hypobaric control of ethylene-induced leaf senescence in intact … · described, andevaluated...

Plant Physiol. (1983) 71, 96-1010032-0889/83/7 1/0096/06/$00.50/0

Hypobaric Control of Ethylene-Induced Leaf Senescence in IntactPlants of Phaseolus vulgaris L.1

Received for publication March 1, 1982 and in revised form September 13, 1982

KARL N. NILSEN- AND CLINTON F. HODGESDepartment of Horticulture, Iowa State University, Ames, Iowa 50011

ABSTRACT

A controlled atmospheric-environment system (CAES) designed to sus-tain normal or hypobaric ambient growing conditions was developed,described, and evaluated for its effectiveness as a research tool capable ofcontrolling ethylene-induced leaf senescence in intact plants of Phaseolusvulgaris L.

Senescence was prematurely-induced in primary leaves by treatmentwith 30 parts per million ethephon. Ethephon-derived endogenous ethylenereached peak levels within 6 hours at 26°C. Total endogenous ethylenelevels then temporarily stabilized at approximately 1.75 microliters perliter from 6 to 24 hours. Thereafter, a progressive rise in ethylene resultedfrom leaf tissue metabolism and release. Throughout the study, the endog-enous ethylene content of ethephon-treated leaves was greater than that ofnontreated leaves.

Subjecting ethephon-treated leaves to atmospheres of 200 millibars,with 02 and CO2 compositions set to approximate normal atmosphericpartial pressures, prevented chlorophyll loss. Alternately, subjecting ethe-phon-treated plants to 200 millibars of air only partially prevented chloro-phyll loss. Hypobaric conditions (200 millibars), with 02 and CO2 at normalatmospheric availability, could be delayed until 48 hours after ethephontreatment and still prevent most leaf senescence. In conclusion, hypobaricconditions established and maintained within the CAES prevented eth-ylene-induced senescence (chlorosis) in intact plants, provided 02 and CO2partial pressures were maintained at levels approximating normal ambientavailability.An unexpected increase in endogenous ethylene was detected within

nontreated control leaves 48 hours subsequent to relocation from wintergreenhouse conditions (latitude, 42°00" N) to the CAES operating atnormal ambient pressure. The longer photoperiod and/or higher tempera-ture utilized within the CAES are hypothesized to influence ethylenemetabolism directly and growth-promotive processes (e.g. response thresh-olds) indirectly.

Leaf senescence represents the final phase of normal leaf de-velopment. Implementation of normal monocarpic, polycarpic, orstress-induced leaf senescence requires concerted phytohormonalparticipation within target tissues. The regulatory participation ofethylene during senescence processes is widely recognized (I) andrecent studies have now confirmed ethylene's role in leaf senes-cence (2, 12).

Endogenous ethylene levels within leaves are primarily regu-lated by the combined contributions of biosynthesis and retention(1, 11, 14, 26). The biosynthetic pathway and regulation of eth-

' Supported by projects 1893, 2001, 2038, and 2308 of the Agricultureand Home Economics Experiment Station, Ames, IA 50011.

2 Present address: 618 16th Street, Ames, IA 50010.

ylene synthesis has received recent clarification (17, 26). Theoverall regulation of biosynthetic rate involves temperature, en-zyme and pathway intermediate levels, selective enzyme activityregulation, and membrane site integrity (3, 14, 18). Endogenousethylene retention varies with temperature, C02:02 ratios, stoma-tal apertures, and ambient atmospheric pressure (1, 24).The precise role of ethylene in the senescence-like plant re-

sponses to certain biotic and abiotic stresses is a current concern(23). Further clarification of this role requires a means of assessingand/or manipulating ethylene status within target tissues. Tech-niques which modify endogenous ethylene status make excellentanalytical companions to direct ethylene determination utilizinggas chromatographic analysis. Conveniently, ethylene is a gasunder natural temperatures and pressures. Hypobaric environ-ments established with air reduce endogenous ethylene levelswithin tissues by means of (a) an immediate drop in partialpressures as gaseous-phase materials vent from leaves in theprocess of establishing the new gas pressure equilibrium, (b) areduction in ethylene biosynthesis due to limited O2 availability(1, 17, 26), and (c) reduced ethylene retention in response to thelow pressure effects on ethylene solubility in tissue fluids (10) andincreased diffusive evacuation from tissue air spaces to the externalambient atmosphere as permitted by the less-dense gaseous mediain accordance with Fick's law (8, 9). It is important to account forthe impact of altered atmospheric compositions on ethylene dif-fusivity. Fortunately, hypobaric conditions will easily offset anydecreased diffusivity of ethylene normally encountered, even un-der 100% 02 (8).

Hypobaric systems have been developed for post-harvest studiesand storage applications (9, 1 1); however, similar environmentalcontrol for studies involving intact growing plants requires greatersystem sophistication. Maintenance of photosynthetic and othergrowth-related processes in hypobaric systems requires the pres-ence of adequate light and control of essential atmospheric com-ponent partial pressures at levels which do not impair normalgrowth and development. Accordingly, we have developed aCAES;3 with hypobaric capabilities (22). This report describes thesystem and its utilization in the control of ethylene-induced leafsenescence of primary leaves of intact plants of Phaseolus vulgarisL.

MATERIALS AND METHODS

Plant Materials and Culture. Phaseolus vulgaris L. cv 'TopCrop' was used for all studies. Plants were established from seedin a 2:1 loam-peat soil mix in square plastic pots (7.5 cm/side)and grown under natural light within a greenhouse providingtemperatures of 18 to 24°C and photoperiods of 8.5 to 9.5 h. Oncethe primary leaves appeared, all subsequently developing shoot

3Abbreviations: CAES, controlled atmospheric-environment system;FID, flame ionization detector.

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HYPOBARIC CONTROL OF LEAF SENESCENCE

FIG. 1. Controlled atmospheric-environment system (CAES). Structural components: (1), control unit stand (Edsal model MB 2442-lW827); (2).CAES chamber(s) (Nalgene 22.24-L transparent polycarbonate vacuum chamber No. 5305-1212) to be placed inside plant growth chamber. Electricalcomponents: (3), power cord (120 v, 30 amp); (4), oil-seal pump switch; (5), water-seal pump switch; (6), humidifier switch. Flow Control components:(7), gas cylinders (N2, 02, 30,000 ,l/l CO2 in 02, C2H4, etc.); (8), two-stage pressure regulators with flowmeters (Matheson 8BF series); (9), gas-proportioning flowmeters (Matheson 7600 series, 150-mm flow tubes, numbers 600 and 602); (10), hypobaric regulator (Matheson model 49 regulator);(11), hypobaric by-pass metering valve (Nupro metering valve B-4LA); (12), flow-metering valve (Nupro metering B-4LA); (13), pump-flow selectorvalve (Whitey 3-way ball valve B43XS4); (14), bleed-metering valve (Nupro metering valve B-4LA) to alleviate cavitation in water-seal pump anddilute gas-mixture entering oil-seal pump; (15), bleed-selector valve (Whitey 3-way ball valve B-43XS4); (16), bleed-flowmeter (Matheson 7231 H, 200Etube); (17), oil-seal pump (Kinney KC-3); (18), water-seal pump (Atlanta Fluidics model A10). Humidification components: (19), humidifier flask (3-LPyrex vacuum flask) to be placed together with the CAES chamber inside plant growth chamber; (20), humidifier controller (YSI Thermistemp modelG3RC); (21), controller temperature sensor (YSI probe No. 651 x 25); (22), thermometer (-10 to 60°C); (23), humidifier heating tape (Cole-ParmerInstrument Co., No. 3112-20, 5 x 61 cm, 115 v, 190 w); (24), humidifier access port.

apices were removed. Plants were used when they were 5 to 6weeks from planting date. Primary leaves had just reached fullexpansion at this stage.

Controlled Atmospheric-Environment System. Experimentsconducted in these and subsequent studies necessitated develop-ment of a CAES with hypobaric capability, which, when utilizedin conjunction with a plant growth chamber, enabled overallenvironmental control (Fig. 1) (22). The CAES was designed toaccommodate placement of intact plants within the transparentCAES chambers (Fig. 1, component 2) which were located withina plant growth chamber and thus remote from the CAES controlunit. Temperature, photoperiod, light quality and levels withinthe CAES chambers were established and maintained utilizing thecontrols of the plant growth chamber.The CAES controls atmospheric pressure, composition, flow

rate, and humidity within the chambers (Fig. 2). The CAES isdesigned to operate from ambient atmospheric pressure (approx-imately 1,000 mbar) to 200 to 250 mbar while maintaining theapproximate normal ambient partial pressures of 02, C0, andsaturated H20 vapor (Table I). These hypobaric conditions areachieved by CAES controls that place on-line either a conven-tional laboratory oil-seal or small industrial water-seal pump. Thewater-seal pump provides maximum safety from fire and/or ex-

plosion in near pure 02 atmospheres and is maintenance-free evenwhen high humidities are maintained. The CAES can be utilizedwithout the pumps for the treatment of plants (at normal ambientpressure) with a wide range of gas compositions and flow rates.The ethylene content of all gas sources was analyzed to detect andthus prevent contamination in the final mixtures.

Ethylene Analysis. Endogenous ethylene was determined byextraction of the internal gases of primary leaves utilizing themethod described by Beyer and Morgan (5). The extraction systemwas modified to accommodate 12 tube-shaped collection flasksplaced within the evacuation chamber (25 cm desiccator). Thismodification enabled the simultaneous extraction of internal gasesfrom 12 pairs of primary leaves. Primary leaves detached forendogenous ethylene analysis were immediately immersed in sat-urated (NH4)2SO4. Internal leaf gases were released and collectedat 120 mbar maintained for 3 min. A 1.0-cc sample of the releasedleaf gases was injected directly into a Varian 3700 GC equippedwith a 2-m activated alumina column (60/80 mesh) and FID. TheGC was operated at injector and detector temperatures of 250°C,an oven temperature of I 10°C, and a carrier gas (He) flow rate of30 cc min-'. The signal of the FID was fed to a Cary model 401electrometer coupled to a Spectra-Physics 4100 computing inte-grator. Subsequent to calibration, sample data were plotted and

97

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NILSEN AND HODGES

Table 1. Controlled Atmosphere Pressure-Composition Settings and the Modification of Partial Pressures byHypobaric Conditions and Water Vapor at 260C

Pressure Partial Pressures"Composition Pressure Dry Gas RHi

Settings Composition N., 02 CO2 Water vapor"mbar % mbar

I 1,000d Air 0 781 210 0.31 02 1,000d Air 95-100 755 203 0.30 333 200 Air 0 156 42 0.06 04 200 Air 95-100 130 35 0.05 335 200 02 + C02 0 0 200 0.30 06 200 02 + C02 95-100 0 167 0.25 33

Determined psychrometrically from wet-bulb and dry-bulb readings."Calculated from calibrated flowmeter-delivered mixing ratios.'Published values for 26°C and respective pressures (I 1, 19)."Ambient approximation.

seaant waste ambent air

FIG. 2. Pressure and flow control schematic of the controlled atmos-pheric-environment system (CAES).

quantified by the computing integrator and expressed as nl I'ethylene.

Chi Analysis. Chl content of primary leaves was determined bya modification of the method described by Knudson et al. (15).Leaves were detached, cut into small pieces, and dried by lyoph-ilization (48 h). After determination of dry weight, individualleaves were placed in 30 ml of 95% ethanol and extracted in thedark for 24 h. The 24-h ethanol extractions were repeated threetimes, combined, and brought to 100 ml. Chl absorbancies weremeasured spectrophotometrically at 665 and 649 nm and appro-priate calculations made to determine Chl a, Chl b, and total Chl(15). Chl concentrations were expressed as ,ug Chl mg-' dry leafwt.

Preliminary Assessment of Endogenous Ethylene and EthephonRates. Prior to initiating specific treatments, it was necessary toassess the effect of environmental relocation on the endogenousethylene content of primary leaves of plants placed within thechambers of the CAES. Nine replicate plants were placed insidethe chambers and held for 48 h at 1,000 mbar with continuous airexchange at a flow rate of 186 cc min-' (Table 1). Plants weresubjected to all combinations of 8- and 12-h photoperiods withtemperatures of 20 and 26°C within the CAES chambers and then

analyzed for endogenous ethylene approximately at mid-photo-period.

Preliminary studies were initiated to determine the appropriatecombination of ethephon and temperature that would inducemeasurable chlorosis in primary leaves within 72 h with minimalabscission. The primary leaves of six replicate plants were brushedwith solutions of ethephon (Ethrel; Amchem Products, Inc., Am-bler, PA) at concentrations of 20, 30, 50, 70, 90, and 100 ppm(0.01% Tween 80) and placed in the CAES chambers at 1,000mbar with an air flow rate of 186 cc min-'. Leaves treated witheach ethephon concentration were evaluated at 20 and 26°Cwithin the CAES chambers under a 12-h photoperiod.

Treatments. Primary leaves to be evaluated for endogenousethylene and for Chl content were treated with 30 ppm ethephon.Ethephon was applied to primary leaves in aqueous solution(0.01% Tween 80) with a soft 2.54-cm paint brush. The solutionwas applied to the adaxial surface of the leaves three times toinsure complete wetting and runoff. Plants were immediatelyplaced in a CAES chamber (Fig. 1, component 2; Fig. 2) at highhumidity to prevent drying of the ethephon solution. Primaryleaves of nontreated control plants were brushed with a 0.01%Tween 80 solution and placed in a second CAES chamber. Inthese and all subsequent studies, plants were held within theCAES chambers for a period of 72 h, at 26°C, with a 12-hphotoperiod and a light level of 145 (± 15) /AE m-2 s-' supplied bycool-white fluorescent lamps.

Ethephon-treated and nontreated plants were subjected to threesets of posttreatment conditions during the 72-h period within theCAES chambers. (a) Treated and nontreated plants were exposedto 1,000 mbar of air (Table I, setting 2) at a flow rate of 186 ccmin-' to determine changes in endogenous ethylene and Chlcontent of primary leaves. Three replicate samples of six pairs oftreated and nontreated primary leaves were each removed fromthe CAES chambers at 6, 12, 24, 48, and 72 h and analyzed forendogenous ethylene. A second series of three replicate samplesof 12 treated and nontreated primary leaves each were removedfrom the CAES chambers at 24, 48, and 72 h and analyzed forChl content. The endogenous ethylene and Chl content ofprimaryleaves of greenhouse plants was determined prior to treatment. (b)Treated and nontreated plants were subjected to 1,000 and 200mbar of air (Table I, settings 2 and 4) at 186 cc min-', and to 200mbar of a mixture of 02 and CO2 (Table 1, setting 6) at 186 ccmin-' adjusted to approximate normal ambient partial pressure of02 and CO2. These conditions were established to determine theeffect of varied O2 and CO2 (partial pressures) under hypobaricconditions (200 mbar) on the total Chl content of primary leaves.Three replicate samples of 12 treated and nontreated primaryleaves were analyzed for Chl content at each pressure-compositionsetting. (c) Treated plants were subjected to an atmospheric pres-

98 Plant Physiol. Vol. 71, 1983




sure-composition exposure sequence consisting of an exposureperiod (h) at 1,000 mbar of air (186 cc min-'), followed by anexposure period at 200 mbar of a mixture of 02 and CO2 (TableI, setting 6) at 186 cc h-'. Specific exposure period sequences of1,000 mbar air followed by 200 mbar 02 + CO2 were 0 + 72, 6+ 66, 12 + 60, 24 + 48, and 72 + 0 (h + h). These conditions wereestablished to determine the capability ofhypobaric pressures (200mbar) of controlled composition (02 + C02) to limit or reverseethephon-induced Chl loss in primary leaves under progressivelylonger initial exposure periods (within 72 h) of the ethephon-treated leaves to 1,000 mbar. Three replicated samples of 12primary leaves for each exposure time combination were analyzedfor Chl content.

RESULTS

Preliminary Endogenous Leaf Ethylene Assessment. Prelimi-nary studies established that temperature and photoperiod sub-stantially influence endogenous ethylene levels within primaryleaves of plants placed in the CAES chambers. The primary leavesof plants maintained at 20°C had relatively low levels of endog-enous ethylene when maintained at either 8- or 12-h photoperiods(Table II). Whereas primary leaves of plants subjected to 26°Cand an 8-h photoperiod showed only a modest increase in endog-enous ethylene, the combination of 26°C and a 12-h photoperiodinduced a sharp rise in ethylene (Table II). Gas chromatographicanalysis of CAES exit gases established that flow rates weresufficient to quickly remove tissue-emitted ethylene from theCAES chambers. These results were therefore not being con-founded by an inadvertent build-up of ambient ethylene.The most appropriate ethephon-temperature combination un-

der 12-h photoperiods was 30 ppm ethephon and 26°C. Thiscombination provided ethylene-induced leaf chlorosis within 72 hwithout significant abscission. Concentrations of ethephon below30 ppm at 26°C failed to consistently induce chlorosis; whereas,concentrations above 30 ppm at 26°C induced abscission within48 h. All concentrations of ethephon evaluated in combinationwith 20°C failed to induce chlorosis while higher concentrationsoften induced abscission of green leaves within 72 h.The foregoing studies established that increasing temperature

(to 26°C) and photoperiod (to 12 h) can induce an elevation inthe level of endogenous ethylene in primary leaves, and that thecombination of 30 ppm ethephon under such environmental con-ditions results in ethylene levels which induce chlorosis of primaryleaves within 72 h with minimal abscission. These parameterswere employed in all subsequent treatments to determine theeffectiveness of hypobaric atmospheric environments at limitingChl loss due to naturally induced endogenous ethylene and tosimulated stress ethylene (ethephon-released).

Table 11. Influence of Temperature and Photoperiod on EndogenousEthylene Levels of Primary Leaves of Phaseolus vulgaris L.

All samples were taken after 48 h exposure to 1,000 mbar air (Table 1,setting 2) within CAES chambers (Fig. 1, component 2). Endogenous gassamples were released from leaves immersed in saturated (NH,)2S0, at120 mbar for 3 min. Ethylene analysis was by GC with flame ionizationdetector.

Temperature Photoperiod Endogenous

°C h nil-'20 + 0.5 8 436c'20±0.5 12 531c26±0.5 8 610b26 ± 0.5 12 3230a

Values followed by the same letter are not significantly different.Duncan's Multiple Range Test (P = 0.05).

40

35

3.0

-5

3- 2.0

1.5

1.0

0.5

¢ 15

EX10

51a

0 6 12 24 72Hors

FIG. 3. Endogenous ethylene levels and Chl content of nontreated andtreated (30 ppm ethephon) primary leaves of P. vulgaris maintained at1,000 mbar of air within CAES chambers. Greenhouse plants were ana-lyzed for ethylene and Chl just prior to placing plants in the CAESchambers. Points followed by the same letter are not significantly different.Duncan's Multiple Range Test. P = 0.05.

15

-

32!.

CL2-2c-)

10

5

n1000 mbar Air 200 mbar Air 200 mbar 02+C02

Atmospheric Pressure and CompositonFIG. 4. Chl content of nontreated and treated (30 ppm ethephon)

primary leaves of P. vulgaris maintained in CAES chambers with atmos-pheric pressure-composition exposures as indicated. The Chl a:b ratios arerepresented by the numbers on each bar of the histogram. Mean total Chlcontents followed by the same letter are not significantly different. Dun-can's Multiple Range Test. P = 0.05.

Ethylene and Chi Content of Primary Leaves at Ambient Pres-sure. Both nontreated and ethephon-treated (30 ppm) primaryleaves of plants exposed to 1,000 mbar air (Table 1, setting 2)showed an increase in endogenous ethylene accompanied by aprogressive loss of Chl for the 72-h exposure period (Fig. 3).Initially, the primary leaves of the greenhouse plants (when firstplaced in the CAES chambers) had an endogenous ethylene levelof 0.25,ul I-' and 15.5 ,ug Chl mg-' dry wt. No significant increase

a Greenhouse Plants

o Untreated Plants

* Treated Plants

a

:a~ ~ ~ ~ 1

- LZJ Untreated Plants

CO~Treated Plants aa

Sc

- 1.18 11 1.52 1.91.20 l.9

I A

ulL

I 711

v

99

48



NILSEN AND HODGES

in endogenous ethylene occurred within the first 24 h amongnontreated plants (Fig. 3); between 24 and 48 h, however, endog-enous ethylene showed a sharp, significant increase. The endoge-nous ethylene level of ethephon-treated leaves, however, showeda rapid significant increase within 6 h of treatment (Fig. 3). Theethylene levels of treated leaves remained relatively constantbetween 6 and 24 h and then increased significantly again between24 and 48 h (Fig. 3). Endogenous ethylene levels of ethephon-treated leaves were consistently greater than those of nontreatedleaves.No significant change occurred in the Chl content of nontreated

leaves within the 72-h observation period (Fig. 3). Ethephon-treated leaves showed a significant decrease in Chl at 48 and 72h compared to that of greenhouse leaves (Fig. 3).

Posttreatment Atmosphere Pressure-Composition Exposuresand Chi Content of Primary Leaves. Nontreated and treatedprimary leaves of plants subjected to 1,000 mbar air in the CAESchambers (Table I, setting 2) for 72 h had Chl contents of 13.4and 7.8 ,ug Chl dry leaf weight-', respectively (Fig. 4). Subjectingthe plants to 200 mbar air (Table I, setting 4) resulted in anonsignificant decrease in the Chl content of nontreated leavesand a significant increase in the Chl content of ethephon-treatedleaves (Fig. 4). The Chl content of primary leaves of nontreatedand treated plants subjected to 200 mbar 02 + CO2 (Table I,setting 6) was about equal and significantly greater than the Chlcontent of nontreated and treated leaves at all other atmosphere-pressure combinations (Fig. 4).The Chl a:b ratios were not different among nontreated and

treated leaves of plants subjected to 1,000 mbar air or 200 mbar02 + CO2 (Fig. 4, numbers printed inside bars). Nontreated andtreated leaves subject to 200 mbar air showed a significant increasein Chl a:b ratio.

Posttreatment Atmospheric Pressure-Composition ExposurePeriod Sequences and the Chl Content of Primary Leaves. Thesooner ethephon-treated primary leaves received exposure to 200mbar O, + CO2 (Table I, setting 6) following initial exposure to1,000 mbar air (Table I, setting 2) the greater the 72-h Chl content.Treated leaves maintained 72 h at 1,000 mbar air showed the mostsevere loss of Chl (Fig. 5). Leaves of plants subjected to exposureperiod sequences of 48 + 24, 24 + 48, and 12 + 60 (h at 1,000mbar air + h at 200 mbar 02 + C02) showed less Chl losscompared to leaves subjected to 72 h at 1,000 mbar air (Fig. 5).Leaves subjected to an exposure period sequence of 6 + 66 showedeven less loss of Chl than that of leaves subject to the sequence of12 + 60.

40

-30-

20

~~~~~~~~bd

10

0+72 6+66 12+60 24+48 48+24 72+0Hours 1000 mbar Ai + Hours 200 mbar 02+CO2

FIG. 5. Chl content of primary leaves of P. vulgaris 72 h after treatmentwith 30 ppm ethephon and exposed to atmospheric pressure-compositionsequences as indicated. Points followed by the same letter are not signifi-cantly different. Duncan's Multiple Range Test. P = 0.05.

DISCUSSION

The data from this study reveal a substantial elevation ofendogenous ethylene within leaves of nontreated plants afterrelocation from greenhouse to CAES (Table II). Because of theplant 'response threshold' concentrations summarized by Burg (7),our data is most unexpected. It is possible that such plant responsethresholds to ethylene are greatly modified and closely synchro-nized with rapid growth and development in vegetative tissue.The plants grown in the greenhouse under a photoperiod of about9 h and a temperature range of 18 to 22°C contained low levels ofendogenous leaf ethylene (Fig. 3). When these plants were placedin the CAES chambers and maintained at 26°C under a 12-hphotoperiod, endogenous ethylene progressively increased withtime (Fig. 3). The higher temperature and longer photoperiod ofthe growth chamber were in all probability growth rate promotive.If further study establishes the levels of endogenous ethylene invegetative tissue to be a close function ofgrowth rate, it is probablethat ethylene response thresholds are also linked to growth rateand hence highly variable. The fact that the progressive rise inendogenous leaf ethylene in nontreated plants failed to induce aloss of Chl (as opposed to ethephon treatment) (Fig. 3) seemssupportive of a variable response threshold. It is apparent fromthis data that studies involving the assessment of endogenous leafethylene must be designed with an appropriate period of environ-mental preconditioning to achieve the stability necessary prior toinitiation of experiments.

Ethephon-released ethylene induces detectable chlorosis by 24h and together with tissue-derived ethylene, induces significantChl loss (senescence) by 72 h (Fig. 3). This data suggests thatsenescence induction in ethephon-treated leaves is a consequenceof either (a) low initial ethylene response thresholds (high sensi-tivity) which may be correlated with and quickly elevated byenhanced growth (1, 18), (b) an ethylene 'exposure-duration'requirement as reported for abscission (16), or (c) an undetectedlarge pulse of ethephon-released ethylene occurring prior to thefirst analysis at 6 h (6, 20). Highly variable ethylene responsethresholds are evident in flowers (21) and reversibility of ethylene-induced responses is a feature of physiologically immature tissuesystems. As tissue systems mature, or become stressed, ethyleneresponse thresholds decline and senescence becomes less reversibledue to rising ABA levels (enhanced tissue sensitivity to ethylene)and declining cytokinin levels (reduced membrane integrity) (12,21).

Subjecting treated plants to hypobaric conditions eliminatedethephon-induced leaf senescence and, in fact, promoted Chllevels above nontreated controls (1,000 mbar ambient) providedambient O2 and CO2 partial pressures were maintained (Fig. 4).In contrast, treated and nontreated plants subjected to 200 mbarof air revealed distorted Chl levels and Chl a :b ratios. The reducedavailability of 02 and/or CO2 may disturb normal O2-dependentsynthesis of Chl b from Chl a (13). The significant elevation ofChl levels at 200 mbar of controlled 02 plus CO2 is of specialinterest (Fig. 4). These data suggest that decreased ethylene reten-tion (not reduced ethylene biosynthesis) is the dominant hypobariceffect and that even in nontreated (control) plants basal ethyleneremoval enhances Chl levels in leaves. The inclusion of ethylenewith O and CO2 in future studies might further clarify this point.The data also support the 'dissociable ethylene-receptor complex'concept of ethylene action (17). It is unlikely, in these studies, thatfurther ethylene metabolism is a central requirement for ethyleneaction on leaf senescence. Oxidation-dependent ethylene action(4) should not be reduced under 200 mbar of 02 plus CO2 becausethe controlled O2 and CO2 partial pressures should sustain priormetabolic redox environments.Chl loss represents a readily determined aspect of the 'gradual

decline' phase of leaf senescence (24). Support for the reversibilityof this process is provided by the data in Figure 5. When compared

Plant Physiol. Vol. 71, 1983100




with the data of Figure 3 (lower panel), Chl loss is terminated assoon as plants are subjected to 200 mbar of 02 plus C02; evenwhen imposed as late as 48 h after ethephon treatment (25). Ifexperimental irradiance levels were higher, enhanced Chl synthe-sis would be expected to produce the actual reversal. Clearly,ethylene levels do not irreversibly 'trip' the onset of leaf senes-cence. Instead, ethylene levels must rise and be sustained aboveresponse thresholds to implement full senescence.The CAES described in this study is effective and reliable.

Three observed limitations are: (a) restricted UVA and UVBtransmission by the polycarbonate chambers; (b) an inability tomaintain 100%o RH at or near leaf surfaces as irradiance levelsrise; and (c) an inability to measure leaf temperatures within theCAES in this study.

LITERATURE CITED

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4. BEYER EM JR, DC BLOMSTROM 1980 Ethylene metabolism and its possiblephysiological role in plants. In F Skoog, ed, Plant Growth Substances 1979.Springer-Verlag. New York, pp 208-218

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17. LIEBERMAN M 1979 Biosynthesis and action of ethylene. Annu Rev Plant Physiol30: 533-591

18. LIEBERMAN M 1979 Role of ethylene in plant growth, development, and senes-cence. In NB Mandava, ed, Plant Growth Substances. American ChemicalSociety, Washington DC, pp 115-134

19. Lisr RJ 1958 Smithsonian Meterological Tables. Rev Ed 6. Smithsonian Miscel-laneous Collection. Vol 114. Smithsonian Institution Press. Washington DC,pp 331-341

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21. MAYAK S. AH HALEVY 1980 Flower senescence. In KV Thimann, ed. Senescencein Plants. CRC Press. Boca Raton, FL, pp 131-156

22. NILSEN KN, CF HODGES 1980 A hypobaric system for intact plant examinationof stress ethylene-related leaf senescence. Hort Science 15: Abstr 429

23. NILSEN KN, CF HODGES 1980 Photomorphogenically defined light and resistanceof Poa pratensis to Drechslera sorokiniana. Plant Physiol 65: 569-573

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