Postharvest Biology and Technology 62 (2011) 133140

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Postharvest Biology and Technology

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Low temperature induced changes in activity and protein levels of the enzymes associated to conversion of starch to sucrose in banana fruit
Roberta Ghedini Der Agopian, Fernanda Helena Gonc alves Peroni-Okita, Claudinia Aparecida Soares, Janana Aparecida Mainardi, Joo Roberto Oliveira do Nascimento, Beatriz Rosana Cordenunsi, Franco Maria Lajolo, Eduardo Purgatto
Universidade de So Paulo, Faculdade de Cincias Farmacuticas, Departamento de Alimentos e Nutric o Experimental, Av. Prof. Lineu Prestes 580, Butant, CEP 05508-000, So Paulo, SP, Brazil

a r t i c l e

i n f o

a b s t r a c t
Storage at low temperature is the most frequently used method to extend the shelf life of banana fruit, and is fundamental for extended storage and transport over long distances. However, storage and transport conditions must be carefully controlled because of the high susceptibility of many commercial cultivars to chilling injury. The physiological behavior of bananas at low temperatures has been studied to identify possible mechanisms of resistance to chilling injury. The aim of this work was to evaluate differences in the starch-to-sucrose metabolism of a less tolerant and susceptible (Musa acuminata, AAA cv. Nanico) and a more tolerant (M. acuminata Musa balbusiana, AAB, cv. Prata) banana cultivar to chilling injury. Fruits of these cultivars were stored in chambers at 13 C for 15 d, at which point they were transferred to 19 C, where they were left until complete ripening. The low temperature induced signicant changes in the metabolism of starch and sucrose in comparison to fruit ripened only at 19 C. The sucrose accumulation was slightly higher in cv. Prata, and different patterns of starch degradation, sucrose synthesis, activity and protein levels of the - and -amylases, starch phosphorylase, sucrose synthase and sucrose phosphate synthase were detected between the cultivars. Our results suggest that starch-to-sucrose metabolism is likely part of the mechanism for cold acclimation in banana fruit, and the cultivar-dependent differences contribute to their ability to tolerate cold temperatures. 2011 Elsevier B.V. All rights reserved.

Article history: Received 14 April 2010 Accepted 24 May 2011 Keywords: Banana cultivars Cold acclimation Ripening Starch mobilization Sucrose synthesis

1. Introduction Banana ripening is associated with susceptibility towards softening, discoloration and decay, thereby making the fruit highly vulnerable to postharvest losses. In order to extend banana shelf life, extensive studies have been carried out with advanced postharvest technologies, such as controlled atmosphere storage methods and cold storage (Chauhan et al., 2006). Cold storage is limited to temperatures above 14 C, due to chilling injuries that lower temperatures can cause on banana fruit. Symptoms of chilling injury include abnormal ripening with a delay in the yellow color development of skin, failure of fruit softening (Jiang et al., 2004), skin pitting and pulp browning caused by cellular membrane damage (Yang et al., 2000; Nguyen et al., 2003). However, the process of chilling injury seems to differ among cultivars and also appears to be related to the genome group. For example, Lichtemberg et al. (2001) noted that the B genome appears to confer greater cold

Corresponding author. Tel.: +55 11 30913656; fax: +55 11 38154410. E-mail address: epurgatt@usp.br (E. Purgatto). 0925-5214/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2011.05.008

resistance to banana than the AAA group both in the eld and during storage. The cultivars that produce fruit with enhanced cold tolerance would be interesting for genetic breeding programs aiming the improvement of banana chilling injury resistance. Several mechanisms have been proposed to explain cold tolerance in plants, including sugar accumulation. Starch-to-sucrose metabolism is accelerated by the increased activity of enzymes, such as amylases (Cottrell et al., 1993), starch phosphorylases (Claassen et al., 1993) and sucrose phosphate synthase (SPS) (Reimholz et al., 1997). However, compared to other vegetable tissues, the role of this metabolism in cold acclimation of banana fruit is poorly understood. The aim of this work was to assess the contribution of starch-tosucrose metabolism as a possible factor conferring cold resistance to some cultivars of banana. For this purpose, we compared the activities and protein levels of enzymes related to the starch-tosucrose metabolism, besides other ripening parameters, in cultivars of bananas that are susceptible (Musa acuminata AAA, subgroup Cavendish, cv. Nanico) and less susceptible (M. acuminata Musa balbusiana AAB, subgroup Prata) to chilling injury (Lichtemberg et al., 2001) after storage at 13 C for 15 d. At 22 C and 85%

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of relative humidity, Nanico fruit achieved peel color index 6 (ideal for commercial sale, skin totally yellow Aurore et al., 2009) at 1216 d after harvest while Prata bananas take between 5 and 7 d to reach the same stage. However, the Prata cultivar, the most consumed in Brazil, is able to tolerate 35 d exposure to 10 C without presenting chilling injury symptoms (Martins et al., 2007). The opposite is observed for Nanico fruit that exhibit several symptoms of chilling injury after 5 d exposure to 10 C (Lichtemberg et al., 2001). 2. Material and methods 2.1. Material Mature green bananas (M. acuminata, AAA, cv. Nanico) and (M. acuminata M. balbusiana, AAB, cv. Prata) were obtained in CEAGESP (Companhia de Entrepostos e Armazns Gerais de So Paulo). The experiment with bananas belong to cv. Nanico was conducted from 29/11/2005 to 27/12/2005 with cv. Prata bananas started at 10/03/2006 and nished at 01/04/2006. The experimental design was totally randomized with the factorial scheme composed by two different temperature treatments. The fruit of each cultivar were separated into two groups and stored in separate chambers at 19 C (control group) or 13 C (cold-stored group). The cold-acclimated fruit were transferred to 19 C after 15 d to complete ripening. At least ve fruit were sampled from each group (control and cold-stored) and were peeled, sliced, frozen in liquid N2 and stored at 80 C for future analysis. Ethylene production and CO2 emission (respiration) was measured daily throughout the experiments and sampling was conducted each two days during preclimacteric phase and daily during the climacteric and postclimacteric phase. 2.2. Ethylene and CO2 emission measurements For ethylene and respiration analyses, bananas were enclosed in 1.5 dm3 jars (three ngers per jar; six jars per treatment). After 1 h, 10 cm3 (for ethylene analysis) and 1 cm3 (for CO2 analysis) samples were taken from the jar headspace using a gas-tight syringe and injected into a gas chromatograph (HP-6890 Agilent Technologies). A ame ionization detector and a thermal conductivity detector were employed for the ethylene and CO2 analyses, respectively. For both gases, an HP-Plot Q (30 m, I.D. 0.53 mm Agilent Technologies) column was used; the injector and detector were set at 250 C, and the runs were performed isothermically at 30 C. The uxes of the helium carrier gas were 0.017 cm3 s1 for ethylene and 0.067 cm3 s1 for CO2. The injections were made in pulsed splitless mode for ethylene and in split mode for CO2 analysis (50:1). Ethylene and CO2 standards in synthetic air (Air Liquid LTD) were used for calibration curves. 2.2.1. Carbohydrate determination Starch from frozen samples (100 mg) was solubilized in 3 cm3 of 0.5 M NaOH. After neutralization with 3 cm3 of 0.5 M acetic acid, an aliquot was precipitated with 4 cm3 of 80% ethanol. The precipitated starch was hydrolyzed with amyloglucosidase (E.C. 3.2.1.3) (28,000 units/l), and the resultant glucose was determined by the glucose-oxidase (E.C. 1.1.3.4) glucose-peroxidase (E.C. 1.11.1.7) glucose-ABTS (2,2 -azinobis[3-ethylbenzthiazoline]sulfonate) system, as described by Cordenunsi and Lajolo (1995). Sucrose was extracted from the fruit pulp frozen in liquid nitrogen and ground to a ne powder in a mortar. For each extraction, 0.5 g of banana frozen powder was mixed, at a ratio of 1:4 (w/v), with 80% ethanol (v/v) at 80 C under agitation (600 s). The homogenate was centrifuged (10,000 g, for 600 s at 25 C), and the supernatant was transferred to a 25 cm3 volumetric ask. The extraction was

repeated twice for each sample, the supernatants were combined and the volume was topped up with 80% ethanol. An aliquot of 1 cm3 of each ethanolic extract was evaporated under vacuum, in a Speedvac system (Savant, Co, Milford, USA). The volume was reconstituted with 1 cm3 of ultrapure water and the sucrose was measured in a HPLC system equipped with a pulsed amperometric detector (Dionex, Sunnyale, USA) and a CarboPac PA1 (4 250 mm) column. Sucrose was eluted at a constant ux of 0.17 cm3 s1 of 18 mM NaOH for 24 min. 2.2.2. Protein extraction For sucrose synthase (SuSy, E.C. 2.4.1.13) and sucrose-phosphate synthase (SPS, E.C. 2.4.1.14) frozen samples were ground in 4 cm3 volumes (w/v) of 100 mM HepesKOH (pH 7.0), 1 mM NaF, 20 mM EDTA, 20 mM cysteine, 1% (w/v) polyvinylpyrrolidone (PVP) 40,000 and 1 mM benzamidine. After centrifugation, the extracts were desalted by dialysis. For -amylase (E.C. 3.2.1.1), the desalted extracts were obtained by the same procedure, using an extraction buffer containing 100 mM HepesKOH (pH 7.0), 0.3% (w/v) NaCl, 20 mM cysteine, 1% (w/v) PVP 40,000 and 1 mM benzamidine. For -amylase (E.C. 3.2.1.2), the extraction buffer contained 50 mM sodium phosphate (pH 7.0) 1% (w/v) soluble PVP 40,000, 1 mM benzamidine and 20 mM cysteine. For starch phosphorylases (E.C. 2.4.1.1), the procedure was the same as amylases, except the concentration of HepesKOH and pH (50 mM and 7.5, respectively), and by addition of 20 mM EDTA (nal concentration). Protein concentrations were measured by the Bradford (1976) method using bovine serum albumin as a standard. 2.2.3. Assay of SuSy, SPS, amylases and starch phosphorylases The SuSy activity was measured in the sucrose synthesis direction using 100 mM TrisHCl (pH 7.6), 1 mM NaF, 10 mM fructose, 5 mM UDP-glucose, 15 mM MgCl2 and freshly desalted extract. The procedure for the SPS assay under saturated conditions utilized 50 mM HepesKOH (pH 7.5), 1 mM NaF, 10 mM UDP-glucose, 5 mM fructose-6-phosphate, 15 mM glucose-6-phosphate, 15 mM MgCl2 and 1 mM NaF. In both cases, the sucrose (SuSy activity) and sucrose-6-phosphate (SPS activity) released was assayed by the thiobarbituric acid method, as described by Cordenunsi and Lajolo (1995). The - and -amylase activities were measured using the Ceralpha Alpha-Amylase and Betamyl kits (Megazyme Int. Ireland Ltd.), respectively, according to the manufacturers instructions. The reaction was stopped after 1 h with 1% Tris. The -nitrophenol released from the substrate was measured at 410 nm. The starch phosphorylases activities were determined by native PAGE in 6% acrylamide/bis-acrylamide (30:0.8) separation gels containing 375 mM TrisHCl pH 8.8 and 0.4% (w/v) oyster glycogen (Sigma). The stacking gels were 3.75% acrylamide/bis-acrylamide (30:0.8), 62.5 mM TrisHCl pH 6.8. The samples (10 g total protein) run at constant current (15 mA) at 4 C. After the run, the gels were washed for 15 min with 40 cm3 washing buffer (100 mM sodium citrateNaOH, pH 6.0) and incubated 1 h at 37 C, without agitation, in the same buffer containing 0.05% of potato soluble starch and 20 mM glucose-1-phosphate. The gels were washed with the buffer citrateNaOH for 15 min at room temperature and the activity bands were revealed with I2 0.67% (w/v)/KI 3.33% (w/v) in deionized water. The gels were digitalized using the GS-700 scanner (Bio-Rad Laboratories) and the images were registered with the Molecular Analyst 1.7 software (Bio-Rad Laboratories) where the brown background derived from the glycogen staining was removed for better visualization of the starch phosphorylase activity bands. 2.3. Total protein extraction and immunoblotting The total protein was extracted, as recommended by Laemmli (1970), and quantied, according to the method of Lowry et al.

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Fig. 1. Ethylene and CO2 levels during ripening at 19 C of Nanico and Prata bananas (left panels) and at low temperature storage (15 d at 13 C) followed by ripening at 19 C (right panels). Each point represents the mean for each parameter and the bars indicate standard deviation (n = 6).

(1951) as modied by Petterson (1977). The extracts were separated with SDS-PAGE and transferred from the gel to a nitrocellulose membrane in a solution of 25 mM Trizma and 192 mM glycine (pH 8.3) at constant voltage (30 V) for 16 h at 4 C. To ensure equal loading of protein in the lanes, the membrane was stained with Ponceau S, according to the method of Sambrook et al. (1989). After destaining in water, the membrane was blocked for 1 h using 50 mM TrisHCl and 150 mM NaCl with 5% skim milk (pH 7.5). Then, the membrane was submitted to a reaction with a specic plastidial -glucan-phosphorylase antibody (Mota et al., 2002) diluted 1:3000 for 10 g of total protein. For SPS and SuSy, anti-serum were diluted 1:1000 for 20 g of protein, as described by Nascimento et al. (1997, 2000). The extract was also subjected to a reaction with the 1:100 dilutions of - and -amylase antibodies, as described by Nascimento et al. (2006) for 30 g protein. Each membrane was washed in TBS buffer (20 mM TrisHCl, pH 8.0, 150 mM NaCl), incubated with the antibody (conjugated with alkaline phosphatase) in a TBS buffer medium with 5% skim milk (diluted 1:30,000), washed again and developed, as described by Sambrook et al. (1989), using a chromogenic substrate. 2.4. Statistical analysis Data analyses were performed using the Tukeys test (p < 0.05) in the Statistical Analysis System (SAS for Windows v.9.0, SAS Institute, Inc., Cary, NC, USA) software package. 3. Results 3.1. The climacteric of Nanico and Prata bananas after cold storage The main differences among the controls of both cultivars (stored at 19 C) were related to the climacteric (Fig. 1). The ethylene and CO2 levels of Prata bananas increased at 2 d after harvest (DAH) and remained high until the end of ripening. The climacteric of Nanico started about 15 d after harvest, and the levels of ethylene were about 3 times lower than those of Prata. On the other

hand, the respiration of Prata bananas was about 2 times lower than Nanico. The appearance of the fruit stored at 19 C after the climacteric was identical to that classied as stage 6 in the peel color index scale (Aurore et al., 2009), i.e., entirely yellow, the pulp texture of the fruit was soft and the aroma was typical for mature bananas. Prata bananas has a postharvest life shorter than Nanico and the measurements were stopped earlier in the Prata group due to the overripe state of the fruit in the two storage conditions (12 DAH at 19 C and 22 DAH at 13 C). Low-temperature (13 C) storage affected the climacteric in both cultivars. Prata presented an evident delay in ripening, but the peak levels of ethylene and CO2 were not affected (Fig. 1). Although the onset of ripening in Nanico was similar to that of the control, the production of ethylene was only about half of that of the control fruit. The visual appearance and softening, after the low temperature storage, of the fruit from both groups were equivalent to the groups stored only at 19 C. However, when Nanico fruit were kept at 10 C for 15 d before transference to 19 C, the bananas developed symptoms of chilling injury (skin browning and unpleasant off-avor development) while Prata fruit ripened as the group kept at 13 C for 15 d (data not shown). 3.2. Starch breakdown and sucrose synthesis of Nanico and Prata bananas Both cultivars accumulated similar amounts of starch during development (20%) (Fig. 2) and at the end of the ripening (4%), but starch degradation in Prata fruit started 10 d before that of Nanico. Sucrose levels were similar in both cultivars. In addition, the carbohydrate amounts were not affected by storage at low temperature but Prata had a substantial delay in the onset of starch degradation and sucrose synthesis, similarly to the ethylene and CO2 proles (Fig. 1). Interestingly, a discrete but persistent increase in sucrose levels of both cultivars was observed during cold storage. 3.3. The activities of - and -amylases in bananas Nanico and Prata Although the levels of starch in both cultivars were comparable, the soluble - and -amylases activities in Prata bananas were 2.4-

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Fig. 2. Starch mobilization and sucrose synthesis during ripening at 19 C of Nanico and Prata bananas (left panels) and the effects of the low temperature storage (15 d at 13 C) followed by ripening at 19 C. Each point represents the mean for each parameter and the bars indicate standard deviation (n = 3).

and 3.0-fold higher, respectively, than those of Nanico (Fig. 3). The activity of -amylase increased consistently as long as the fruit remained in low temperature. By contrast, the -amylase activity presented another peak in Prata bananas but not in Nanico. The protein amount, as assessed by immunoblotting, reected different levels of amylase activities in the cultivars (Fig. 4), as stronger signals were observed for Prata. In addition, the intensity of the bands for - and -amylases was well correlated to the soluble activities observed for the control and cold-stored fruits of this cultivar. The additional peak of -amylase activity observed at day 6 in cold-stored Prata bananas was also coincident to an intense band revealed in the immunoblotting.

3.4. The activities of cytosolic and plastidial starch phosphorylases Cold storage also promoted signicant differences in the activities of plastidial and cytosolic forms of starch phosphorylase in both cultivars (Fig. 5). The differentiation of the phosphorylases isoforms in the native gel containing glycogen is based on the different afnities towards the branched glucan. The cytosolic form has high afnity towards the glucan and this is recognized as a band at the top of the gel while the plastidial form exhibits less afnity towards glycogen and is detected as a band in the middle of the gel (Sonnewald et al., 1995; Zeeman et al., 2004). While the

Fig. 3. Activity proles of starch hydrolases in Nanico and Prata bananas ripened at 19 C (control group) and fruit stored at low temperature (13 C) for 15 d and transferred to 19 C for ripening. Each point represents the mean for each enzyme assay and the bars indicate standard deviation. The assays were done in triplicate.

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Fig. 4. Protein levels of starch hydrolases from Nanico and Prata bananas ripened at 19 C (control groups) and stored 15 d at 13 C and transferred to 19 C for ripening.

activities of the two phosphorylase forms of Nanico bananas were negatively affected by cold, the opposite effect was seen for Prata. More intense activity bands for the cytosolic form were noticed in extracts from cold-stored Prata. The same pattern was observed for the plastidial form, which revealed an intense band at day 5. The corresponding protein levels of the plastidial form were increased for the Prata cultivar (control or low-temperature storage). 3.5. Activity and expression of sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) The activities and protein levels of SuSy and SPS were measured during ripening of the control and cold-stored fruit. The control group of the Nanico cultivar (stored at 19 C) presented changes in SuSy and SPS activities (Fig. 6) and protein levels (Fig. 7), in accordance to previous observations (Cordenunsi and Lajolo, 1995; Nascimento et al., 1997, 2000). Although there are no previous reports of SuSy for Prata bananas, the observed prole was similar to that of Nanico, while SPS was quite different between the cultivars, In Prata bananas stored at 19 C, the SPS activity the SPS

activity on day 1 was almost three times higher than that of Nanico. In both cultivars, the SPS activity increased almost three fold during ripening, but in Prata the highest activity level was twice that observed in Nanico. In contrast, when the fruit were kept at low temperature, the proles between the cultivars were very different. While Nanico fruit stored at low temperature presented no changes from their respective controls, the Prata fruit exhibited a quite different SuSy activity prole, peaking at day 5 (DAH) and remaining high until the day 15. The Prata fruit presented a higher SPS activity than Nanico (Fig. 6), and this difference persisted even in the cold-acclimated bananas. In addition, there was also a remarkable peak of activity at day 5 in Prata stored at 13 C. According to the immunoblottings, the protein levels of SuSy and SPS in Prata bananas were correlated to the observed changes in activity (Fig. 7). The bands related to SuSy indicated a continuous decrease in protein levels during ripening at 20 C, but a signicant increase in the intensity of the reactive bands when the fruit were kept in cold. For SPS, a more intense band was also noticed at day 5 followed by a discrete increase through ripening.

Fig. 5. Gel activity assay for cytosolic and plastidial starch phosphorylases. The bands are differentiated by the migration pattern in polyacrylamide gel containing glycogen. The cytosolic form has high afnity towards the glucan and remains at the top of the gel while the plastidial form has low afnity and is detected in the middle of the gel. Bands are detected by the ability of the enzymes to synthesize long glucan chains using starch as a glucan primer (Sonnewald et al., 1995) and are revealed by iodine staining. The upper panels are the activities of phosphorylases detected in control (19 C) and low temperature stored fruit (13 C) of Nanico and Prata bananas. The lower panels (indicated as protein levels) show the immunoblottings of the plastidial form of starch phosphorylase.

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Fig. 6. Activity proles of SuSy and SPS in Nanico and Prata bananas ripened at 19 C (control group) and fruit stored at low temperature (13 C) for 15 d and transferred to 19 C for ripening (as indicated by the dashed line). Each point represents the mean for each enzyme assay and the bars indicate standard deviation. The assays were done at least in triplicate.

4. Discussion The Prata bananas are valued in the Brazilian market due to their attractive organoleptic characteristics (delicate taste in comparison to the fruit of the cultivars from the group Cavendish). The cold tolerance, even when exposed to low temperatures (10 C), contributes to the potential for these fruit to withstand long-term storage, typical in long distance transport. The onset of ripening was remarkably different between the control groups (19 C) of Prata and Nanico (Fig. 1). These differences might be explained by the capacity of each banana cultivar for endogenous ethylene production or their sensitivity to this hormone. The short time for ripening of Prata fruit suggests that this cultivar is more responsive or sensitive to ethylene for the initiation of the autocatalytic synthesis of the hormone.

Low temperatures delayed ripening and ethylene and respiration peaks were slightly decreased at the climacteric of Nanico (Jiang et al., 2004). In contrast, Prata fruit exhibited similar ethylene and respiration production, indicating that this cultivar recovered very well after exposure to low temperatures. Starch degradation being delayed by low temperature was more evident in Prata bananas. Although no signicant changes in polysaccharide levels were detected during cold storage, there was signicant sucrose accumulation in both cultivars. This accumulation was most evident in Prata, and the sucrose amount accumulated in the cold temperature was almost twice that of Nanico. The importance of soluble carbohydrates in freezing tolerance induced by cold acclimation has been reported in red raspberry plants (Palomen et al., 2000), tomato leaves (Keller and Steffen, 1995), potato tubers (Hill et al., 1996) and melon fruit (Burger et al.,

Fig. 7. Protein levels of SuSy and SPS from Nanico and Prata bananas ripened at 19 C (control groups) and stored 15 d at 13 C and transferred to 19 C for ripening (indicated as 13 C).

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2000). In these produce, the effects of low (12 C) and high (18 C) temperatures in sucrose accumulation were proposed as being part of the cold acclimation mechanism. In the present experiments, the temperature of 13 C was chosen because it is the limit for avoiding chilling injury in Nanico fruit and in other bananas from the Cavendish group. However, it is well-documented that Prata bananas are resistant to temperatures as low as 8 C without showing symptoms of chilling injury. In fact, Prata bananas did not show any visible sign of chilling injury (data not shown) when stored at 10 C in other experiments. Therefore, the high resistance of Prata could be attributed, at least in part, to the cryoprotective effect of sucrose. In bananas, a rise in soluble sugar content under cold stress would be the consequence of the stimulation of starch mobilization. Starch may be degraded either hydrolytically or phosphorolytically (Smith et al., 2005), and the products exported to the cytosol would be converted to sucrose via sucrose phosphate synthesis (SPS) (Krause et al., 1998). The amylolytic activities in the pulp of both cultivars at 19 C were correlated to starch degradation (Fig. 3), but there was a consistent increase in -amylase activity in both cultivars and a transient -amylase peak in Prata fruit stored at low temperature. Previous studies with fruit of the Nanico cultivar (Bassinello et al., 2002; Nascimento et al., 2006) have suggested that gene expression is an important component of the regulation of amylases. The transient induction of -amylase in cold-acclimated Prata bananas suggests that this enzyme can be synthesized in response to ethylene (Nascimento et al., 2006) or low temperature (Kaplan et al., 2006), which could be part of the mechanism of cold tolerance of Prata fruit. Although Nanico also exhibited an increase in -amylase activity, no signicant changes were observed for amylase. Transient expression of -amylase was seen in apple fruit at low temperatures (Wegrzyn et al., 2000), and it was reported that cold temperatures can induce the expression of different isoforms of amylase and an increasing up to ve-fold in activity in potato tubers (Nielsen et al., 1997). Its product, maltose, has also been reported to have an important role in the cold tolerance of Arabidopsis thaliana and sweet potatoes (Lu and Sharkey, 2006). Indeed, when Arabidopsis plants were kept at 4 C for 12 h, the plastid-localized -amylase (BAM3) transcript levels increased and were associated with maltose accumulation in the leaves (Kaplan and Guy, 2004). In some cases, sugar accumulation in cold-stressed tissues is not completely correlated to amylases (Ohyama et al., 1996; Deiting et al., 1998) and likely involves starch phosphorylases. In potato tubers, phosphorylase activity has been implicated as another component of cold acclimation in vegetable tissues (Claassen et al., 1993). The results for the starch phosphorylases activity in Prata bananas indicated an overall increase in the expression of plastidial isoforms during cold storage, although a similar pattern was not seen for Nanico fruit. The involvement of plastidial starch phosphorylase in resistance against abiotic stresses was raised when mutant lines of Arabidopsis, in which the phosphorylase expression was eliminated, developed serious damage in the leaf blade under exposure of the plants to low humidity or acute salt stress (Zeeman et al., 2004). In some accessions of Solanum tuberosum ssp. andigena, drought stress induced the expression of plastidial starch phosphorylase (Watkinson et al., 2008). Despite the high protein level of plastidial phosphorylase in the Nanico cultivar, the activity remained low when the fruit was stored at cold temperatures. The different results for Prata and Nanico phosphorylases indicate changes in the activation state of the enzyme, as previously suggested (Mota et al., 2002; Mainardi et al., 2006). In fact, the redox system (Balmer et al., 2006). Ferredoxin/thioredoxin has been proposed as a possible regulatory mechanism involved in the control of phosphorylase activity.

The activities of sucrose-metabolizing enzymes SuSy and SPS were affected by temperature in both cultivars, but remarkable differences were noticed in Prata fruit stored at 13 C. The high levels of SuSy activity and transient peaks for Susy and SPS at day 5 suggest a contribution of these enzymes to the development of cold tolerance in fruit of the Prata cultivar. SuSy is an important element in the starch biosynthetic pathway, and its involvement in futile cycles of synthesis and degradation of sucrose has been implicated in regulating carbon partitioning and starch levels (Geingenberger and Stitt, 1993). SPS is a key enzyme in sucrose accumulation in ripening bananas (Cordenunsi and Lajolo, 1995; Nascimento et al., 1997), and high SPS activity can contribute to a carbon gradient ux from starch to sucrose in banana fruit pulp. In spinach, SPS activity signicantly increased as a result of low temperature treatment (Guy et al., 1992), and there is evidence that SPS transcript levels are regulated by abiotic factors, such as low temperature and water decit (Ingram et al., 1997; Langenkamper et al., 1998). Our results indicated a remarkable activation of the starchmetabolizing enzymes in cold-acclimated bananas. The coldrelated transient induction of -amylase and plastidial phosphorylase could provide the soluble carbohydrates responsible for the chilling tolerance of the Prata fruit. Interestingly, the concerted induction of the starch-metabolizing enzymes could account for only a marginal decrease in starch content. Therefore, other elements are likely necessary for the massive conversion of starch to soluble sugars during normal ripening, or there could be a structural limitation to the degradation of starch granules before the climacteric event. The concerted increase of both SuSy and SPS in cold-stored Prata fruit could be part of a mechanism that contributes to the starch mobilization and sucrose synthesis involved in cold acclimation, a non-climacteric related condition where sucrose is needed for its cryoprotective effect. Further studies with other banana cultivars susceptible and resistant to low temperature long-term storage would be useful to evaluate if the hypothesis raised by the present work can be extended to other banana cultivars. Acknowledgments The authors thank BRASNICA and MAGRIO Companies for banana samples, and FAPESP, CNPq, and CAPES for supporting this work. References
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