J Sci Food Agric 1998, 76, 215È220

Effect of Exogenous Calmodulin and ATP on theActivity of Ethylene Forming Enzyme Obtainedfrom Tomatoes and Green Pea PodsCharles K Njoroge,1,* Eduardo L Kerbel1 and Donald P Briskin2

1 Department of Horticulture, University of Illinois, Urbana-Champaign, 1201 W Gregory Drive, Urbana,IL 61801, USA2 Department of Crop Sciences, University of Illinois, Urbana-Champaign, 1201 W Gregory Drive,Urbana, IL 61801, USA

(Received 28 March 1996 ; revised version received 2 May 1997 ; accepted 19 June 1997)

Abstract : Ethylene forming enzymes (EFE) from both peas and tomatoes wereprepared. EFE from both sources were inhibited by adenosine triphosphate(ATP). Exogeneous calmodulin had no e†ect on EFE activity either in the pres-ence or absence of ATP. The presence of gramicidin in pea microsomal mem-brane EFE did not a†ect EFE activity in the presence of ATP. This suggests thatATP is inhibiting EFE activity through phosphorylation and not through trans-membrane proton Ñux. However, inhibition of EFE activity by ATP was par-tially reversed by 60 lM N-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide(W7) and 15 lM triÑuoperazine (TFP) but not by 60 lM N-6(aminohexyl)-1-naphthalenesulphonamide (W5), which suggests that the ATP inhibitory a†ecton EFE is probably controlled by calmodulin. 1998 SCI.(

J Sci Food Agric 76, 215È220 (1998)

Key words : calmodulin ; adenosine triphosphate (ATP) ; adenosine diphosphate(ADP) ; inorganic phosphate (Pi) ; triÑuoperazine (TFP) ; N-6(aminohexyl)-1-naphthalenesulphonamide (W5) ; N-6(aminohexyl)-5-chloro-1-napthalenesul-phonamide (W7) ; ethylene forming enzyme (EFE)

INTRODUCTION

EFE oxidises 1-aminocyclopropane-1-carboxylic acid(ACC) to ethylene which is the Ðnal step in ethylenebiosynthesis (Kende 1990). This is one of the keyenzymes in ethylene biosynthesis whose study, untilrecently, had been limited by the inability to obtainactive EFE in vitro. However, Ververidis and John(1991) isolated active EFE in a cell-free system frommelons.

In related work, we have demonstrated that EFE isthe step at which calcium inhibits ethylene biosynthesis(Njoroge and Kerbel 1995) and this e†ect of calcium onEFE activity was thought to be mediated through cal-modulin.

* To whom correspondence should be addressed at EgertonUniversity, P.O. Box 536, Njoro, Kenya.

Calmodulin is the most frequently reported intracel-lular receptor for Ca2`. It is a highly conserved proteincharacteristic of eukaryotic organisms, and has beenimplicated as a key regulatory protein in animal andplant cells (Means et al 1984 ; Allan and Trewavas1987). The concentration of calmodulin in most tissuesvaries between 2 and 40 lmol kg~1 fresh weight whichtranslates to 10~6È10~7 M in plant cells (Poovaiah andReddy 1987). This is well above the amount necessaryfor the establishment of maximum e†ective concentra-tion of the calciumÈcalmodulin complex. Calmodulinconcentration, therefore, does not seem to be a limitingfactor for the reactions in which it participates(Poovaiah and Reddy 1987).

CalciumÈcalmodulin mediated processes can be con-trolled by the level of calcium (Blowers and Trewavas1988) or by the level of calmodulin (Coccuci andNegrini 1988), the involvement of which is suggested by

2151998 SCI. J Sci Food Agric 0022-5142/98/$17.50. Printed in Great Britain(

216 C K Njoroge, E L Kerbel, D P Briskin

the action of calcium chelators and ionophores (Perdueet al 1988) and calmodulin antagonists (Jacoby andRudich 1987).

The intracellular levels of calcium in resting plant andanimal cells is 10~8È10~7 M (Allan and Trewavas 1987).In the presence of the primary signals in plant cells suchas light and gravity, the intracellular calcium concentra-tion in the cell is increased to 10~6È10~5 M, which isadequate for it to bind to calmodulin. Intracellularlevels of calcium could also be increased by exogenousintroduction of calcium into the plant cell, as done inthis work. In either case, calcium is bound by calmodu-lin, forming calciumÈcalmodulin complex. The calciumÈcalmodulin complex is bound to calmodulin-bindingproteins, which are mainly enzymes which could eitherbe activated or inactivated. The calciumÈcalmodulincomplex may produce the physiological responsedirectly or through regulation of protein kinase(s) whichphosphorylate(s) the enzymes, hence modulating theiractivity (Allan and Trewavas 1987). As intracellularcalcium levels become too high, membrane locatedCa2`-ATPases pump calcium back out through theplasma membrane or into organelles, until the cellreturns to its normal resting level and the calciumÈcalmodulin regulated processes are shut o† as calmodu-lin no longer binds calcium.

Since the actual mechanism of calcium e†ect on EFEactivity is not clearly understood, this work was done toinvestigate the e†ect of calmodulin and ATP on EFEactivity in both peas and tomatoes. This study attemptsto explain the mode of action of inhibition of EFEactivity by Ca2`.

MATERIALS AND METHODS

Plant materials

Tomatoes (L ycopersicon esculentum, cv Caruso) weregrown at the University of Illinois greenhouses. Thegrowth conditions were 18É5¡C (night) and 26¡C (day).The lighting regime was 16 h of light and 8 h of dark-ness. Fresh green pea pods were purchased from aproduce store. Pea tissue was chosen as an experimentalmaterial because it was the only tissue in which in vitroEFE activity had been observed in a cell-free systembefore Ververidis and John (1991) extracted it frommelons. In this work, active in vitro microsomal mem-brane EFE was required, hence the choice of pea tissue.Tomatoes were chosen as experimental materials as thiswas part of a bigger study addressing the postharvestquality of tomatoes.

Extraction of ethylene forming enzyme (EFE) from peas

The microsomal membrane fraction (MMF) from peapods was obtained according to Mayak et al (1981) and

McRae et al (1982), with modiÐcations. The extractionbu†er contained 55 mM N-[2-hydroxyethyl] piperazine-N-[3-propane sulphonic acid] (EPPS), pH 7É5, and4 mM dithiothreitol (DTT). The pea pods were choppedinto cold bu†er (1 g tissue per 2 ml of bu†er). The tissuewas homogenised in a blender for three 20 s-periodswith 30 s rest periods. The homogenate was Ðlteredthrough four layers of cheesecloth and the Ðltrate wascentrifuged at 13 000] g for 20 min in a Sorval RC2-Bsuperspeed centrifuge, using an SS34 rotor. The super-natant was further centrifuged at 80 000 ] g for 45 minin a Beckman L7-55 ultracentrifuge using a TY35 2349rotor. The pellet was resuspended in 2 mM EPPS (pH7É5) bu†er (1 ml bu†er for every 50 g of plant tissue)using a camel hair brush and homogenised in a downceglass homogeniser to give the microsomal membranefraction (MMF) EFE from pea pods. The supernatantwas dicarded as it had been observed to have no EFEactivity. The soluble EFE from peas was obtainedaccording to Ververidis and John (1991), with somemodiÐcations. Fresh pea pods were frozen in liquidnitrogen. The bu†er used contained ; 100 mM N-[2-hydroxyethyl] piperazine-N-[ethane sulphonic acid](HEPES), pH 7É5, 30 mM L-ascorbic acid, and 10% glyc-erol. The bu†er was cooled and then degassed invacuum to remove air which was replaced with nitrogengas. The frozen pea pods were ground in bu†er withpestle and mortar until a smooth slurry was obtained(2 ml bu†er per 1 g of tissue). The slurry was degassedin vacuum to further remove incorporated air andreplaced with nitrogen gas. The degassed slurry was Ðl-tered through four layers of cheesecloth and the Ðltrateobtained centrifuged at 13 000] g for 20 min in aSorval RC2-B superspeed centrifuge using SS34 rotor.Some of the supernatant obtained was used as theenzyme preparation while the rest was centrifuged at80 000] g for 45 min as before to obtain microsomalmembrane fraction (MMF). The pellet was resuspendedin the extraction bu†er as earlier described.

Extraction of EFE from tomato fruit

Soluble EFE from tomatoes was obtained as for peasabove. Tomato tissue at pink stage was frozen in liquidnitrogen. It was then ground in the extraction bu†erwith pestle and mortar. The extraction bu†er used wassimilar to the one used for peas, but it had a pH of 8É25.All other operations were similar to those used for peasabove. Microsomal membrane fraction (MMF) and themicrosomal supernatant from tomatoes were obtainedin the same way as in peas above.

Assay of EFE

EFE assay was similar for both pea and tomatoenzymes. The MMF were assayed for EFE activity by

Calmodulin AT P and EFE activity in tomatoes and peas 217

mixing, in order : 55 mM EPPS (pH 7É5) bu†er, 1 mM

ACC and 0É1 ml MMF (EFE), to a total volume of 1 mlin 7 mm] 50 mm test tubes. The test tubes werecapped with rubber septum stoppers and incubated at30¡C for 2 h in a shaking water bath at the end ofwhich, 2 ml of the head space gas from each test tubewas injected into a gas chromatograph (model CARLHACH AGC 400 series) equipped with an FID detectorand an alumina column for ethylene determination.

The soluble EFE was assayed in a reaction mixturecontaining ; 1 mM ACC, 0É1 mM and 0É5 ml ofFeSO4the enzyme (EFE) extract. Other speciÐc reagents foreach assay are given below. The total assay volume wasincreased to 3 ml with the extraction bu†er containing30 mM L-ascorbic acid, 100 mM HEPES (pH 7É5) bu†erand 10% glycerol. Ethylene was determined as above.In the case of tomatoes, a pH of 8É25 was used.

E†ect of calmodulin and ATP on pea EFE activity

The e†ect of calmodulin on EFE activity was deter-mined in a reaction mixture containing : 55 mM EPPS(pH 7É5) bu†er, 1 mM ACC, 1 mM 5 mM ATPCaCl2 ,(pH 7É5), 0É1 ml (MMF) EFE and increasing amounts ofcalmodulin. In the experiment, the calmodulin levelsused were 0É00, 0É38, 0É57, 0É76 and 0É95 lM. The e†ectof Mg2` was tested by incorporating 5 mM inMgCl2the reaction mixture. Ethylene determination was asdescribed above.

E†ect of gramicidin on EFE activity

This was determined by incorporating gramicidin at aconcentration of 12 lM in the reaction mixture contain-ing : 55 mM EPPS (pH 7É5) bu†er, 1 mM ACC, 5 mM

5 mM ATP and 1 mM EFE assay wasMgCl2 , CaCl2 .then done as above.

E†ect of ATP, ADP and on pea EFE activityPi

The e†ect of ATP, ADP and on EFE activity in thePiabsence of exogenous calmodulin was determined in areaction mixture containing 55 mM EPPS (pH 7É5)bu†er, 1 mM ACC, 0É1 ml MMF (EFE), 5 mM ATP,1É25 mM ADP and 1É25 mM and increasing levels ofPi ,calcium to a maximum of 5 mM At each level ofCaCl2 .calcium the EFE assay was done in triplicate. Thecontrol treatment had the same reaction mixturewithout ATP, ADP or Pi .

E†ect of ATP, ADP and on tomato EFE activityPi

The e†ect of ATP, ADP and on EFE activity fromPitomato fruit was determined in a reaction mixture con-taining 1 mM ACC, 0É1 mM 5 mM 1 mMFeSO4 , MgCl2 ,

and 0É5 ml of the tomato enzyme preparationCaCl2(EFE) for the control. The treatments had either 5 mM

ATP, 1É25 mM ADP or 1É25 mM The total reactionPi .volume was made up to 3 ml using the extraction bu†ercontaining 30 mM L-ascorbate, 100 mM HEPES (pH8É25) and 10% glycerol. The EFE assay was then doneas above.

E†ect of ATP and calmodulin antagonists on tomatoEFE activity

The e†ect of calmodulin antagonists on EFE activitywas determined in a reaction mixture containing 1 mM

ACC, 1 mM 0É1 mM 0É1% (v/v) 95%CaCl2 , FeSO4 ,ethanol, 5 mM ATP and 0É5 ml of the enzyme extract,for the control. The other treatments contained either,60 lM N-6(aminohexyl)-1-naphthalenesulphonamide(W5), or 60 lM N-6(aminohexyl)-5-chloro-1-naphthale-nesulphonamide (W7), which were dissolved in 95%ethanol, such that the Ðnal ethanol concentration in thereaction mixture was 0É1% (v/v).

E†ect of calmodulin antagonists in the absence ofATP was carried out as above using W7 and tri-Ñuoperazine (TFP). In the case of TFP, a concentrationof 15 lM was applied and dimethylsulphoxide was usedas the TFP solvent to a level of 0É1% (v/v) in the reac-tion mixture.

In all the assays of EFE activity from tomato fruit,each treatment was carried out in Ðve replicates. Boththe data from tomato and pea EFE were subjected tostatistical analysis using Statgraphics StatisticalPackage.

RESULTS

E†ect of calmodulin and ATP on EFE activity

Figure 1 shows that calmodulin has no e†ect on theactivity of microsomal membrane fraction EFE frompea pods. However, 5 mM ATP reduced EFE activity(Fig 1).

Fig 1. E†ect of calmodulin and ATP on the activity of micro-somal membrane ethylene forming enzyme (EFE) from peas

obtained under aerobic conditions.

218 C K Njoroge, E L Kerbel, D P Briskin

Fig 2. E†ect of ATP on the activity of ethylene formingenzyme (EFE) from peas obtained under anoxic conditions.

Combined use of calcium and magnesium gave ahigher EFE activity/than calcium used separately. Thiswas observed in all situations where calcium and mag-nesium were combined and reasons were not clear.However, even in this case, calmodulin had no signiÐ-cant e†ect on EFE activity (Fig 1). In another experi-ment, calmodulin was incorporated at 1É5 lM in tomatoEFE reaction mixture. The results obtained were 2É91(0É1) and 2É81 (0É07) nl g~1 h~1 for control and calmo-dulin treatments, respectively, which show that even athigh concentrations, calmodulin had no observablee†ects on EFE activity. Figures in brackets representthe standard error.

Figures 2 and 3 show that ATP reduces the activityof soluble EFE from peas and tomatoes respectively, asit does in EFE from MMF of peas.

Di†erent levels of calcium did not seem to have anye†ect on EFE activity (Figs 4 and 5).

E†ect of ADP, ATP and on EFE activityPi

Figure 4 shows the e†ect of ATP, ADP and on EFEPifrom MMF of peas, relative to control. and controlPitreatments had similar EFE activity, ADP treatmenthad a slightly lower activity, while ATP treatment hadthe lowest EFE activity.

For tomato EFE, the results obtained were 1É48(0É08), 2É12 (0É06), 1É48 (0É13) and 1É04 (0É09) nl g~1 h~1

Fig 3. E†ect of ATP on the activity of ethylene formingenzyme (EFE) from tomatoes obtained under anoxic condi-

tions.

Fig 4. E†ect of ATP, ADP and on the activity of micro-Pisomal membrane ethylene forming enzyme (EFE) from peasobtained under anoxic conditions.

for control, ADP and ATP treatments, respectively.Pi ,Figures in brackets indicate standard errors for eachvalue. Both ADP and the control had similar EFEactivities but it was not clear why treatment gave aPihigher EFE activity. Again ATP treatment had signiÐ-cantly lower EFE activity than the other treatments.

All the treatments with ATP had similar EFE activitywhether the reaction mixture had Ca2` alone orCa2`] Mg2`. As before, combined use of 1 mM Ca2`and 5 mM Mg2` resulted in higher EFE activity thanuse of 1 mM Ca2` alone (Figs 1 and 5).

E†ect of gramicidin on ATP-inhibited EFE activity

This experiment was carried out on the microsomalmembrane EFE from peas, as gramicidin only acts onintact membranes. Figure 5 shows that ATP inhibitedEFE activity in the presence or absence of gramicidin.Di†erent levels of calcium did not seem to have anye†ect on EFE activity.

E†ect of W7 and TFP on EFE activity

In this experiment, the results obtained were 0É6 (0É02),0É72 (0É03) and 2É25 (0É09) nl g~1 h~1 for control TFPand W7 treatments, respectively. Figures in bracketsrepresent standard error. TFP and W7 resulted inincreased EFE activity, relative to the control. TFPresulted in only a slight increase while W7 resulted in a

Fig 5. E†ect of Ca2`, Mg2`, gramicidin and ATP on theactivity of the microsomal membrane ethylene formingenzyme (EFE) from peas obtained under aerobic conditions.

Calmodulin AT P and EFE activity in tomatoes and peas 219

four-fold increase as W7 is a more potent calmodulinantagonist than TFP.

E†ect of W5, W7 and ATP on EFE activity fromtomato fruit

The results obtained in this experiment were 2É24 (0É07),2É28 (0É07) and 2É82 (0É10) nl g~1 h~1 for control, W5and W7 respectively. Figures in brackets represent stan-dard error. W5 gave similar EFE activity to the control.W7 had a signiÐcantly higher EFE activity relative toW5 and the control.

DISCUSSION

The inhibition of ethylene biosynthesis by calcium hasbeen observed in tomatoes (Wills and Tirmazi 1979)and in apples (Sams and Conway 1987). However, theactual mechanism through which calcium inhibits ethyl-ene biosynthesis is not known. Calcium has been knownto inÑuence enzyme activity through calmodulin byforming a calciumÈcalmodulin complex. This complexinÑuences the enzyme action directly by binding ontothe target enzymes thus changing their structural con-formation which alters substrate binding thus inÑu-encing the enzyme activity. The calciumÈcalmodulincomplex could also inÑuence the enzyme activitythrough protein kinases which phosphorylate the targetenzymes thus inÑuencing their activity (Allan and Tre-wavas 1987). Calcium could also inÑuence enzymeactivity by acting as the co-factor for the enzyme(Bouzayen et al 1991). In an attempt to elucidate thee†ect of calcium, we have shown that this inhibitionoccurs at the EFE step of ethylene biosynthesis(Njoroge and Kerbel 1995). This work attempts toinvestigate a possible calmodulin involvement incalcium inhibited ethylene biosynthesis by determiningthe e†ect of calmodulin on EFE.

The exogeneous incorporation of calmodulin intomicrosomal membrane EFE from peas does not a†ectits activity (Fig 1). As indicated in the results, incorpor-ation of exogeneous calmodulin at a concentration of1É5 lM into EFE obtained from tomato fruits had noe†ect on EFE activity. Higher levels of calmodulin (upto 4É54 lM in tomato EFE) gave similar results.However, this observed ine†ectiveness of calmodulindoes not rule out the inÑuence of calmodulin on EFEactivity as the level of calmodulin in plant tissues is atsaturating amounts and, therefore, calmodulin modu-lated enzymes may already be at optimum levels(Poovaiah and Reddy 1987). The amount of calmodulinin plant cells ranges between 10~6 and 10~7 M

(Poovaiah and Reddy 1987) and, for optimum enzymemodulation by calmodulin, only 1 lM calmodulin(Salimath and Marme 1983) is required. It is thereforeprobable that the modulation of EFE by calmodulinwas already at its maximum. In certain cases some

enzymes have been observed to have an integral calmo-dulin molecule within their structure and, therefore,their modulation is independent of the addition of exog-enous calmodulin to the reaction mixture (Harper et al1991). This too could be a possible reason for notobserving responses to addition of calmodulin.

The results show that calmodulin antagonistsenhanced EFE activity, recording a four-fold and aslight increase relative to the control for W7 and TFP,respectively. W7 is a more potent calmodulin antagonistthan TFP. This is in agreement with our earlier workon whole tomatoes and conÐrms that EFE activity iscontrolled by calmodulin.

On observing that calmodulin inÑuenced EFE activ-ity, we set out to Ðnd the possible mode of action. Wesuspected that calmodulin could be inÑuencing EFEactivity through protein kinase(s), hence ATP wasincorporated into the reaction mixture to test this e†ect.

This work shows that ATP reduces EFE activity.However, this ATP e†ect was calmodulin independentat the calmodulin concentrations used in the experiment(Fig 1). Similarly, the activity of EFE was reduced byATP in the absence of calmodulin in microsomal mem-brane EFE from peas (Fig 4) or in soluble EFE fromeither peas (Fig 2) or tomatoes (Fig 3). As suggestedabove, the levels of the natural calmodulin in the EFEextracts could be adequate to inÑuence enzyme activity.

The involvement of calmodulin in this ATP e†ect onEFE activity, was investigated by use of W7 and W5 inthe presence of ATP. W5 and W7 were used at the sameconcentration (60 lM). The results show that, in thepresence of W5, there was no di†erence in EFE activityrelative to the control. W5 is less hydrophobic than W7and its binding to calmodulin is minimal hence it ismainly used as a non-binding control for W7. However,W5 and W7 have a very similar structure and are,therefore, expected to produce similar non-speciÐce†ects in a reaction mixture. In the presence of W7,there was an increase in EFE activity relative to bothW5 and control. This would suggest that calmodulin isinvolved in the ATP-inhibited EFE activity becausechelation of calmodulin by W7 resulted in an increasein EFE activity.

The results show that the ATP e†ect is also calciumindependent. For calcium dependent enzymes, modula-tion occurs at calcium concentrations ranging between0É1 lM and 10 lM as observed in zucchini hypocotyls(Salimath and Marme 1983) and in protein kinase fromsoybeans (Harmon et al 1987). The observed calcium-independence of ATP-inactivation of EFE could beexplained by the fact that calcium in the EFE extractwas at saturation levels for enzyme activity and furtheraddition of calcium did not have any additional e†ectson the enzyme activity.

Ethylene biosynthesis has been thought to be coupledto a transmembrane electrogenic proton Ñux (John1983). This proton Ñux requires ATP and sealed cell

220 C K Njoroge, E L Kerbel, D P Briskin

membranes for its maintenance (John 1983) and inclu-sion or exclusion of ATP in a reaction involving sealedcell membranes would test this possible e†ect of ATPon ethylene biosynthesis.

In sealed microsomal membrane EFE from peas,ATP was thought to be exerting its e†ect either bymaintaining a transmembrane proton Ñux (John 1983)or through protein kinase(s) that could have exerted itse†ect through phosphorylation (Ranjeva and Boudet1987 ; Blowers and Trewavas 1988). Figure 5 shows thatgramicidin, which collapses transmembrane proton Ñux,did not reverse the inhibitory e†ect of ATP on EFEactivity. If ATP inhibited EFE activity by maintaining aproton Ñux, its e†ect would have been reversed bygramicidin. However, that was not observed and themost likely mode of action of ATP inhibition of EFEactivity was through phosphorylation of EFE byprotein kinase(s), thus controlling its activity, as hasbeen observed in other enzymic plant systems (Blowersand Trewavas 1988 ; Poovaiah and Friedman 1991).

As ATP breaks down to ADP and on hydrolysis,Piand because there could be active phosphatases in theEFE extract that could hydrolyse ATP to ADP and Pi ,it was necessary to investigate the e†ect of ADP and Pion EFE activity. The results from microsomal mem-brane EFE from peas (Fig 4) suggest that they had noe†ect. Inorganic phosphate did not a†ect EFE(Pi)activity (Fig 4). ADP reduced EFE activity but not tothe same extent as ATP (Fig 4). Tomato EFE showedsimilar results where ADP did not a†ect EFE activity,while increased EFE activity. The e†ect of ATP was,Pitherefore, not due to its hydrolysis products. It was con-cluded that ATP was exerting its e†ect directly, mostprobably through phosphorylation by a protein kinase.Such protein kinases have been identiÐed in planttissues (Ladror and Zieleski 1989 ; Li et al 1991).

CONCLUSION

In conclusion, EFE from both peas and tomatoes seemto be inhibited by calcium through calciumÈcalmodulincomplex. The actual e†ect of ATP is not clear, but itwas suspected to be mediated through phosphorylationby protein kinase(s), some of which may be calciumÈcalmodulin dependent.

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