Plant Physiol. (1989) 91, 669-6730032-0889/89/91 /0669/05/$01 .00/0

Received for publication April 20, 1989and in revised form June 12, 1989

Ethylene-Like Activity of Isocyanides'

Jeanette M. Quinn* and Shang Fa YangMann Laboratory, Department of Vegetable Crops, University of California, Davis, California 95616

ABSTRACT

Benzyl isocyanide, cyclohexyl isocyanide, benzyl isocyanate,methyl isocyanate, benzyl isothiocyanate, and methyl isothiocy-anate were each tested for ethylene-like activity in a pea (Pisumsativum) growth assay. Only the isocyanides gave an ethylene-like response; the concentration that gave a half-maximal re-sponse was approximately 10 to 15 microliters per liter for eachisocyanide, and this activity was inhibited by norbomadiene, a

competitive inhibitor of ethylene action. Since the isocyanides didnot promote endogenous ethylene production, it was concludedthat the isocyanides acted directly to give an ethylene-like re-sponse. The isocyanides were further shown to elicit ethylene-like activity in a potato (Solanum tuberosum) tuber respirationassay, in a camation (Dianthus caryophyllus) senescence assayand in a carrot (Daucus carota L.) isocoumarin formation assay.The feasibility of employing azido derivatives of benzyl isocyan-ide to photoaffinity label ethylene receptors in vivo is discussed.

Ethylene is an important plant hormone regulating a widevariety of plant processes (1). The biochemical mechanism bywhich ethylene exerts its effect is still unknown; however, itis generally believed that ethylene acts via a proteinaceousreceptor which contains a transition metal (4, 12, 19).Employing in vitro binding assays, two groups have identi-

fied and partially purified ethylene binding proteins (2, 13-15, 17). However, the physiological relevance ofthese proteinsto ethylene action in intact plants is unknown. An alternativeapproach is to identify ethylene binding components in vivoby covalently linking ethylene receptors in vivo to an affinitylabel and using the label to facilitate purification and charac-terization of receptors. Although ethylene itself absorbs atshort UV wavelengths (- 170 nm) (6), but at longer UVwavelengths (228 nm, 270 nm) when complexed with cop-

per(I) (5), an attempt to use radiolabeled ethylene as a photo-affinity probe in intact plant tissue resulted in little or no

specific labeling (10). This was probably due to the ethyleneonly absorbing at very short UV wavelengths (- 170 nm) withlow absorptivity (6) and low reactivity. Therefore, it is desir-able to use alternate photoaffinity probes which absorb atlonger UV wavelengths.

Previously, Sisler (12) reported that a number ofpi-acceptorcompounds, including some aliphatic and aromatic isocya-nides, elicited an ethylene-like response in an abscission assayor a respiration assay. In this investigation we further char-acterized and confirmed that benzyl isocyanide and cyclo-

'This research was supported by U.S. Department of Agriculturegrant 87-CRCR-1-2419 I.

669

hexyl isocyanide mimic ethylene action. We have additionallytested several other related compounds for their ability toelicit ethylene-like responses.

MATERIALS AND METHODS

Materials

Benzyl isocyanide, cyclohexyl isocyanide, benzyl isocya-nate, benzyl isothiocyanate, methyl isocyanate, methyl isothi-ocyanate and NBE2 were purchased from Aldrich (Milwau-kee, WI), and pea seeds were from Atlee Burpee Co. (War-minster, PA).

Bioassays

Biological activity of ethylene and other compounds wasassayed based on growth inhibition of pea (Pisum sativumvar. Alaska) seedlings (16), on promotion of respiration inpotato (Solanum tuberosum var. Russett) tubers (18), onpromotion of senescence of carnation (Dianthus caryophyllusL. cv White Sim) flowers (9), and on the promotion ofisocoumarin formation in carrots (Daucus carota L. var.Packer 83 or var. Dominator) (7).For the pea growth assay, 3 d old etiolated pea seedlings

were exposed for three days to compounds as previouslydescribed ( 16).For the respiration assay, potato tubers harvested from the

field were enclosed (three tubers/treatment) under an 02atmosphere (18) in 17 L chambers containing KOH pelletsto absorb CO2 produced. Compounds to be tested were in-jected via rubber septa into the chambers. After incubationfor 24 h at 25°C, individual tubers were transferred to 0.5 Ljars, and their respiration rates under oxygen during thesubsequent 1 to 2 h were determined by measuring the CO2accumulated in the jars, using an Horiba infrared analyzer.For flower senescence, carnations harvested at normal com-

mercial maturity were cut to 30 cm in length. Each lot ofthree flowers in a flask containing 300 mL ofwater was placedin a 34 L chamber which contained KOH pellets to absorbCO2 produced. Chambers were sealed, and compounds to betested were injected via rubber septa onto filter papers insidethe chamber. The chambers were held at 1 5°C, and the timeto the onset of senescence as indicated by the inrolling of thepetals was taken (9).For isocoumarin accumulation, carrots were placed in 9 L

chambers at 15°C and exposed to ethylene or isocyanide.After treatment for the indicated time periods, three carrots

2Abbreviations: NBE, norbornadiene; ACC, I-aminocyclopro-pane- l-carboxylic acid.

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Plant Physiol. Vol. 91,1989

from each treatment were removed, and 3 g of peel from eachcarrot were extracted with 15 ml spectrophotometric gradehexane overnight at room temperature. The hexane extractswere extracted with an equal volume of 80% ethanol, and theisocoumarin content was determined from the absorption ofthe ethanol layer at 267 nm, using a molar absorptivity of14,800 and a partition recovery factor of 84% (7).

Determination of Intemal Concentration of Ethylene inPea Seedlings

Internal concentrations of ethylene in pea seedlings aftertreatment with various compounds for 3 d were determinedeither immediately or after 30 min of diffusion by a vacuumextraction method as described by Beyer and Morgan (3). Theethylene concentration was measured using a gas chromato-graph equipped with an alumina column and a flame ioni-zation detector.

Determination of ACC Content in Pea Seedlings

ACC was extracted from pea seedlings that had been ex-posed to either ethylene or isocyanide and measured as pre-viously described (8).

RESULTS

Effect of Isocyanides, Isocyanates and Isothiocyanateson Pea Seedling Elongation

Upon exposure to ethylene or its analogs, etiolated peaseedlings exhibit the triple response of shortening, bending,and thickening of their epicotyls (1, 4). The inhibition ofelongation caused by ethylene or compounds that mimicethylene action has been used as a quantitative assay forethylene-like activity (16). When benzyl isocyanide, cyclo-hexyl isocyanide, benzyl isocyanate, methyl isocyanate, ben-zyl isothiocyanate, and methyl isocyanate were tested usingthis assay, only benzyl isocyanide and cyclohexyl isocyanideinhibited epicotyl elongation (Table I) and promoted bendingand thickening of the epicotyls (Fig. 1). The triple responsecaused by benzyl isocyanide was relieved by norbornadiene(16), indicating that the benzyl isocyanide elicited ethylene-like action. The concentration of benzyl isocyanide or cyclo-hexyl isocyanide resulting in half-maximal inhibition ofgrowth was estimated to be 10 to 15 ,uL/L. The concentrationof ethylene causing half-maximal inhibition was approxi-mately 0.3 ,uL/L (Table I). Although the isothiocyanates andthe isocyanates also inhibited elongation at certain concentra-tions (Table I), no bending or thickening of epicotyls wasobserved. At higher concentrations, the isocyanates and iso-thiocyanates were toxic to the seedlings, as indicated by thediscoloration and wilted appearance of the seedlings, suggest-ing that any inhibition of elongation caused by these com-pounds is due to their toxicity.

Effect of Ethylene and Isocyanides on the Internal ACCand Ethylene Concentrations in Pea Seedlings

To determine whether the effect of isocyanides on theinhibition of the pea seedling elongation was direct or via

Table I. Effects of Isocyanides and Related Compounds on PeaSeedling Elongation

Twenty 3-d-old seedlings were incubated with each test compoundfor 3 d. Average length of epicotyls prior to incubation was 31 ± 7mm.

Compound Concentration Increase in EpicotylCompound Cocentration Length ± SE

sLIL mm

Ethylenea 0 76 ± 4CH2=CH2 0.3 36 ± 2

1.0 20±13.0 8±1

Benzyl isocyanidea 0 77 ± 45 48± 2

QC CH2-N=C 10 36 ± 325 31±240 15± 1100 8± 1

Cyclohexyl isocyanidea 0 74 ± 510 38±1

N=C 100 4 ±1

Benzyl isocyanate 0 76 ± 4100 57+ 3b

C) CH2-N=c-O 250 22 ± 2b500 Dead

Benzyl isothiocyanate 0 79 ± 210 29±3b

(DOSN) CH2-N=C=S 20 17 ± 2b30 Dead

Methyl isocyanate 0 81 ± 1CH3-N=C=O 100 54 ± 2b

400 39 ± 2b600 Dead

Methyl isothiocyanate 0 85 ± 4CH3-N=C--S 10 54 ± 3b

20 60 ± 4b40 Dead

a Seedlings show a typical triple response. b Epicotyls were notbent or thickened.

stimulation ofendogenous ethylene production, pea seedlingswere treated for 3 d with ethylene or isocyanide, and theirintercellular ethylene concentration was determined. If thelatter is the case, it is expected that the internal ethyleneconcentrations in the tissues treated with an isocyanide at 10and 100 gL/L should be about 0.3 and 3 uL/L, respectively,because isocyanides at 10 and 100 gL/L exert an effectequivalent to that exerted by 0.3 and 3 ALIL ethylene (TableI). Intercellular gas was extracted either immediately afterremoval from the treatment chamber to test the reliability ofthe method, or after 30 min diffusion to allow accumulatedethylene to escape so that the true internal ethylene concen-tration under ethylene-free air could be reached. As shown inTable II, immediate extraction (approximately 20 to 30 selapsed between transferring the seedlings from the chamberto the extraction apparatus) of seedlings exposed to 0.3 and1.0 fL/L ethylene results in internal ethylene concentrationsof 0.26 and 0.97 gL/L, respectively; these values are close to

670 QUINN AND YANG

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ETHYLENE-LIKE ACTIVITY OF ISOCYANIDES

Table II. Internal Ethylene Concentration of Pea Seedlings WhichHave Been Pretreated with Ethylene or Isocyanides for 3 d

Internal ethylene concentrations were determined either immedi-ately or 30 min after removal from the treatment chambers to allowdiffusion.

Internal EthyleneConcentration

Compound, Concentration Immediately AfterafterAfe

transfer 30 miAL/L AL/L

None (control) 0.12 0.08Ethylene,

0.3 0.26 0.051.0 0.97 0.23

Benzyl isocyanide,10 0.11 0.06

100 0.10 0.03Cyclohexyl isocyanide,

10 0.16 0.07100 0.19 0.06

Table Ill. ACC Content in Pea Seedlings Treated with Ethylene orIsocyanides

Three-day-old etiolated pea seedling were treated in enclosedchambers with ethylene or isocyanide. After treatment for 3 d, ACCcontent in the seedlings was assayed. The values represent the meanACC content ± SD for six replicates.

Compound, Concentration ACC Content

ALIL nmoles/g tissueNone (control) 0.79 ± 0.38Ethylene, 0.3 0.58 ± 0.20Benzyl isocyanide,

10 0.57 ± 0.13100 0.93 ± 0.09

Cyclohexyl isocyanide,10 0.74 ± 0.08

100 0.94 ± 0.15

Figure 1. A, Effect of increasing concentrations of benzyl isocyanideon the epicotyl elongation in pea seedlings. The benzyl isocyanidetreated seedlings show a "triple response" similar to that of ethylene-treated seedlings. B, The response to benzyl isocyanide is inhibitedby 1000 AL/L NBE, an inhibitor of ethylene action.

the expected values and thus verify the validity of the method.When the treated seedlings were allowed to stand for 30 minbefore extraction, the internal ethylene concentrations ex-

tracted from pea seedlings treated with benzyl isocyanide or

cyclohexyl isocyanide are not significantly different fromthose of control seedlings (Table II), indicating that the iso-cyanides do not promote endogenous ethylene production. Ifisocyanides exerted ethylene action by promoting ethyleneproduction, it is expected that isocyanide treatment would

cause an elevation in the amount of endogenous ACC. Wehave therefore determined the endogenous ACC levels in peaseedlings treated with ethylene or isocyanide. The results ofTable III indicate that the levels of ACC in control and inisocyanide treated seedlings are not significantly different.These observations indicate that the isocyanides exert theireffect on pea seedling elongation directly and not via pro-motion of endogenous ethylene production.

Effect of Ethylene and isocyanides on Potato TuberRespiration

Storage organs including potato tubers are known to exhibitan increase in respiration in response to exogenous ethylene,and this increase is further enhanced if the exposure to eth-ylene is done under an 02 atmosphere (18). Based on thisresponse, we have examined the effect of isocyanides on thepromotion of respiration in potato tubers. Both benzyl iso-cyanide and cyclohexyl isocyanide caused a rise in tuberrespiration (Table IV). Although the apparent maximum

A

B

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Plant Physiol. Vol. 91, 1989

Table IV. Effect of Ethylene and Isocyanides on Potato TuberRespiration

Potato tubers were treated for 24 h under 02 with ethylene orisocyanide and their respiration rates during the subsequent 1 to 2 hwere measured under 02. Three tubers per treatment were used.

Compound, Concentration Respiration Rate ± SD

filLIL mg C02/kg h

None (control) 12.8 ± 2.0Ethylene,

0.2 22.6 ± 2.30.4 36.2 ± 3.30.8 46.7 ± 8.71.6 75.2 ± 6.93.2 103.8 ± 5.85.0 95.6 ± 10.3

10.0 95.0 ± 7.520.0 90.0 ± 7.0

Ethylene, 0.4 + NBE, 1000 13.5 ± 0.4Ethylene, 0.4 + NBE, 5000 17.1 ± 0.6Benzyl isocyanide,

10 15.6 ± 0.930 17.3 ± 1.7100 18.1 ±3.6200 22.0 ± 4.1400 22.7 ± 1.6600 38.1 ± 4.1

Benzyl isocyanide, 600 + NBE, 1000 19.8 ± 2.2Cyclohexyl isocyanide,

10 18.8 ± 1.530 19.1 ± 1.8100 24.3 ± 3.4250 69.8 ± 0.8500 52.0 ± 8.8

Cyclohexyl isocyanide, 250 + NBE, 21.5 ± 0.75000

response exerted by the isocyanides was smaller than thatcaused by ethylene, the isocyanide-induced rise in respirationwas inhibited by norbornadiene, indicating that the isocya-nides mimic ethylene action.

Effect of Ethylene and Isocyanides on CarnationSenescence

The onset ofsenescence in cut carnations can be acceleratedby exposure to exogenous ethylene (9), and this accelerationis dependent on the concentration of ethylene applied. Figure2 shows the effect of 25 ,uL/L benzyl isocyanide, 25 ,uL/Lcyclohexyl isocyanide, and 0.3 ,uL/L ethylene on the senes-

cence of carnation flowers. After 7 d of exposure, the controlflowers still looked fresh, whereas the flowers treated withethylene or isocyanide showed extensive petal inrolling, whichis a measure of senescence. Flowers exposed to 0.3 MtL/Lethylene began showing inrolling of petals by d 2, whileflowers exposed to 10, 25, and 50 ,uL/L of benzyl isocyanideor cyclohexyl isocyanide began showing petal inrolling by d5, d 4, and d 2, respectively (data not shown). This concen-

tration dependent acceleration of senescence by the isocya-nides is similar to the effect of ethylene and again indicatesthese isocyanides exert ethylene-like action, except that a 100-

Figure 2. Effect of isocyanides on the inrolling of carnation petals.Flowers were exposed to air (control), 0.3 AL/L ethylene, 25 MAL/Lbenzyl isocyanide or 25 AL/L cyclohexyl isocyanide. The photographwas taken after 7 d of exposure.

fold greater concentration of isocyanide is required to givethe same response caused by ethylene.

Effect of Ethylene and Isocyanides on IsocoumarinAccumulation in Carrots

Carrots exposed to ethylene during storage produce higherlevels of isocoumarin than carrots held in ethylene free air,and the level of this response varies between cultivars (7). Theeffect of isocyanides on isocoumarin formation was thereforeexamined. Carrots (cv. Packer 83) were treated (three carrotsper treatment) for 8 d with cyclohexyl isocyanide, benzylisocyanide, or ethylene at 15'C. The level of isocyanide incontrol carrots was 3.5 ± 0.3 mg/100 g peel. Of the concen-trations tested, 5 tiL/L ethylene, 1000 ttL/L benzyl isocyanide,or 100 MtL/L cyclohexyl isocyanide caused the greatest accu-mulations of isocoumarin in treated carrots with levels of 45± 16, 182 ± 27, and 140 ± 18 mg isocoumarin/100 g peel +SD, respectively. Cultivar Dominator carrots treated with 5,uL/L ethylene, 1000 uL/L benzyl isocyanide, or 100 ,uL/Lcyclohexyl isocyanide had isocoumarin levels of 17 ± 4, 14 +3 and 36 ± 10 mg/100 g peel, respectively.

DISCUSSION

Of the compounds tested in the pea growth assay, onlybenzyl isocyanide and cyclohexyl isocyanide were capable ofeliciting a triple response similar to that caused by ethylene.The isocyanates and isothiocyanates tested had no effect onthe seedling growth up to concentrations where they becametoxic. Benzyl isocyanide and cyclohexyl isocyanide exhibitethylene-like activity in four different bioassay systems(Tables I and IV, Figs. 1 and 2). In the pea growth assay, theconcentration which gave half-maximal activity was approx-imately 10 to 15 ,AL/L for each isocyanide as compared to0.3 qL/L for ethylene (Table I). Thus, in terms of concentra-tions required to elicit ethylene action, isocyanides are 40times less active than ethylene. However, it should be notedthat the most active ethylene analog known to elicit ethylene

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ETHYLENE-LIKE ACTIVITY OF ISOCYANIDES

action is propylene, which is 100 times less active than eth-ylene (4). Thus, isocyanides should be considered to be as

active, if not more active, than propylene in eliciting ethyleneaction in this assay system. In the senescence assay and theisocoumarin assay, the concentrations of isocyanide givinghalf-maximal activity were not determined. Although isocy-anides were effective in promoting respiration in the respira-tion assay, and the rise in respiration caused by the isocyanideswas inhibited by norbornadiene, an inhibitor of ethyleneaction (Table IV), the increase in respiration achieved withthe isocyanides was less than the maximal respiration ratecaused by 3.2 ,L/L ethylene. These observations suggest thatthe putative receptor responsible for inhibition of pea hypo-cotyl elongation is different from that responsible for thepromotion of respiration in potato tubers. In the pea elonga-tion system, the isocyanides and ethylene exhibited differentaffinities for the receptor but elicited similar maximal re-

sponses. However, in the potato respiration system, the iso-cyanides and ethylene exhibited not only different affinities,but also different maximal responses.The possibility that the isocyanides may have elicited eth-

ylene-like action by stimulating ethylene production was ex-

cluded based on the observation that there was no significantincrease in internal ethylene concentration resulting from theapplication of these compounds to pea seedlings (Table II).This conclusion was further supported by the observation thatthese compounds did not cause an increase in the level ofACC in this tissue (Table III).

It is interesting that compounds like isocyanides are capableof eliciting an ethylene-like response as they are structurallyvery dissimilar from ethylene. The isocyanide group is highlycharged and connected to a bulky group in both benzylisocyanide and cyclohexyl isocyanide. In contrast, substitu-tion of a bulky group onto an olefin results in a reduction or

elimination of ethylene-like activity (4). However, isocyanidedoes meet certain requirements for ethylene action as pre-

scribed by Burg and Burg (4) in that it contains an unsaturatedbond adjacent to a terminal carbon atom which is not posi-tively charged. Additionally, isocyanides and other com-

pounds which mimic ethylene action tend to be pi-acceptormolecules as discussed by Sisler ( 12).The verification of benzyl isocyanide as a compound with

ethylene-like activity allows us to now consider its usefulnessas a photoaffinity label for the identification of in vivo ethylenereceptors. Since an azido derivative of benzyl isocyanidewould have an absorption maximum between 250 and 255nm (1 1), and a high molar absorptivity, the possibility of usingazidobenzyl isocyanide as a photoaffinity probe for ethylenereceptors is currently being investigated.

ACKNOWLEDGMENTS

The authors thank Dr. Herman Timm, Department of VegetableCrops, University of California, Davis, for providing the potatotubers, and Meng-Jen Wu, Environmental Horticulture, Universityof California, Davis, for providing the carnations used in this study.

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