ewPostharvest Biology and Technology 44(2007)
34–41
Cloning and expression analysis of phenylalanine ammonia-lya in
relation 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,Chine Academy of Sciences,Guangzhou 510650,The People’s Republic of China
c Guangdong Key Laboratory for Postharvest Science,Guangzhou 510642,The People’s Republic of China
Received 20June 2006;accepted 9November 2006
Abstract
Bananas are highly susceptible to chilling injury (CI)and phenylalanine ammonia-lya (PAL),as a ke
y enzyme involved in plant phenyl-propanoid metabolism,has been associated with low temperature stress in plant tissues.However,little is known about the role of PAL (including PAL activity,gene and protein expression)in postharvest chilling tolerance of banana fruit.Two partial cDNAs quences (MaPAL1and MaPAL2)with about 760bp were cloned from banana pulp by RT-PCR.Western and northern hybridizations were ud to investigate expression of PAL protein and PAL genes in fruit stored for 10days at 7◦C (chilling temperature)and then transferred to 22◦C (room temperature).The effects of propylene (a functional ethylene analog)on their expression in relation to CI were also examined.Northern and western blot analys revealed that mRNA transcripts of MaPAL1and MaPAL2and PAL protein levels in banana fruit during storage incread,reaching a peak at about day 8,and finally decread at chilling temperature.Prior to low temperature storage,pretreatment with propylene could alleviate CI and enhance PAL activity,protein amount and mRNA transcripts of MaPAL1and MaPAL2.Moreover,changes in PAL activity,protein amount and accumulation of MaPAL1and MaPAL2exhibited almost the same patterns.The results suggest that the induction of PAL in banana fruit during low temperature storage is regulated at transcriptional and translational levels,and is related to reduction in CI symptoms.
©2006Elvier B.V .All rights rerved.
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Keywords:Banana;Chilling injury;PAL;Ethylene;Activity;Expression
1.Introduction
Banana fruit are highly susceptible to chilling injury (CI)(Pantastico et al.,1990).Storage at low temperatures (<13◦C)results generally in peel pitting,discoloration and abnormal fruit ripening.CI is a limiting factor in extending storage life and is responsible for substantial postharvest loss in many areas of cultivation.Rearch has been undertaken into understanding CI mechanisms,which,in turn,helps develop effective methods to prevent the occurrence of CI in banana fruit.Phenylalanine ammonia-lya (PAL,EC 4.3.1.5),a key enzyme involved in phenylpropanoid metabolism,catalys
∗Corresponding author.Tel.:+862085280229;fax:+862085282107.E-mail address:wjlu@scau.edu (W.-J.Lu).1
The authors contributed equally to this work.
phenylalanine to trans -cinnamic acid,which is the first step in the biosynthesis of phenylpropanoids,leading to a diver group of plant condary metabolites,including lignins,phy-toalexins,and flavonoids.In addition,PAL is induced by various biotic (e.g.infection by virus,bacteria
tedaand fungi)and abiotic (e.g.low and high temperatures,UV-B light and wounding)stress (Hahlbrock and Scheel,1989;Dixon and Paiva,1995;Strack,1997).The findings suggest that PAL plays an esntial role in modulating the resistance of plant tissues to such stress.
Ripening of banana fruit is associated with a sharp increa in ethylene production (Seymour,1993;Golding et al.,1998),and is initiated either by the natural production of endogenous ethylene as banana fruit reach full physiological maturity or by u of commercial exogenous ethylene ripening proce-dures (Wills et al.,2001).Application of exogenous ethylene
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劝学 翻译>grow什么意思Y.Wang et al./Postharvest Biology and Technology44(2007)34–4135
or propylene(a functional ethylene analog)at a non-chilling temperature prior to storage at low temperature alleviates the development of CI symptoms in citrus fruit(Lafuente et al., 2001)and banana fruit(Wang et al.,2006).Moreover,treat-ment with exogenous ethylene stimulates PAL activity in the flavedo of mature grapefruit(Riov et al.,1969),while the nsitivity of mandarins to chilling correlates with low con-stitutive levels of PAL mRNA and activity upon exposure of the fruit to low te
mperature(Sanchez-Ballesta et al.,2000b). Additionally,the increa in PAL activity and PAL mRNA levels,but not in ethylene,stimulated by chilling is affected by the age of the fruit(Lafuente et al.,2003).Thefindings indicate that more molecular details about the roles of PAL and ethylene in relation to CI in harvested fruit require further investigation.
To the best of our knowledge,there has been no report on expression profiles of PAL genes and PAL protein in relation to CI in banana fruit.The objectives of the prent work were to clone PAL genes from banana fruit and investigate the relationships between PAL and CI,and then evaluate the effect of exogenous propylene on activity,protein levels and mRNA transcripts of PAL in relation to CI.
2.Materials and methods
2.1.Plant material
Fruit of mature pre-climacteric banana(Musa sp.cv. Williams,Cavendish sub-group AAA)were obtained from a local commercial plantation near Guangzhou.Hands were parated into individualfingers.Individual fruit were dipped for2min in a500L/L Sportak(a.i.prochloraz)fungicide solution to control dia(Wade et al.,1993).They were then allowed to air-dry at25◦C for2h.Fruit were lected for freedom from visual defects and for uniformity of weight and shape and then divide
d at random into two groups.Fruit from thefirst group were placed into unaled plastic boxes (150fingers/box)and then pre-treated with1000L/L propy-lene(a functional ethylene analog)at22◦C for16h.The cond group of150fruitfingers were placed into another unaled plastic boxes and left ungasd(0L/L propylene, control).Both control and pre-treated fruit were subquently stored at7◦C(chilling temperature)for10days and sam-pled every2or3days for measurements.For another further sampling,fruit from the two different treatment groups were transferred to22◦C after8days of storage at7◦C.All of the fruit were then treated with1000L/L propylene for 16h and held at22◦C to ripen for5days,prior to sampling every1or2days for analys.For each sample,peel and pulp of10banana fruit were parated,then frozen imme-diately in liquid nitrogen andfinally stored at−80◦C prior to u.Each treatment consisted of three independent repli-cates.In this study,two incubators(Sanyo MIR553model, Gunma,Japan)were ud for fruit storage at7and22◦C, respectively.2.2.Assay for PAL activity
PAL activity was measured as previously described by Solecka and Kacperska(2003)with a minor modification. All experimental procedures were carried out at4◦C.Briefly, peel or pulp tissues(2g)were homogenized with mortar and pestle in6mL of extraction buffer[50mM Tris–HCl buffer, pH8.8,15mM-mercaptoethanol,5mM EDTA,5mM ascorbic acid,10M leupeptin,1mM PMSF and0.
15% (w/v)PVP].The homogenate wasfiltrated through four lay-ers of cheecloth and centrifuged at12,000×g for20min at4◦C.The supernatant was ud as the crude enzyme.The reaction mixture(3mL)contained16mM l-phenylalanine, 50mM Tris–HCl buffer(pH8.8),3.6mM NaCl and0.5mL of enzyme solution.Incubation was performed at37◦C for 1h and the reaction was stopped by the addition of500L of6M HCl.The reaction mixture was then centrifuged for 10min at12,000×g to pellet the denatured protein.The absorbance was measured at290nm before and after incuba-tion.One unit of enzyme activity was defined as the amount of PAL that produced1mol of cinnamic acid within1h and was expresd asmol cinnamic acid/mg protein/h.Protein was estimated according to Bradford(1976)using BSA as a standard.
2.3.Protein extraction and Western blot analysis
Total proteins were extracted according to Campos-Vargas et al.(2005)with a minor modification.The extraction buffer contained50mM Tris–HCl(pH8.8),5mM ascorbic acid, 5mM EDTA,1mM PMSF,14mM-mercaptoethanol and 0.15%(w/v)PVP.The extracted proteins were parated by SDS-PAGE on a12%polyacrylamide gel as described by Laemmli(1970).The same amount(10g)of protein was loaded per lane.After electrophoresis,the proteins were electro-transferred to nitrocellulo(0.45m,Amersham LIFE SCIENCE)using a transfer apparatus(Bio-Rad)by the method
of Isla et al.(1998).After rinsing in TBS buffer [10mM Tris–HCl(pH7.5)and150mM NaCl],the mem-brane was blocked for2h with3%(w/v)BSA in0.05%(v/v) Tween20and TBS,and then incubated with gentle shaking for3h in a1000-fold diluted solution of a primary poly-clonal PAL antibody(kindly provided by Prof.N.Amrhein)at 25◦C.Following extensive washes with TBST buffer[TBS, 0.05%(v/v)Tween20],the membrane was incubated with goat anti-rabbit IgG-alkaline phosphata conjugate(Sigma, St.Louis;1:1000diluted in TBST)at25◦C for1h and again washed with the above TBST buffer.The membrane was stained with a10mL solution containing nitro blue tetrazolium(NBT)and5-bromo-4-chloro-3-indolyl phos-phate(BCIP)in the dark.The reaction was terminated by the addition of double distilled water.The amount of PAL protein was quantified by scanning the NC membranes after immunoblotting with a densitometer using ImageQuant software.
36Y.Wang et al./Postharvest Biology and Technology44(2007)34–41
2.4.RNA extraction and cloning of banana PAL cDNAs
Total RNA from banana fruit was extracted using the hot borate method(Wan and Wilkin,1994).Frozen tissues(10g) from the peel or pulp of banana fruit were ground to afine powder in a mortar using a pestle in the prence of liquid nitrogen.Two ends of six individual bananafingers
were dis-carded.The mid-part of about5cm in length was peeled,and peel and pulp were then cut parately into small pieces.The peel and pulp samples were well mixed andfinally parated into three replicates.
Total RNA extracted from banana pulp was ud as templates for the RT-PCR.The product(thefirst-strand cDNA)was subjected to PCR amplification. Degenerate primers were designed with reference to the conrved amino acid quences of : 5 -TANGGNGTYACNACNGGNTTYG-3 and antin: 5 -GGNCCNARCCAN TGNGGNGANC-3 (N=A,C,G or T).Reactions for the RT-PCR were subjected to one cycle of 94◦C for3min,35cycles(94◦C for1min,50◦C for2min and72◦C for2min)and one cycle of72◦C for10min.PCR products of the predicted size(about760bp in length)were purified and cloned into pMD-18T vector(TaKaRa,Shiga, Japan).The nucleotide quences of the cDNA inrts were determined using a DNA quencer(Model ABI377,USA) with either-21M13or M13quencing primers,according to the manufacturer’s instructions.
2.5.Northern blot analysis
Total RNA(10g)was parated on a1.2%agaro–formadehyde gel and capillary blotted onto the PVDF mem-brane(Biodyne®B0.45m,PALL).The membrane was blot-dried and cross-linked with U
V at280nm.DIG-labeled specific probes of3 -untranslated regions of MaPAL1and MaPAL2were made using the PCR DIG probe synthesis Kit (Roche Applied Science,Mannheim,Germany)and primers for MaPAL1(n:5 -ATCTCGTCCCGCTGTCTTAC-3 and antin:5 -TCCCCTCCAGTATGT GCTCCA-3 )and for MaPAL2(n:5 -TTCCCCTCCGTGGCACTATC-3 and antin:5 -GCTTGTGGGTGAGGTGGTCG-3 ).The membrane was hybridized with the DIG-labeled probe for 16h at45◦C in high-SDS buffer(7%SDS,5×SSC, 50mmol/L sodium-phosphate,pH7.0,2%blocking reagent and0.1%N-lauroylsarcosine)containing50%deionized for-mamide(V/V)(Roche,Germany).Blots were washed twice at37◦C in2×SSC and0.1%SDS for10min,followed by washing twice at62◦C in0.1×SSC and0.1%SDS for 30min.The membranes were then subjected to immuno-logical detection following the manufacturer’s instructions (Roche Applied Science).
外教英语1对1哪家好2.6.Statistics
The experiments were arranged in a completely random-ized design,and each treatment comprid three replicates.Data were prented as means±standard errors(S.E.).Least significant differences(LSD)were calculated to compare significant effects at the5%level.
All treatments were repeated at least three times while all samples were analyzed three times.Mean
s and standard errors were calculated from pooled data.In thefigures,the vertical line associated with each point reprents the stan-dard error.
3.Results
3.1.CI symptoms
The relationship between CI symptoms and the ethylene production rate of banana fruit was shown in our previ-ous report(Wang et al.,2006).Banana fruit began to show CI symptoms evident as pitting and brown patches on the skin after4days of storage at7◦C.CI symptoms incread markedly when fruit were removed from7to22◦C.Pre-treatment with propylene,prior to storage at7◦C,delayed the appearance of CI symptoms by3days.Moreover,the propy-lene pre-treated fruit that were stored for8days at7◦C and transferred to22◦C with propylene treatment for initiation of ripening,had less CI symptoms.
3.2.PAL activities of banana peel and pulp as affected
by propylene treatment
PAL activities of banana peel and pulp tissues tended to increa during storage at7◦C(Fig.1).Moreo
ver,the increa in PAL activity was greater in the propylene-treated fruit than in control fruit both when stored at7◦C or
trans-Fig.1.Effect of propylene treatment on PAL activity in banana peel and pulp tissues.Fruit were
pre-treated with0(control)or1000L/L propy-lene for16h at22◦C,prior to storage at7◦C,and then divided into two groups.Thefirst group of fruit was stored at7◦C for10days.The cond group was stored for8days at7◦C,transferred to22◦C and treated again with1000L/L propylene and then held at22◦C for5days to ripen.Each value reprented the mean±S.E.of three replicates.An asterisk indicates significant differences between control and treatment at the5%level.
manager什么意思Y.Wang et al./Postharvest Biology and Technology 44(2007)34–41
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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 1000L/L propylene for 16h 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 10days.The cond group was stored for 8days at 7◦C,transferred to 22◦C and treated again with 1000L/L propylene and then held at 22◦C for 5days to ripen.Equal protein amounts (10g)were subjected to SDS-PAGE and transferred to a nitrocellulo membrane.Thereafter,the PAL amount was immunodetected with the specific antibody.Image (a and c)and quantitative analysis (b and d)of immunoblotting bands.Lanes 1–8in (a)control fruit:after 0,3,6,8and 10days of storage at 7◦C,and subquent 1,3and 5days of storage at 22◦C after 8days of storage at 7◦C.Lanes 1–8in (c)propylene-treated fruit:after 0,3,6,8and 10days of storage at 7◦C,and subquent 1,3and 5days of storage at 22◦C after 8days of storage at 7◦C.
ferred to 22◦C and treated again with propylene and then held at 22◦C to ripen.This is in agreement with the report by Kamo et al.(2000),who found that ethylene treatment could increa PAL activity in banana fruit.The maximum level of PAL activity was obtained after 8days of storage at 7◦C.The PAL activities of peel and pulp tissues of propylene-treated fruit incread 1.18-and 1.73-fold,respectively,compared to the control fruit.After storage at 7◦C,the PAL activity of peel tissues beg
an to decrea during subquent stor-age at 22◦C while that of pulp tissues changed only slightly (Fig.1).
3.3.Amounts of PAL protein of banana peel and pulp tissues affected by propylene treatment
A peptide of 70kDa was specifically detected with the anti-parsley PAL polyclonal antibody,indicating that PAL proteins exist both in peel and pulp tissues of propylene-treated and control banana fruit (Figs.2and 3).The immune signal in the peel tissues (Fig.2)was noticeably stronger than in the pulp tissues (Fig.3),while the immune signal in the propylene-treated fruit (including peel and pulp tissues)was also stronger than that in control
fruit.
Fig.3.Western blot analysis of PAL in pulp of control (a and b)and propylene treatment (c and d)of banana fruit.Fruit were pre-treated with 0or 1000L/L propylene for 16h,prior to storage at 7◦C,and stored at 7◦C for 10days.Some control and pre-treated fruit were transferred to 22◦C after storage at 7◦C for 8days,and treated again with 1000L/L propylene,then held at 22◦C for 5days to ripen.Equal amounts of protein (10g)were subjected to SDS-PAGE and transferred to a nitrocellulo membrane.Thereafter,the PAL amounts were immunodetected with the specific antibody.Image (a and c)of immunoblotting and quantitative analysis (b and d)of immunoblotting bands.Lanes 1–8in (a)control fruit:after 0,3,6,8and 10days of cold storage,after1,3and 5days removed from 7to 22◦C after cold storage.Lanes 1–8in (c)propylene-treated fruit:after 0,3,6,8and 10days of cold storage,after1,3and 5days removed from 7to 22◦C after cold storage.
38Y.Wang et al./Postharvest Biology and Technology 44(2007)
34–41音乐之声主题曲
Fig.4.Alignment of the predicted protein quences of the two cDNA fragments of PAL isolated from pulp tissues of banana fruit using the method of RT-PCR.Two rines,at positions 96and 103,which were conrved at the active site of all known PAL quences (Schuster and Retey,1995)are indicated.
批准英文PAL protein amounts in peel tissues of control and propylene-treated fruit incread,then reached a maximum after 8days of storage at 7◦C,and finally decread slightly after 10days (lanes 1–5in Fig.2a and c).PAL protein amounts of fruit that were treated with 1000L/L propy-lene and then held at 22◦C for 5days to ripen after storage at 7◦C decread and were lowest after 5days of storage at 22◦C (lanes 6–8in Fig.2a and c).Fig.2further shows that the immune signal in the propylene-treated peel tissues was obviously stronger than that in control fruit at any pha (Fig.2b and d).
As with peel tissues,PAL protein amounts in pulp tissues of control and propylene-treated fruit showed almost the same pattern (lanes 1–8in Fig.3a and b and lanes 1–8in Fig.3c and d).
3.4.Isolation and quence analysis of cDNAs of PAL from banana fruit
Two partial cDNA fragments about 760bp in length were cloned using total RNA from pulp tissues by RT-PCR.A BLAST arch of GenBank revealed that MaPAL1shared 84%identity with ZmPAL while
MaPAL2shared 83%iden-tity with OsPAL .Thus,the two fragments were considered to be cDNA fragments of PAL ,named as MaPAL1and MaPAL2,respectively (Fig.4).As a partial MaPAL cDNA quence registered as AJ555536in GenBank was located in the different regions with the two PAL cDNA quences of this study in the PAL full length quence,it is difficult
to know whether AJ555536is different from MaPAL1and MaPAL2.
In addition,MaPAL1and MaPAL2shared 81%identity in nucleotide quence and 87%identity in amino acids.Deduced amino acid quences of MaPAL1and MaPAL2contained two rines,at positions 96and 103,which are suggested to be conrved at the active site of all known PAL genes (Schuster and Retey,1995).
3.5.Accumulation of MaPAL1and MaPAL2mRNAs of banana peel and pulp tissues affected by propylene treatment
PAL genes are expresd in all plant tissues investigated (Dixon and Paiva,1995).In this study,mRNAs of MaPAL1and MaPAL2could be detected in both peel and pulp tissues of banana fruit,while accumulation of the MaPAL1transcript was slightly higher than that of MaPAL2(Figs.5and 6).Accu-mulation of MaPAL1and MaPAL2transcripts in peel and pulp tissues of banana fruit at 7◦C in
cread,then reached a peak at about day 8and finally decread (Fig.5),which was in accor-dance with activities and protein amounts of PAL (Figs.1–3).Therefore,it might be suggested that the chilling temperature induced the expression of MaPAL1and MaPAL2.Moreover,the pretreatment with propylene stimulated the expression of MaPAL1and MaPAL2in the peel and pulp tissues of fruit stored at 7◦C (Fig.5)or in fruit transferred to 22◦C and treated again with propylene and then held to ripen (Fig.6).In addition,the mRNA transcripts of MaPAL1and MaPAL2
