Postharvest Biology and Technology 25 (2002) 301–309 www.elsevier.com/locate/postharvbio

Relationship between the levels of ammonia andco-ordination of phenylalanine ammonia-lyase and

phosphoenolpyruvate carboxylase in Annona cherimolastored under different conditions

Roberto Maldonado, Marıa I. Escribano, Carmen Merodio *Departamento de Ciencia y Tecnologıa de Productos Vegetales, Instituto del Frıo, Consejo Superior de In�estigaciones Cientıficas,

Ciudad Uni�ersitaria, 28040 Madrid, Spain

Received 17 April 2001; accepted 29 October 2001

Abstract

The levels of ammonia in cherimoya (Annona cherimola, Mill) fruit changed in response to different storageconditions. The highest amount of ammonia was observed in fruit stored at a chilling temperature, and the lowestammonia level was found in CO2-treated fruit at ambient temperature. These fruit presented the highest amount ofnitrogen incorporated in polyamines, mainly in spermidine and spermine. Moreover, the fact that the trend inpolyamines and ammonia content reverted when treated fruit were transferred to air would appear to confirmpreferential reassimilation of ammonia under high CO2 treatment. The increase in phenylalanine ammonia-lyase(PAL) activity observed in ripening fruit at ambient temperature was substantially reduced in fruit stored at a chillingtemperature. Our results suggest that PAL activity increases in fruit with a high demand of ammonia for furtherassimilation processes, and that this activity decreases in fruit where ammonia reassimilation does not take place.Additionally, in the case of CO2-treated fruit, ammonia availability is apparently promoted by the deamination ofamino acids, which is reflected in a significant decrease in free amino acid content without a concomitant rise in totalprotein levels. We observed that the patterns of change in phosphoenolpyruvate carboxylase (PEPC) and PALactivities were opposite. Moreover, coordination between these enzymes was related to the metabolic endogenouslevels of ammonia. The role of ammonia levels as a significant metabolic signal for controlling carbon and nitrogenmetabolism is discussed. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Cherimoya; Amino acids; Ammonia; Fruit; High CO2 levels; PAL; PEPC; Polyamines; Proteins; Reassimilation;Temperature

1. Introduction

In sink tissues, ammonia can be released duringbiochemical processes such as protein catabolism,amino acid deamination and some specific biosyn-

* Corresponding author. Tel.: +34-91-544-5607; fax: +34-91-549-3627.

E-mail address: merodio@if.csic.es (C. Merodio).

0925-5214/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

PII: S0 925 -5214 (01 )00196 -X

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thetic reactions such as those involving phenyl-propanoids and lignin biosynthesis (Lam et al.,1996 and references therein). Phenylalanine ammo-nia-lyase (PAL, EC 4.3.1.5.) catalyses the elimina-tion of ammonia from L-phenylalanine to yieldtrans-cinnamic acid, the first step in the phenyl-propanoid pathway. PAL activity has been foundin some fungi and in all higher plants analyzed butnot in animals. PAL, at a branch point betweenprimary metabolism and natural product biosyn-thesis, is a key step in regulation of the pathway.While many reports have demonstrated a correla-tion between changes in the levels of the enzymeand accumulation of major pheynylpropanoidproducts (Bate et al., 1994), little attention has beenpaid to the role of PAL as a source of ammonia(Singh et al., 1998). In previous work (Assis et al.,2001) we observed that PAL activity increased earlyin cherimoya fruit ripening at 20 °C and that suchan increase in PAL activity did not have a corre-sponding impact on the changes in the majorproduct, lignin, or on the total polyphenol content.However, it is clear that some compounds must bereleased in the PAL-catalyzed reaction from theconversion of phenylalanine to cinnamate. Ourworking hypothesis is that, given a high level ofPAL activity during cherimoya fruit ripening, am-monia must be continuously formed during thisprocess. We argue that the purpose of the increasein PAL activity early on in storage in air at 20 °Cis to ensure an adequate supply of available ammo-nia for further ripening metabolic processes.Clearly, ammonia released by these metabolic reac-tions must be rapidly reassimilated into the aminoacids and amides needed for building some specificripening proteins (Montero et al., 1995), and intosome nitrogenous compounds integrated in differ-ent responses triggered after harvest. We havepreviously reported a relationship between the risein putrescine content (with no variation in sper-midine and spermine levels) and the ripening pro-cess in cherimoya fruit (Escribano and Merodio,1994). Moreover, the ripening-related rise in pu-trescine levels was regulated via arginine decar-boxylase-activity-mediated synthesis and not by therelease of the polyamine from conjugated forms(Escribano et al., 1996). Since polyamines andethylene have antagonistic effects and share a

common intermediate in their biosynthetic path-ways, the availability of S-adenosylmethionine hasbeen the driver of most of the studies aboutpolyamines. But polyamines, which in some in-stances comprise up to 20% of the total solublenitrogen in the plant, are also part of a defencemechanism against environmental stress(Bouchereau et al., 1999). We have reported anincrease in nitrogen compounds involved in theadaptive response of plant tissues to the cytosolicacidification in CO2-treated fruit caused by storageat chilling temperatures (Merodio et al., 1998).Also, it has been proposed that polyamine accumu-lation is a result of detoxification of ammoniawhich is released in plant cells after exposure toseveral kinds of stress (Lovatt, 1990). However,little is known about the interaction between am-monia availability and polyamine levels in fruitduring ripening and under stress storage condi-tions.

In order to produce dicarboxylic acids as carbonskeletons for ammonia assimilation, the enzymephosphoenolpyruvate carboxylase (PEPC, EC4.1.1.31) plays an important role (Guy et al., 1989;Moraes and Plaxton, 2000). This enzyme has anadditional metabolic impact on ammonia reassim-ilation as a modulator of the levels of phospho-enolpyruvate (PEP). Since PEP is also a precursorfor the synthesis of phenylalanine, the substrate forthe phenylpropanoid pathway initiated by PAL, aco-ordinate regulation of PAL and PEPC must beoperating. Despite the involvement of these en-zymes in the well-interconnected nitrogen and car-bon metabolic pathways (Huppe and Turpin,1994), comparative analyses of PAL and PEPCactivities from the same fruit tissues have not beendescribed.

The aim of the present study is to highlight therelationship between the activation/deactivation ofphenylpropanoid pathways and ammoniaavailability in cherimoya fruit stored under differ-ent conditions. Evidence concerning the reassimila-tion of ammonia in cherimoyas was obtained byanalyzing the content of nitrogen sequestered inpolyamines. Changes in the levels of total proteinand free amino acids as reflecting biochemicalprocesses involved in the liberation of ammoniawere also analyzed. Comparative analyses of PALand PEPC activities from fruits under different

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conditions were also performed. We conclude thatthere is an interrelation between ammonia assimi-lation and activation of PAL, and that it is neces-sary for the ripening process. The results arediscussed in light of the possible involvement ofammonia as a metabolic signal for controllingnitrogen and carbon metabolism.

2. Materials and methods

2.1. Plant material

Cherimoya (Annona cherimola Mill. cv. Fino deJete) fruit from early in the season (October) wereharvested in Almunecar (Granada) and selectedfor freedom from defects. At the Instituto del Frıo(Madrid), fruit (named pre-stored) were randomlydivided into three groups of 15 cherimoyas each,placed in a 22 l glass jar and held at either 20 °C(two groups, named control and CO2-treated) or6 °C (one group, named chilling temperature).The CO2-treated group was maintained under acontinuous stream of a gas mixture containing20% CO2+20% O2+60% N2 with a flow rate of100 ml min−1. After 3 days of treatment, a groupof seven fruit was transferred to air and held at20 °C for 1 day (named CO2-removed). Cheri-moyas stored at both 20 and 6 °C without CO2

treatment were kept under a continuous stream ofair. After 3 days in each of the storage conditions,cherimoyas were sampled. Also, fruit were ana-lyzed 1 day after transfer to air. Each sampleconsisted of three cherimoyas randomly collected,peeled and quick-frozen in liquid nitrogen andstored at −80 °C. The remaining fruits wereused in determinations and assessment of fruitripeness and quality.

2.2. Polyamine analysis

The method described by Escribano and Mero-dio (1994) was used to perform free polyamineanalysis. Briefly, extracts for polyamine analysiswere prepared by homogenizing frozen tissues in5% (v/v) ice-cold perchloric acid (PCA) and 1.6-hexanediamine (125 nmol g−1 fresh weight) wasadded as an internal standard. After 1 h on ice the

homogenate was centrifuged at 27 000×g for 30min at 4 °C. The supernatant was removed andused for polyamine determination. Polyamineswere derivatized with dansyl chloride and ana-lyzed by HPLC. Samples were injected onto areverse phase C18 column (150×4.6 mm, 5 �mparticle diameter) and eluted with a programmedwater:methanol (v/v) solvent gradient progressingfrom 60 to 95% of methanol in 21 min at a flowof 0.9 ml min−1. Dansyl-polyamines were de-tected by a fluorescence spectrophotometer (exci-tation wavelength, 350 nm; emission wavelength,495 nm; model SFM 25, Kontron Instruments),and the peaks, areas and retention times werecomputer-recorded and calculated with Integra-tion Pak Program software (KontronInstruments).

To estimate the overall efficiency of nitrogenreassimilation in polyamines and to rapidly com-pare the effect of each treatment, the total numberof moles of nitrogen which were sequestered inpolyamines was calculated using the followingformula: total nitrogen in putrescine=2[Put] andtotal nitrogen in spermidine plus spermine=3[Spd]+4[Spn] (Turano and Kramer, 1993).

2.3. Ammonia and total amino acid measurement

Ammonia and total amino acid content wereanalyzed in the 5% ice-cold PCA supernatantobtained from polyamine extraction. For ammo-nia, 1 ml aliquots of supernatant were mixed with1 ml of 0.1 M Hepes buffer, pH 7.0, containing7% (w/v) PVPP and neutralized with 0.1–0.2 MKOH up to a pH value between 7.0 and 7.5. Aftercentrifugation at 10 000×g for 10 min, ammoniacontent in the supernatant was derived from thespectrophotometrically measured NADH oxida-tion produced by glutamate dehydrogenase in acoupled reaction assay (Boehringer Mannheim,Germany).

Amino acids were determined spectrophotomet-rically at 340 nm following the reaction between�-amino groups and o-phthaldialdehyde (OPA) inthe presence of �-mercaptoethanol, using glycineas a standard (Church et al., 1983).

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2.4. Total protein determination

Total protein content was analyzed in the 5%(v/v) ice-cold PCA precipitated extract obtainedfrom polyamine extraction. The pellet was resus-pended in 1 N NaOH and centrifuged, and theprotein content was measured by the method ofLowry et al. (1951) using bovine serum albumin(BSA) as a standard.

2.5. Phenylalanine ammonia-lyase acti�ity

The procedure described by Assis et al. (2001)was used for extraction and assay of PAL activity(PAL, EC 4.3.1.5.). Briefly, protein extract wasobtained by homogenizing 0.5 g acetone powderprepared from frozen pulp, in 5 ml 0.1 M sodiumborate buffer, pH 8.8, containing 5 mM �-mer-captoethanol, 2 mM EDTA and 4% (w/v) PVP at4 °C. After 1 h, the homogenate was centrifugedat 27 000×g for 30 min at 4 °C. The reactionmixtures contained 10 mM of L-phenylalanine, 30mM sodium borate buffer, pH 8.8, and 1 ml crudeextract in a total volume of 3 ml. The substratewas added after 10 min preincubation and thereaction was stopped with 0.1 ml 6 N HCl. PALactivity was determined by the production of cin-namate for 90 min at 30 °C under continuousshaking, measured by the absorbance change at290 nm (Zucker, 1965). Specific enzyme activitywas defined as nmol cinnamic acid per h and permg of protein.

2.6. Phosphoenolpyru�ate carboxylase acti�ity

Protein extracts for the PEP carboxylase activ-ity (PEPC, EC 4.1.1.31) assay were obtained byhomogenizing ground, frozen cherimoya meso-carp tissue (2.5 g fresh weight) at 4 °C in 7.5 mlof 50 mM Tris–HCl, pH 7.8, containing 10 mMMgCl2, 5 mM NaHCO3, 2 mM DTT, 0.25 mMEDTA and 2% (w/v) PVPP. The homogenate wascentrifuged at 20 000×g for 30 min at 4 °C.PEPC activity was determined spectrophotometri-cally at 340 nm by coupling the reaction toNADH oxidation in the presence of malate dehy-drogenase (MDH) as described by Munoz et al.(2001b). The standard assay medium contained

0.2 ml of crude enzyme extract (which containedMDH activity), 0.14 mM NADH, 10 mM MgCl2,12 mM NaHCO3, 10 mM DTT, 0.25 mM EDTAand 50 mM Tris–HCl buffer (pH 7.8). The reac-tion was started by the addition of PEP at 4 mMfinal concentration in the spectrophotometer cu-vette containing the assay mixture at 27 °C. Spe-cific enzyme activity was expressed as �mol ofPEP consumed per min per mg of protein.

Protein concentration in both enzymatic ex-tracts was measured by the Bradford (1976)method using a protein-dye reagent (Bio-Rad)and BSA as a standard.

2.7. Statistical analyses

Data from at least three replicates per samplewere processed by one-way analysis of variance(ANOVA) using the least significant test (Stat-graphics program, STSC, Rockville, MD, USA)to determine the level of significance at P�0.05.The results presented here represent data fromthree or more experiments performed insuccession.

3. Results

3.1. Changes in the le�els of free ammonia andpolyamine-nitrogen

The free ammonia content was analyzed incherimoyas under the different storage conditions.The values obtained, shown in Fig. 1, confirmthat storage at 20 °C for 3 days produced asignificant decrease (P�0.05) in the ammoniapool. As compared with control fruit, this de-crease was greater in cherimoyas treated withCO2,, where the lowest free ammonia levels werefound. However, when treated fruit were trans-ferred to air, the ammonia content increased. Incontrast, when cherimoyas were stored for 3 daysat chilling temperature, the free ammonia contentincreased sharply, reaching levels of 2.06 �molg−1 f.w.

Fig. 2 shows the total number of nmoles ofnitrogen per g of fresh weight which are incorpo-rated in polyamines (putrescine and spermidine

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Fig. 1. Endogenous levels of ammonia in cherimoya fruitstored under different conditions. Pre-stored (after harvest),control (3 days in air at 20 °C), CO2-treated (3 days in 20%CO2 at 20 °C), CO2-removed (CO2-treated transferred to airfor 1 day), and chilling temperature (3 days in air at 6 °C).Data are means of two separate experiments (n=6) and S.E.are shown by vertical bars.

Fig. 3. Total free amino acid levels in cherimoya fruit underdifferent storage conditions. The storage conditions and barsymbols are described in Fig. 1. Data are means of twoseparate experiments (n=6) and SE are shown by verticalbars.

gen was incorporated mainly into Spd+Spm. Areverse effect was found when these fruit weretransferred to air. After 3 days of storage atchilling temperature, the polyamine nitrogen lev-els declined sharply to just over 61% of the pre-stored value. Although the total nitrogencontained as polyamines was lower than that con-tained as free ammonia (e.g. 250 nmol polyaminesvs. 400 nmol ammonia, in the case of CO2-treatedfruit), the pattern of changes in polyamine nitro-gen reassimilation was consistent with the contentof free ammonia in fruit under the different treat-ments (Fig. 1).

3.2. Changes in the total titres of free aminoacids and proteins

Total free amino acid contents in fruit underdifferent storage conditions were also determined(Fig. 3). Total amino acids titres were 1.5 timeshigher in cherimoyas of CO2-removed and controlgroups than in pre-stored fruit. However, freeamino acids decreased significantly (P�0.05) infruit after 3 days of high CO2 levels. CO2-treatedfruit presented the lowest levels of free aminoacids. Quantitative measurements of total protein(Fig. 4) showed that whereas there was a signifi-cant increase in the accumulation of protein incontrol fruit, high CO2-treated fruit contained asimilar amount of protein to pre-stored fruit.

plus spermine). Total polyamine nitrogen in-creased in control, CO2-treated and CO2-removedgroups of fruit over the levels of pre-stored fruit,although the distribution among specificpolyamines was different between the differentstorage conditions. In control fruit, whereas thelevels of spermidine and spermine (Spd+Spm)decreased, putrescine increased almost 3-fold. Un-der CO2 treatment on the other hand, the nitro-

Fig. 2. Changes in the amount of nitrogen incorporated in freepolyamines (total titres and putrescine vs. spermidine plusspermine levels) in cherimoya fruit stored under differentconditions. The storage conditions are described in Fig. 1.Data are means of two separate experiments (n=6).

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After transfer to air, protein content increased,reaching similar values to the control. In the caseof fruit stored for 3 days at low temperature,there was non-significant variation in the initialamount of total protein.

3.3. Changes in PAL and PEPC acti�ities

Fig. 5 shows PAL and PEPC activities in cheri-moyas under different storage conditions. PALactivity increased to a similar extent in both con-trol fruit (up to 2.4-fold) and in cherimoyas sub-jected to high-CO2 treatment (up to 2.1-fold).When CO2 was removed, the level of PAL activityin these cherimoyas declined to a value similar tothat observed for pre-stored fruit. After 3 days ofstorage at 6 °C, PAL activity was found to beconsiderably lower than in fruit stored at ambienttemperature and even lower than in fruit immedi-ately after harvest.

The pattern of changes in PEPC activity (Fig.5) was exactly the reverse of that described forPAL. Pre-stored fruit exhibited high PEPC activ-ity (25.94�3.3 �mol min−1 mg−1 protein). Stor-age at ambient temperature produced a decreasein PEPC activity contrasting with the high enzymeactivity detected in fruit stored at chilling temper-ature. At the end of 3 days of CO2 treatment,PEPC activity was similar to that of control fruit;however, this activity increased after transfer toair.

Fig. 5. Changes in PAL and PEPC activities in cherimoya fruitunder different storage conditions. The storage conditions andbar symbols are described in Fig. 1. Data are means of twoseparate experiments (n=6) and SE are shown by verticalbars.

4. Discussion

The term ‘ammonia’ is used in this study torefer to the compound, without defining its stateof protonation, as in Kleiner (1981). The presentwork showed changes in ammonia levels in cheri-moya fruit in response to storage under differentconditions. King and Morris (1994) also reporteddifferences in ammonia content in the varioussections of broccoli during postharvest senes-cence. Since one of the routes of ammonia reas-similation runs via glutamine, the urea cycle andultimately into polyamines, the total number ofmoles of nitrogen incorporated in polyamines wascalculated to estimate the overall efficiency ofammonia reassimilation. Our data show a sharpdecrease in ammonia content paralleling an in-crease in total polyamine nitrogen in cherimoyasstored at 20 °C, either in air or under high levelsof CO2. However, in the case of fruit stored atchilling temperature, there was a massive accumu-

Fig. 4. Total protein levels in cherimoya fruit under differentstorage conditions. The storage conditions and bar symbolsare described in Fig. 1. Data are means of two separateexperiments (n=6) and SE are shown by vertical bars.

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lation of ammonia. Accumulation of ammoniumhas also been reported in stressed plants (Laz-cano-Ferrat and Lovatt, 1988). The accumulationof ammonia in fruit stored at chilling temperaturecould be explained by an active polyaminecatabolism together with some restriction on am-monium reassimilation. On the other hand, thelow levels of both ammonia and amino acids andthe high levels of polyamines found in CO2-treated fruit suggest that ammonia reassimilationis activated by CO2 treatment at ambient temper-ature. Furthermore, the observed decrease inpolyamine content accompanying the increase inthe amino acid pool and ammonia levels whenCO2-treated fruit were transferred to air, seems toconfirm the positive correlation between reassimi-lation of ammonia into polyamines and high-CO2

treatment. The accumulation of polyamines ob-served in CO2-treated fruit may exert a protectivefunction that could be related to some of the rolesattributed to these compounds, such as free radi-cal scavenging or pH-balancing cations (Smith,1985).

The suppression of ripening-associated gene ex-pression has been reported in fruit subjected to ahigh CO2 concentration (Rothan et al., 1997), butthe present results prompt the speculation thatpreferential use of amino acids for non-proteinnitrogenous compounds through CO2 treatmentcould help to prevent the increase in proteincontent observed in ripening control fruit. How-ever, when CO2 was removed and fruit was ableto ripen, there was an increase in the turnover ofprotein, the same as in control fruit stored atambient temperature. Furthermore, non-synthesisof specific ripening proteins may be responsiblefor the delay observed in ripening process inCO2-treated fruit (Del Cura et al., 1996).

The specific metabolic reaction catalyzed byPAL, involving conversion of L-phenylalanine totrans-cinnamate, is also an important source ofammonia in plant tissues. Several reports haveshown that PAL regulation is complex, and thatexpression and activity are responsive to environ-mental and development signals (Liang et al.,1989; Solecka and Kacperska, 1995; Sanchez-Ballesta et al., 2000). It has also been reportedthat PAL activity is inhibited by products of the

phenylpropanoid pathway (Shields et al., 1982).Our results suggest that PAL activity increased infruit having a high ammonia demand and de-creased in fruit having a low rate of ammoniareassimilation, reflecting the metabolic status im-posed by storage in different conditions. It isinteresting to note that since assimilation/detoxifi-cation of ammonia is a highly energy-intensiveprocess (Givan, 1979), the low rate of ammoniareassimilation in chilled fruit is consistent with themaintenance of ATP values previously observedin storage of cherimoya fruit at chilling tempera-ture (Munoz et al., 2001b).

The involvement of carbon metabolism in theassimilation of nitrogen, and conversely the effectof this on the rate and route of carbonmetabolism is well known (Huppe and Turpin,1994). The integration of these two importantmetabolic processes must involve extensive regula-tion and coordination between regulatory en-zymes. PEPC catalyses the irreversiblecarboxylation of PEP and bicarbonate and playsan important role in carbon economy. Since PEPis also involved in the synthesis of phenylalanine,the substrate for the phenylalanine pathway ini-tiated by PAL, regulation of these two enzymescan be expected to be coordinated. On analyzingthe effect of different storage conditions, we ob-served that PAL activity increased in cherimoyasexhibiting low PEPC activity. We suspect thatPAL activity increased in fruit with high PEPavailability as a result of non utilization by PEPC.These metabolic characteristics occur in fruitstored at ambient temperature in air and underhigh CO2-treatment. When CO2-treated fruit weretransferred to air, PEPC was activated and PALactivity decreased. It is possible that the increaseddemand for carbon skeletons created by an ele-vated carbon flux to amino acids was met by thediversion of fixed carbon to the anaplerotic path-way, through activation of PEPC and a conse-quent decrease in the content of PEP available forPAL. In addition, in the case of PEPC extractedfrom cherimoyas treated with high CO2 levels, wepreviously reported (Munoz et al., 2001a) changesin the kinetic constant (Km for PEP) and sensitiv-ity to malate in order to adapt its activity to thedemand of C4-carbon skeletons imposed by highCO2 treatment

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Furthermore, the co-ordinate response of PALand PEPC must be mediated by regulatory signalsshared among these enzymes. The variations inthe amount of ammonia parallel to those in thePEPC and opposed to those in PAL activitysuggest that ammonia levels are probably a sig-nificant metabolic signal for controlling nitrogenand carbon metabolism in fruit. The role of am-monia as a signalling molecule is poorly under-stood (Coruzzi and Bush, 2001), and the way inwhich ammonium is sensed requires further study.The accumulation of free ammonia in cherimoyasstored at chilling temperature concurs with a re-ported decrease in cytoplasmic pH (Munoz et al.,2001b), and, therefore, a pH-dependent signal canbe expected. Moreover, PEPC and PAL activitiesare modulated by changes in pH value (Hagen-doorn et al., 1990; Mateos et al., 1993; Echevarriaet al., 1994)

In conclusion, this paper reports the activationof phenylalanine deamination in fruit having ahigh ammonia demand for further reassimilation.Our results also confirm that the patterns ofchange in PAL activity under different storageconditions are the reverse of patterns of change inPEPC activity, reflecting the interrelationship be-tween carbon and nitrogen metabolism. More-over, levels of ammonia, whether directly orthrough changes in pH value, may constitute asignificant metabolic signal for the co-ordinatedresponse required to meet the demands created bythe ripening process. Our current research contin-ues to focus determining the role of ammonia inregulation of PAL.

Acknowledgements

This work was supported by a grant fromSpain, CICYT (ALI99-0954-C03-01). We thankDr Molina for his helpful suggestions to improvethe manuscript.

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