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Postharvest Biology and Technology 44 (2007) 34–41

Cloning and expression analysis of phenylalanine ammonia-lyase inrelation to chilling tolerance in harvested banana fruit

Yong Wang a,b,1, Jian-Ye Chen a,c,1, Yue-Ming Jiang b, Wang-Jin Lu a,c,∗a College of Horticultural Science, South China Agricultural University, Guangzhou 510642, The People’s Republic of China

b South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, The People’s Republic of Chinac Guangdong Key Laboratory for Postharvest Science, Guangzhou 510642, The People’s Republic of China

Received 20 June 2006; accepted 9 November 2006

bstract

Bananas are highly susceptible to chilling injury (CI) and phenylalanine ammonia-lyase (PAL), as a key enzyme involved in plant phenyl-ropanoid metabolism, has been associated with low temperature stress in plant tissues. However, little is known about the role of PALincluding PAL activity, gene and protein expression) in postharvest chilling tolerance of banana fruit. Two partial cDNAs sequences (MaPAL1nd MaPAL2) with about 760 bp were cloned from banana pulp by RT-PCR. Western and northern hybridizations were used to investigatexpression of PAL protein and PAL genes in fruit stored for 10 days at 7 ◦C (chilling temperature) and then transferred to 22 ◦C (roomemperature). The effects of propylene (a functional ethylene analog) on their expression in relation to CI were also examined. Northernnd western blot analyses revealed that mRNA transcripts of MaPAL1 and MaPAL2 and PAL protein levels in banana fruit during storagencreased, reaching a peak at about day 8, and finally decreased at chilling temperature. Prior to low temperature storage, pretreatment withropylene could alleviate CI and enhance PAL activity, protein amount and mRNA transcripts of MaPAL1 and MaPAL2. Moreover, changes

n PAL activity, protein amount and accumulation of MaPAL1 and MaPAL2 exhibited almost the same patterns. The results suggest thathe induction of PAL in banana fruit during low temperature storage is regulated at transcriptional and translational levels, and is related toeduction in CI symptoms.

2006 Elsevier B.V. All rights reserved.

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eywords: Banana; Chilling injury; PAL; Ethylene; Activity; Expression

. Introduction

Banana fruit are highly susceptible to chilling injury (CI)Pantastico et al., 1990). Storage at low temperatures (<13 ◦C)esults generally in peel pitting, discoloration and abnormalruit ripening. CI is a limiting factor in extending storageife and is responsible for substantial postharvest loss in

any areas of cultivation. Research has been undertaken intonderstanding CI mechanisms, which, in turn, helps develop

ffective methods to prevent the occurrence of CI in bananaruit. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), a keynzyme involved in phenylpropanoid metabolism, catalyses

∗ Corresponding author. Tel.: +86 20 85280229; fax: +86 20 85282107.E-mail address: wjlu@scau.edu.cn (W.-J. Lu).

1 These authors contributed equally to this work.

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henylalanine to trans-cinnamic acid, which is the first stepn the biosynthesis of phenylpropanoids, leading to a diverseroup of plant secondary metabolites, including lignins, phy-oalexins, and flavonoids. In addition, PAL is induced byarious biotic (e.g. infection by viruses, bacteria and fungi)nd abiotic (e.g. low and high temperatures, UV-B light andounding) stresses (Hahlbrock and Scheel, 1989; Dixon andaiva, 1995; Strack, 1997). These findings suggest that PALlays an essential role in modulating the resistance of plantissues to such stresses.

Ripening of banana fruit is associated with a sharp increasen ethylene production (Seymour, 1993; Golding et al., 1998),

nd is initiated either by the natural production of endogenousthylene as banana fruit reach full physiological maturity ory use of commercial exogenous ethylene ripening proce-ures (Wills et al., 2001). Application of exogenous ethylene

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r propylene (a functional ethylene analog) at a non-chillingemperature prior to storage at low temperature alleviates theevelopment of CI symptoms in citrus fruit (Lafuente et al.,001) and banana fruit (Wang et al., 2006). Moreover, treat-ent with exogenous ethylene stimulates PAL activity in theavedo of mature grapefruit (Riov et al., 1969), while theensitivity of mandarins to chilling correlates with low con-titutive levels of PAL mRNA and activity upon exposure ofhe fruit to low temperature (Sanchez-Ballesta et al., 2000b).dditionally, the increase in PAL activity and PAL mRNA

evels, but not in ethylene, stimulated by chilling is affectedy the age of the fruit (Lafuente et al., 2003). These findingsndicate that more molecular details about the roles of PALnd ethylene in relation to CI in harvested fruit require furthernvestigation.

To the best of our knowledge, there has been no report onxpression profiles of PAL genes and PAL protein in relationo CI in banana fruit. The objectives of the present workere to clone PAL genes from banana fruit and investigate

he relationships between PAL and CI, and then evaluate theffect of exogenous propylene on activity, protein levels andRNA transcripts of PAL in relation to CI.

. Materials and methods

.1. Plant material

Fruit of mature pre-climacteric banana (Musa sp. cv.illiams, Cavendish sub-group AAA) were obtained fromlocal commercial plantation near Guangzhou. Hands were

eparated into individual fingers. Individual fruit were dippedor 2 min in a 500 �L/L Sportak (a.i. prochloraz) fungicideolution to control disease (Wade et al., 1993). They werehen allowed to air-dry at 25 ◦C for 2 h. Fruit were selectedor freedom from visual defects and for uniformity of weightnd shape and then divided at random into two groups. Fruitrom the first group were placed into unsealed plastic boxes150 fingers/box) and then pre-treated with 1000 �L/L propy-ene (a functional ethylene analog) at 22 ◦C for 16 h. Theecond group of 150 fruit fingers were placed into anothernsealed plastic boxes and left ungassed (0 �L/L propylene,ontrol). Both control and pre-treated fruit were subsequentlytored at 7 ◦C (chilling temperature) for 10 days and sam-led every 2 or 3 days for measurements. For another furtherampling, fruit from the two different treatment groups wereransferred to 22 ◦C after 8 days of storage at 7 ◦C. All ofhe fruit were then treated with 1000 �L/L propylene for6 h and held at 22 ◦C to ripen for 5 days, prior to samplingvery 1 or 2 days for analyses. For each sample, peel andulp of 10 banana fruit were separated, then frozen imme-iately in liquid nitrogen and finally stored at −80 ◦C prior

o use. Each treatment consisted of three independent repli-ates. In this study, two incubators (Sanyo MIR 553 model,unma, Japan) were used for fruit storage at 7 and 22 ◦C,

espectively.

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d Technology 44 (2007) 34–41 35

.2. Assay for PAL activity

PAL activity was measured as previously described byolecka and Kacperska (2003) with a minor modification.ll experimental procedures were carried out at 4 ◦C. Briefly,eel or pulp tissues (2 g) were homogenized with mortar andestle in 6 mL of extraction buffer [50 mM Tris–HCl buffer,H 8.8, 15 mM �-mercaptoethanol, 5 mM EDTA, 5 mMscorbic acid, 10 �M leupeptin, 1 mM PMSF and 0.15%w/v) PVP]. The homogenate was filtrated through four lay-rs of cheesecloth and centrifuged at 12,000 × g for 20 mint 4 ◦C. The supernatant was used as the crude enzyme. Theeaction mixture (3 mL) contained 16 mM l-phenylalanine,0 mM Tris–HCl buffer (pH 8.8), 3.6 mM NaCl and 0.5 mLf enzyme solution. Incubation was performed at 37 ◦C forh and the reaction was stopped by the addition of 500 �Lf 6 M HCl. The reaction mixture was then centrifuged for0 min at 12,000 × g to pellet the denatured protein. Thebsorbance was measured at 290 nm before and after incuba-ion. One unit of enzyme activity was defined as the amountf PAL that produced 1 �mol of cinnamic acid within 1 h andas expressed as �mol cinnamic acid/mg protein/h. Proteinas estimated according to Bradford (1976) using BSA as a

tandard.

.3. Protein extraction and Western blot analysis

Total proteins were extracted according to Campos-Vargast al. (2005) with a minor modification. The extraction bufferontained 50 mM Tris–HCl (pH 8.8), 5 mM ascorbic acid,mM EDTA, 1 mM PMSF, 14 mM �-mercaptoethanol and.15% (w/v) PVP. The extracted proteins were separatedy SDS-PAGE on a 12% polyacrylamide gel as describedy Laemmli (1970). The same amount (10 �g) of proteinas loaded per lane. After electrophoresis, the proteins were

lectro-transferred to nitrocellulose (0.45 �m, AmershamIFE SCIENCE) using a transfer apparatus (Bio-Rad) by

he method of Isla et al. (1998). After rinsing in TBS buffer10 mM Tris–HCl (pH 7.5) and 150 mM NaCl], the mem-rane was blocked for 2 h with 3% (w/v) BSA in 0.05% (v/v)ween 20 and TBS, and then incubated with gentle shakingor 3 h in a 1000-fold diluted solution of a primary poly-lonal PAL antibody (kindly provided by Prof. N. Amrhein) at5 ◦C. Following extensive washes with TBST buffer [TBS,.05% (v/v) Tween 20], the membrane was incubated withoat anti-rabbit IgG-alkaline phosphatase conjugate (Sigma,t. Louis; 1:1000 diluted in TBST) at 25 ◦C for 1 h andgain washed with the above TBST buffer. The membraneas stained with a 10 mL solution containing nitro blue

etrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phos-hate (BCIP) in the dark. The reaction was terminated

y the addition of double distilled water. The amount ofAL protein was quantified by scanning the NC membranesfter immunoblotting with a densitometer using ImageQuantoftware.

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PAL activities of banana peel and pulp tissues tendedto increase during storage at 7 ◦C (Fig. 1). Moreover, theincrease in PAL activity was greater in the propylene-treatedfruit than in control fruit both when stored at 7 ◦C or trans-

Fig. 1. Effect of propylene treatment on PAL activity in banana peel andpulp tissues. Fruit were pre-treated with 0 (control) or 1000 �L/L propy-lene for 16 h at 22 ◦C, prior to storage at 7 ◦C, and then divided into two

6 Y. Wang et al. / Postharvest Bio

.4. RNA extraction and cloning of banana PAL cDNAs

Total RNA from banana fruit was extracted using the hotorate method (Wan and Wilkin, 1994). Frozen tissues (10 g)rom the peel or pulp of banana fruit were ground to a fineowder in a mortar using a pestle in the presence of liquiditrogen. Two ends of six individual banana fingers were dis-arded. The mid-part of about 5 cm in length was peeled, andeel and pulp were then cut separately into small pieces. Theeel and pulp samples were well mixed and finally separatednto three replicates.

Total RNA extracted from banana pulp was useds templates for the RT-PCR. The product (the first-trand cDNA) was subjected to PCR amplification.egenerate primers were designed with reference to the

onserved amino acid sequences of PALs, i.e. sense:′-TANGGNGTYACNACNGGNTTYG-3′ and antisense:′-GGNCCNARCCAN TGNGGNGANC-3′ (N = A, C, G or). Reactions for the RT-PCR were subjected to one cycle of4 ◦C for 3 min, 35 cycles (94 ◦C for 1 min, 50 ◦C for 2 minnd 72 ◦C for 2 min) and one cycle of 72 ◦C for 10 min. PCRroducts of the predicted size (about 760 bp in length) wereurified and cloned into pMD-18T vector (TaKaRa, Shiga,apan). The nucleotide sequences of the cDNA inserts wereetermined using a DNA sequencer (Model ABI 377, USA)ith either -21M13 or M13 sequencing primers, according

o the manufacturer’s instructions.

.5. Northern blot analysis

Total RNA (10 �g) was separated on a 1.2% agarose–ormadehyde gel and capillary blotted onto the PVDF mem-rane (Biodyne®B 0.45 �m, PALL). The membrane waslot-dried and cross-linked with UV at 280 nm. DIG-labeledpecific probes of 3′-untranslated regions of MaPAL1 andaPAL2 were made using the PCR DIG probe synthesis Kit

Roche Applied Science, Mannheim, Germany) and primersor MaPAL1 (sense: 5′-ATCTCGTCCCGCTGTCTTAC-3′nd antisense: 5′-TCCCCTCCAGTATGT GCTCCA-3′) andor MaPAL2 (sense: 5′-TTCCCCTCCGTGGCACTATC-3′nd antisense: 5′-GCTTGTGGGTGAGGTGGTCG-3′). Theembrane was hybridized with the DIG-labeled probe for

6 h at 45 ◦C in high-SDS buffer (7% SDS, 5× SSC,0 mmol/L sodium-phosphate, pH 7.0, 2% blocking reagentnd 0.1% N-lauroylsarcosine) containing 50% deionized for-amide (V/V) (Roche, Germany). Blots were washed twice

t 37 ◦C in 2× SSC and 0.1% SDS for 10 min, followed byashing twice at 62 ◦C in 0.1× SSC and 0.1% SDS for0 min. The membranes were then subjected to immuno-ogical detection following the manufacturer’s instructionsRoche Applied Science).

.6. Statistics

The experiments were arranged in a completely random-zed design, and each treatment comprised three replicates.

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d Technology 44 (2007) 34–41

ata were presented as means ± standard errors (S.E.). Leastignificant differences (LSD) were calculated to compareignificant effects at the 5% level.

All treatments were repeated at least three times whilell samples were analyzed three times. Means and standardrrors were calculated from pooled data. In the figures, theertical line associated with each point represents the stan-ard error.

. Results

.1. CI symptoms

The relationship between CI symptoms and the ethyleneroduction rate of banana fruit was shown in our previ-us report (Wang et al., 2006). Banana fruit began to showI symptoms evident as pitting and brown patches on the

kin after 4 days of storage at 7 ◦C. CI symptoms increasedarkedly when fruit were removed from 7 to 22 ◦C. Pre-

reatment with propylene, prior to storage at 7 ◦C, delayed theppearance of CI symptoms by 3 days. Moreover, the propy-ene pre-treated fruit that were stored for 8 days at 7 ◦C andransferred to 22 ◦C with propylene treatment for initiationf ripening, had less CI symptoms.

.2. PAL activities of banana peel and pulp as affectedy propylene treatment

roups. The first group of fruit was stored at 7 ◦C for 10 days. The secondroup was stored for 8 days at 7 ◦C, transferred to 22 ◦C and treated againith 1000 �L/L propylene and then held at 22 ◦C for 5 days to ripen. Eachalue represented the mean ± S.E. of three replicates. An asterisk indicatesignificant differences between control and treatment at the 5% level.

Y. Wang et al. / Postharvest Biology and Technology 44 (2007) 34–41 37

Fig. 2. Western blot analysis of PAL in banana peel tissues of control fruit (a and b) and propylene-treated fruit (c and d). Fruit were pre-treated with 0 (control)or 1000 �L/L propylene for 16 h at 22 ◦C, prior to storage at 7 ◦C, and then divided into two groups. The first group of the fruit was stored at 7 ◦C for 10 days.The second group was stored for 8 days at 7 ◦C, transferred to 22 ◦C and treated again with 1000 �L/L propylene and then held at 22 ◦C for 5 days to ripen.Equal protein amounts (10 �g) were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Thereafter, the PAL amount was immunodetectedw d) of imd fter 8 da8 at 22 ◦C

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ith the specific antibody. Image (a and c) and quantitative analysis (b andays of storage at 7 ◦C, and subsequent 1, 3 and 5 days of storage at 22 ◦C aand 10 days of storage at 7 ◦C, and subsequent 1, 3 and 5 days of storage

erred to 22 ◦C and treated again with propylene and theneld at 22 ◦C to ripen. This is in agreement with the report byamo et al. (2000), who found that ethylene treatment could

ncrease PAL activity in banana fruit. The maximum level ofAL activity was obtained after 8 days of storage at 7 ◦C. TheAL activities of peel and pulp tissues of propylene-treatedruit increased 1.18- and 1.73-fold, respectively, compared

o the control fruit. After storage at 7 ◦C, the PAL activityf peel tissues began to decrease during subsequent stor-ge at 22 ◦C while that of pulp tissues changed only slightlyFig. 1).

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ig. 3. Western blot analysis of PAL in pulp of control (a and b) and propylene trearopylene for 16 h, prior to storage at 7 ◦C, and stored at 7 ◦C for 10 days. Some codays, and treated again with 1000 �L/L propylene, then held at 22 ◦C for 5 days to

ransferred to a nitrocellulose membrane. Thereafter, the PAL amounts were immund quantitative analysis (b and d) of immunoblotting bands. Lanes 1–8 in (a) conemoved from 7 to 22 ◦C after cold storage. Lanes 1–8 in (c) propylene-treated fruirom 7 to 22 ◦C after cold storage.

munoblotting bands. Lanes 1–8 in (a) control fruit: after 0, 3, 6, 8 and 10ys of storage at 7 ◦C. Lanes 1–8 in (c) propylene-treated fruit: after 0, 3, 6,after 8 days of storage at 7 ◦C.

.3. Amounts of PAL protein of banana peel and pulpissues affected by propylene treatment

A peptide of 70 kDa was specifically detected with thenti-parsley PAL polyclonal antibody, indicating that PALroteins exist both in peel and pulp tissues of propylene-reated and control banana fruit (Figs. 2 and 3). The immuneignal in the peel tissues (Fig. 2) was noticeably stronger than

n the pulp tissues (Fig. 3), while the immune signal in theropylene-treated fruit (including peel and pulp tissues) waslso stronger than that in control fruit.

tment (c and d) of banana fruit. Fruit were pre-treated with 0 or 1000 �L/Lntrol and pre-treated fruit were transferred to 22 ◦C after storage at 7 ◦C forripen. Equal amounts of protein (10 �g) were subjected to SDS-PAGE andnodetected with the specific antibody. Image (a and c) of immunoblottingtrol fruit: after 0, 3, 6, 8 and 10 days of cold storage, after1, 3 and 5 dayst: after 0, 3, 6, 8 and 10 days of cold storage, after1, 3 and 5 days removed

38 Y. Wang et al. / Postharvest Biology and Technology 44 (2007) 34–41

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PAL protein amounts in peel tissues of control andropylene-treated fruit increased, then reached a maximumfter 8 days of storage at 7 ◦C, and finally decreased slightlyfter 10 days (lanes 1–5 in Fig. 2a and c). PAL proteinmounts of fruit that were treated with 1000 �L/L propy-ene and then held at 22 ◦C for 5 days to ripen after storaget 7 ◦C decreased and were lowest after 5 days of storaget 22 ◦C (lanes 6–8 in Fig. 2a and c). Fig. 2 further showshat the immune signal in the propylene-treated peel tissuesas obviously stronger than that in control fruit at any phase

Fig. 2b and d).As with peel tissues, PAL protein amounts in pulp tissues

f control and propylene-treated fruit showed almost the sameattern (lanes 1–8 in Fig. 3a and b and lanes 1–8 in Fig. 3cnd d).

.4. Isolation and sequence analysis of cDNAs of PALrom banana fruit

Two partial cDNA fragments about 760 bp in length wereloned using total RNA from pulp tissues by RT-PCR. ALAST search of GenBank revealed that MaPAL1 shared4% identity with ZmPAL while MaPAL2 shared 83% iden-ity with OsPAL. Thus, the two fragments were consideredo be cDNA fragments of PAL, named as MaPAL1 and

aPAL2, respectively (Fig. 4). As a partial MaPAL cDNAequence registered as AJ555536 in GenBank was locatedn the different regions with the two PAL cDNA sequencesf this study in the PAL full length sequence, it is difficult

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PAL isolated from pulp tissues of banana fruit using the method of RT-PCR.known PAL sequences (Schuster and Retey, 1995) are indicated.

o know whether AJ555536 is different from MaPAL1 andaPAL2.In addition, MaPAL1 and MaPAL2 shared 81% identity

n nucleotide sequence and 87% identity in amino acids.educed amino acid sequences of MaPAL1 and MaPAL2

ontained two serines, at positions 96 and 103, which areuggested to be conserved at the active site of all known PALenes (Schuster and Retey, 1995).

.5. Accumulation of MaPAL1 and MaPAL2 mRNAs ofanana peel and pulp tissues affected by propylenereatment

PAL genes are expressed in all plant tissues investigatedDixon and Paiva, 1995). In this study, mRNAs of MaPAL1nd MaPAL2 could be detected in both peel and pulp tissuesf banana fruit, while accumulation of the MaPAL1 transcriptas slightly higher than that of MaPAL2 (Figs. 5 and 6). Accu-ulation of MaPAL1 and MaPAL2 transcripts in peel and pulp

issues of banana fruit at 7 ◦C increased, then reached a peak atbout day 8 and finally decreased (Fig. 5), which was in accor-ance with activities and protein amounts of PAL (Figs. 1–3).herefore, it might be suggested that the chilling temperature

nduced the expression of MaPAL1 and MaPAL2. Moreover,he pretreatment with propylene stimulated the expression of

aPAL1 and MaPAL2 in the peel and pulp tissues of fruittored at 7 ◦C (Fig. 5) or in fruit transferred to 22 ◦C andreated again with propylene and then held to ripen (Fig. 6).n addition, the mRNA transcripts of MaPAL1 and MaPAL2

Y. Wang et al. / Postharvest Biology and Technology 44 (2007) 34–41 39

Fig. 5. Changes in mRNA accumulation of MaPAL1 and MaPAL2 in peel and pulp tissues for banana fruit pre-treated with 0 or 1000 �L/L propylene for 16 hand then stored at 7 ◦C for 10 days. Total RNA (10 �g per lane) was used for RNA gel blot analysis and hybridized with DIG- labeled PAL probes. Ethidiumbromide staining of rRNA is shown as the loading control.

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ig. 6. Changes in mRNA accumulation of MaPAL1and MaPAL2 in peel antored for 8 days at 7 ◦C, transferred to 22 ◦C and treated again with 1000ane) was used for RNA gel blot analysis and hybridized with DIG- labeled

ncreased firstly and then decreased when fruit stored at 7 ◦Cor 8 days were transferred to 22 ◦C and treated again with000 �L/L propylene to ripen. Greater expression of MaPAL1nd MaPAL2 was observed in the peel than in the pulp tissuesoth when fruit were stored at 7 ◦C (Fig. 5) or transferred to2 ◦C and treated again with 1000 �L/L propylene and theneld to ripen (Fig. 6), which was in agreement with the facthat PAL exhibited higher activity in the peel than in the pulpissues (Fig. 1).

. Discussion

Propylene is a functional ethylene analog and, at higheroncentrations, has the same effect in alleviating CI symp-oms and accelerating ripening of banana fruit as ethyleneStavroulakis and Sfakiotakis, 1997). In this study, applica-

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tissues for banana fruit pre-treated with 0 or 1000 �L/L propylene for 16 h,opylene and then held at 22 ◦C for 5 days to ripen. Total RNA (10 �g perobes. Ethidium bromide stained rRNA is shown as the loading control.

ion of exogenous propylene (1000 �L/L) prior to storage at◦C delayed the appearance of CI symptoms by 3 days onanana fruit. Moreover, the propylene-pretreated fruit thatere stored at 7 ◦C for 8 days, then transferred to 22 ◦C and

reated with propylene again for initiation of ripening had lessI symptoms, which reinforced the suggestion that ethylenenhances tolerance of banana fruit to chilling temperatures.his observation is in agreement with the reports of Zhou etl. (2001) and Lafuente et al. (2001, 2004), who found thatthylene plays a positive role in alleviating CI symptoms ofectarines and citrus fruit.

PAL is the key enzyme between the shikimate pathwaynd secondary, phenylpropanoid metabolism (Dixon et al.,

992). The latter is also related to the plant defense sys-em (Dixon and Paiva, 1995). In previous investigations,AL activity could be induced by various stresses, suchs chilling (Sanchez-Ballesta et al., 2000a; Lafuente et al.,

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003), wounding (Campos-Vargas et al., 2005), UV-B lightTeklemariam and Blake, 2004), ozone (Sgarbi et al., 2003),athogen invasion (Jones, 1984) and plant hormones includ-ng jasmonic acid, salicylic acid and abscissic acid (Wen etl., 2005; Chen et al., 2006). In addition, there are a feweports on PAL activity involved in chilling susceptibilitynd ethylene production in harvested fruit (Martınez-Telleznd Lafuente, 1997; Lafuente et al., 2001, 2003, 2004). Thenduction of PAL in response to low temperature reduces theevelopment of CI symptoms in citrus fruit (Lafuente et al.,001). Furthermore, the low temperature-induced PAL acti-ation is concomitant with the rise in ethylene productionhen mandarin fruit are exposed to cold stress (Martınez-ellez and Lafuente, 1997). The present work showed thatpplication of 1000 �L/L propylene induced a rapid and sub-tantial increase in PAL activity in banana fruit at the chillingemperature, especially after 8 days of storage (Fig. 1), andlleviated development of CI symptoms, as showed in ourrevious report (Wang et al., 2006). The results further rein-orce the suggestion that an increase in PAL activity maye involved in reducing CI and that the reduction of the CIan be achieved by applying exogenous ethylene. However, aigher correlation in banana peel tissues was found betweenhe change in PAL activity and CI development. Nguyen et al.2003, 2004) reported that modified atmosphere packagingeduced CI symptoms of banana fruit, which was possiblyue to lower PAL activity. Thus, such contradictory resultsoncerning the precise role of PAL in regulating CI of bananaruit are yet to be fully explained.

PAL activity is most affected by enhanced gene tran-cription or translation (Bolwell, 1992; Thulke and Conrath,998; Campos-Vargas et al., 2005), or slow turnover (Crosbynd Vayda, 1991). In ‘Romanine’ lettuce tissues, woundingnduces PAL activity by increasing the turnover of the inducedAL protein (Campos-Vargas et al., 2005). The same results also observed in grape berries, where salicylic acid couldctivate PAL activity by enhancing the synthesis of new PALrotein (Wen et al., 2005; Chen et al., 2006). In the presenttudy, a 70 kDa peptide of banana fruit was specificallyetected both in propylene-treated and control fruit usinghe anti-parsley PAL polyclonal antibody on SDS-PAGEel. Furthermore, propylene treatment or storage at chillingemperature enhanced the accumulation of the 70 kDa pep-ide, particularly at day 8 of storage at chilling temperatureFigs. 2 and 3). These results are consistent with the change inAL activity (Fig. 1). Thus, it is suggested that the PAL acti-ation by propylene or chilling temperature may be attributedo the synthesis of new PAL protein.

In most plants, PAL is encoded by a small multi-gene fam-ly with two to six members (Zhu et al., 1995), while its family

embers are differentially expressed in plant tissues as wells in the response to different stress conditions (Liang et al.,

989). Sanchez-Ballesta et al. (2000a, 2000b) found that lowemperature could induce the accumulation of PAL mRNAn chilling-sensitive citrus fruit. In this study, two partialDNAs sequences (MaPAL1 and MaPAL2) of about 760 bp in

D

d Technology 44 (2007) 34–41

ength were cloned from banana pulp tissues, and propylenereatment or storage at chilling temperature enhanced mRNAranscripts of MaPAL1 and MaPAL2, particularly at day 8 oftorage at chilling temperature (Fig. 5). Furthermore, greaterxpression of MaPAL1 and MaPAL2 in the peel tissues wasbserved than in pulp tissues (Fig. 5). Therefore, it is sug-ested that PAL activity was regulated at the transcriptionalevel by low temperature or ethylene.

This study has provided some new information to under-tanding of the role of PAL in CI of banana fruit in relationo ethylene effects. Previous results indicated that the induc-ion of PAL in ‘Fortune’ mandarins could be a protectiveesponse of the fruit to chilling temperature (Lafuente et al.,001, 2003), while other reports suggested that the accumu-ation of PAL transcript may serve as a molecular markeror chilling tolerance in citrus fruit (Sanchez-Ballesta etl., 2000b). This study on bananas showed that ethyleneould enhance the activity and protein amount of PAL, andRNA transcripts of MaPAL1 and MaPAL2 of banana fruit

uring storage at chilling temperature, and it is suggestedhat PAL is positively related with tolerance of banana fruito CI.

cknowledgments

The authors thank Dr. Nikolaus Amrhein (Institute of Plantciences, Swiss Federal Institute of Technology) for the gen-rous gift of parsley PAL polyclonal antibody. We also thankhe two referees for their valuable suggestions. This workas supported by the National Natural Science Foundationf China (grant nos. 30571299, 30425040 and 30430490),hina Postdoctoral Science Foundation and Guangdong Sci-nce Foundation (06200670).

eferences

olwell, G.P., 1992. A role for phosphorylation in the down regulation ofphenylalanine ammonia-lyase in suspension cultured cells of Frenchbean. Phytochemistry 31, 4081–4086.

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