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Plant Physiology and Biochemistry 61 (2012) 123e130

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Purification and chemical characterisation of a cell wall-associatedb-galactosidase from mature sweet cherry (Prunus avium L.) fruit

Carmela Gerardi*, Federica Blando, Angelo Santino*

Institute of Sciences of Food Production, C.N.R. Unit of Lecce, via Monteroni, 73100 Lecce, Italy

a r t i c l e i n f o

Article history:Received 26 June 2012Accepted 21 September 2012Available online 8 October 2012

Keywords:b-GalactosidaseCell wallFruit ripeningSweet cherry

* Corresponding authors. Tel.: þ390832422606; faxE-mail addresses: carmela.gerardi@ispa.cnr.it (C. G

cnr.it (A. Santino).

0981-9428/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.09.012

a b s t r a c t

Using four different chromatographic steps, b-galactosidase was purified from the ripe fruit of sweetcherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunusavium b-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of fourdifferent active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and b-galactosidase-activity staining. The active polypeptides were individually excised from the gel andsubjected to SDS-PAGE. Each of the four native enzymes showing b-galactosidase activity was composedof two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjectedto N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry b-galactosidaseshowed a 43% identity with the 44 kDa subunit of persimmon and apple b-galactosidases and the 48 kDasubunit of carambola galactosidase I. The sweet cherry b-galactosidase exhibited a strict specificitytowards p-nitrophenyl b-D-galactopyranoside, a pH optimum of 4.0 and Km and Vmax values of 0.42 mMand 4.12 mmol min�1 mg�1 of protein respectively with this substrate. The enzyme was also activetowards complex glycans. Taken together the results of this study prompted a role for this class ofenzymes on sweet cherry fruit ripening and softening.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Fruit maturation and softening are often associated withextensive changes in the structure and composition of cell wallwhich include increasing hydration and changes in the cohesion ofthe pectin gel, a breakdown and dissolution of the pectin-richmiddle lamella and a reduction in cell-to-cell adhesion [1]. All ofthese physiological changes contribute to the final texture of fruit.The disassembly of cell wall components during ripening is broughtabout by a range of tightly regulated enzyme activities. One of theearliest changes occurring in fruit ripening is the loss of pectingalactan side-chains that increases pectin solubilisation byaffecting rhamnogalacturonan solubility with a consequentincrease in cell wall porosity and the access of pectin methylesterases (PME; EC 3.1.1.11) and polygalacturonases (PG; EC 3.2.1.15)to pectin substrates [1]. Other enzymes, such as glucanases (EC3.2.1.4), xyloglucanases (EC 3.2.1.151), mainly focus their activity tothe hemicellulose fraction. However, the biochemical processeswhich regulate changes in fruit firmness are not yet fully

: þ390832422620.erardi), angelo.santino@ispa.

son SAS. All rights reserved.

understood. The application of molecular biology techniques tostudy the role played by each class of enzymes in fruit softeningindicated that, at least in some plant species, this physiologicalprocess is even more complex than that hypothesised on the basisof chemical/biochemical studies. In the emerging scenario,different isoforms of the main enzymes, whose expression is tightlyregulated both temporally and spatially during fruit development,play different roles in fruit ripening and softening. In the case oftomatoes, the plant species in which fruit ripening and softeninghas been studied in better details, the down-regulation of poly-galacturonase failed to delay fruit softening [2], thus indicating thatother hydrolases, together with PG, may play a role in the structuralchanges of cell wall that occur during tomato fruit ripening [3]. Onepossible candidate is represented by b-galactosidases (EC 3.2.1.23),since tomato fruit development is accompanied by a net loss ofgalactosyl residues throughout fruit ripening [4e7]. The expressionprofiling of b-galactosidase genes during tomato fruit developmentshowed that seven different genes are expressed, with each dis-playing a different spatial and temporal expression profile [8].Furthermore, the antisense suppression of one of these genes,which is highly expressed during the early stages of tomato fruitdevelopment, resulted in an increase in fruit cracking and inducedsome morphological changes, such as a reduced locular space anda thicker fruit cuticle [9]. These findings suggest that the

A

B

Fig. 1. Glycosidase activities detected in the water soluble protein fraction (WSPF; A)and salt-soluble protein fraction (SSPF; B) extracted from the acetone powder of cherryfruits (cv Lapins) at four ripening stages: stage I (unripe), stage II (turning), stage III(ripe) stage IV (over-ripe). Results are the mean � SD of values obtained from fourseparate acetone powder preparations.

C. Gerardi et al. / Plant Physiology and Biochemistry 61 (2012) 123e130124

degradation of galactosyl residues is involved in pectin depoly-merisation and solubilisation, thus indicating that b-galactosidasesplay an essential role in fruit softening.

b-Galactosidases are a class of widespread glycosyl hydrolases.In higher plants, b-galactosidases have been detected in varioustissues. Increased gene expression and b-galactosidase activityhave been reported during fruit ripening in a number of plantspecies, such as tomato [8], apple [10], muskmelon [11], kiwi fruit[12], orange [13], strawberry [14], coffee [15], mango [16], peach[17] and papaya [18]. Since endo-galactanases have not beendetected in higher plants, the enzyme activity responsible fordegradation of cell wall b-galactan can be identified as an exo-b-D-galactosidase (EC 3.2.1.23), which catalyses the hydroxylation ofterminal, non-reducing b-D-galactosyl residues from b-D-galacto-sides. b-D-galactosidases have been purified and characterised fromseveral different plant fruits [7,11,16,19] and in certain cases, such asapple [10], tomato [8,20], strawberry [14], banana [21] and pear[22], the corresponding cDNAs have been reported.

To date, the biochemical processes involved in cell wall firmnessreduction during cherry fruit ripening are poorly understood.Batisse et al. [23] reported that pectin changes are very limitedduring fruit ripening and, consequently, fruit softening should notdepend on pectin depolymerisation but, more probably, to theaction of other cell wall hydrolases acting on other cell wallcomponents. Moreover, Batisse et al. [24] showed that texturaldifferences between crisp and soft cherry fruits were not due to thedepolymerisation of pectins, but most likely due to the degree ofpolymerisation of pectin side-chains. In a previous study, this groupshowed that a cytosolic b-glucosidase (EC 3.2.1.21), also showing b-galactosidase and b-fucosidase activities towards syntheticsubstrates, is abundantly accumulated during fruit ripening and isalso associated, to a low extent, with cell wall of ripe fruit [25].However, the presence of other cell wall-associated hydrolases hasnot yet been reported in cherry fruits.

With the aim of investigating the presence and relevance ofother cell wall-degrading enzymes in sweet cherry fruit softening,the main hydrolytic activities of cell wall-associated protein frac-tions from different ripening stages were assayed. A cell wall-associated b-galactosidase was purified and characterised usingboth synthetic and natural substrates. The role of b-galactosidasesin sweet cherry fruit ripening and softening is also discussed.

2. Results and discussion

2.1. Glycosidase activities during cherry ripening

Acetone powder prepared from sweet cherry fruits at differentripening stages was used for the extraction of total proteins and theassay of glycosidase activities in cherry fruits during ripening.Acetone powder can be considered a good starting materialcompared to fresh fruits as demonstrated in a previous study [25].With the aim to study cell wall glycosidases during sweet cherryripening, a procedure was set up to reduce the presence of watersoluble proteins (WSPF) in the salt-soluble protein fraction (SSPF),comprising cell wall-associated proteins. As described in theMaterial and Methods section, the acetone powders obtained fromdifferent ripening stages were first subjected to water extraction toremoveWSPF and were then exhaustively washed with a low ionicstrength buffer before their resuspension in a high ionic strengthbuffer. After centrifugation, the solubilised proteins were used forglycosidase activity assays. The protein yield in WSPF showed a netincrease during ripening stages (from about 3 mg/g FW recorded atstage I, to 5.4; 12.5 and 28.5 at stages from II to IV). Fig. 1A reportsthe glycosidase activities detected in WSPF extracted from cherryfruits at four ripening stages (unripe: stage I; turning: stage II; ripe:

stage III; over-ripe: stage IV). Among the detected glycosidases, b-D-fucosidase (EC 3.2.1.38) and b-D-glucosidase showed the highestactivity with a sharp increase from stage III to IV. A previous studyof this group, showed that a b-D-glucosidase abundantly expressedin ripe sweet cherry fruits accounts for most of this enzymaticactivity [25]. Out of the other water soluble glycosidases testedhere, b-D-galactosidase and a-L-arabinopyranosidase showeda slight increase from stage III to IV. Conversely, a-D-galactosidase(EC 3.2.1.22) was stable during fruit development.

The same enzymatic assays were also carried out on the SSPFsamples from different ripening stages. This allowed the detectionof glycosidase activities specific to the SSPF (Fig. 1B). Differentlyfrom the WSPF, the protein yield in this fraction was fairly stableduring fruit ripening (about 26.4; 22.2; 25.8 and 22.9 mg/g FWwererecorded from stage I to IV). The highest activity among salt-solubleglycosidases was represented by b-D-galactosidase, which showeda constant increase during ripening from stage I to III and decreasedfrom stage III to IV. b-D-fucosidase, showed a trend similar to that ofb-D-galactosidase activity, although with lower levels. a-D-man-nosidase (EC 3.2.1.25), b-D-glucosidase and a-L-arabinopyr-anosidase activities were also recorded with a similar but muchlower levels. By contrast, a-D-galactosidase activity decreased fromstage I to stage II and then was stable up to stage IV, while b-D-xylosidase, and a-L-rhamnosidase (EC 3.2.1.40) were not detected inSSPF samples.

2.2. Cell wall-associated glycosidase (ionically- and covalently-linked) during cherry ripening

Previous studies demonstrated that hydrolases can be eitherfreely soluble in low ionic strength buffers or bound to wall poly-mers [26]. To discriminate between these two fractions, enzymat-ically active cell walls were isolated from the flesh of cherry fruits atthe four ripening stages and were incubated with appropriatesubstrates to provide an estimation of ionically- and covalently-

C. Gerardi et al. / Plant Physiology and Biochemistry 61 (2012) 123e130 125

linked activities. Among the tested glycosidases, the most activewere b-fucosidase, b-glucosidase and a-galactosidase, followed byb-galactosidase, a-mannosidase and a-L-arabinopyranosidase(Fig. 2) in decreasing order of activity. Noteworthy, all the enzy-matic activities showed a similar trend with a net decreasethroughout fruit development and an almost undetectable activityrecorded in the IV stage (Fig. 2).

Fig. 3. Purification of b-galactosidase from SSPF of sweet cherry fruits by cationexchange chromatography (A) and hydrophobic interaction chromatography (B). (A)About 5 mg of protein were applied to S-Hyper D column and eluted with a step 0e500 mM gradient of NaCl ( ). The solid line ( ) shows the protein elution asA280, b-galactosidase activity is expressed U/ml of recovered fractions ( ). (B)About 0.58 mg of the active fraction recovered after ion exchange chromatographywere loaded on a TSK phenyl 5PW column and eluted with a linear 1.5e0 M gradient ofammonium sulphate ( ). The solid line ( ) shows the protein elution as A280,b-galactosidase activity is expressed as U/ml of recovered fractions ( ).

2.3. b-Galactosidase purification and characterisation

The results reported in Fig. 1B indicated that b-D-galactosidaseand b-D-fucosidase are the main glycosidases among SSPF of ripesweet cherry fruit, and could play a role in the cell wall changesoccurring during fruit ripening and softening. Furthermore, theresults of this study indicated that the activity of these enzymesincreased throughout fruit ripening up to the ripe stage (stage III)and decreased in the over-ripe stagewhere these enzymes aremostlikely released in the soluble fraction due to the cell walls re-arrangements and loosening.

In a previous study, it was shown that a 68 kDa b-glucosidase,abundantly expressed in ripe sweet cherry fruit and responsible formost of b-D-glucosidase, b-D-fucosidase and b-D-galactosidaseactivities detected in the WSPF samples of ripe fruit, is also asso-ciated, to amuch lower extent, with cell wall of ripe fruit. Therefore,it was hypothesised that b-glucosidase may account, at least in part,for the enzymatic activities found in SSPF. With the aim of verifyingthe presence of other b-galactosidases in this fraction, the proteinswere first precipitated with 90% ammonium sulphate and thenseparated by anion exchange chromatography. Two different peaksof b-galactosidase activity were recovered: the first eluting withunbound proteins and another one at about 200 mMNaCl (Fig. 3A).The first peak showed mainly b-D-galactosidase activity, whereasthe latter, as expected from previous results [25], contained the68 kDa b-glucosidase showing b-D-glucosidase, b-D-fucosidase andb-D-galactosidase activities (data not shown). With the aim ofpurifying and better characterising the b-galactosidase detected inthe unbound protein fraction of the anion exchange chromatog-raphy, this fraction was purified by a combination of cationexchange chromatography, hydrophobic interaction chromatog-raphy and size exclusion chromatography (Table 1). In all of thesesteps, a single peak of b-galactosidase activity was always recov-ered (Fig. 3B). At the end of the reported purification procedure thesweet cherry b-galactosidase was purified up to 131-fold witha recovery of about 5% of the total activity recorded in the SSPF

Fig. 2. Glycosidase activities detected in cell walls obtained from cherry fruits (cvLapins) at four ripening stages: stage I (unripe), stage II (turning), stage III (ripe) stageIV (over-ripe). Results are the mean � SD of values obtained from three separate cellwall preparations.

sample (Table 1). The low recovery of b-galactosidase activity wasmost likely due to the low solubility of the enzyme in the low ionicstrength buffers used in ion exchange chromatography and theweak stability of the purified enzyme. Other authors have also re-ported the instability of purified b-galactosidases [12,27,28].

The Prunus avium b-galactosidase (Pab-Gal) showed an apparentmolecular mass of about 100 kDa as estimated either by sizeexclusion chromatography or ND-PAGE (data not shown). SDS-PAGE carried out on the active fractions collected after size exclu-sion chromatography indicated the presence of two main poly-peptides of about 54 and 33 kDa, together with some minor bandsof about 70 and 40 kDa (Fig. 4). The protein fraction containing thepartially purified Pab-Gal (shown in Fig. 4 lane 5) was also sub-jected to native IEF and b-galactosidase-activity staining. As re-ported in Fig. 5, four different active polypeptides, with estimatedpIs of about 7.9, 7.4, 6.9 and 6.4, were positive to the 6-bromo-2naphthyl-b-D-galactoside staining thus representing slightlydifferent isoforms of the Pab-Gal. The active bands were individu-ally excised from the gel and subjected to SDS-PAGE. Each of thefour native enzymes showing b-galactosidase activity was

Table 1Purification of b-galactosidase from sweet cherry fruits. The enzyme was extractedand purified as reported in “materials and methods”.

Purification steps Totalprotein(mg)

Totalactivity(U)

Specificactivity(U mg�1

prot)

Yield(%)

Purificationfactor

Salt-solubleproteins

28.30 10.69 0.38 100 1.0

90% (NH4)SO4

precipitation16.34 9.47 0.58 88 1.5

Anionic exchangechromatography

4.20 7.67 1.83 72 4.8

Cationic exchangechromatography

0.58 4.89 8.43 46 22.2

Hydrophobicinteractionchromatography

0.07 1.32 18.86 12 49.6

Size exclusionchromatography

0.01 0.50 50.00 5 131.6

Fig. 5. The purified b-galactosidase was subjected to native IEF and stained by b-galactosidase-activity staining with 6-bromo-2-napthyl-b-D-galactoside. Afterwards,each of the four active bands (pIs: 7.9, 7.4, 6.9, 5.4; highlighted by the boxes) weredenatured, subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. MWM,molecular weight markers from top to bottom: phosphorylase b (97 kDa); serumalbumin (66.2 kDa); ovalbumin (45 kDa); carbonic anhydrase (31 kDa); trypsininhibitor (21.5 kDa); lysozyme (14.4 kDa).

C. Gerardi et al. / Plant Physiology and Biochemistry 61 (2012) 123e130126

composed of the two 54 and 33 kDa polypeptides (Fig. 5). Both ofthese polypeptides were subjected to N-terminal amino acidsequence analysis (Table 2). A 43% identity was found between the54 kDa polypeptide of Pab-Gal and the 44 kDa subunit ofpersimmon [29] and apple [10] b-galactosidases and the 48 kDasubunit of carambola galactosidase I [30]. The same degree ofidentity was also found between the Pab-Gal N-terminus andamino acids 31e44 of the strawberry b-galactosidase 1, as deducedby the reported cDNA sequence [14]. A lower degree of identity wasfound between Pab-Gal and the N-terminus of pepper [7] andtomato TBG5 b-galactosidase [8] (Table 2). As far as the 33 kDapolypeptide of Pab-Gal is concerned, its N-terminal amino acidsequence showed a 22% identity (44% similarity) with the 36 kDasubunit of the carambola b-galactosidase. A lower (5%) degree ofidentity was found with the 34 kDa subunit of persimmon b-galactosidase (Table 3).

2.4. Enzymological properties of sweet cherry b-galactosidase

Pab-Gal exhibited low levels of activity against p-nitrophenyl a-L-arabinopyranoside (10%) and p-nitrophenyl b-D-fucopyranoside

Fig. 4. SDS-PAGE analysis of b-galactosidase purification steps: lane 1, total cell wallprotein extracted from acetone powder (about 20 mg); lane 2, anion exchange chro-matography not bound proteins (about 20 mg); lane 3, 90% (NH4)2SO4 precipitatedproteins (about 20 mg); lane 4, active fraction recovered after cation exchange chro-matography (about 20 mg); lane 5, active fraction recovered after hydrophobic inter-action chromatography (about 20 mg); lane 6, active fraction recovered after sizeexclusion chromatography (about 5 mg); MWM, molecular weight markers from top tobottom: phosphorylase b (97 kDa); serum albumin (66.2 kDa); ovalbumin (45 kDa);carbonic anhydrase (31 kDa); trypsin inhibitor (21.5 kDa).

(8%) when compared with its activity towards p-nitrophenyl b-D-galactopyranoside (100%), indicating that b-galactosidase hasa high specificity against the b-galactosyl linkage. No activity wasfound towards other synthetic substrates (Table 4).

The partially purified Pab-Gal has a pH optimum of 4.0 and Km

and Vmax values of 0.42 mM and 4.12 mmol min�1 mg�1 of protein,respectively, using p-nitrophenyl b-D-galactopyranoside asa substrate.

Finally, whether Pab-Gal was active towards complex glycanswas verified. With this aim, total cell wall was extracted from ripesweet cherry and about 4 mg of total cell wall were used to test thehydrolytic activity of Pab-Gal (about 10 mg) in a total volume of1 mL. In these experimental conditions, it was found that Pab-Galwas able to release about 1.1 mg of D-galactose mg substrate�1.

3. Discussion

The results reported here indicate that b-galactosidase was themain glycosidase activity detected in the SSPF purified from sweetcherry fruit at different ripening stages. This increased approxi-mately 1.6-fold during ripening and was one of the main cell wall-associated glycosidases, together with the b-glucosidase activitythat had been previously purified and characterised [25]. Reportsfrom other groups pointed to the role of b-galactosidases in fruitripening through their action on pectins [11,31]. To better clarify therole of these enzymes in sweet cherry fruit ripening and softening,the biochemical purification and characterisation of the main b-galactosidase detected in the SSPF of ripe fruit was carried out. TheP. avium b-galactosidase (Pab-gal) has a molecular mass of about100 kDa and consists of two main polypeptides of about 54 and33 kDa.

Overall, our results imply that the original 100 kDa polypeptideundergoes some post-translational modifications. Among these isthe proteolytic cleavage responsible for the formation of the two

Table 2N-terminal sequence of the 54 kDa subunits of sweet cherry b-galactosidase was compared to the sequence of other related plant b-galactosidase.

Enzyme Amino acid sequence Residues Identity(%)

Cherry b-galactosidase subunits of 54 kDa e L V K Y I V V A L P Y D G 1e14 e

Persimmon b-galactosidase 44 kDa A e V T Y D H R A L V I D G 1e14 43Apple b-galactosidase 44 kDa A K V T Y D H R A L V I D G 1e14 43Strawberry Fabgal2 T T V S Y D H R A L V I D G 31e44 43Carambola 48 kDa A G V T Y D H R A L V I D G 1e14 43Pepper b-galactosidase (PBG1) e N V S Y D D R A I V I N G 1e14 29Tomato TBG5 A N V T T D H R A L V V D G 31e44 29

C. Gerardi et al. / Plant Physiology and Biochemistry 61 (2012) 123e130 127

subunits forming the active Pab-Gal. In this context, amino acidicsequences alignment revealed that a significant homology occurredbetween the N-termini of the major subunit of sweet cherry,persimmon fruit and carambola b-galactosidases, and theN-termini of a number other plant b-galactosidases whose fulllength sequences have been deduced from the correspondingcDNAs. Sequence alignment analyses carried out on the N-terminusof the minor subunits, indicated that these polypeptides pairedwith the middle of b-galactosidases full length sequences [29,30].

Other b-galactosidases purified from a number of plant speciesare generally composed of two subunits of about 42e45 kDa and31e35 kDa [10,12,29]. In the case of apple b-galactosidase, theN-terminus sequence information of the 44 kDa subunit allowedthe isolation of the corresponding cDNA clone and the molecularmass of the mature apple b-galactosidase, deduced by its cDNA,was 78.5 kDa [10]. On the basis of sequence information from otherplant b-galactosidase genes, two groups with different molecularmasses were identified [8]: a first group comprising b-galactosi-dases reported from apple, carnation, papaya and tomato TBG4with a molecular mass ranging from 80.5 to 82.8 kDa, and a secondgroup comprising asparagus and the other tomato b-galactosidases(TBG1eTBG3, TBG5eTBG7) characterised by a higher molecularmass (89.8e99.9 kDa). Themolecular mass for Pab-gal estimated bysize exclusion chromatography and ND-PAGE was about 100 kDa,so it could be ascribed to the 89.9e99.9 kDa b-galactosidase group.

As determined by isoelectric focussing, active Pab-gal wasshown to possess four different values of pIs: 6.4, 6.9, 7.4, and 7.9(see Fig. 5). The different isoelectric points of the Pab-Gal might bedue to some post-translational modifications occurring to specificamino acids or to N-linked oligosaccharide chains. Indeed, plantb-galactosidases often contain more than one putative glycosyla-tion site [14]. Multiple pI values were reported for b-galactosidasesisolated from a number of plant species, i.e. barley, corn, rye, jackbean, spinach, etc. [32]. For example, the three pIs reported for jackbean b-galactosidase (6.9, 7.7, 8.3) were similar to those of Pab-gal.As far as fruit b-galactosidases is concerned, neutral/basic pHranges were observed for carambola, kiwi fruit, tomatoes, redpepper and papaya [30,33e35]. By contrast, acidic pI values werereported for avocado, mature green pepper and papaya [33,35,36].

Monomeric galactose was the only product detected when thepurified Pab-gal was incubated with native cell walls. Therefore, itcan be concluded that the enzyme functions as an exo-b-galacto-sidase, removing single galactosyl residues from the non-reducingend of the polysaccharides. In a previous study [25], it was shown

Table 3N-terminal sequence of the 33 kDa subunits of sweet cherry b-galactosidase was compa

Enzyme Amino acid sequence

Cherry b-galactosidase subunit of 33 kDa X V G L G T P VCarambola b-galactosidase subunit of 36 kDa W S Y I N E P VPersimmon b-galactosidase subunit of 34 kDa X S X F N Q P I

that the sweet cherry b-glucosidase was able to hydrolyse either p-nitrophenyl b-D-glucopyranoside or p-nitrophenyl b-D-fucopyr-anoside to a similar extent. A lower activity (about 50%) wasrecorded using p-nitrophenyl b-D-galactopyranoside. A large vari-ability in the substrate specificity of plant b-galactosidases appearsevident when comparing results from other previous studies.Indeed, b-galactosidases purified from kiwi fruit, apple and peppershowed a strict specificity towards p-nitrophenyl b-D-galactopyr-anoside [7,10,12]. The same enzyme purified from persimmon fruitshowed a wider specificity and was also able to hydrolysep-nitrophenyl a-L-arabinopyranoside together with p-nitrophenylb-D-galactopyranoside [31].

Of particular note, the results of this study indicated that thePab-gal was also active towards complex glycans and the rate offree galactose (1.1 mg of D-galactose mg substrate�1) recorded here,using cell wall extracts from ripe sweet cherry as a substrate, washigher than that reported for some isoforms of Japanese pearb-galactosidase (where a similar procedure of native cell wallextraction and free galactose determination was used), where thegalactose released ranged from 0.10 to 3.12 mg/mL reaction mixture[37]. Previous reports [38,39] showed the presence of galactose indifferent fractions of cell walls isolated from Prunus species. Note-worthy, galactose was the second most abundant sugar afterarabinose.

Understanding the role of b-galactosidases during fruit ripeningmay help to shed light on this complex and tightly regulatedmechanism. The release of galactosyl residues and other sugars, i.e.glucose, fucose and mannose, from side-chains of pectin couldaffect the cell wall structure and the content in these components.In this context, it’s noteworthy that all the main glycosidaseactivities (b-galactosidase, b-fucosidase, b-glucosidase) decreasedin the SSPF and were released in the WSPF at the over-ripe stage.The synergistic effects among different cell wall-modifyingenzymes on cell wall disassembly during fruit ripening has beenalready reported in other plant species [40,41]. Therefore, it is likelythat either the Pab-gal, here characterised, and the b-glucosidase,we previously reported [25], can influence fruit firmness eitherdirectly or indirectly, by improving pectin accessibility to otherenzymes, such as endo-polygalacturonases.

In conclusion, the results from this work and previous reports[7,18] point to an important role of b-galactosidases in the earlyevents of fruit ripening and softening. Understanding themolecularbasis of fruit softening will help to develop new strategies for futureprograms aimed at the improvement of the shelf-life of fruits.

red to the sequence of other related plant b-galactosidase.

Residues Identity(%)

Similarity(%)

P X X X X X T I A K 1e18 e e

G I S K D D T I X X 9e26 22 44G I S S X N A e e e 6e20 5 44

Table 4Substrate specificity studies of the purified b-galactosidase against syntheticsubstrates. Enzyme activity was assayed as reported in “materials and methods”.Activities are expressed as percentage of the activity calculated with p-nitrophenylb-D-galactopyranoside towards which the enzyme showed the best performance.ND, not detected.

Substrates Enzymaticactivity (%)

p-nitrophenyl b-D-galactopyranoside 100p-nitrophenyl a-L-arabinopyranoside 10p-nitrophenyl b-D-fucopyranoside 8p-nitrophenyl b-D-glucopyranoside NDp-nitrophenyl a-D-galactopyranoside NDp-nitrophenyl a-D-mannopyranoside ND

C. Gerardi et al. / Plant Physiology and Biochemistry 61 (2012) 123e130128

4. Materials and methods

4.1. Plant material

Sweet cherry (P. avium L., cv Lapins) fruits were collected inspring 2010 from a local orchard. Fruits were harvested from threeplants at four different ripening stages: unripe (stage I), turning(stage II), ripe (stage III) and over-ripe (stage IV).

4.2. Acetone powder preparation

Acetone powder was obtained from 65 g of cherry fruit flesh atdifferent ripening stages, after extraction with 32.5 ml of coldacetone (�20 �C). The mixture was homogenised twice for 45 s ina blender at maximum speed. The homogenate was filteredthrough filter paper in a Buchner funnel and washed with twovolumes of cold acetone (�20 �C). The powder was dried at roomtemperature and stored at �20 �C under N2 atmosphere.

4.3. Protein extraction and b-glycosidases activity assay

Proteins were extracted from acetone powder and the freshflesh of cherry fruits cv Lapins at the above indicated ripeningstages, with ten volumes of H2O containing 40 mM 2-b-mercap-toethanol (buffer A) and stirred for 1 h at 4 �C. After centrifugationat 19,800�g for 30min, the supernatant was retained (H2O extract),the pellet was washed with 30 volumes of buffer A and resus-pended in ten volumes of 50 mM sodium acetate (pH 4.5) con-taining 40 mM 2-b-mercaptoethanol (buffer B) and stirring for 1 hat 4 �C. After centrifugation, the supernatant was retained and thepellet was washed with 30 volumes of buffer B, resuspended in 5volumes of buffer B containing 1 M NaCl and stirred for 2 h at 4 �C.After centrifugation at 19,800�g for 30 min, the supernatant wasretained (salt extract).

Glycosidase activities were assayed as described by Ross et al.[12]. Different amountsof protein samples in salt extractweremixedwith 25 mM sodium acetate (pH 4), containing 0.3% 2-b-mercap-toethanol, and 2mM p-nitrophenyl glycopyranoside (p-nitrophenylb-D-glucopyranoside, p-nitrophenyl a-D-galactopyranoside, p-nitrophenyl b-D-fucopyranoside, p-nitrophenyl b-D-galactopyrano-side, p-nitrophenyl a-D-mannopyranoside, p-nitrophenyl a-L-ara-binopyranoside, p-nitrophenyl b-D-xylopyranoside, p-nitrophenyla-D-fucopyranoside, p-nitrophenyl a-D-rhamnopyranoside, p-nitrophenyl b-D-mannopyranoside) (Sigma, St. Louis, MO). After60 min incubation at 30 �C in microtiter plates, the reactions werestopped by the addition of 200 ml of 200mMNa2CO3 and absorbanceat 405 nm was measured using a microtiter plate reader (Bio-Rad,Hercules, CA, USA). One unit (U) of glycosidase activity was definedas the amount of protein able to release 1 mmol of p-nitrophenolper min.

4.4. b-Galactosidase purification

Salt-soluble proteins were extracted as described above from50 g of acetone powder obtained from the ripe (stage III) cherryfruit of cv Lapins. Ammonium sulphate was added to the extract to90% saturation. The precipitated proteins were resuspended in10 mM triethanolamine pH 8 (buffer C) and dialysed for 24 hagainst the same buffer. Protein purification was carried out witha Biosys 510 Protein Purification System (Beckman, Fullerton CA) byanion exchange column chromatography on a Q HyperD� 20column (Beckman 0.46� 10 cm) pre-equilibratedwith buffer C. Thecolumn was washed with the same buffer and the b-galactosidaseactivity was eluted with the unbound proteins. b-Galactosidaseactivity was assayed as described above for glycosidase activity.Ammonium sulphate was added to the active unbound proteins to90% saturation. The precipitated proteins were resuspended in50 mM sodium acetate pH 4.5 (buffer D) and dialysed for 24 hagainst the same buffer. Further b-galactosidase purification wascarried out on an S-Hyper D� 20 column (Beckman 0.46 � 10 cm)pre-equilibrated with buffer D. The column was washed with thesame buffer and the bound proteins eluted with a 0e500 mMgradient of NaCl with a flow of 1 ml/min. One millilitre fractionswere collected and assayed for b-galactosidase activity. The activefractions were pooled and purified further by hydrophobic inter-action chromatography on a TSK phenyl 5PW column (Bio-Rad75 � 7.5 mm). The column was equilibrated with 50 mM sodiumacetate (pH 4.5) added with 1.5 M ammonium sulphate, and thebound proteins were eluted with a linear 1.5e0 M gradient ofammonium sulphate with a flow of 0.5 ml/min. The active fractionswere pooled and applied to a TSK 2000 column (TOSO Haas, Phil-adelphia, PA, 0.75 � 30 cm) for size exclusion chromatography(SEC). The columnwas equilibratedwith buffer D and proteins wereeluted at flow rate of 0.5 ml/min.

4.5. Electrophoresis

SDS-PAGE was carried out on 15% polyacrylamide gels. NativePAGE was performed on linear gradient (10e30%) acrylamide slabgels. The molecular weight of proteins was determined using 97e14 kDa and 200e6.5 kDamolecular weight marker proteins (Sigma,St. Louis, MO). Native isoelectric focussing (IEF) was performed on5% acrylamide slab gels as described by Robertson et al. [42].Isoelectric points were calculated using IEF protein standards from9.3 to 3.55 (Sigma, St. Louis, MO). Activity staining after nativeisoelectric focussing was performed according to Spielman andMowshowitz [43] using 6-bromo-2 naphthyl-b-D-galactoside(Sigma, St. Louis, MO) as a substrate.

4.6. b-Gal peptide sequencing

Amino terminal sequence analysis of the purified b-galactosi-dase was performed with a Pulse liquid-phase protein sequencer(477A, Applied Biosystems-Perkin Elmer, Foster City, CA). Proteins,separated by SDS-PAGE, were electroblotted onto a polyvinylidenedifluoride membrane (Nycomed, Amersham Biosciences, SanFrancisco, CA, USA). The blotted polypeptide was excised from themembrane and arranged in the sequencer cartridge. Phenyl-thiohydantoin (PTH)-amino acids from Edman degradation phaseswere identified by HPLC with a PTH analyser (ABI-Perkin ElmerMod 120A) on line with the sequencer.

4.7. Enzyme characterisation

The pH optimum of the purified enzyme against p-nitrophenylb-D-galactopyranoside was determined in acetate buffer at a pH

C. Gerardi et al. / Plant Physiology and Biochemistry 61 (2012) 123e130 129

range from 3.5 to 5.5; phosphate buffer at a pH range from 6.0 to 8.0and TriseHCl buffer at a pH range from 8.5 to 9.0. The purifiedenzyme was assayed in the presence of 0.1e20 mM p-nitrophenylb-D-galactopyranoside, p-nitrophenyl b-D-fucopyranoside andp-nitrophenyl a-L-arabinopyranoside, and the values of Km andVmax determined for each substrates.

4.8. Substrate specificity

The activities of b-galactosidase and other glycosidases weredetermined using the corresponding p-nitrophenyl glycosidesubstrates. The assay was performed as described above.

4.9. Preparation of total cell walls from ripe sweet cherry fruit

Fruit flesh was boiled for 10 min in methanol and homogenisedin one volume of 40 mM HEPESeNaOH pH 7.5, plus 0.5 M KCl. Thehomogenate was filtered through Miracloth and the filtratecentrifuged at 800�g for 10 min. The supernatant was discardedand the pellet washed three times with 40 mM HEPESeNaOH pH7.5, twice with chloroformemethanol (1:1) and twice with coldacetone. The pellet was dried under vacuum and saved as a total cellwall fraction.

4.10. Enzymatic assays on total cell wall fraction

Hydrolytic activity towards purified cell wall fraction wasassayed as described by Kitagawa et al. [37]. Each reaction mixture(400 ml) contained 4 mg of total cell walls in 50 mM sodium acetate(pH 5.0), along with 50 mM NaCl and 8 ml of toluene as a bacte-riostat. Purified b-galactosidase (10 mg) was added to the reactionmixture and allowed to react for 72 h at 37 �C. The reactions werestopped by boiling at 100 �C for 3 min. After centrifugation at16,000�g for 20 min, the amount of released D-galactose and D-glucose was analysed using an enzymatic kit for glucose andgalactose determination (Boehringer Mannheim GmbH, Man-nheim, DE). Control samples were run with the heat-denaturedprotein.

4.11. Enzymatically active cell wall preparation

Enzymatically active cell walls were prepared from flesh ofcherry fruits at four ripening stages (unripe, turning, ripe andover-ripe) as described by Fernández-Bolanos et al. [44]. To 100 gof flesh 10 mM sodium phosphate buffer (pH 7.4, 200 ml) wasadded with 1 mM DTT, 2 mM sodium metabisulphite and 0.5 Msucrose. The mixture was homogenised in a Waring blender for2 min at maximum speed and then stirred for 1 h in an ice-bath.The homogenate was centrifuged at 20,000�g for 30 min (4 �C)and the supernatant was discarded. The pellet was filteredthrough a fine-mesh (40 mm) nylon filter and washed with 2 l of10 mM sodium phosphate (pH 7.4) containing 1 mM dithio-threitol. Finally, the residue was washed with 500 ml of coldacetone (�20 �C). The cell walls were dried at room temperatureunder vacuum, collected, and stored in a sealed containerat �20 �C. Aliquots of cell walls were used to assay enzymaticactivities in cell wall incubations.

4.12. Glycosidase activities in enzymatically active cell walls

Glycosidase activities were determined by measuring theamount of p-nitrophenyl released from the respective p-nitro-phenol glycopyranoside (Sigma, St. Louis, MO) substrates. Thereaction mixtures were composed of 5 mg of cell walls and 25 mMsodium acetate pH 4.0, containing 0.3% 2-b-mercaptoethanol and

2 mM p-nitrophenyl glycopyranoside (p-nitrophenyl b-D-gluco-pyranoside, p-nitrophenyl a-D-galactopyranoside, p-nitrophenylb-D-galactopyranoside, p-nitrophenyl b-D-fucopyranoside andp-nitrophenyl a-L-arabinopyranoside). After 60 min incubation at30 �C, the reaction mixtures were stopped by the addition of 200 mlof 0.2 M Na2CO3 and centrifuged at 15,000�g for 10 min. Theliberated p-nitrophenol was measured in the supernatant byabsorbance at 405 nm. Enzymatic activity was expressed in nkat(nmol p-nitrophenol liberated s�1) per g of cell wall. Blanks wereprepared by stopping the reaction prior to the addition of thesubstrate.

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