-nol. Lipid samples, usually equivalents of 25 to 50 rib segments,were dissolved in 50 ul base reagent and 50 ,ul benzene, andkept at 80 C for 20 min. Thereafter, 150 ,ul water and 150 ,uldiethyl ether were added. After thorough mixing, the phaseswere allowed to separate; the lower aqueous phase was re-moved, and the ether phase was washed with 100 ,ul water anddried over Na2SO4. Aliquots of 1 ,ul were injected into a Perkin-Elmer gas chromatograph model F 11, equipped with a column

('/8 inch x 6 ft) (10% DEGS-PS on 80-100 mesh Supelcoportpurchased from Supelco, Bellefonte, Pa). The chromatographicanalyses were performed isothermically at 195 C.Measurement of Radioactivity. The radioactivity of 33P was

determined with a Packard Tri-Carb scintillation spectrometer,using the following scintillant: 3 liters toluene, 12 g PPO, 0.3 gdimethyl-POPOP (Research Products International Corp., ElkGrove Village, Ill.), and 300 ml Bio-Solv-BBS-3 (BeckmanInstruments, Fullerton, Calif.). The counting efficiency for MPwas 86%. Aliquots of lipid solutions were dried down in scintil-lation vials and redissolved in scintillation fluid. Radioactivity inchromatogram spots was determined by scraping the silica gelfrom the plate and suspending it in scintillation fluid.

RESULTS

Phospholipid Levels during Normal Aging. Rib segmentswere cut on day -1, incubated overnight on 5 mm KCI, andtransferred on day 0 into Erlenmeyer flasks containing agar. Thephospholipid level of these rib segments declined slowly andsteadily from day -1 until about noon of day 0 (Fig. 1). Asrolling up of the rib segments and ethylene formation com-menced, the rate of phospholipid loss increased, reaching at 5PM a rate which was about six times higher than the rate duringthe previous night. In parallel experiments, segments excised onday -1 were incubated overnight on 5 mm KCl solution contain-ing KH233PO4 (2 ,uCi/ml). In order to chase the radioactivephosphate, the ribs were floated on the morning of day 0 for 2 hron 20 mm K-phosphate buffer (pH 6) before being placed onagar. The analysis of lipid phosphorus and of radioactivity indi-cated that total phospholipids and radioactivity in phospholipidsdeclined in parallel (Fig. 2).

Phospholipid Ratios. In order to investigate whether or not allphospholipid species were lost at the same rate, phospholipidswere analyzed by two-dimensional TLC. The analysis of tissueprior to senescence (day 0, 9:00 AM) and at an advanced stage ofsenescence (day 0, 9:00 PM) showed no striking change in theratio of the major components PC,3 PE, and PI. The minorcomponents, PA and PG, were present in such low concentra-tions that no changes could be detected with adequate accuracyusing our methods. In tissue which was treated with 3P andchased as described above, we found that PC consistently re-tained relatively more radioactivity during aging than all otherphospholipids (Fig. 3).

Analysis of Fatty Acids. The esterified fatty acids were con-verted to methyl esters from fraction 3 of the silicic acid columnand were analyzed by gas chromatography. During senescencethere was a 40% decrease in the level of fatty acids esterified tophospholipids which corresponded to the loss of phospholipidsduring the same period; no significant changes were found in theratio of the fatty acid components (Table I). The level andcomposition of the free fatty acids which were extracted fromfraction 1 of the silicic acid column remained unchanged duringsenescence.Treatment with Exogenous Ethylene. Rib segments, cut on

day -1 and incubated overnight on 5 mm KCl, were treated inthe morning of day 0 with 10 ,ul/l ethylene for 90 min. After this,the Erlenmeyer flasks were opened, flushed thoroughly withethylene-free air, and recapped. The treatment with ethyleneaccelerated rolling up and endogenous ethylene production asdescribed earlier (9). The rate of phospholipid loss was alsosignificantly increased, but the effect of the gas on this processwas less pronounced than that on rolling up and endogenousethylene production (Fig. 4).

3 Abbreviations: PA: phosphatidic acid; PC: phosphatidylcholine;PE: phosphatidylethanolamine; PG: phosphatidylglycerol; PI: phospha-tidylinositol.

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890

8

I.-zw

0

co 6m

0

o.4

-i

2

BEUTELMANN AND KENDE

1600 2000 2400 400 800 IZ.00 16;00 2000

TIME OF DAY

FIG. 1. Phospholipid content, ethylene production, and rolling up ofrib segments cut on day -1, incubated overnight on 5 mM KCI, andtransferred on day 0 (8:00 AM) onto agar in Erlenmeyer flasks. Lipidphosphorus (@ *); ethylene production (A--- -A); rolling up

(0- -*- .0)

coX

0

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LO=a o10c ,

E

0.

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0

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TIME OF DAY

FIG. 2. Pulse-chase experiment with `3P. Segments were cut on day-1, incubated overnight in KH233PO4 (2 ,pCi/ml) and floated on 20 mMphosphate buffer on day 0 (7:00-9:00 AM), and then transferred to agar.Lipid phosphorus (@ -); radioactivity in phospholipids (O- - -0);ethylene production (A A); rolling up (0- - --O).

Incubation on Benzyladenine. Rib segments cut on day -1were incubated on 5 ,uM BA in 5 mm KCI overnight andtransferred in the morning of day 0 to agar in Erlenmeyer flasks.As observed earlier (9), treatment of the tissue with the cytoki-nin caused a delay in both rolling up and ethylene production(Fig. 5). Treatment with BA also reduced the loss of phospholip-ids. The effect of BA on phospholipid loss was already evident inthe morning of day 0, when rolling up and the rise in ethyleneproduction had not yet started. Similar results were obtained inthree experiments.

Incubation on AgNO3. Overnight incubation on 10 ,UM

Plant Physiol. Vol. 59, 1977

AgNO3 solution prevented rolling up of the rib segments almostcompletely. Even incubation on 5 ,iM AgNO3 inhibited rollingup by 80% (Fig. 6). However, phospholipid loss and ethylene

300 production of silver-treated and control samples were almostidentical (Fig. 6), and there was no difference in the time courseof color change from blue to purple (data not shown). In the

280 morning of day 0, silver-treated rib segments were always rolledj slightly backward, i.e. in the opposite direction of the normal

260 > rolling up.n In experiments where rib segments were incubated on

240 Z AgNO3, it was interesting to note that Ag+ inhibited rolling up:i without affecting ethylene biogenesis (Fig. 6). For this reason,0

we investigated the effect of silver ions on another ethylene220 response, namely ethylene-induced ethylene synthesis. As in

previous experiments, rolling up was severely inhibited in200

C

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33 incorporated

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FIG. 3. Phospholipid composition and distribution of 33P radioactiv-ity in phospholipids. Rib segments were cut on day -1, incubated on

KH233PO4 (2 ,Ci/ml) overnight, floated on 20 mm phosphate buffer (pH6) on day 0 (7:00-9:00 AM), and then transferred to agar. Determina-tions were made at 9:00 AM and at 9:00 PM on day 0.

Table I. Fatty Acid Comrposition of Phospholipids in Rib Segmentsprior to Senescence and at an Advanced Stage of Senescence

Fatty Acid % of major esterified fatty acid inphospholipids1

day -1, 6:00 PM day 0, 9:00 PM

Palmitic acid (16:0) 30.3 26.7Stearic acid (18:0) 2;9 4.5Oleic acid (18:1) 1.5 1.6Linoleic acid (18:2) 28.6 28.3Linolenic acid (18:3) 36.7 38.9

'Average values of three experiments which gave very similarresults.

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c)J i fII I-Ethylene

A/ I/- 6

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Plant Physiol. Vol. 59, 1977 MEMBRANE LIPIDS AND AGING

12:00 16:00Time of Day

20:00

500

0 oE E

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400 ID - 'o o*Xa 0P. OD,

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FIG. 4. Effect of ethylene treatment on phospholipid content, rollingup, and ethylene production. Rib segments were cut on day -1 andmeasurements were taken on day 0. The ethylene treatment lasted from9:00 to 10:30 Am on day 0. Lipid phosphorus (0-O); ethyleneproduction (A--- -A); rolling up 0- -L-I. 0 A 0: control; * A U:ethylene treatment.

-P(BA)

ROLLING (C)....

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, #to-./

o~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~l

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CP

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Time of Day

FIG. 6. Effect of AgNO3 on phospholipid content, ethylene produc-tion, and rolling up. Rib segments were cut on day -1, incubated on 5jLM AgNO3 overnight, and transferred to agar on day 0 (8:00 AM). Lipidphosphorus (0 O); ethylene production (A--- -A); rolling up(0- - - -0). 0 A 0: control; 0 A *: AgNO3 treatment.

O000

I-0

CPc

c-400

0

101ob.SC

cr300 0

200

ISO

Time of Day

FIG. 5. Effect of BA on phospholipid content, ethylene production,and rolling up. Segments were cut on day -1, incubated on 5 ,UM BAovernight, and transferred to agar on day 0 (8:00 AM). Lipid phosphorus(0-0); ethylene production (A- - -A); rolling up (L- - - --O0 A O control; 0 A *: BA treatment.

AgNO3-treated segments, and exogenously applied ethylene didnot overcome this inhibition. Neither the rate nor the timecourse of ethylene-induced ethylene formation was affected bypreincubation on AgNO3 (Fig. 7).

Incubation on CoC12. Overnight incubation of rib segments on500 AM CoC12 reduced the total amount of ethylene producedduring day 0 and also rolling up of segments by over 80% (Fig.8). As observed in earlier experiments with AgNO3, the ribsegments showed first "reversed rolling" (a< 1800) which turnedto values above 1800 during the day. The rate of phospholipidloss was markedly reduced by Co2+, but a significant difference

0-

0E0co.0

0L..

0C0C

ew

C

:00

Time of DayFIG. 7. Effect of AgNO3 on ethylene-induced ethylene synthesis and

rolling up. Rib segments were cut on day -1 and incubated overnight on

10 ELM AgNO3 or 5 mm KCI. They were transferred to agar on day 0,and either exposed for 90 min (9:00-10:30 AM) to 10 p1/I ethylene or toethylene-free air. After this period, all flasks were vented and sealedagain. Top: rolling up; bottom: ethylene production. : no ethylenetreatment; - - -: ethylene treatment; OI A: overnight incubation on KCI;

A: overnight incubation on AgNO3.

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BEUTELMANN AND KENDE

8

7

a

t -iEE

Cl)U)

o

cgir -

J-2%.

4

P(co) /

ROLLING (C) /

/.I

/.

/ ROLLING (Co )ff ~~~~I *- .

__

, 'lETHYL ENE

18:00 8goO Oo00 OO 2000

Time of Day

FIG. 8. Effect of CoC12 on phospholipid content, ethylene produc-tion, and rolling up. Rib segments were cut on day -1, incubated on 500,UM CoC12 overnight, and transferred to agar on day 0 (8:00 AM). Lipidphosphorus (0 O); ethylene production (A--- -A); rolling up

(0-.-.- -0); 0 A 0: control; * A *: CoC12 treatment.

between the Co2+-treated and control tissue could not be ob-served before 5:00 PM on day 0 (Fig. 8).

In a separate set of experiments, we investigated whetherethylene could overcome the inhibitory effect of Co2+ on rollingup. The rolling up of rib segments which had been incubatedovernight on CoC12 (500 ,UM) and which were transferred toErlenmeyer flasks without ethylene at 8:30 AM on day 0 reached2200 by 8:30 PM of the same day. The rolling up Co2+-treatedsegments which were transferred to Erlenmeyer flasks with 101.lIl ethylene present during the entire 12-hr incubation periodreached a value of 2100. In the same experiment, ethyleneadvanced rolling up of rib segments without Co2+ pretreatmentas compared to control segments kept in air. In either case,

rolling up was completed by 8:30 PM (a>360'). This evidenceindicates that the inhibition of rolling up by Co2+ is not reversibleby treatment with ethylene.

DISCUSSION

The level of phospholipids in excised flower tissue of . tricolorwas found to decrease at a slow rate before any signs of senes-cence were evident. The rate of phospholipid loss acceleratedconsiderably as rolling up of rib segments and ethylene produc-tion commenced. Pulse-chase experiments using 33P as a markershowed that the loss of radioactivity from phospholipid paral-leled the loss of total phospholipid and confirmed that the levelsof all major phospholipid species declined. It is not possible todetermine from our experiments whether the decline in phos-pholipid levels is a result of reduced synthesis, increased break-down, or both. Ethylene applied to rib segments on day 0consistently accelerated phospholipid loss in comparison to con-trol tissue without ethylene treatment, but this effect was quanti-tatively less striking than the effect on other parameters of aging,such as rolling up, ethylene evolution, and loss of cellular com-partmentation. Even more importantly, ethylene-stimulated lossof phospholipids often lagged behind other ethylene-inducedsymptoms of aging. This may indicate that the reduction inphospholipid levels is not one of the primary ethylene-controlledreactions leading to increased membrane permeability. The ana-tomical characteristics of the rib tissue and limitations of ouranalytical methods prevent unequivocal separation of cause andeffect, i.e. whether applied ethylene increases membrane perme-ability by enhancing phospholipid loss or whether phospholipid

/ETHYLENE(C)

loss is a consequence of other ethylene-controlled catabolicprocesses which affect membrane integrity. Rib segments arecomposed of several cell types, and the earliest ethylene re-sponse appears to be confined to relatively few cells in the tworidges of each rib. The rolling up of the ribs is driven by thosetwo ridges, at least in part through loss of turgor on the adaxialside and possibly also through turgor-mediated growth on theabaxial side. It is doubtful that we could have observed phospho-lipid loss in a small fraction of the total population of rib cellsduring the earliest response to applied ethylene. In the course ofnatural aging, as opposed to premature ethylene-induced aging,there may be a far greater synchrony in the metabolic state of allcells of the rib tissue which may explain the close temporalcoincidence of phospholipid loss and other symptoms of floweraging.The level of fatty acids esterified to phospholipids fell in

parallel with the decrease in the level of phospholipids. The ratioof the major fatty acid components of phospholipids stayedconstarqt during senescence. There was no increase in the level ofunsaturated fatty acids, such as might be expected in senescingtissue (16). The levels of free fatty acids, glycolipids, and ste-roids remained unchanged during aging of the rib segments(unpublished data).

In quantitative terms, our results are in agreement with thoseof other investigators (2, 3, 18) who also found decreasing levelsof membrane lipids in senescing tissue. However, in contrast tothese authors, we did not find significant qualitative changes inlipid composition during aging of flower tissue. This is mostlikely because earlier experiments (2, 3, 18) were performedwith yellowing leaf tissue where degradation of chloroplastmembranes with distinct lipid composition predominated duringearly phases of senescence. The effect of ethylene on lipidmetabolism was investigated by Irvine and Osborne (7) whofound a decreased incorporation of '4C-glycerol into neutrallipids and phospholipids of pea stem sections following ethylenetreatment. They concluded that ethylene inhibited biosynthesisof all lipids which contained glycerol. Since possible changes inthe pool sizes of glycerol were not taken into account, theseexperiments cannot be interpreted unequivocally.The results of our experiments with AgNO3 were surprising

inasmuch as silver ions inhibited one ethylene response in ribsegments (rolling up) while not affecting others (ethylene syn-thesis and phospholipid loss). Beyer (1), who first found thatsilver ions abolished the effect of ethylene in a number of plantsystems, postulated that Ag+ interfered with the action of ethyl-ene at its receptor site. If this were true, one would have toassume the existence of at least two sites of ethylene action inflower tissue of Ipomoea, one of which is Ag+-sensitive and onewhich is not. Alternatively, Ag+ may inhibit rolling up of ribsegments at a step subsequent to the initial interaction of ethyl-cne with its binding site.Lau and Yang (10) found that Co2+ inhibited the production

of ethylene in mung bean hypocotyls and in apple tissue, pre-sumably by blocking the conversion of methionine to ethylene.Our experiments also showed an inhibition by Co2+ of ethylenesynthesis and of other symptoms of aging. However, exoge-nously applied ethylene did not overcome the Co2+-mediatedinhibition of rolling up. In view of this, Co2+ cannot be regardedas a specific inhibitor of ethylene biosynthesis in flower tissue ofIpomoea, even though methionine serves as precursor of ethyl-ene in this system as well (6).Our experiments were undertaken to elucidate further the

chain of events which leads to senescence of the morning gloryflower. According to our hypothesis, the sharp increase in ethyl-ene generation is not the event that triggers senescence, but isthe consequence of earlier metabolic reactions, probably degra-dative in nature, which lead to increased membrane permeabil-ity. The resultant loss in cellular compartmentation (5) isthought to cause mixing of previously sequestered components

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MEMBRANE LIPIDS AND AGING

of the ethylene-generating system, thereby stimulating ethyleneevolution. Once formed, ethylene enhances its own synthesisand accelerates aging by further increasing membrane permea-bility. Support for the view that ethylene generation is a fairlylate event in the senescence process comes from two lines ofevidence presented in this communication. (a) Loss of phospho-lipids consistently preceded ethylene evolution and was alreadydetectable during the night between day -1 and day 0. (b) BA,which delays senescence of rib segments when given during thenight between day -1 and day 0, retarded the loss of phospho-lipid, an effect which was already evident early in the morning ofday 0. We suggest that phospholipid loss is a marker for degra-dative processes which affect membrane integrity and whichultimately lead to a breakdown of cellular compartmentation, asharp increase in ethylene synthesis, and senescence of theflower tissue. While ethylene undoubtedly increases membranepermeability (5), it is still an open question whether it does so bydirectly affecting phospholipid metabolism.

Acknowledgment -We thank R. deZacks for her help in the growth and preparation of plantmaterial.

LITERATURE CITED

1. BEYER EM JR 1976 A potent inhibitor of ethylene action in plants. Plant Physiol 58: 268-271

2. DRAPER SR 1969 Lipid changes in senescing cucumber cotyledons. Phytochemistry 8: 1641-1647

3. FERGUSON CHR, EW SInON 1973 Membrane lipids in senescing green tissues. J Exp Bot79: 307-316

4. FOLCH J, M LEES, GH SLOANE-STANLEY 1957 A simple method for the isolation andpurification of total lipids from animal tissues. J Biol Chem 226: 497-509

5. HANSON AD, H KENDE 1975 Ethylene-enhanced ion and sucrose efflux in morning gloryflower tissue. Plant Physiol 55: 663-669

6. HANSON AD, H KENDE 1976 Methionine metabolism and ethylene biosynthesis in senes-cent flower tissue of morning-glory. Plant Physiol 57: 528-537

7. IRVINE RF, DJ OSBORNE 1973 The effect of ethylene on 11-'4Clglycerol incorporation intophospholipids of etiolated pea stems. Biochem J 136: 1133-1135

8. KENDE H, B BAUMGARTNER 1974 Regulation of aging in flowers of Jpomoea tricolor byethylene. Planta 116: 279-289

9. KENDE H, AD HANSON 1976 Relationship between ethylene evolution and senescence inmorning-glory flower tissue. Plant Physiol 57: 523-527

10. LAU O-L, SF YANG 1976 Inhibition of ethylene production by cobaltous ion. Plant Physiol58: 114-117

11. LUDDY EF, RA BARFoED, SF HERa, B MAGIDMAN 1968 A rapid and quantitative procedurefor the preparation of methyl esters of butteroil and other fats. J Am Oil Chem Soc 45:549-552

12. MAcRoBBIE EAC, J DAINTY 1958 Sodium and potassium distribution and transport in theseaweed Rhodymenia palmata (L.) Grev. Physiol Plant 11: 782-801

13. PrrmAN MG 1963 The determination of the salt relations of the cytoplasmic phase in cells ofbeetroot tissue. Aust J Biol Sci 16: 647-668

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