Copyright © Physiologia Plantarum 2001PHYSIOLOGIA PLANTARUM 113: 50–58. 2001Printed in Ireland—all rights reser�ed ISSN 0031-9317

Involvement of polyamines in the control of fruitlet physiologicalabscission in grapevine (Vitis �inifera)

Aziz Aziza,*, Oliver Brunb and Jean-Claude Audrana

aLaboratoire de Biologie et Physiologie Vegetales, UPRES EA 2069 URVVC, Europol’Agro, Uni�ersite de Reims Champagne Ardenne,B.P. 1039, F-51687 Reims Cedex 2, FrancebMumm – Perrier-Jouet, Vignobles et Recherches, 11 A�. Champagne, BP. 186, F-51206 Epernay Cedex, France*Corresponding author, e-mail: aziz.aziz@uni�-reims.fr

Received 13 November 2000; revised 26 March 2001

increased free and conjugated PA levels in the inflorescencesThe potential contribution of polyamines (PAs) in the regula-tion of physiologically induced fruitlet abscission was investi- and markedly inhibited abscission. �-Difluoromethylarginine,

an inhibitor of arginine decarboxylase, but not �-difluoro-gated in cuttings from two cultivars of Vitis �inifera L., Pinotnoir (PN) and Merlot (MRT). Abscission was higher in MRT methylornithine, an inhibitor of ornithine decarboxylase, low-

ered PA levels and increased abscission. Treatment with cy-than in PN and was preceded by a decrease in free PA levels.clohexylamine or �-hydroxyethylhydrazine as potentThis decline was more pronounced in inflorescences than ininhibitors of Spd synthase and PA oxidases, respectively,leaves of the sensitive cultivar. Soluble conjugated PA showed

an opposite trend in both cultivars. This suggests a cause-ef- reduced the Spd and/or spermine levels and enhanced free Putin the inflorescences, inducing an increased abscission of floralfect relationship between free and/or conjugated PA levels in

floral organs and susceptibility to abscission. Spermidine organs shortly after anthesis. These data suggest that PAs,(Spd), but not putrescine (Put) or diaminopropane, supplied at particularly Spd, could be involved in the regulation of0.5–1 mM to the nutritive medium prior to the anthesis, grapevine fruitlet physiological abscission.

in Galston et al. 1997, Martin-Tanguy 1997), little isknown about the involvement of these compounds in thecontrol of the flower or young fruit abscission in perennialplant species.

There is evidence that the diamine putrescine (Put) andthe PAs spermidine (Spd) and spermine (Spm), as well astheir biosynthetic enzymes, arginine decarboxylases (ADC;EC 4.1.1.19) and ornithine decarboxylase (ODC; EC4.1.1.17), are involved in normal growth and developmentin higher plants (reviewed in Galston et al. 1997, Waldenet al. 1997). PAs occur as free cations and as conjugateswith hydroxycinnamic acids and macromolecules (Galstonand Sawhney 1990). Many observations suggest that thesesubstances may play a role as endogenous regulators inplants and be involved in the synthesis of proteins, RNA,and DNA and in the stabilization of membranes (Altman

Introduction

One of the most frequent problems in crop plants isfruitlet abscission, which often occurs just after floweringeven under normal environmental conditions, thereby re-ducing potential yield. In various perennial plants, thisphenomenon called ‘physiological abscission’ is largelyconsidered to be associated with metabolic disorders anddepletion in the nutrient levels feeding to the inflorescence(Kaska 1989, Osborne 1989, Gonzalez-Carranza et al.1998). It is well known that fruit set requires positivegrowth signals generated during pollination and/or laterafter fertilization (Gillaspy et al. 1993). Changes inamounts of phytohormones, resulting from alterations inmetabolism and/or transport, could be involved in fruitletabscission (Sexton and Roberts 1982, Ruperti et al. 1998).However, despite numerous studies relating polyamine(PA) metabolism to flowering of annual plants (reviewed

Abbre�iations – ADC, arginine decarboxylase; Agm, agmatine; CHA, cyclohexylamine; DAO, diamine-oxidase; Dap, 1,3-diaminopropane;DFMA, �-difluoromethylarginine; DFMO, �-difluoromethylornithine; HEH, �-hydroxyethylhydrazine; MRT, Merlot; PA, polyamine;PAO, polyamine-oxidase; PN, Pinot noir; Put, putrescine; Spd, spermidine; Spm, spermine..

Physiol. Plant. 113, 200150

and Bachrach 1981, Slocum et al. 1984, Smith 1985). PAsare also implicated in environmental stress responses (Flo-res and Galston 1984, Aziz et al. 1998, 1999) and in thecontrol of senescence (Tiburcio et al. 1994, Larher et al.1998) and morphogenesis (Evans and Malmberg 1989,Galston and Sawhney 1990).

The changes in the metabolism of PAs are correlatedwith floral development, and their conjugates accumulatein shoot apices upon floral initiation (Perdrizet and Pre-vost 1981). Early work showed that exogenous applicationof PAs during fruit set increased the number and the sizeof the fruit that set (reviewed in Evans and Malmberg1989). Previous genetic evidence comes from the isolationof certain mutants of tobacco for a role of PAs in plantsexual reproduction (Malmberg and McIndoo 1983).These mutants, which are deficient in PAs, show alterationin floral morphology such as anthers partially turned intopetals and ovules transformed into stamens. Conversely,Petunia mutants with irregular floral development displayabnormal PA levels (Gerats et al. 1988) and male sterilemutants of maize lack conjugated PA accumulation in theanthers (Flores et al. 1989). A recent report (Applewhiteet al. 2000) indicates a close connection between Spd andreproductive development in several lines of Arabidopsismutants.

The relative contribution of biosynthesis enzymes to themodulation of PA levels is dependent on plant species andgrowth process (Martin-Tanguy 1997). Some reports sug-gest that only ADC is involved in PA synthesis duringfruit set in pea (Perez-Amador et al. 1995), whereas bothADC and ODC activities are present in tomato ovariesand ODC is up-regulated during fruit set (Alabadi et al.1996). Tobacco, carrying an oat ADC gene under the con-trol of an ABA-inducible promoter, showed acceleratedgrowth and development and increased flower and capsulenumbers (Limami et al. 1999). Spd synthase (EC 2.5.1.16)activity was also detected in pea ovaries and one of thetwo genes encoding this enzyme was highly expressed inyoung leaves and flowers at anthesis (Alabadi and Car-bonell 1999). The metabolites resulting from thecatabolism of PAs may also be responsible for the ob-served events during development. Martin-Tanguy (1997)suggested that in male sterile flowers diamine-oxidase ac-tivity could be involved in the regulation of the amineconcentrations during sexual differentiation and/or theirtransport at the subcellular level.

Accumulating evidence from transgenic and mutantplants prompted us to investigate the possible involvementof PAs in the control of flower or fruitlet abscission. Inthe present work, we report the time courses of free andconjugated polyamine concentrations in leaves and infl-orescences of fruiting cuttings from two cultivars ofgrapevine. We compare PA levels in relation to abscissionin both cultivars, since these cultivars were characterizedby different sensitivity to fruitlet abscission. The effects ofexogenous di- and polyamines, as well as those of appro-priate inhibitors on endogenous PA levels and fruitlet ab-scission, were also investigated.

Materials and methods

Plant material and growth conditions

Three-node-long cuttings of two cultivars of Vitis �iniferaL., Pinot noir (clone 521) and Merlot (clone 181) werecollected from 6-year-old plants. Cuttings were surface-sterilized with cryptonol and rooted as described byMullins and Rajasekaran (1981). Selected rooted cuttingswere placed in plastic pots filled with vermiculite substrateand grown in a culture chamber (16-h photoperiod, tem-perature 25/20°C day/night, irradiance of 200 �mol m−2

s−1 (fluorescent lights Philips TLD36W/83), relative hu-midity 75/90% day/night). Only a single flowering stemwas allowed to develop on each plant during growth.Plants were constantly fertilized with Hoagland’s solution(Hoagland and Arnon 1938).

Polyamine and inhibitor treatments

Putrescine, spermidine and 1,3-diaminopropane wereadded to the nutritive solution at various concentrations(from 0.1 to 1 mM) 7 days before anthesis. �-Difl-uoromethylarginine (DFMA), �-difluoromethylornithine(DFMO), cyclohexylamine (CHA) and �-hydroxyehtyl-hydrazine (HEH) were also supplied separately at con-centrations ranging from 0.5 to 2 mM in the nutritivesolution. Plants were grown under culture chamber condi-tions as described above. Plants were divided at differentdevelopmental stages into roots, leaves and flowers orfruitlets, then weighed and frozen in liquid nitrogen beforeanalysis. All experiments were replicated at least threetimes.

Polyamine extraction and analysis

Frozen samples were powdered and mixed with 1 M HCl(0.2/1, w/v). The homogenates were kept for 1 h at 2°Cand centrifuged for 20 min at 24000 g to obtain freepolyamines. An aliquot of the supernatant was hydrolyzedwith 6 M HCl for 12 h at 105°C (Aziz and Larher1995). This fraction contained free PAs as well as acid-soluble conjugates. The pellet was washed three timeswith 1 M HCl and hydrolyzed with 6 M HCl for 12 hat 105°C. This fraction contained acid-insoluble PA con-jugates. High-performance liquid chromatography (HPLC)and a fluorescence spectrophotometer detector wereused to separate and quantify polyamines prepared astheir dansyl derivatives according to the method ofFlores and Galston (1982). The column was a reversephase hypersil C18 (particle size 5 �m, 4.6×250 mm, Su-pelco). Dansylated polyamines were eluted with aprogrammed methanol:water solvent gradient, changingfrom 60 to 95% over 23 min at a flow rate of 1 mlmin−1; elution was completed in 7 min (Aziz et al. 1997).The column was washed with 100% methanol for 5 min.For dansylated polyamine analysis, an excitation wave-length of 365 nm was used with an emission wavelengthof 510 nm.

Physiol. Plant. 113, 2001 51

Results

Changes in free and conjugated PA levels duringdevelopment

The most abundant free PAs in grapevine were Spd, Spmand Dap (a product of Spd and/or Spm oxidation), whilePut and Agm were present at low levels. Fig. 1 shows thetime course of free PA content in the inflorescences andleaves of PN and MRT. During development, free PAcontents were found to be higher before anthesis in PN thanin MRT organs, excepted for Dap. In the inflorescences ofPN (Fig. 1a), free Spd and Spm amounts increased duringdevelopment until anthesis, then decreased gradually andremained significant even after full bloom. While Agm andDap contents decreased in the inflorescences of PN, nochange was observed at the level of Put. However, in theinflorescences of MRT (Fig. 1b), the content of all free PAsdecreased earlier (before anthesis) and remained very lowduring flowering.

In leaves, free PA levels were also higher in PN than inMRT at different developmental stages (Fig. 1c,d). In PN(Fig. 1c), the Spd and Dap levels decreased before anthesis,but remained high during flowering, while Put and Spmcontents were enhanced. However, in the leaves of MRT(Fig. 1d), free Put, Spd, Spm and Dap decreased before full

flowering. No major change was observed in Agm in eithercultivar.

The conjugated PAs in grapevine organs were mainlypresent as acid-soluble forms of Put, Spd and Dap. Whenconsidering total PAs, changes in free and soluble-conju-gated PA contents followed opposite trends before anthesisin all organs examined of both cultivars (Fig. 2). In contrastto PN (Fig. 2a,c), MRT showed that the decrease in totalfree PA content was gretaer in inflorescences than in leaves(Fig. 2b,d). However, the level of conjugated PAs in PNincreased to a maximum value in the inflorescences at theanthesis, then decreased gradually (Fig. 2a). Change in thesecompounds was not highly significant in leaves (Fig. 2c).The level of soluble-conjugated PA increased in both in-florescences and leaves of MRT until anthesis (Fig. 2b,d),then their content declined in leaves, while it remainedrelatively constant in inflorescences.

Abscission patterns during development

Floral organs started to drop before anthesis for both PNand MRT fruiting cuttings (Fig. 3). A substantial increase inabscission rate was observed just after full flowering (7 daysafter anthesis) in MRT, while it occurred gradually in PN.By 15 days after anthesis, more than 45% of flowers on

Fig. 1. Time course of freepolyamine contents in inflorescences(a,b) and leaves (c,d) of fruitingcuttings from two grapevinecultivars, Pinot noir (a,c) and Merlot(b,d), during development. Put (�),Agm (�), Spd (�), Spm (�), Dap(�). Data are mean�SE, n=5.

Physiol. Plant. 113, 200152

Fig. 2. Time course of total free and acid-soluble conjugatedpolyamine contents in inflorescences (a,b) and leaves (c,d) of fruit-ing cuttings from two grapevine cultivars, Pinot noir (a,c) andMerlot (b,d), during development. Total free polyamines (�), acid-soluble conjugated polyamines (Put+Spd+Dap, �). Data aremean�SE, n=5.

Fig. 4. The relationship between the amount of free polyamines inthe inflorescences and the abscission level during development offruiting cuttings from Merlot. Data are means from 13 batches ofcuttings treated simultaneously, each consisting of 5 cuttings.

Fig. 5. Effects of putrescine (�), spermidine (�) and di-aminopropane (�) on abscission in two grapevine cultivars, Pinotnoir (PN) and Merlot (MRT). Polyamines were added separately at0.1–1 mM, 7 days before anthesis and the abscission was deter-mined at fruit set (25 days after anthesis). Data are means�SE,n=5. * indicates that the percentage of abscission is significantlydifferent between Spd treatment and its control at P�0.05 byStudent’s t-test.

MRT had abscised against about 20% on PN. Abscissioncontinued to increase until fruit setting, reaching about 25and 60% in PN and MRT, respectively. From the presentresults it is clear that MRT was more sensitive to abscissionthan PN.

Fig. 3. Abscission pattern of flowers and fruitlets in fruiting cut-tings of two grapevine cultivars, Pinot noir (PN, �) and Merlot(MRT, �). Data are mean�SE, n=6.

Physiol. Plant. 113, 2001 53

Fig. 6. Effect of exogenous spermidine(Spd) on free polyamine levels in roots(�), leaves (�) and inflorescences (�)of two grapevine cultivars, Pinot noir(a,b) and Merlot (c,d). Control (a,c);treated with Spd (b,d). Spd was addedat 0.5 mM, 7 days before anthesis andthe endogenous polyamines wereanalysed 7 days after anthesis (fullflowering). Data are means�SE, n=5.

Relationships between free PA concentrations and abscission

As shown in Fig. 4, the increased percentage of originalflowers abscised in MRT was associated with a decrease infree PA levels in the corresponding inflorescences. It becameapparent that the two processes are correlated starting froma total free PA concentration of less than 0.7 �mol g−1

fresh mass. Thus, abscission potential or fruit set coulddepend on free PA levels in the floral organs before anthesis.

Effects of exogenous PAs on the abscission level

The level of abscission in both grapevine cultivars wasinfluenced by the addition of PAs to the nutritive medium(Fig. 5). Thus, application of Spd at 0.5 mM, 7 days prioranthesis, reduced abscission by about 20 and 35% in PN andMRT, respectively. However, Put and Dap addition at thesame concentration did not cause any significant change inthe abscission levels of both cultivars.

Changes of PA concentration after treatment with Spd

When fruiting cuttings were treated 7 days before anthesiswith 0.5 mM Spd, they exhibited changes in their endoge-nous PAs particularly in leaves and inflorescences. Fig. 6shows the main changes in free PA titers occurring in roots,leaves and inflorescences at full flowering of PN and MRT.Compared to the control, Spd increased substantially onlyin the inflorescences of PN, whereas it increased in bothleaves and inflorescences of MRT. Feeding of Spd markedlyaugmented Put and Spm levels in the inflorescences of both

cultivars. Spd treatment also reduced Agm and Dap levels inleaves and inflorescences of PN and MRT (Fig. 6). How-ever, PA levels remained lower in roots and were notaffected by Spd treatment. Spd application also led to adoubling or almost so in the level of acid-soluble conjugatedPut and Spd in the inflorescences of both cultivars (resultsnot shown).

Effects of inhibitors of PA metabolism on the abscission level

Abscission increased with increasing concentrations ofDFMA, a specific inhibitor of ADC (Fig. 7). With 1 mMDFMA, abscission reached maximum values of about 40and 20% in PN and MRT, respectively, compared to thecontrol. In contrast, abscission level remained relativelyunchanged in both cultivars after application of DFMO, aspecific inhibitor of ODC.

The abscission percentage also increased in response toCHA, an inhibitor of Spd synthase, added in the externalmedium. It reached values of about 60 and 80% in PN andMRT, respectively, with 1 mM CHA (Fig. 7). Similarly,HEH, a potent inhibitor, of PAO, strongly increased abscis-sion of floral organs in both cultivars. The highest value ofabscission was obtained with 1 mM HEH.

Changes in PA concentrations after treatment withinhibitors

The effects of inhibitors of PA metabolism were investigatedonly in PN, which exhibited a low susceptibility to fruitletabscission. The application of DFMA at 1 mM caused a

Physiol. Plant. 113, 200154

Fig. 7. Effects of �-difluoromethylarginine (DFMA), �-difl-uoromethylornithine (DFMO), inhibitors of arginine decarboxylaseand ornithine decarboxylase, respectively, cyclohexylamine (CHA,inhibitor of spermidine synthase) and �-hydroxyethylhydrazine(HEH, inhibitor of polyamine oxidases) on abscission in twograpevine cultivars, Pinot noir (PN) and Merlot (MRT). Inhibitorswere added at 0.5–2 mM, 7 days before anthesis and abscission wasdetermined at fruit set. DFMA (�), DFMO (�), CHA (�), HEH(�). Data are means�SE, n=3 for DFMA and n=5 for the otherinhibitors. * indicates that the percentage of abscission is signifi-cantly different between a treatment and its control at P�0.05 byStudent’s t-test.

Discussion

Polyamines are alleged to have a regulatory role in floweringand physiological fruit abscission in grapevine plants. Thisnotion is based on the observations: (1) that the low level offree and conjugated PAs in the inflorescences is correlatedwith abscission sensitivity, (2) that free PA levels decreasedin both leaves and inflorescences of grapevine before thestart of floral organ abscission and (3) that exogenous Spdinhibited abscission, while the application of appropriateenzyme inhibitors lowered PA levels and strongly increasedabscission. Certainly, these observations must lead to fur-ther investigations of the connection between PA and fruitsetting, as suggested by Galston et al. (1997).

Our results indicate that the level of PAs in floral organsdepends on the developmental stage of the cuttings. Hence,before anthesis, free PA mobilization appeared more pro-nounced in inflorescences than in leaves of the ‘abscising’cultivar. This suggests that PA partitioning between sourceand sink organs might be a critical factor controlling plantproductivity. Increased fruitlet number could be achievedindirectly through an increase in flower number as a resultof better flower initiation or by a direct effect on fruit set. Inthis context, PAs are assumed to be involved in floralinitiation, floral evocation and reproduction (Galston et al.1997, Martin-Tanguy 1997).

The pre-anthesis depletion of free PA levels in grapevinecuttings was also associated with production of soluble-con-jugated PAs in foliar and inflorescence tissues, while theinsoluble forms were present at low levels during develop-ment (data not shown). It appeared that the high level ofsoluble-conjugated PAs in the inflorescences was correlatedwith a low sensitivity to abscission. These adjustments mightresult from a conversion of free PAs to conjugated formsthrough an activation of polyamine transferases in both leafand floral tissues, leading to their accumulation in theinflorescences. It has been reported that soluble-conjugatedPAs move from leaves to the young floral buds of Sinapisalba (Havelange et al. 1996). These forms are assumed to bemarkers for female reproductive organ fertility (Martin-Tan-guy 1997). For instance, it seems that the inflorescences ofgrapevine must receive at least part of their PAs, in freeand/or conjugated forms, from leaves needed for reproduc-tive organ stability and/or fertility, as reported for otherplants (Bagni and Torrigiani 1992).

The PA status of the cutting seems to be responsible forthe control of physiological abscission in grape. Indeed, Spd(not Put or Dap) application led to an increase in free andconjugated Put and Spd levels in both leaves and inflores-cences of grape and inhibited abscission. In this respect, theeffect of Spd might be mediated by the enhanced level ofSpd itself through its preferential accumulation in the infl-orescences or by the Spd-induced changes in the content ofendogenous free and soluble conjugates. It is suggested thatfruit set could be positively influenced by the limitation offree Put accumulation and the increase in free and soluble-conjugated Spd in the floral organs.

Additionally, perturbation of PA metabolism by treatinggrapevine cuttings with DFMA, an inhibitor of ADC,caused a dramatic inhibitory effect on the level of free PAs

marked decrease in free PA levels in all organs examined atthe end of flowering (Fig. 8). However, DFMO slightlyreduced Put levels in the leaves but increased Spd in bothroots and inflorescences. Agm and Spm levels were alsoincreased by DFMO in leaves and inflorescences. This effectcould be related to the stimulation of ADC activity byDFMO as reported earlier (Burtin et al. 1989).

In cuttings treated with 1 mM CHA, Agm and Putcontents increased in both roots and leaves, while Spd, Spmand Dap decreased in all organs (Fig. 8). In the inflores-cences, Spd and Spm levels decreased by 80 and 50%respectively. Those of Agm, Put and Dap remained rela-tively unchanged. The application of HEH in the externalmedium caused also a decrease in Spm and Dap levels inboth leaves and inflorescences (Fig. 8). The Spd level re-mained, however, unchanged, while Put increased in allorgans in response to HEH treatment.

Physiol. Plant. 113, 2001 55

Fig. 8. Effects of �-difluoromethylarginine (DFMA), �-difluoromethylornithine (DFMO), cyclohexylamine (CHA) and �-hydroxyethylhy-drazine (HEH) on free polyamine levels in roots (�), leaves (�) and inflorescences (�) of Pinot noir. Inhibitors were added at 1 mM, 7days before anthesis and the endogenous polyamines were analysed 7 days after anthesis (full flowering). Data are means�SE, n=3 forDFMA and n=5 for the other inhibitors.

and increased floral organ abscission. Conjugated PA con-tent also declined in floral organs (data not shown). Incontrast, DFMO, an inhibitor of ODC, led to a slightproduction of free PAs without significant effects onfruitlet abscission. These results suggest that PA synthesisbefore anthesis might require ADC, rather than ODC ac-tivity. Furthermore, the effects of DFMA on both PAlevels and abscission were greater in plants of low sensitiv-ity. This strengthens the hypothesis that the PA pool pro-duced from ADC may counteract floral organ abscission.However, the effect of DFMO on PA content could berelevant to its stimulatory effect on ADC activity (Burtinet al. 1989), since the Agm level was enhanced in responseto DFMO treatment. It was reported that, during vegeta-tive growth and floral bud formation, ADC but not ODCactivity was the main source of the PA synthesis (Tiburcioet al. 1988). Subsequent development, however, appears tobe dependent on ODC activity. Similarly, in pea plants,gene expression of the ADC is high in young developingtissues, like shoot tips and flower buds (Perez-Amador etal. 1995). Our results are consistent with the hypothesis

that fruitlet abscission could be induced, at least in part,by depressing PA synthesis, the target being the ADCpathway.

Fruitlet abscission seems to be extremely sensitive toCHA, an inhibitor of Spd synthase, and to HEH, aninhibitor of PAO. The addition of CHA or HEH revealeda possible contribution of the Spd synthase and PAO path-ways in modulating PA concentrations in both leaves andinflorescences. These results support the contention thatboth Spd synthesis and catabolism are tightly coordinatedand both of these processes could be required for an opti-mal level of Spd, and subsequently for regular fruitletabscission. This is also in accordance with the findings ofBurtin et al. (1991) showing that inhibition of Spd synthe-sis in tobacco caused malformation of anthers and infertil-ity, and Applewhite et al. (2000), suggesting a role for Spdin the bolting and flowering in a delayed-flowering mutantof Arabidopsis. The balance between PA and ethylene syn-thesis may also be one of the major determinants in regu-lating the abscission process, since Spd synthase requiresan aminopropyl group donor (decarboxylated SAM). A

Physiol. Plant. 113, 200156

recent report indicated that increased ethylene productionduring floral development was accompanied by increasedfruitlet abscission in peach (Ruperti et al. 1998). In contrast,treatment with inhibitors of ethylene production stimulatedfree and conjugated PA accumulation (Martin-Tanguy et al.1993).

We can conclude that the susceptibility of grapevine toflower or fruitlet abscission is greatly dependent on themodulation of PA concentrations in the floral organs earlyduring development. It is likely that the use of PA mutantsfrom other plants (Malmberg and Watson 1996, Applewhiteet al. 2000) is now necessary in order to understand physio-logical mechanism of PAs in the control of fruitletabscission.

Acknowledgements – The authors wish to thank Dr D. Tepfer,INRA Versailles, France, for critical reading the manuscript andcorrecting English. Thanks are also due to Merrell-Dow ResearchLaboratories, Cincinnati, OH, USA for providing us with DFMOand DFMA. This work was supported by the Mumm–Perrier-JouetSociety, Epernay, France.

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