Ethylene and preharvest drop the effect of AVG and NAA on fruit abscission in apple

ORIGINAL PAPEREthylene and preharvest drop:the effect of AVG and NAA on fruit abscission in apple (Malus domestica L.Borkh)Valeriano Dal Cin ÆMarcello Danesin ÆAlessandro Botton ÆAndrea Boschetti ÆAlberto Dorigoni ÆAngelo RaminaReceived:17January 2008/Accepted:19July 2008/Published online:3August 2008ÓSpringer Science+Business Media B.V.2008Abstract L -Aminoethoxyvinylglycine (AVG)and 1-naphthylacetic acid (NAA)are known to affect preharvest fruit drop,fruit quality and fruit maturation in Golden Delicious apples (Malus 9domestica Borkh).Experiments were carried out on GD/M9trees treated at three different developmental stages (41,28and 17days before the beginning of the commercial harvest)with AVG and NAA.Both chemicals signif-icantly reduced fruit drop without significantly affecting the fruit weight.Background colour devel-opment and ripening were both delayed by AVG,whereas NAA significantly enhanced yellowing with-out affecting the evolution of ripening.Ethylene evolution and transcription profiles of genes involved in ethylene biosynthesis (MdACS1and MdACO1)and action (MdETR1,MdERS1and MdCTR1)were mon-itored in cortex from the date of the first treatment until the beginning of fruit drop in the control trees (21days after the beginning of commercial harvest).AVGblocked or efficiently reduced the ethylene evolution.This effect was paralleled by a down-regulation of MdACS1,MdACO1,MdETR1and MdERS1.NAA at the second and third date of application enhanced the onset of ethylene evolution,although,at the end of the experiment,no difference was found between control and treated fruits.The chemical applied in the first date significantly down-regulated the transcription of the genes at the end of the experiment.MdCTR1expres-sion,basically unaffected by AVG and NAA,appeared to be transiently down-regulated.The initial down-regulation is under developmental control,whereas the late regain of transcript accumulation paralleled the ethylene evolution.Keywords MdACO1ÁMdACS1ÁMdCTR1ÁMdERS1ÁMdETR1ÁStreif indexAbbreviations ACS 1-Aminocyclopropane-1-carboxylatesynthaseACO 1-Aminocyclopropane-1-carboxylateoxidaseETR Ethylene resistant ERS Ethylene response sensor AVG L -Aminoethoxyvinylglycine DAB Days after bloom GD Golden deliciousLBPA Laser-based photoacoustic NAA 1-Naphthylacetic acidV.Dal Cin ÁM.Danesin ÁA.Botton ÁA.Ramina (&)Department of Environmental Agronomy and Crop Science,University of Padova,Agripolis-Vialedell’Universita`16,35020Legnaro (Padova),Italy e-mail:angelo.ramina@unipd.itA.BoschettiCNR Institute of Photonics and Nanotechnologies,ITC,38050Povo Trento,ItalyA.DorigoniExperimental Institute for Agriculture,Via Mach 2,San Michele all’Adige,38010Trento,ItalyPlant Growth Regul (2008)56:317–325DOI 10.1007/s10725-008-9312-5IntroductionPreharvest fruit drop is an important cause of fruit loss in apple industry.Fruit shedding is caused by a surge of endogenous ethylene which is generally considered to be the main factor controlling fruit drop(Kende1993). Chemicals inhibiting ethylene biosynthesis and signal cascade,sprayed during the late part of the fruit developmental cycle,have been proved to efficiently reduce preharvest drop and delay ripening(Rath et al. 2006;Yuan and Carbaugh2007).Exogenous auxin has also been used to counteract the effect of endogenous ethylene.In particular,NAA was effective in delaying preharvest apple fruit drop,and repeated applications of the chemical were more effective than a single one (Marini et al.1993;Yuan and Carbaugh2007). Besides reducing fruit drop,NAA may enhance background colour development and fruit softening, under circumstantial environmental conditions(Yuan and Carbaugh2007).In fruit species the preharvest drop is a conse-quence of the onset of ripening which,in climacteric fruit,is a syndrome developmentally regulated and strictly controlled by ethylene(Yang and Hoffman 1984;Oetiker and Yang1995;Schaller and Kieber 2002;Giovannoni2004;Sato et al.2004;Genard and Gouble2005).The biosynthetic pathway of this hormone is mainly controlled by two enzymes:ACS (1-aminocyclopropane-1-carboxylate synthase)and ACO(1-aminocyclopropane-1-carboxylate oxidase) (Bleecker and Kende2000),which in apple as in other species are encoded by multigene families, whose different members are involved in the regula-tion of several physiological processes besides ripening(Dal Cin et al.2006;Harada et al.2000; Oraguzie et al.2004;Sunako et al.1999;Dal Cin et al.2007a;Wakasa et al.2006;Atkinson et al. 1998;Costa et al.2005).Ethylene is perceived by receptors encoded by a multigene family in which,besides constitutively expressed members,some genes up-regulated during the onset of ripening are present(Chang and Stadler 2001;Bleecker1999;Klee and Tieman2002). Downstream ethylene receptors,CTR1,a MAPKKK encoded by a multigene family both in Arabidopsis (Huang et al.2003)and tomato(Adams-Phillips et al.2004),acts as a negative effector.The purpose of this research was to investigate the relationship between preharvest fruit drop and the expression of ethylene-related genes by means of L-aminoethoxyvinylglycine(AVG)and1-naphthyl-acetic acid(NAA)treatments.Materials and methodsPlant material and treatmentsThe research was carried out on9-year-old apple trees(cv Golden Delicious/M9)in a private orchard located in Val di Non(Denno,Trento,Italy,340m a.s.l.).In2004,70homogenous trees were identified and divided into seven groups.Three of them were sprayed with ReTainÒ(AVG,4.15%w/w)and three with Obsthormon24a(NAA,7.5%w/w)until run off at three dates corresponding to41,28or17days before the beginning of the commercial harvest, respectively.One group was kept as untreated control.The timing of experiments was chosen according to previousfield trials and corresponded to the switch of ethylene biosynthesis from system1 to system2(Barry et al.2000).Experiments were performed from116days after full bloom(DAB), when fruit size fully developed,until178DAB,when the ripening syndrome and the natural fruit drop in the control trees were well advanced.The effect of treatments was monitored throughout the commercial harvest window,occurring from157 to170DAB,by assessing fruit drop,average fruit weight,colour index and Streif index.Thefirst picking date was decided according to a Streif index, assessed on the control fruits,around0.08±0.009, which is suitable for long storage of the Golden Delicious fruits grown in the Trentino area(Delong et al.1999).The cumulative preharvest drop was assessed by summing the number of fruits shed at each harvest date and compared to the total.Values are reported as an average per tree.Fruit weight,ground colour and Streif index were monitored along the three harvest dates and expressed as average per tree.Ground colour was measured by Greefa grading machine and expressed as index according to the following scale: \3.6=green; 3.6–3.9=green–yellow; 3.9–4.2= yellow–green;[4.2=yellow.Streif index is calcu-lated as follows:Firmness/(Soluble solids*Starch). Firmness was monitored with a penetrometer(TR S.n.c.,Forlı`,Italy)using an11mm probe.In order to avoid the large variability often encountered within fruit populations at each devel-opmental stage,the fruit average size(cross diameter) was assessed on200fruits for each treatment and the standard error(SE)determined before performing the sampling.Ethylene measurements and molecular analyses were performed on fruits displaying a cross diameter within the±SE.Ethylene determination and sample collection were carried out onfive fruits for each treatment and the means and standard deviations calculated.Ethylene biosynthesis analysis was per-formed with the laser based photoacoustic(LBPA) technique as previously described(Dal Cin et al. 2005b).For molecular analyses fruit was peeled and samples of cortex immediately frozen in liquid nitrogen and stored at-80°C.Expression analysisThe expression analysis was performed by semiquan-titative RT-PCR on agarose gel as previously described(Dal Cin et al.2007a).Total RNA was obtained following the protocol described by Dal Cin et al.(2005a).cDNA was prepared as previously described(Dal Cin et al.2005b).One microlitre of first-strand cDNA was used for the RT-PCR that was performed in three replicates.Transcript accumulation of genes under investiga-tion MdACS1(U89156),MdACO1(AB030859), MdETR1(AF032448),MdERS1(AY083169)and MdCTR1(AY670703)was evaluated with specific primers,as reported by Dal Cin et al.(2007b),in25l l reactions using0.025U l l-1of AmpliTaq Gold (Applied Biosystem)with a Gene Amp PCR system 9700(Applied Biosystems,Branchburg,NJ).For each reaction a set of a different number of cycles ranging between20and35was tested to choose those corresponding to the exponential phase.Each cycle consisted of30s denaturation at94°C,30s annealing at63°C and30s extension at72°C;cycles were preceded by5min denaturation at94°C and followed by afinal7min extension at72°C.The PCR products were electrophoresed in2%agarose gels,stained with ethydium bromide,and images obtained via UV exposition elaborated using the KODAK1D 3.6 software(Scientific Imaging Systems-Eastman Kodak Company).The expression level was evaluated, normalized with the housekeeping gene(ubiquitin, DQ438989)and expressed in arbitrary units. ResultsEffect of NAA and AVG on fruit drop and ripeningThe effect of the chemicals on fruit drop and ripening are shown in Table1.AVG was effective in reducing the preharvest fruit drop,regardless of the time of application.Nevertheless,the most effective treatment was the last one,performed at140DAB.NAA reduced fruit abscission to a lower extent than AVG,being theTable1Effect of AVG(L-aminoethoxyvinylglycine)and NAA(1-naphthylacetic acid)applied at41(1),28(2)and17(3)days before the beginning of commercial harvest on fruit drop,fruit weight,colour index a and Streif index bCumulative fruit drop(%)Fruit weight(g)Colour index Streif indexControl untreated 6.0a224.2a 3.73b0.053b AVG1 1.8c220.5a 3.50c0.060a AVG2 2.7c220.5a 3.61c0.058a AVG3 1.5c220.5a 3.57c0.061a NAA1 3.3bc220.6a 3.79a0.051b NAA2 4.0b220.7a 3.81a0.054b NAA3 4.3b220.7a 3.82a0.053bThe harvest was performed in three picks(157,164and170DAB)corresponding to the commercial window.Data reported in the table refer to the average of the three picks,with the exception of fruit drop,expressed as a cumulative value.Different letters within columns correspond to statistically different values(P B0.05)using the LSD testa Ground colour was measured by Greefa grading machine and expressed as index according to the following scale:\3.6=green;3.6–3.9=green–yellow;3.9–4.2=yellow–green;[4.2=yellowb Streif index is calculated as follows:Firmness/(Soluble solids*Starch)first application time (116DAB)the most effective.The chemicals did not significantly influence fruit weight,although treated trees constantly produced fruits with an average weight slightly lower than the control.As far as the ground colour is concerned,the two chemicals showed opposite effects:AVG reduced the colour development at all application time,whereas NAA significantly increased it.Taking into account the Streif index,AVG significantly delayed ripening,whereas NAA was ineffective.Effect of NAA and AVG on transcription of genes involved in ethylene biosynthesisEthylene evolution measured during late fruit devel-opment and ripening displayed a basal level until140DAB,the last date of NAA and AVG application (Fig.1).The hormone biosynthesis in the control fruits started to increase at 150DAB,reached a small peak at 164DAB,then slightly declined at 171DAB and dramatically increased thereafter,reaching a very high level at the end of the experiment.The fruits of the AVG treated trees were characterized by very low ethylene production until 164DAB,then ethylene biosynthesis slightly increased,more consistently in the fruits treated at the second date.However,ethylene production remained always at a much lower level than the control.In the NAA treated fruits ethylene evolution was similar to the control until ter on (164and 171DAB),fruits treated at the second and at the third date produced more ethylene than the control.At the end oftheFig.1Effect of AVG (left)and NAA (right)on ethylene evolution andexpression profiles of genes involved in the hormone biosynthesis (MdACS and MdACO1),during apple (GD/M9)fruit development and ripening.Ethylene was determined by the laser photo acoustic system on five fruits.Mean values are reported with barsrepresenting standard error.Transcript quantification was performed through RT-PCR in three replicates.Mean values are reported,with bars representing standard deviations.Chemicals (dotted line)were applied at three dates corresponding to 41(1),28(2)or 17(3)days before the predicted harvest of the control (continuous line).Control:square;First date of application:triangle;Second date of application:upside-down triangle;Third date of application:circleexperiment the ethylene level still increased,although the difference among treatments disappeared,with the exception of the last spray showing higher value than the control(Fig.1).MdACS1transcript amount in control fruits was almost constant until157DAB,then it started to increase with a little peak at164DAB and afinal burst at178DAB,similarly to what observed for ethylene production.In AVG-treated fruits,the transcripts did not differ significantly from control at164and 171DAB,but were dramatically lower at178DAB. Analogously to what observed for ethylene,fruits of the second AVG spray displayed a transcript level slightly higher than the other AVG treatments but still much lower than the control.In general,regarding fruits from NAA-treated trees,MdACS1transcripts slightly differed from the control already at150DAB. More precisely,the last two treatments determined a weak increase at150and157DAB,whereas no difference was observed in the fruits of the most precocious ter on,transcript amount of the second and third NAA treatment was similar to the control whereas that of thefirst one was lower, especially at the end of the experiment(Fig.1).MdACO1transcript amount in control fruits showed a small peak at129DAB but it started to increase rapidly only after150DAB.Concerning AVG,this small peak was absent in fruits treated at thefirst date.Thereafter,transcripts level was fairly basal and increased only to a limited extent at171 and178DAB.In NAA-treated fruits,the small peak at129DAB persisted.In the following dates,the transcript accumulation patterns in the different treatments were similar to what previously reported for MdACS1,although the difference among treat-ments was broader(Fig.1).Effect of NAA and AVG on transcription of genes involved in ethylene signal transductionMdERS1transcript accumulation in control fruits displayed a small peak at129DAB,then declined at 141DAB,and thereafter constantly increased up to the end of the experiment.Thefirst AVG treatment nullified the early little peak and completely inhibited thefinal dramatic increase in transcript accumulation. NAA applications had no effect on the early peak but slightly affected the level of transcripts at later dates, without any significant modification of the pattern.At the end of the experiment,compared to the control, the transcript accumulation was higher in fruits of the last two applications and lower in those of thefirst one(Fig.2).MdETR1transcripts in control fruits increased at constant rate,reaching a maximum at157and 164DAB,and thereafter decreased.AVG-treated fruits showed a transcript accumulation generally lower than the control.In particular,the earlier the treatment the higher the difference.Nevertheless,by the end of the experiments no significant difference was found among treatments.NAA-treated fruits were characterized by a transcript accumulation lower or equal to the control up to159DAB. Thereafter the effect of the chemicals appeared to be variable(Fig.2).MdCTR1transcript amount in control fruits was constant from116to129DAB,then declined reaching the lowest level at164DAB andfinally increased reaching a value similar to the beginning. AVG and NAA did not largely modify the pattern observed in the control(Fig.2).DiscussionEffects of AVG as well as NAA on preharvest fruit drop reconfirm information previously reported by Yuan and Carbaugh(2007)and Rath et al.(2006) showing the effectiveness of both chemicals in reducing apple fruit abscission,although AVG was more effective than NAA.Since applications were performed at a stage in which fruit is almost completely mature,the differences observed in weight,though marginal,may be due to an effect on latefilling.Indeed,although the growth period was extended consequent to the delay of ripening,the fruits might have undergone competition due to the higher fruit number present within the tree.Never-theless,as far as AVG is concerned,a direct negative effect of the chemical on fruit growth cannot be ruled out.The installation of ripening was definitely delayed by AVG treatments as documented by the colour and the Streif indexes,which are positively and negatively related to the progression of ripening, respectively.Concerning NAA,a stimulation of the background colour development was observed,in spite of any significant effect on the Streif index (Table1).Regarding ethylene evolution,the chemical appli-cations had an opposite effect.AVG reduced the transient small increase in ethylene evolution and impressively delayed the final ethylene burst.On the other hand,NAA seemed to have the opposite effect determining in general higher level of ethylene biosynthesis.Taking into account the AVG action,it has been demonstrated that the chemical binds to ACS inhibiting its activity (Capitani et al.2002)and dou-bling the half-life of the enzyme (Yoshii and Imaseki 1982).The long lasting effect of the first application in our experiments (at least 62days)and in other published works (Bramlage et al.1980;Autio and Bramlage 1982)contrasts with the short half-life of the enzyme and with the fact that AVG should be metabolized and newly synthesized ACS may be active and able to produce ACC.It may be then assumed that the AVG treatment also slows down development by reducing ethylene production.Indeed,it has been demonstrated in tomato that ethylene applications determined a degradation of ethylene receptors (Kevany et al.2007),which are negative regulators (Wilkinson et al.1997).The analysis of the gene expression patterns indicated that the up-regula-tion of ACO preceded the onset of ethylene evolution,whereas the increase in ACS transcripts was concurrent with it (Fig.1).At this stage,the gene expression appeared to be developmentally regulated.A correla-tion between the transcripts level and ethylene evolution,as pointed out in several other systems (Zheng et al.2005),was assessed only in the late stage of ripening when the system 2of ethylene biosynthesis is largely established.During this phase MdACS1and MdACO1are up-regulated by ethylene.Nevertheless,Fig.2Effect of AVG (left)and NAA (right)onexpression profiles of genes involved in ethylene action (MdERS1,MdETR1and MdCTR1),during apple (GD/M9)fruit development and ripening.Transcript quantification wasperformed through RT-PCR in three replicates.Mean values are reported,with bars representing standard deviations.Chemicals(dotted line)were applied at three dates corresponding to 41(1),28(2)or 17(3)days before the predicted harvest of the control (continuous line).Control:square;First date of application:triangle;Second date of application:upside-down triangle;Third date of application:circleMdACS1expression appeared to be more tightly related than that of MdACO1to ethylene,taking into account the dramatic increase in the transcript accu-mulation of the former occurring in correspondence of the ethylene burst(Fig.1).Concerning ethylene receptors,MdERS1transcript accumulation preceded the onset of ethylene evolution,whereas the levels of MdETR1expression transiently increased reaching a maximum at157and164DAB(Fig.2).As far as MdCTR1expression is concerned,its down-regulation sequentially preceded the onset of MdERS1,MdACO1 and MdACS1expression,and the ethylene burst.The minimum level of expression was reached at 164DAB.At the end of the experiment,the initial level was restored concurrently with the maximum ethylene evolution and the highest MdACS1,MdACO1 and MdERS1transcript accumulation(Figs.1and2).AVG,besides negatively affecting ethylene evo-lution,blocked transcript accumulation of MdACS1, MdACO1and MdERS1.Moreover,the chemical transiently down-regulated MdETR1at a different extent according to the time of application.The transcription profile of MdCTR1was slightly affected by both thefirst and the second application of AVG, enhancing the transient down-regulation observed throughout the experiment.The effect of NAA changed according to the application time.Thefirst treatment was effective in down-regulating the expression pattern of MdACS1and MdACO1 throughout the last part of the experimental period, without any significant effect on ethylene evolution. The second and third applications caused a marked anticipation of the onset of ethylene evolution, paralleled by an up-regulation of MdACS1,MdACO1 and MdERS1.A general negative effect was exerted on MdETR1transcription,whereas an effect similar but stronger than that of AVG was observed on MdCTR1transcript accumulation.As general remarks,the effect of AVG and NAA in reducing preharvest fruit drop is reconfirmed,in agreement with the direct block of ethylene biosyn-thesis exerted by AVG and the known action of auxin (i.e.NAA)in regulating the sensitivity of abscission zone tissues to ethylene(Meir et al.2006).Further-more,the effect of AVG is consistent with the molecular data,pointing out a general shift of the expression pattern of all the genes analysed,except for MdCTR1,whose expression might be proba-bly perturbed by the poisoning action exerted by L-aminoethoxyvinylglycine on cell metabolism.On the other hand,the effect of NAA on fruit drop is not apparently coherent with the expression data,reveal-ing an earlier increase in MdACS1and MdACO1 transcript accumulation concurrently with the antic-ipation of the ethylene burst.However,these results fully agree with the stimulation of background colour development,as reported by others and also detected in auxin-treated loquat fruits(Agusti et al.2003). These observations strengthen the hypothesis that, while ethylene positively triggers both ripening and abscission possibly through the same transductive pathway,two different mechanisms depending on the target tissues and/or organs might be involved in mediating the NAA action.Interestingly,the effect of NAA at this stage of fruit development is the opposite of that found during the physiological drop in which the chemical actually enhances abscission(Bangerth 2000;Dal Cin et al.2007c).It would be interesting to study the differential response of the system related to development.A further important aspect concerns the MdCTR1 transcript accumulation profile,characterized by an initial down-regulation that appears to be develop-mentally controlled,followed by a regain of the gene transcription concurrent with the burst in ethylene biosynthesis.These data are consistent with those previously reported in tomato(Leclercq et al.2002) and apple(Dal Cin et al.2007c).From a practical point of 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