Activity and Expression of Banana Starch Phosphorylases

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O R I G I N A L A R T I C L E

Renata V. da Mota Beatriz R. CordenunsiJoa o R.O. do Nascimento Eduardo PurgattoMaria R.M. Rosseto Franco M. Lajolo

Activity and expression of banana starch phosphorylasesduring fruit development and ripening

Received: 3 March 2002 / Accepted: 1 July 2002 / Published online: 3 September 2002 Springer-Verlag 2002

Abstract Two main forms of starch phosphorylase (EC2.4.1.1) were identified and purified from banana (Musaacuminata Colla. cv. Nanica o) fruit. One of them, des-ignated phosphorylase I, had a native molecular weight

of 155 kDa and subunit of 90 kDa, a high affinity to-wards branched glucans and an isoelectric point around5.0. The other, phosphorylase II, eluted at a higher saltconcentration from the anion exchanger, had a low af-finity towards branched glucans, a native molecularweight of 290 kDa and subunit of 112 kDa. Kineticstudies showed that both forms had typical hyperboliccurves for orthophosphate (Pi) and glucose-1-phos-phate, and that they could not react with substrates witha blocked reducing end or a-1,6 glucosidic bonds. An-tibodies prepared against the purified type-II form andcross-reacting with the type-I form showed that therewas an increase in protein content during development

and ripening of the fruit. The changes in protein levelwere parallel to those of phosphorylase activity, in boththe phosphorolytic and synthetic directions. Consideringthe kinetics, indicating that starch phosphorylases arenot under allosteric control, it can be argued that proteinsynthesis makes a contribution to regulating phosphor-ylase activity in banana fruit and that hormones, likegibberellic acid and indole-3-acetic acid, may play aregulating role. For the first time, starch phosphorylasesisoforms were detected as starch-granule-associatedproteins by immunostaining of SDSPAGE gels.

Keywords Fruit ripening Musa Starch

Starch-granule-associated proteinsStarch phosphorylase

Abbreviations daa: days after anthesis GA3: gibberellicacid Glc-1-P: glucose-1-phosphate IAA: indole-3-acetic acid IEF: isoelectric focusing Pi: orthophos-phate SGAP: starch-granule-associated protein

Introduction

Starch phosphorylases (EC 2.4.1.1) catalyze the revers-ible conversion of the a-1.4-glucosidic bonds of glucanpolymers into glucose-1-phosphate (Glc-1-P) in thepresence of orthophosphate (Pi). In the synthetic direc-tion, a glucosyl unit is transferred from Glc-1-P intoa-1,4-glucans of increasing chain length. In the phos-phorolytic reaction, Glc-1-P is released from a-1,4-glu-cans of decreasing chain length. Because of thereversibility of the conversion it is possible for these

enzymes to contribute to both the synthesis and degra-dation of starch.

Two forms of the enzyme have been purified andcharacterized in a variety of plant tissues, e.g. spinachleaves (Steup and Scha chtele 1981), pea cotyledons (vanBerkel et al. 1991), potato tubers (Mori et al. 1991),potato leaves (St-Pierre and Brisson 1995) and Viciafaba cotyledons (Buchner et al. 1996).

It is believed that the plastidic form, which has highaffinity for glycogen and a subunit of 90 kDa, may beinvolved in starch synthesis and/or degradation due toits localization next to the starch granule, while abranched glucan with a high degree of polymerization,

present in the cytoplasm, may be the substrate for acytosolic phosphorylase that has low affinity for glyco-gen and a subunit of >100 kDa (Buchner et al. 1996).Based on experiments in which the expression of cyto-solic phosphorylase was inhibited in potato plants,Duwenig et al. (1997) concluded that it does not par-ticipate in starch metabolism, but is connected to theregulation of plant growth and development.

In many climacteric fruits, starch, the primary en-ergy reserve, is synthesized in the amyloplasts duringdevelopment and is degraded during ripening.

Planta (2002) 216: 325333DOI 10.1007/s00425-002-0858-6

R.V. da Mota B.R. Cordenunsi J.R.O. do NascimentoE. Purgatto M.R.M. Rosseto F.M. Lajolo (&)Laborato rio de Qumica, Bioqumica e BiologiaMolecular de Alimentos, Departamento de Alimentosa Nutricao Experimental, FCF, Universidadede Sao Paulo, Avenida Prof Lineu Prestes 580,Bloco 14, 05508-900, Sao Paulo, SP, BrasilE-mail: [email protected]: +55-11-38154410


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Hydrolytic and phosphorolytic enzymes are involvedin this process although it is not known whichmechanism triggers the process of granule degradation(Areas and Lajolo 1981; Garcia and Lajolo 1988;MacRae et al. 1992).

Banana fruit has a high amount of starch, which israpidly degraded into sugars during ripening, and bothamylolytic and phosphorolytic activities have alreadybeen detected. Areas and Lajolo (1981) described acomplex pattern of phosphorolytic activity duringbanana fruit ripening. Iyare and Ekwukoma (1992)also observed an increase in phosphorylase and in-vertase activities, besides amylases, during the ripeningof plantain. Similar to Garcia and Lajolo (1988), theyobserved a sharp increase in amylase activity,suggesting that hydrolysis would be more importantthan phosphorolysis in starch degradation, but therewas no strong evidence to establish the predominantpathway.

Despite the results indicating a hydrolytic pathway,the requirement for ATP for the synthesis of solublesugars in banana and kiwi fruits suggests that at least

some part of the carbohydrate exported from the amy-loplast should be in the form of hexose phosphate(MacRae et al. 1992; Hill and ap Rees 1994). Thephosphorolysis process may keep the amount of ATP ata sufficient level to account for sucrose synthesis.However, the overall contribution of each pathway ofstarch-degradative activity, their regulation at themolecular and genetic levels, and the result of theirconcerted action have not been accessed.

Because of the lack of information about phosphor-ylase activity, expression and regulation in fruits duringthe starch degradation process, the aim of this work wasto characterize and estimate the activity and expression

of starch phosphorylases during the development andripening of banana fruit. Based on the literature indi-cating that starch phosphorylase is not regulated by al-losteric control in higher plants and that the expressionof other metabolizing enzymes can be affected by planthormones, indole-3-acetic acid (IAA) and gibberellicacid (GA3) were infiltrated into banana slices to checktheir influence on starch degradation, and phosphory-lase activity was also studied.

Materials and methods

Plant material

Unripe immature banana (Musa acuminata Colla. cv. Nanicao)fruits were harvested at different stages of development. Unripemature fruits, harvested at 120 days after anthesis (daa), were leftto ripen under a controlled temperature (19 C) and the ripeningprocess was followed via the starch content, enzymatically deter-mined as described before by Nascimento et al. (2000). Samplestaken at different days after harvesting were peeled, sliced, frozen inliquid N2 and stored at 80 C. For isolation of granule starch andthe extraction of granule-bound protein, bananas were used atthree stages: immature green (80 daa), mature green (110 daa) andclimacteric (ca. 10% starch).

Protein extraction and phosphorylase activities

One part of the frozen sample was extracted in 6 parts of50 mM HepesKOH (pH 7.5) containing 20 mM EDTA, 20 mMcysteine, 1% polyvinylpyrrolidone MW 40,000 (PVP-40) and1 mM benzamidine. The supernatant obtained after centrifuga-tion at 8,000 g for 50 min was considered to be the crude ex-tract. Starch phosphorylase activity was measured in thephosphorolytic and synthesis directions, as described by Kumarand Sanwal (1982). In the phosphorolytic direction, the assaysystem consisted of 50 mM Mes buffer (pH 6.0), 1% freshly

prepared soluble starch, 5 mM sodium fluoride, enzyme extractand 50 mM Na2HPO4 in a total volume of 500 ll. The reactionwas carried out at 30 C and stopped by heating the mixture inwater bath for 1 min. The released Glc-1-P was determined in acoupled enzyme reaction using phosphoglucomutase and Glc-6-Pdehydrogenase according to Bergmeyer (1984). In the synthesisdirection, the assay was performed in 50 mM Mes buffer (pH6.0), 5 mM sodium fluoride, 1% freshly prepared starch, enzymeextract and 15 mM Glc-1-P in a total volume of 500 ll. Thereaction was carried out at 30 C and stopped by the addition of1 vol. of 10% trichloroacetic acid (TCA) and 4 vol. of 0.1 Nsodium acetate. The Pi formed was quantified by using a mo-lybdate-based assay (Lowry and Lopez 1946). The occurrence oftwo forms (I and II) of the enzyme in banana fruits was checkedby their differential affinity towards branched glucans in a nativepolyacrylamide gel containing glycogen (25 lg/ml gel) according

to Sonnewald et al. (1995). Protein content was determined ac-cording to Bradford (1976) or by the Lowry method as modifiedby Peterson (1977).

Enzyme purification

Crude extracts of bananas at 110 daa were fractionated with am-monium sulfate (3060% saturation) and the recovered pellet(8,000 g/10 min.) was dialyzed in 20 mM Tris buffer (pH 7.5)containing 1 mM benzamidine (buffer A). The dialyzed fractionwas directly applied to DEAE-cellulose, and after being washedwith buffer A plus 0.15 M NaCl to remove unbound proteins, thephosphorylase fractions were eluted with 0.7 M NaCl in buffer A.After concentration by ammonium sulfate (85% saturation) thedialyzed extract was loaded onto an HR 5/5 Mono Q column

(Amersham Pharmacia Biotech) equilibrated with buffer A, andprotein was eluted with a linear KCl gradient (00.5 M) in the samebuffer. Fractions containing phosphorylase activity were pooledand loaded onto a Sephacryl S-300 column, previously equilibratedwith buffer A containing 0.15 M NaCl. The eluted fractions pre-senting phosphorylase activity were pooled and used for subse-quent analysis.

Molecular weight and isoelectric point (pI)

Native molecular weight was determined by gel-filtration chroma-tography in a Sephacryl S-300 column (30 cm 1 cm i.d.), previ-ously calibrated with molecular weight standards (29660 kDa).SDSPAGE (7.5%) gels, calibrated with molecular weight markersranging from 29 to 205 kDa was used to determine the subunitmolecular weight, while pI was determined by isoelectric focusing(IEF) gels containing 2% ampholytes (pH 310) and proteinstandards with pI ranging from 4.6 to 7.2.

Antiserum preparation

Based on their activities on native PAGE gels, phosphorylasebands were electro-eluted and submitted to SDSPAGE. Theprotein bands related to phosphorylase I and phosphorylase IIwere removed from the gel and used for immunization of malerabbits according to Jurd and Bog-Hansen (1990). For the westernblots, samples were separated by SDSPAGE and electro-blotted

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onto nylon membranes. Incubations of filters and color develop-ment by alkaline phosphatase activity were performed according toSambrook et al. (1989).

Isolation of starch granules and extraction of starch-boundproteins

Starch granules were isolated essentially as described by Ritte et al.(2000). Total starch-granule-associated protein (SGAP), granulesurface protein and granule-enclosed protein were extracted as

described by Ritte et al. (2000), with a modification in SDS (1% to10%) concentration, as recommended by Rahman et al. (1995). Inorder to increase the amount of protein loaded in SDSPAGEwells, extracted proteins were precipitated with 12% TCA and,after stepwise washing with cold ethanol-ether-chloroform (2:2:1,by vol.), acetone and ether, the precipitate was recovered in SDSPAGE sample buffer. Protein separation and western blots weredone as described above.

Preparation and infiltration of banana slices

Banana slices were prepared and infiltrated as already described byPurgatto et al. (2001), using 0.1 mM IAA or 0.1 mM GA3 in120 mM mannitol.

Results

Purification and molecular characterizationof phosphorylases

Two main protein peaks with phosphorylase activitywere eluted from the Mono-Q columns. The form ob-tained at lower salt concentrations (from 0.125 to 0.2 Mof KCl) was named phosphorylase I while the formeluted at higher salt concentrations (from 0.25 to 0.35 Mof KCl) was named phosphorylase II. On native PAGEgels containing glycogen (Fig. 1A), phosphorylase I was

the most active with high affinity towards the branchedglucan (in the top of the gel), while phosphorylase IIconsisted of three proteins with less affinity towards theglycogen.

With the degree of purification achieved after gel fil-tration, it was possible to isolate the phosphorylaseprotein by elution from native PAGE gels. Polyclonalantiserum raised against phosphorylase II confirmed theidentity of the proteic bands since it was able to com-pletely remove the starch phosphorylase activity frombanana extracts in the immunoprecipitation assay.Western blotting (SDSPAGE) of the phosphorylases ateach of the purification steps (Fig. 1B) showed the

enrichment of two bands, with subunits of 90.6 kDa(phosphorylase I) and 112 kDa (phosphorylase II).

Based on the elution volumes with phosphorylaseactivity from the Sephacryl S-300 column, phosphory-lase I is apparently a 155-kDa enzyme while phosphor-ylase II has a molecular weight around 290 kDa. Whenthe pools eluted from the gel-filtration column weresubmitted to IEF, activity staining allowed the identifi-cation of proteins at pH 5.2 and 5.3 from the pool ofphosphorylase I and proteins at pH 6.4, 6.6, 7.6 and 7.8from the pool of phosphorylase II.

Kinetic constants for Glc-1-P and Pi

The kinetic properties of banana phosphorylase were

assayed at different substrate concentrations, both in thesynthetic and degradative directions. When starch wasused as substrate for the phosphorolytic assay with dif-ferent concentrations of Pi or as a primer for the syn-thetic assay using Glc-1-P, typical MichaelisMentencurves were obtained for both phosphorylase forms. Theaffinity of phosphorylase II for Glc-1-P (Km 0.6 mM)was higher than that of phosphorylase I (Km 2.4 mM),and likewise for Pi: 4.4 mM versus 11.2 mM. On theother hand, the values for Vmax obtained at differentconcentrations of Pi and Glc-1-P were higher for phos-phorylase II than for phosphorylase I.

Kinetic constants for glucans

Table 1 summarizes the kinetic parameters for malto-triose, maltopentaose and amylopectin of phosphory-lase I compared with phosphorylase II, and also theresults obtained for the latter using starch, amylose andmaltohexaose.

Km values for maltotriose were similar for both formsbut phosphorylase II showed higher specificity (estimatedby Vmax/Km): 5.8 versus 1.8. On the other hand, phos-phorylase I exhibited lower affinity than phosphorylase II

Fig. 1AC Electrophoretic profiles of banana (Musa acuminata)phosphorylases I (pho I) and II (pho II). A Iodine staining for thesynthetic activity of the pho I and pho II bands on a native PAGE(6%) gel containing glycogen (25 lg/ml). B Coomassie-bluestaining of the protein bands separated by 7.5% SDSPAGE. CImmunostaining of phosphorylase bands separated by 7.5% SDSPAGE, using the antiserum against pho II. Lanes: 1 crude extract,2 3060% ammonium sulfate, 3 pool DEAE, 4 85% ammoniumsulfate, 5 pool PHO I from the Mono Q column, 6 pool PHO Ifrom the Sephacryl S-300 column, 7 pool PHO II from theMono Q column, 8 pool PHO II from the Sephacryl S-300 column.The bands corresponding to pho I and pho II are indicated on theright

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for linear dextrins, such as maltopentaose (Km 0.3 versus0.1). With this substrate, phosphorylase II also showedhigher specificity since the apparent Vmax/Km ratio wasalmost 7 times that of phosphorylase I.

When the two phosphorylase forms were assayedwith a branched glucan like amylopectin, the resultsobtained clearly indicated that isoenzyme 1 had higheraffinity and specificity for this kind of substrate as shownby Km, Vmax and Vmax/Km values.

Phosphorylase activity and expressionduring development and ripening

Figure 2 shows an increase of almost 50% in starchcontent during the last 80 days before harvest andconcomitantly an increase in enzyme activity regardlessof the direction assayed. During this period the activityincreased approximately 130 and 120%, respectively, inthe phosphorolytic and synthetic directions. Westernblotting, using the specific antiserum (Fig. 2A), similarlyshowed the accumulation of the 112-kDa subunit of

phosphorylase II besides smaller amounts of the 90-kDasubunit of phosphorylase I, and also a cross-reactingband around 100 kDa.

When phosphorylase activities were assayed duringfruit ripening, similar results were obtained in bothphosphorolytic and synthetic reactions (Fig. 2B). At thebeginning of starch degradation there was an increase inenzyme activity concomitant with starch degradation,peaking at day 7 of ripening. Afterwards phosphorylasesactivities were reduced almost to the initial level atday 10. As fruit ripening proceeded, a new increase inphosphorylase was detected around day 12 in both di-rections. This behavior was repeatedly observed in sev-

eral different experiments, and it usually happenedduring the late stages of ripening when minimal amountsof starch were present in the pulp.

Western-blot analysis showed an increase in theamounts of phosphorylase I and II subunits, and alsothe 100-kDa band, peaking around days 79 (Fig. 2B).This behavior was consistent with the overall increase inphosphorylase activity and it was a clear indication ofaccumulation of phosphorylase protein during bananaripening even though it did not closely follow the ob-served activity changes.

Table 1 Kinetic parametersof starch phosphorylasespurified from banana (Musaacuminata) fruit

Phosphorylase I Phosphorylase II

Km Vmax Vmax/Km Km Vmax Vmax/Km

Glc-1-P 2.39 6.86 2.8 0.64 9.22 14.4Pi 11.24 9.60 0.8 4.38 21.50 4.9Maltotriose 2.9 5.1 1.8 2.5 14.4 5.8Maltopentaose 0.3 9.1 30.3 0.1 21.1 211Maltohexaose 0.6 59.2 98.7Amylose 1.5 22.2 14.8

Amylopectin 0.1 8.9 89.0 3.2 26.4 8.3Starch 0.4 5.1 12.8

Fig. 2 Time-courses of phosphorylase activity during bananadevelopment (A) and ripening (B), and the corresponding westernblots (below). Starch contents (open squares) and phosphorylaseactivity measured in the phosphorolytic (open circles) and synthetic( filled circles) directions are shown. Data are means SD (n=3).Banana samples were harvested at the time of commercial harvest(day zero) and 80, 50, 25, and 10 days before (A), or at days 1, 5, 7,9, 10 and 12 after harvesting (B). Molecular weights (kDa)corresponding to the phosphorylase proteins are indicated on theright of each blot

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The results obtained for GA3-infiltrated banana sliceswere similar to those observed with IAA (Fig. 5). Adelay of 2 days in starch degradation was observed forthe slices infiltrated with GA3, a pattern that was alsofollowed by the activity profile. In spite of the minordecrease in starch content on the first days after treat-ment, the onset of degradation was impaired by GA3,which was paralleled by a delay in the starch phos-phorylase activity increase. When the extracts wereprobed with the specific serum in western blots (Fig. 5),minor differences were observed between the profiles ofphosphorylase proteins in control and GA3-infiltratedslices.

Discussion

With the procedure employed for purification it waspossible to isolate two forms of starch phosphorylasefrom banana fruit. They differed in subunit size and af-finity for glucans. The phosphorylase I isoform (MW155 kDa, subunit 90.6 kDa) showed high affinity towardsbranched glucans, while phosphorylase II (native MW

290 kDa, subunit 112 kDa) exhibited low affinity towardsbranched glucans. Therefore, like the enzymes from otherplant sources (Nighojkar and Kumar 1997), both forms ofbanana phosphorylase can be considered as dimers.

The Km and Vmax values obtained using differentsubstrates indicated that the phosphorylases also differin relation to affinity and specificity for glucans. In itshigh affinity and specificity for amylopectin, bananaphosphorylase I is similar to the cytosolic phosphory-lases from other plant tissues (Steup and Schachtele1981; Mori et al. 1991; van Berkel et al. 1991; St-Pierreand Brisson 1995; Buchner et al. 1996). As the chlo-roplast envelope membrane is not permeable to high-

molecular-weight polysaccharides, there are two possibleexplanations for phosphorylase I activity: an indepen-dent pool of carbohydrates in the cytosol, or a disinte-gration of the chloroplast envelope during bananaripening, allowing the cytosolic phosphorylase to par-ticipate in the starch degradation process. Both possi-bilities may occur in banana tissue during ripening.Transmission electron microscopy of banana tissueduring the ripening process showed a disorganization ofthe amyloplast membrane (not shown). In addition,preliminary tests in our laboratory pointed to a poly-saccharide in banana fruit similar to that found by Yangand Steup (1990) in higher plants. They purified a gly-

cogen-like polysaccharide with strong affinity for cyto-solic phosphorylase, suggesting that it could be apossible in vivo substrate for the isoenzyme. Phosphor-ylase II showed the highest affinity and specificity formaltopentaose and maltohexaose, for which the Km andVmax values are far from those obtained for maltotrioseand amylopectin. As observed by Witt and Sauter (1995)who found that starch phosphorylase II was able toattack the starch granule when associated with a-amy-lases, these substrates with a low degree of polymeriza-tion would be present in vivo as products of theenzymatic action of a-amylases on the starch granule.The apparent lower affinity and specificity for maltotri-

ose indicate that there is a minimum size for a glucan asa phosphorylase substrate.When glucans of higher molecular weight were tested,

starch showed the highest affinity. However, the lowVmax could be a consequence of the a-1,6 bonds presentin the structure that are not degraded by phosphorylaseaction. Both the molecular and kinetic properties allowus to classify phosphorylase I as a type I or H andphosphorylase II as a type II or L described in otherplants. In this way phosphorylase II would be a com-partmentalized form in the amyloplast, and would be

Fig. 5 Upper panels The effect of GA3 infiltration on starchcontent and phosphorylase activity during banana ripening. Thestarch contents (squares) and phosphorylase activities (circles) weredetermined for control slices infiltrated with mannitol (filledsymbols), and GA3-treated slices (open symbols). Data are means SD (n=3). Lower panels Western blots of bananas sampled atdays 4, 17, 18, 20, 21 and 23 after GA3 infiltration. Molecularweights (kDa) corresponding to the phosphorylase proteins areindicated on the right

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non-allosteric enzymes (Segel 1975, p. 957), suggestingthat banana starch phosphorylase activity is not underallosteric control. This idea is reinforced by the fact thatcovalent and allosteric regulatory mechanisms have notbeen observed for plant phosphorylases, only for thoseof animals. Besides ethylene, other plant hormones, likeauxins and gibberellins, appear to be triggering agentsfor expression of several important genes. In relation tostarch-metabolizing enzymes, the effects of GA3 ona-amylase expression and secretion from the aleuronelayer of cereals are well known (Rogers and Rogers1999). However, there is lack of information for fruits,regarding starch-metabolizing enzymes and plant hor-mones during ripening. In bananas, the accumulation ofb-amylase transcripts was delayed by infiltration of IAA(Purgatto et al. 2001), but neither the activities of otherstarch-metabolizing enzymes nor the effects of otherhormones were evaluated. In this way the infiltration ofplant hormones that can affect starch metabolism couldgive an insight about the regulatory mechanism ofphosphorylase activity.

Figure 4 shows that infiltration of IAA promoted a

2-day delay in starch degradation when compared withcontrol slices. The typical pattern of fluctuations instarch phosphorylase activity showed minor differencesin the level of activity between days 18 and 22, but it wasnot so strong as to justify the delayed starch degrada-tion. Wang et al. (1993) observed the presence of twonew starch phosphorylase isoforms in callus tissue ofsweet potato growing in a medium without auxin. Thewestern-blot analysis did not indicate any appreciabledifference between the profiles of phosphorylase proteinof control and treated slices. Even the smaller and lessabundant phosphorylase bands accumulated to similarextents in the two groups.

It is not ruled out that the fluctuations of phosphory-lase activity during the starch-mobilization period couldbe due to changes in activity of a specific isoform of theenzyme, and it is possible that phytohormones can alterthis equilibrium, leading to impaired starch degradation.These effects seem to be more evident with GA3.

The results obtained for GA3-treated banana slices(Fig. 5) led to the assumption that GA3 could in partregulate starch phosphorylase activity, or at least affectthe mechanism of regulation, since there was a delay ofat least 2 days in the increase in starch phosphorylaseactivity which was parallel to the delay in starch de-gradation. There are several descriptions of the promo-

tion ofa-amylase expression by GA3 in cereals but thereare no data about GA3 and starch phosphorylases. Asdiscussed previously, there is no direct correlation be-tween phosphorylase content and its activity fluctuationduring ripening, which suggests the existence of a morecomplex regulatory mechanism. When the levels ofphosphorylase proteins were evaluated a slight accu-mulation of phosphorylase bands was observed, as evi-denced by the appearance of the smaller bands. Thisenrichment of phosphorylase forms peaked just at theonset of starch degradation, as observed previously in

the whole fruit (Fig. 2). A similar evaluation for theGA3-infiltrated slices showed no evident change in theamount of phosphorylase proteins. The specific antise-rum detected only equal amounts of the abundantphosphorylase II form and no traces of the smallerbands of 90 kDa and 70 kDa.

Even considering that a refinement of the study of theeffects of GA3 on phosphorylase regulation is needed,the results of GA3 treatment are indirect evidence thatthe changes in activity are precisely adjusted to the re-quirements of the starch degradation process duringripening. Whether the hormone has a direct effect onphosphorylase or causes a perturbation in the metabolicequilibrium that affects phosphorylase activity remainsto be determined.

Acknowledgements The authors thank FAPESP for supportingthis work and CAPES for the scholarship.

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Activity and Expression of Banana Starch Phosphorylases