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Changes on the cell wall composition of tree-ripened “Bartlett” pears (Pyrus communis L.)
Postharvest Biology and Technology 73 (2012) 72–79
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Changes on the cell wall composition of tree-ripened “Bartlett” pears (Pyrus communis L.) María D. Raffo a , Nora M.A. Ponce b , Gabriel O. Sozzi c , Carlos A. Stortz b , Ariel R. Vicente d,e,∗ a
Instituto Nacional de Tecnología Agropecuaria (INTA), EEA Alto Valle de Río Negro, Ruta Nac. 22 Km. 1190, 8332 Allen, Argentina Departamento de Química Orgánica-CIHIDECAR, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, 1428 Buenos Aires, Argentina c CONICET, Av. B. Rivadavia 1917, 1033 Buenos Aires, Argentina d Centro de Investigación y Desarrollo en Criotecnología de Alimentos, Facultad de Ciencias Exactas, CONICET-UNLP, 47 y 116, La Plata 1900, Argentina e Cátedra de Agroindustrias, Facultad de Ciencias Agrarias y Forestales, Calle 60 y 119, La Plata 1900, Argentina b
a r t i c l e
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Article history: Received 29 March 2012 Accepted 1 June 2012 Keywords: Pectin Pear Firmness Ripening Texture
a b s t r a c t Most of the European pear cultivars fail to develop a desirable texture when ripening on the tree. In order to identify some biochemical anomalies that might be associated with this phenomenon, the main differences in cell wall polysaccharides occurring during “Bartlett” pear on-tree ripening were evaluated. Fruit was harvested at 3 different ripening stages (119, 147 and 161 days after anthesis, DAA) namely unripe (UR) mid-ripe (MR) both developing normal texture upon ripening, and late-ripe (LR), which did not result in buttery and juicy fruit. The characterization of cell wall structure was accomplished by determining the monosaccharide composition, yields, and solubility of uronic acids and neutral sugars in fractions extracted by water (WSF), 0.05 M CDTA (CSF), 0.1 M Na2 CO3 (NSF), 1 M and 4 M KOH (1KSF and 4KSF). The size exclusion profiles in all the isolated fractions were also determined. In the transition from the UR to the MR stage, glucose from the 4KSF and Ara from all other fractions became markedly solubilized. A slight increase in water-soluble pectin content and depolymerization of the 4KSF and CSF were also found during this period. From the MR to the LR stage, a glucan probably originating in cellulose turnover was found in the 4KSF, and (though at a lower rate) Ara solubilization continued. In contrast, the depolymerization of all wall fractions after day 147 was minimal, and a shift of water soluble pectins toward the CSF was detected. An imbalance in polyuronide degradation, including decreased depolymerization of pectin backbones, together with continuing removal of Ara from RG I, and/or other modifications increasing ionic interactions, may lead fruit which yield an abnormal, mealy texture upon ripening. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Proper maturity at harvest is crucial for most fruit, since it can greatly affect resistance to decay, effectiveness of postharvest treatments and susceptibility to storage disorders (Chen and Mellenthin, 1981; Calvo and Sozzi, 2004; Silva et al., 2010). Non-climacteric commodities should be picked close to the organoleptic maturity, since premature harvest results in inferior eating quality. For most climacteric fruit, there is a broader harvest period depending on the intended destination (rapid commercialization after few weeks,
∗ Corresponding author at: Cátedra de Agroindustrias, Facultad de Ciencias Agrarias y Forestales, Calle 60 y 119, La Plata 1900, Argentina. Tel.: +54 221 423 6758x441; fax: +54 221 423 6758x441. E-mail addresses: [email protected] (M.D. Raffo), [email protected] (N.M.A. Ponce), [email protected] (G.O. Sozzi), [email protected] (C.A. Stortz), [email protected] (A.R. Vicente). 0925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.06.002
short- or long-term storage) (Kader, 2002). Strikingly, most Euro˜ and pean pears show some resistance to ripening (Villalobos-Acuna Mitcham, 2008), may become mealy if held on the tree (Murayama et al., 2006a) and attain better quality if picked at the mature green stage and ripened after harvest. It has been shown that progressive dismantling of cell wall material is responsible, at least in part, for the extensive softening occurring during ripening in many fruit (Brummell and Harpster, 2001; Brummell, 2006). In pears, several studies conducted to date have focused on the evaluation of wall metabolism during storage, and on the identification of different patterns of disassembly among genotypes. Early work showed that postharvest softening occurred in association with dynamic changes in both pectin and cross-linking glycans (Jermyn and Isherwood, 1956). Ahmed and Labavitch (1980) reported that the main modifications accompanying firmness loss of stored pears included the solubilization of large amounts of arabinose and galacturonic acid. Murayama et al. (2002) investigated the postharvest changes in wall components
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of European pears having normal and abnormal ripening behavior and showed that pectin solubilization was reduced in fruit developing mealy texture. Stored European “La France” pears which yielded a melting and juicy texture also showed higher polyuronide solubilization than non-melting firm Chinese “Yali” pear (Hiwasa et al., 2004). Murayama et al. (1998) concluded that the maturity of “’Marguerite Marillat” and “La France” pears at harvest affected the solubility of cell wall polysaccharides and that fruit harvested 14 or 28 d after the optimum time, did not become buttery and juicy. More recently the same authors (Murayama et al., 2006b) reported that girdling on the branch above the abscission zone, partially restored “Bartlett” pear softening and wall degradation. To complement these studies, and in order to gain further insight regarding the changes in the cell wall composition of tree-ripened “Bartlett” pears, we analyzed 3 different developmental stages (119, 147 and 161 DAA) namely unripe (UR) mid-ripe (MR) developing normal texture upon ripening, and late-ripe (LR) pears, which did not result in buttery and juicy fruit. 2. Materials and methods
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2.5. Free juice Free juice was determined according to Crisosto and Labavitch (2002) with slight modifications: harvested fruit was held at 20 ◦ C until ripe. The pears were peeled and 4 wedge-shape sectors were cut from the pulp. Tissue (50 g) from eight different fruit was compressed with a manual press and filtered through cloth. The slurry was centrifuged at 3400 × g for 10 min and the supernatant weighed. The percentage of free juice was calculated. 2.6. Fruit sensory evaluation Eleven assessors were recruited for a sensory panel, based on their ability to discriminate taste and aroma attributes. Eleven pears were selected (one for each evaluator). Each pear was cut, unpeeled, into 8 pieces using a fruit cutter. Each panelist received two pieces from opposite sides of the same pear at a time, being instructed to eat only the pulp. Texture attributes were developed based on the Texture Profile Method (Civille and Szczesniak, 1973). Individual attributes were measured using a structured linear scale from 0 to 10 where 0 = none, 10 = extreme. The evaluation was repeated three times.
2.1. Plant material The ‘Bartlett’ pear (Pyrus communis cv Bartlett) trees used for the experiments were located in the Río Negro Upper Valley, Argentina (39◦ 01 00 S, 67◦ 40 00 W, 242 m above sea level). Fruit from the outer canopy, fully exposed to the northern side was harvested at 3 developmental stages, unripe (UR, 119 DAA) and mid-ripe (MR, 147 DAA) and late-ripe (LR, 161 DAA). At each sampling date, 30 fruit were taken and used to determine ethylene production, color and firmness. An additional 30 pears were peeled and after removal of the core, the pulp was sliced into pieces, frozen in liquid nitrogen, and stored at −50 ◦ C until use for cell wall analysis. 2.2. Ethylene production Ethylene production was assessed by placing the pears in a 3 L glass container tightly sealed with a lid carrying a silicone septum. One milliliter of the headspace gas was extracted after 1 h at 20 ◦ C. Ethylene was quantified on a gas chromatograph (GC-14A, Shimadzu, Japan). The injector, oven and detector (FID) temperatures were 100, 40 and 240 ◦ C respectively. Helium was used as the carrier gas. Three independent composite samples (of three healthy pears each) per stage were evaluated. 2.3. Firmness Compression tests were done with an electronic penetrometer (FTA, Güss, South Africa) with a 7.9-mm diameter probe. Fruit with removed skin were punctured to a depth of 10 mm. The maximum force during the test was registered, and results were expressed in newtons (N). Two opposite spots at the equatorial plane of the fruit were punctured, averaged and considered a replicate. Thirty replicates per developmental stage were evaluated. 2.4. Color Color was determined with a Minolta chroma meter CR-300 (Minolta, Osaka, Japan). The L*, a*, and b* values were obtained using CIE illuminant C lighting conditions. The hue and chroma were calculated as 180 − tg b/a and (a2 + b2 )1/2 respectively. Thirty pears per condition were evaluated.
2.7. Preparation and fractionation of the alcohol-insoluble residue Cell wall preparation and fractionation were performed as previously described (Raffo et al., 2011). The alcohol insoluble residue (AIR) was air-dried in a vacuum desiccator overnight and then weighed. AIR content was expressed in grams per kg of fresh fruit. The starch content of the AIR was estimated using an enzymatic method (Karkalas, 1985) involving ␣-amylase, amyloglucosidase, and o-dianisidine using a kit provided by Sigma (St. Louis, MO). Results were calculated in grams of starch per 100 g of AIR. For fractionation 1 g of AIR was stirred for 24 h at room temperature with 100 mL of 0.02% (w/v) thimerosal aqueous solution and filtered. The suspension was filtered, and the filtrate was saved and designated the water-soluble fraction (WSF). Sequential extraction of the pellet with 0.05 M CDTA in 0.05 M NaOAc/HOAc buffer, pH 6, containing 0.02% (w/v) thimerosal (24 h), 0.1 M Na2 CO3 /0.02 M NaBH4 (24 h), 1 M KOH/0.02 M NaBH4 (24 h), and 4 M KOH/0.02 M NaBH4 (24 h) yielded the CDTA-soluble fraction (CSF), Na2 CO3 soluble fraction (NSF), 1 M and 4 M KOH-soluble fractions (1KSF and 4KSF), respectively. The supernatants were recovered after centrifugation at 13,000 × g for 10 min. In the case of the KOH-soluble fractions, the pH was adjusted to 5 with glacial acetic acid. All fractions were dialyzed (MW cutoff 6000–8000 Da) against tap water for 2 days and against distilled water for another day at 4 ◦ C. The fractions were recovered by lyophilization. 2.8. Uronic acid, total carbohydrate, and neutral sugar measurements Uronic acids were quantitated by the m-hydroxybiphenyl method (Filizzetti-Cozzi and Carpita, 1991) using galacturonic acid as standard and expressed as anhydro units. Total carbohydrates were determined by the phenol–H2 SO4 method (Dubois et al., 1956) using glucose as standard. The proportion of neutral sugars was determined after subtracting the uronic acid content from that of total carbohydrates. For this purpose, the phenol-H2 SO4 reaction was also carried out with a galacturonic acid standard, which showed an absorbance ratio of 0.28 against the same glucose weight (Raffo et al., 2011). Measurements were done in triplicate and results were expressed in g of glucose or galacturonic acid for NS and UA respectively per kg of AIR.
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2.9. Size exclusion chromatography (SEC) In order to examine the size distributions of polymers in CSF and NSF, 3 mg of lyophilized samples from each fraction were dissolved in 0.8 mL of 0.4 mg/mL imidazole containing 0.2 mL of 1 M ammonium acetate (pH 5). Solutions were centrifuged and then chromatographed on a low-pressure SEC by employing a 300 mm × 9 mm i.d. Sepharose CL-2B column (Sigma Chemical Co., St. Louis, MO) eluted at room temperature with 0.2 M ammonium acetate, pH 5. Fractions were collected, and aliquots were assayed for total carbohydrates (Ponce et al., 2010). Samples from the WSF and 1KSF and 4KSF were dissolved in 0.1 M NaOH, cleaned up by centrifugation, and chromatographed on a 300 mm × 9 mm i.d. Sepharose CL-6B column (Sigma Chemical Co., St. Louis, USA) eluted at room temperature with 0.1 M NaOH. Fractions were collected, and aliquots were assayed for total carbohydrates (Dubois et al., 1956). 2.10. Neutral sugar composition Each fraction (ca. 3 mg) was hydrolyzed with 1 mL of 2 M trifluoroacetic acid (TFA) for 90 min at 120 ◦ C in closed-cap vials. The TFA was eliminated by evaporation, and the resulting monosaccharides were reduced to alditols using NaBH4 , converted to alditol acetates as previously reported (Ponce et al., 2010) and subsequently analyzed using a Hewlett-Packard 5890 gas chromatograph (Agilent Technologies Inc., Santa Clara, CA) fitted with a capillary column 30 m, 0.25 mm i.d., 0.20 m, SP-2330 (Supelco Inc., Bellefonte, PA) and equipped with a FID operated at 240 ◦ C. The injector temperature was 240 ◦ C, and the oven temperature was kept isothermally at 220 ◦ C. Nitrogen was used as the carrier gas at a flow rate of 1 mL min−1 . Samples were injected with a split ratio of 80:1. Myoinositol was used as the internal standard, and the different alditol acetates were identified by comparison with authentic standards. The percentage of the different monosaccharides was calculated by considering that the FID responses are proportional to the molecular weight of the alditol acetates. Results were expressed in g of glucose or galacturonic acid for NS and UA respectively per kg of AIR. 2.11. Statistical analysis For uronic acid content, neutral sugar content, and other physical measurements, statistical significance was determined by one-way ANOVA with the PC-SAS software package (SAS Institute Inc., Cary, NC). The model assumptions of homogeneity of variance and normality were tested by means of the Levene and Shapiro–Wilk tests, respectively. Treatment means were compared using Tukey’s studentized range test (P < 0.05). 3. Results and discussion 3.1. Ethylene production and color and firmness Ethylene production at day 119 was low (95 nL kg−1 h−1 ) and did not change significantly during on-tree ripening (Fig. 1A). Ethylene is important in the regulation of pear ripening (Defilippi et al., 2011), and it has been suggested that reduction on its biosynthesis might result in abnormal ripening of pears (Fonseca et al., 2005). Fruit peel lightness was 56 at day 119 and increased as yellow surface color developed; reaching values close to 58 at the MR stage (Fig. 1B). No changes were detected afterwards. The hue angle decreased from 121 at day 119 to 117 at the MR stage, accompanying chlorophyll degradation (Fig. 1C). Subsequently, the reduction of the hue continued but at a lower rate until the LR stage. Fruit chroma showed no variations from UR to MR stage but later on
it increased slightly (Fig. 1D). Firmness decreased throughout ontree ripening (Fig. 1E) reaching 60 N at day 147 and to 30 N at the LR stage. No differences in free juice were found between UR and MR fruit. In contrast, significantly less juice was extracted from pears left on the tree until day 161 (Fig. 1F). We also performed a sensory analysis test with a panel of assessors able to discriminate taste and aroma. Results for juiciness are shown in Fig. 1G, whereas a plot for other attributes is depicted in Fig. S1. Fruit harvested early showed lower acidity and sweetness scores than pears from the later harvests (Fig. S1). The most prominent differences detected in the pears held on the tree until the latest ripening stage (161 DAA) were lower juiciness (Fig. 1G) and acidity (Fig. S1). Overall, results on fruit physical and chemical analysis showed that ripening progressed as the fruit was held on the tree. However, LR pears did not develop full juiciness and/or buttery texture upon ripening. Terasaki et al. (2006) by using a non-destructive vibrometer found a loss of elasticity in fruit developing textural disorders. In order to prevent this, pears may be harvested at the mature green stage and ripened off the tree. A number of protocols to induce ripening, including a period of low temperature storage and exposure to ethylene have been developed for the different varieties (Kondo ˜ and Mitcham, 2008). and Takano, 2000; Villalobos-Acuna
3.2. Cell wall solubilization of pectin and cross-linking glycans We evaluated the changes in the composition of cell wall components to identify some uncommon disassembly events that may be associated with the failure to develop normal texture. The AIR yield dropped 33% from the UR to the MR stage (Fig. 1H). A reduction in total AIR is a common feature observed during ripening, resulting from wall turnover and also fruit growth and starch degradation (Raffo et al., 2011). The starch content represented 15% of the AIR and did not change from the UR to the MR stage. Subsequently, there was a decrease in starch which accounted for 5% of the AIR at day 161. The highly lignified thick-walled stone cells may also increase the ambiguity of the AIR (Jermyn and Isherwood, 1956). No further decrease of AIR content was observed when the fruit was maintained in the tree for more than 146 DAA. We then fractionated the cell wall components based on their ease of extraction. The water soluble uronic acids (UA) increased from the UR to the MR stage (Fig. 2A). An accumulation in water soluble polyuronides and a reduction in insoluble pectin were found when pear fruit ripens off the tree (Ben-Arie and Sonego, 1979; Yoshioka et al., 1992). The fruit held on the tree 161 days, developing abnormal texture upon ripening, presented lower water soluble UA than that at the MR and even the UR stage. This is unexpected, since a common feature accompanying firmness loss of many fruit is an increase in water extractable pectin (Brummell, 2006). Pears with altered texture had less water-soluble polyuronides than fruit developing a buttery and juicy texture (Murayama et al., 1998). The UA content of the NSF and 1KSF increased also until day 146, but no changes were observed afterwards. This differs from studies on detached pears, which showed that tightly bound pectins show large reductions as normal textural properties develop (Murayama et al., 1998, 2006b). As expected, the 4KSF fraction represented less than 2% of total UA and showed no changes at any ripening stage analyzed. The solubilization of pectin was not completely arrested during the transition from stages MR to LR. In fact, a marked increase was detected in the CSF, which represented almost 40% of total cell wall UA of LR pears. The increase of CDTA-soluble pectins likely occurred from insolubilization of water soluble polyuronides. The degree of esterification of pectins decreases during pear ripening (Yoshioka et al., 1992). The generation of calcium binding sites by polymethylesterase-mediated demethylation of polyuronides together with the maintenance of a proper molecular size to allow
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Fig. 1. (A) Ethylene, (B) lightness, (C) hue, (D) chroma, (E) firmness, (F) free juice, (G) juiciness profile and (H) yields of the alcohol insoluble residue (AIR) from “Bartlett” pears at unripe (UR, 119 DAA), mid-ripe (MR, 147 DAA) and late-ripe stage (LR, 161 DAA). The standard deviations are shown. Different letters indicate significant differences based on a Tukey test at a level of significance of P < 0.05.
the formation of ionic bridges with calcium, may be involved in the observed shift, but this should be further evaluated. Neutral sugars (NS) at the UR stage were more abundant in the 4KSF (ca. 37%), followed by the 1KSF, NSF and CSF with 24, 20 and 13% of the total each (Fig. 2B). The water soluble NS at this stage were very low. A large reduction in the 4KSF, and an accumulation in the WSF were found in NS from the unripe to the mid-ripe stage. In MR fruit the NS were quite evenly distributed in all the wall fractions. The water soluble-neutral sugars continued increasing throughout development. At the LR stage the WSF contained the
highest content of NS followed by the CSF. The rise in NS at this stage seemed to result from 4 M KOH insoluble material associated with ␣-cellulose. The similar starch contents at the UR and MR stages suggests that the modifications observed in the AIR were mostly related to changes in cell wall polysaccharides. Between MR and LT some starch was degraded and this may be reflected mostly in the changes in the KOH soluble fractions, where this component usually appears (Alayón-Luaces et al., 2012). This should have not affected the reduction in the solubility of water soluble pectins observed in LR pears.
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Fig. 2. (A) Uronic acids (UA) and (B) neutral sugar (NS) distribution of cell wall fractions from “Bartlett” pears at unripe (UR, 119 DAA), mid-ripe (MR, 147 DAA), and late-ripe stage (LR, 161 DAA). Different letters indicate significant differences based on a Tukey test at a level of significance of P < 0.05.
3.3. Monosaccharide composition of pectin and cross-linking glycans At all the developmental stages studied arabinose (Ara) followed by galactose (Gal) and glucose (Glc) were the most abundant NS in the WSF (Fig. 3). The sugar content increased markedly from the
UR to the LR stage. Ara accumulated 8-fold followed by Gal, which rose 5-fold from the initial to the final ripening stage. The accumulation of Ara in the WSF took place mainly between UR and MR stages. In contrast, the level of Gal in the fraction soluble in water increased at a same rate throughout development. An RGI with large and highly branched arabinan side chains has already been identified in “Bartlett” pear (Dick and Labavitch, 1989). RG-I is degraded in ripening pears, with the initial loss of much of its arabinose (Dick and Labavitch, 1989). The CSF showed a similar pattern to that described for the WSF with Ara and Gal as the main NS. The NSF was the fraction containing the highest Ara contents. Before ripening, it accounted for more than 50% of total NS, interestingly (as observed for the water- and CDTA-soluble fractions) its content also increased as ripening progressed, suggesting that some of the Ara-rich polysaccharides were strongly associated to cross-linking glycans or cellulose. The 1KSF showed more prominence of other sugars besides Ara, such as Glc, Gal, Xyl (xylose) and Man (mannose) (Fig. 4). In this case, Ara content also increased from the UR to the MR stages, together with a rapid loss of Glc from UR to MR. Extensive loss of Glc was also detected between these stages in the 4KSF, but later on Glc content increased in both glycan-rich fractions. Overall, results show that Ara is the most dynamic cell wall NS in “Bartlett” pear ripening. Ahmed and Labavitch (1980) found that substantial amounts of galacturonic acid and arabinose were lost during “Bartlett” pear ripening. A more detailed evaluation of all fractions indicated that the solubilization of this sugar occurred more markedly between the UR and MR stages and that it may have been released at least in part from the KOH-insoluble material.
3.4. Depolymerization pectin and cross-linking glycans Ahmed and Labavitch (1980) reported that the molecular weight of pear homogalacturonan (HGA) decreased during ripening. We evaluated the modifications of molecular weight distribution of the different fractions by SEC (Fig. 5). In stored Red “Bartlett” pear the water soluble polyuronides were depolymerized (Yoshioka et al., 1992). In contrast in the present study, no changes in polymer size were observed on the WSF and NSF throughout on tree
Fig. 3. Sugar composition (g kg AIR−1 ) of the water-, CDTA-, and Na2 CO3 -soluble fractions (WSF, CSF and NSF, respectively) from “Bartlett” pears at unripe (UR, 119 DAA), mid-ripe (MR, 147 DAA), and late-ripe stage (LR, 161 DAA).
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Fig. 4. Sugar composition (g kg AIR−1 ) of the 1 M KOH- (1KSF) and 4 M KOH-soluble fraction (4KSF) from “Bartlett” pears at unripe (UR, 119 DAA), mid-ripe (MR, 147 DAA), and late-ripe stage (LR, 161 DAA).
ripening. The CSF showed some depolymerization from the UR to the MR stage, but subsequently no further changes were detected. Pectin depolymerization of stored pears has been positively correlated with polygalacturonase (PG) activity (Yoshioka et al., 1992).
Fonseca et al. (2005) also found that physical or chemical factors reducing the resistance of pears to ripening (ethylene or chilling) induce the expression of several cell wall-degrading proteins including polygalacturonase and expansin. The patterns of
Fig. 5. Size exclusion chromatography profiles of the WSF, CSF, NSF, 1KSF and 4KSF of “Bartlett” pears at unripe (UR, 119 DAA), mid-ripe (MR, 147 DAA), and late-ripe stage (LR, 161 DAA), on Sepharose CL-6B (WSF, 1KSF and 4KSF) or CL-2B (CSF and NSF) columns. V0 , void volume; Vt , total volume.
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and Agencia Nacional de Promoción Científica y Tecnológica (PICT 2006-01267 and PICT 2009-0059) for financial support. N.M.A.P., G.O.S., C.A.S. and A.R.V. are Research Members of the National Research Council of Argentina (CONICET). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.postharvbio.2012.06.002. References
Fig. 6. Sequence of wall changes in tree ripened “Bartlett” pears at unripe (UR, 119 DAA) or mid-ripe (MR, 147 DAA) developing normal texture upon ripening, and at late-ripe stage (LR, 161 DAA) which did not result in buttery and juicy fruit.
transcript accumulation for the pectin-depolymerizing enzymes polygalacturonase (PcPG1 and PcPG3) genes showed good association with fruit softening in pear fruit ripened on and off the tree (Murayama et al., 2006a). Girdling treatments on the branch over the abscission zones resulted in increased expression of the mentioned PG genes, and a partial recovery of wall solubilization. However, the changes in polymer size of the fruit were not directly studied. A shift toward lower molecular weights was also observed in the 4KSF between the UR and MR stages, but no changes were detected afterwards. Our results show that depolymerization of polysaccharides from tree ripened “Bartlett” pears takes place at intermediate stages. The limited pectin depolymerization when the fruit is maintained in the tree is at least unusual since polyuronide hydrolysis has been shown to be a late ripening event in most fruits (Yoshioka et al., 1992; Brummell, 2006). 4. Conclusion “Bartlett” pears ripened on-tree, are less juicy and develop mealy texture upon ripening. The cell wall analysis showed arabinose solubilization as one of the clearest features, occurred at a higher rate from the UR to the MR stage, but proceeded throughout on-tree ripening (Fig. 6). Water soluble pectins increased from the UR to the MR but later on decreased in association with the accumulation of CDTA-soluble polyuronides in LR fruit. Both crosslinking glycans solubilization and depolymerization took place mainly at intermediate stages, and some cellulose-derived glucans appear to solubilize in the last stage. In contrast, pectin depolymerization, which is normally described as a late ripening event, was not observed in any of the fractions if fruit is held on the tree after the MR stage. As for an explanation for the suboptimal ripening of LR fruit, fewer differences than might have been expected have emerged. However, it seems clear that some insolubilization of pectin results in LR fruit as well as a modification of arabinose metabolism. An imbalance in pectin degradation, including decreased pectin backbone depolymerization, together with continuing removal of Ara from RG-I, and/or other modifications increasing ionic interactions, may lead to fruit that losses firmness but yields an abnormal mealy texture upon ripening. Acknowledgements We thank Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 0353), Universidad de Buenos Aires (UBACyT Program),
Ahmed, A.E.R., Labavitch, J.M., 1980. Cell wall metabolism in ripening fruit. I. Cell wall changes in ripening ‘Bartlett’ pears. Plant Physiology 65, 1009–1013. Alayón-Luaces, P., Ponce, N.M.A., Mroginski, L.A., Stortz, C.A., Sozzi, G.O., 2012. Compositional changes in cell wall polysaccharides from apple fruit callus cultures modulated by different plant growth regulators. Plant Science 185–186, 169–175. Ben-Arie, R., Sonego, L., 1979. Changes in pectic substances in ripening pears. Journal of the American Society for Horticultural Science 104, 500–505. Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Molecular Biology 47, 311–340. Brummell, D.A., 2006. Cell wall disassembly in ripening fruit. Functional Plant Biology 33, 103–119. Calvo, G., Sozzi, G.O., 2004. Improvement of postharvest storage quality of ‘Red Clapp’s’ pears by treatment with 1-methylcyclopropene at low temperature. Journal of Horticultural Science & Biotechnology 79, 930–934. Chen, P.M., Mellenthin, W.M., 1981. Effects of harvest date on ripening capacity and postharvest life of ‘d’Anjou’ pears. Journal of the American Society for Horticultural Science 106, 38–42. Civille, G., Szczesniak, A., 1973. Guidelines to training a texture profile panel. Journal of Texture Studies 4, 204–223. Crisosto, C.H., Labavitch, J.M., 2002. Developing a quantitative method to evaluate peach (Prunus persica) flesh mealiness. Postharvest Biology and Technology 25, 151–158. Defilippi, B.G., Manríquez, D., Robledo, P., 2011. Use of 1-methylcyclopropene (1MCP) as a strategy to improve post-harvest life of ‘Abate Fetel’ pears. Acta Horticulturae 909, 739–744. Dick, J.A., Labavitch, J.M., 1989. Cell wall metabolism in ripening fruit. IV. Characterization of the pectic polysaccharides solubilised during softening of ‘Bartlett’ pear fruit. Plant Physiology 89, 1394–1400. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350–356. Filizzetti-Cozzi, T.M.C.C., Carpita, N.C., 1991. Measurement of uronic acids without interference from neutral sugars. Analytical Biochemistry 197, 157–162. Fonseca, S., Monteiro, L., Barreiro, M.G., Pais, M.S., 2005. Expression of genes encoding cell wall modifying enzymes is induced by cold storage and reflects changes in pear fruit texture. Journal of Experimental Botany 56, 2029–2039. Hiwasa, K., Nakano, R., Hashimoto, A., Matsuzaki, M., Murayama, H., Inaba, A., Kubo, Y., 2004. European, Chinese and Japanese pear fruit exhibit differential softening characteristics during ripening. Journal of Experimental Botany 55, 2281–2290. Jermyn, M.A., Isherwood, F.A., 1956. Changes in the cell wall of the pear during ripening. Biochemical Journal 64, 123–132. Kader, A.A. (Ed.), 2002. Postharvest Technology of Horticultural Crops, Publication 3311. , third ed. University of California, Agriculture and Natural Resources, p. 535. Karkalas, J.J., 1985. An improved enzymic method for the determination of native and modified starch. Journal of the Science of Food and Agriculture 36, 1019–1027. Kondo, S., Takano, Y., 2000. Cell wall metabolism and induction of ripening capacity in ‘La France’ pears as influenced by 2,4-DP. Journal of the American Society for Horticultural Science 125, 242–247. Murayama, H., Takahashi, T., Honda, R., Fukushima, T., 1998. Cell wall changes in pear fruit softening on and off the tree. Postharvest Biology and Technology 14, 143–149. Murayama, H., Katsumata, T., Horiuchi, O., Fukushima, T., 2002. Relationship between fruit softening and cell wall polysaccharides in pear fruit after different storage periods. Postharvest Biology and Technology 26, 15–21. Murayama, H., Katsumata, T., Endou, H., Fukushima, T., Sakurai, N., 2006a. Effect of storage period on the molecular-mass distribution profile of pectic and hemicellulosic polysaccharides in pears. Postharvest Biology and Technology 40, 141–148. Murayama, H., Sekine, D., Yamauchi, Y., Gao, M., Mitsuhashi, W., Toyomasu, T., 2006b. Effect of girdling above the abscission zone of fruit on ‘Bartlett’ pear ripening on the tree. Journal of Experimental Botany 57, 3679–3686. Raffo, M.D., Ponce, N.M.A., Sozzi, G.O., Vicente, A.R., Stortz, C., 2011. Compositional changes in ‘Bartlett’ pears (Pyrus comunnis L.) cell wall polysaccharides as affected by sunlight conditions. Journal of Agricultural and Food Chemistry 59, 12155–12162.
M.D. Raffo et al. / Postharvest Biology and Technology 73 (2012) 72–79 Ponce, N.M.A., Ziegler, V.H., Stortz, C.A., Sozzi, G.O., 2010. Compositional changes in cell wall polysaccharides from Japanese plum (Prunus salicina Lindl) during growth and on-tree ripening. Journal of Agricultural and Food Chemistry 58, 2562–2570. Silva, F.J.P., Gomes, M.H., Fidalgo, F., Rodrigues, J.A., Almeida, D.P.F., 2010. Antioxidant properties and fruit quality during long-term storage of rocha pear: effects of maturity and storage conditions. Journal of Food Quality 33, 1–20. Terasaki, S., Sakurai, N., Zebrowski, J., Murayama, H., Yamamoto, R., Nevins, D.J., 2006. Laser Doppler vibrometer analysis of changes in elastic properties of ripening
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‘La France’ pears after postharvest storage. Postharvest Biology and Technology 42, 198–207. ˜ M., Mitcham, E.J., 2008. Ripening of European pears: the chilling Villalobos-Acuna, dilemma. Postharvest Biology and Technology 49, 187–200. Yoshioka, H., Aoba, K., Kashimura, Y., 1992. Molecular weight and degree of methoxylation in cell wall polyuronide during softening in pear and apple fruit. Journal of the American Society for Horticultural Science 117, 600–606.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Changes on the cell wall composition of tree-ripened “Bartlett” pears (Pyrus communis L.) María D. Raffo a , Nora M.A. Ponce b , Gabriel O. Sozzi c , Carlos A. Stortz b , Ariel R. Vicente d,e,∗ a
Instituto Nacional de Tecnología Agropecuaria (INTA), EEA Alto Valle de Río Negro, Ruta Nac. 22 Km. 1190, 8332 Allen, Argentina Departamento de Química Orgánica-CIHIDECAR, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, 1428 Buenos Aires, Argentina c CONICET, Av. B. Rivadavia 1917, 1033 Buenos Aires, Argentina d Centro de Investigación y Desarrollo en Criotecnología de Alimentos, Facultad de Ciencias Exactas, CONICET-UNLP, 47 y 116, La Plata 1900, Argentina e Cátedra de Agroindustrias, Facultad de Ciencias Agrarias y Forestales, Calle 60 y 119, La Plata 1900, Argentina b
a r t i c l e
i n f o
Article history: Received 29 March 2012 Accepted 1 June 2012 Keywords: Pectin Pear Firmness Ripening Texture
a b s t r a c t Most of the European pear cultivars fail to develop a desirable texture when ripening on the tree. In order to identify some biochemical anomalies that might be associated with this phenomenon, the main differences in cell wall polysaccharides occurring during “Bartlett” pear on-tree ripening were evaluated. Fruit was harvested at 3 different ripening stages (119, 147 and 161 days after anthesis, DAA) namely unripe (UR) mid-ripe (MR) both developing normal texture upon ripening, and late-ripe (LR), which did not result in buttery and juicy fruit. The characterization of cell wall structure was accomplished by determining the monosaccharide composition, yields, and solubility of uronic acids and neutral sugars in fractions extracted by water (WSF), 0.05 M CDTA (CSF), 0.1 M Na2 CO3 (NSF), 1 M and 4 M KOH (1KSF and 4KSF). The size exclusion profiles in all the isolated fractions were also determined. In the transition from the UR to the MR stage, glucose from the 4KSF and Ara from all other fractions became markedly solubilized. A slight increase in water-soluble pectin content and depolymerization of the 4KSF and CSF were also found during this period. From the MR to the LR stage, a glucan probably originating in cellulose turnover was found in the 4KSF, and (though at a lower rate) Ara solubilization continued. In contrast, the depolymerization of all wall fractions after day 147 was minimal, and a shift of water soluble pectins toward the CSF was detected. An imbalance in polyuronide degradation, including decreased depolymerization of pectin backbones, together with continuing removal of Ara from RG I, and/or other modifications increasing ionic interactions, may lead fruit which yield an abnormal, mealy texture upon ripening. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Proper maturity at harvest is crucial for most fruit, since it can greatly affect resistance to decay, effectiveness of postharvest treatments and susceptibility to storage disorders (Chen and Mellenthin, 1981; Calvo and Sozzi, 2004; Silva et al., 2010). Non-climacteric commodities should be picked close to the organoleptic maturity, since premature harvest results in inferior eating quality. For most climacteric fruit, there is a broader harvest period depending on the intended destination (rapid commercialization after few weeks,
∗ Corresponding author at: Cátedra de Agroindustrias, Facultad de Ciencias Agrarias y Forestales, Calle 60 y 119, La Plata 1900, Argentina. Tel.: +54 221 423 6758x441; fax: +54 221 423 6758x441. E-mail addresses: [email protected] (M.D. Raffo), [email protected] (N.M.A. Ponce), [email protected] (G.O. Sozzi), [email protected] (C.A. Stortz), [email protected] (A.R. Vicente). 0925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.06.002
short- or long-term storage) (Kader, 2002). Strikingly, most Euro˜ and pean pears show some resistance to ripening (Villalobos-Acuna Mitcham, 2008), may become mealy if held on the tree (Murayama et al., 2006a) and attain better quality if picked at the mature green stage and ripened after harvest. It has been shown that progressive dismantling of cell wall material is responsible, at least in part, for the extensive softening occurring during ripening in many fruit (Brummell and Harpster, 2001; Brummell, 2006). In pears, several studies conducted to date have focused on the evaluation of wall metabolism during storage, and on the identification of different patterns of disassembly among genotypes. Early work showed that postharvest softening occurred in association with dynamic changes in both pectin and cross-linking glycans (Jermyn and Isherwood, 1956). Ahmed and Labavitch (1980) reported that the main modifications accompanying firmness loss of stored pears included the solubilization of large amounts of arabinose and galacturonic acid. Murayama et al. (2002) investigated the postharvest changes in wall components
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of European pears having normal and abnormal ripening behavior and showed that pectin solubilization was reduced in fruit developing mealy texture. Stored European “La France” pears which yielded a melting and juicy texture also showed higher polyuronide solubilization than non-melting firm Chinese “Yali” pear (Hiwasa et al., 2004). Murayama et al. (1998) concluded that the maturity of “’Marguerite Marillat” and “La France” pears at harvest affected the solubility of cell wall polysaccharides and that fruit harvested 14 or 28 d after the optimum time, did not become buttery and juicy. More recently the same authors (Murayama et al., 2006b) reported that girdling on the branch above the abscission zone, partially restored “Bartlett” pear softening and wall degradation. To complement these studies, and in order to gain further insight regarding the changes in the cell wall composition of tree-ripened “Bartlett” pears, we analyzed 3 different developmental stages (119, 147 and 161 DAA) namely unripe (UR) mid-ripe (MR) developing normal texture upon ripening, and late-ripe (LR) pears, which did not result in buttery and juicy fruit. 2. Materials and methods
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2.5. Free juice Free juice was determined according to Crisosto and Labavitch (2002) with slight modifications: harvested fruit was held at 20 ◦ C until ripe. The pears were peeled and 4 wedge-shape sectors were cut from the pulp. Tissue (50 g) from eight different fruit was compressed with a manual press and filtered through cloth. The slurry was centrifuged at 3400 × g for 10 min and the supernatant weighed. The percentage of free juice was calculated. 2.6. Fruit sensory evaluation Eleven assessors were recruited for a sensory panel, based on their ability to discriminate taste and aroma attributes. Eleven pears were selected (one for each evaluator). Each pear was cut, unpeeled, into 8 pieces using a fruit cutter. Each panelist received two pieces from opposite sides of the same pear at a time, being instructed to eat only the pulp. Texture attributes were developed based on the Texture Profile Method (Civille and Szczesniak, 1973). Individual attributes were measured using a structured linear scale from 0 to 10 where 0 = none, 10 = extreme. The evaluation was repeated three times.
2.1. Plant material The ‘Bartlett’ pear (Pyrus communis cv Bartlett) trees used for the experiments were located in the Río Negro Upper Valley, Argentina (39◦ 01 00 S, 67◦ 40 00 W, 242 m above sea level). Fruit from the outer canopy, fully exposed to the northern side was harvested at 3 developmental stages, unripe (UR, 119 DAA) and mid-ripe (MR, 147 DAA) and late-ripe (LR, 161 DAA). At each sampling date, 30 fruit were taken and used to determine ethylene production, color and firmness. An additional 30 pears were peeled and after removal of the core, the pulp was sliced into pieces, frozen in liquid nitrogen, and stored at −50 ◦ C until use for cell wall analysis. 2.2. Ethylene production Ethylene production was assessed by placing the pears in a 3 L glass container tightly sealed with a lid carrying a silicone septum. One milliliter of the headspace gas was extracted after 1 h at 20 ◦ C. Ethylene was quantified on a gas chromatograph (GC-14A, Shimadzu, Japan). The injector, oven and detector (FID) temperatures were 100, 40 and 240 ◦ C respectively. Helium was used as the carrier gas. Three independent composite samples (of three healthy pears each) per stage were evaluated. 2.3. Firmness Compression tests were done with an electronic penetrometer (FTA, Güss, South Africa) with a 7.9-mm diameter probe. Fruit with removed skin were punctured to a depth of 10 mm. The maximum force during the test was registered, and results were expressed in newtons (N). Two opposite spots at the equatorial plane of the fruit were punctured, averaged and considered a replicate. Thirty replicates per developmental stage were evaluated. 2.4. Color Color was determined with a Minolta chroma meter CR-300 (Minolta, Osaka, Japan). The L*, a*, and b* values were obtained using CIE illuminant C lighting conditions. The hue and chroma were calculated as 180 − tg b/a and (a2 + b2 )1/2 respectively. Thirty pears per condition were evaluated.
2.7. Preparation and fractionation of the alcohol-insoluble residue Cell wall preparation and fractionation were performed as previously described (Raffo et al., 2011). The alcohol insoluble residue (AIR) was air-dried in a vacuum desiccator overnight and then weighed. AIR content was expressed in grams per kg of fresh fruit. The starch content of the AIR was estimated using an enzymatic method (Karkalas, 1985) involving ␣-amylase, amyloglucosidase, and o-dianisidine using a kit provided by Sigma (St. Louis, MO). Results were calculated in grams of starch per 100 g of AIR. For fractionation 1 g of AIR was stirred for 24 h at room temperature with 100 mL of 0.02% (w/v) thimerosal aqueous solution and filtered. The suspension was filtered, and the filtrate was saved and designated the water-soluble fraction (WSF). Sequential extraction of the pellet with 0.05 M CDTA in 0.05 M NaOAc/HOAc buffer, pH 6, containing 0.02% (w/v) thimerosal (24 h), 0.1 M Na2 CO3 /0.02 M NaBH4 (24 h), 1 M KOH/0.02 M NaBH4 (24 h), and 4 M KOH/0.02 M NaBH4 (24 h) yielded the CDTA-soluble fraction (CSF), Na2 CO3 soluble fraction (NSF), 1 M and 4 M KOH-soluble fractions (1KSF and 4KSF), respectively. The supernatants were recovered after centrifugation at 13,000 × g for 10 min. In the case of the KOH-soluble fractions, the pH was adjusted to 5 with glacial acetic acid. All fractions were dialyzed (MW cutoff 6000–8000 Da) against tap water for 2 days and against distilled water for another day at 4 ◦ C. The fractions were recovered by lyophilization. 2.8. Uronic acid, total carbohydrate, and neutral sugar measurements Uronic acids were quantitated by the m-hydroxybiphenyl method (Filizzetti-Cozzi and Carpita, 1991) using galacturonic acid as standard and expressed as anhydro units. Total carbohydrates were determined by the phenol–H2 SO4 method (Dubois et al., 1956) using glucose as standard. The proportion of neutral sugars was determined after subtracting the uronic acid content from that of total carbohydrates. For this purpose, the phenol-H2 SO4 reaction was also carried out with a galacturonic acid standard, which showed an absorbance ratio of 0.28 against the same glucose weight (Raffo et al., 2011). Measurements were done in triplicate and results were expressed in g of glucose or galacturonic acid for NS and UA respectively per kg of AIR.
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2.9. Size exclusion chromatography (SEC) In order to examine the size distributions of polymers in CSF and NSF, 3 mg of lyophilized samples from each fraction were dissolved in 0.8 mL of 0.4 mg/mL imidazole containing 0.2 mL of 1 M ammonium acetate (pH 5). Solutions were centrifuged and then chromatographed on a low-pressure SEC by employing a 300 mm × 9 mm i.d. Sepharose CL-2B column (Sigma Chemical Co., St. Louis, MO) eluted at room temperature with 0.2 M ammonium acetate, pH 5. Fractions were collected, and aliquots were assayed for total carbohydrates (Ponce et al., 2010). Samples from the WSF and 1KSF and 4KSF were dissolved in 0.1 M NaOH, cleaned up by centrifugation, and chromatographed on a 300 mm × 9 mm i.d. Sepharose CL-6B column (Sigma Chemical Co., St. Louis, USA) eluted at room temperature with 0.1 M NaOH. Fractions were collected, and aliquots were assayed for total carbohydrates (Dubois et al., 1956). 2.10. Neutral sugar composition Each fraction (ca. 3 mg) was hydrolyzed with 1 mL of 2 M trifluoroacetic acid (TFA) for 90 min at 120 ◦ C in closed-cap vials. The TFA was eliminated by evaporation, and the resulting monosaccharides were reduced to alditols using NaBH4 , converted to alditol acetates as previously reported (Ponce et al., 2010) and subsequently analyzed using a Hewlett-Packard 5890 gas chromatograph (Agilent Technologies Inc., Santa Clara, CA) fitted with a capillary column 30 m, 0.25 mm i.d., 0.20 m, SP-2330 (Supelco Inc., Bellefonte, PA) and equipped with a FID operated at 240 ◦ C. The injector temperature was 240 ◦ C, and the oven temperature was kept isothermally at 220 ◦ C. Nitrogen was used as the carrier gas at a flow rate of 1 mL min−1 . Samples were injected with a split ratio of 80:1. Myoinositol was used as the internal standard, and the different alditol acetates were identified by comparison with authentic standards. The percentage of the different monosaccharides was calculated by considering that the FID responses are proportional to the molecular weight of the alditol acetates. Results were expressed in g of glucose or galacturonic acid for NS and UA respectively per kg of AIR. 2.11. Statistical analysis For uronic acid content, neutral sugar content, and other physical measurements, statistical significance was determined by one-way ANOVA with the PC-SAS software package (SAS Institute Inc., Cary, NC). The model assumptions of homogeneity of variance and normality were tested by means of the Levene and Shapiro–Wilk tests, respectively. Treatment means were compared using Tukey’s studentized range test (P < 0.05). 3. Results and discussion 3.1. Ethylene production and color and firmness Ethylene production at day 119 was low (95 nL kg−1 h−1 ) and did not change significantly during on-tree ripening (Fig. 1A). Ethylene is important in the regulation of pear ripening (Defilippi et al., 2011), and it has been suggested that reduction on its biosynthesis might result in abnormal ripening of pears (Fonseca et al., 2005). Fruit peel lightness was 56 at day 119 and increased as yellow surface color developed; reaching values close to 58 at the MR stage (Fig. 1B). No changes were detected afterwards. The hue angle decreased from 121 at day 119 to 117 at the MR stage, accompanying chlorophyll degradation (Fig. 1C). Subsequently, the reduction of the hue continued but at a lower rate until the LR stage. Fruit chroma showed no variations from UR to MR stage but later on
it increased slightly (Fig. 1D). Firmness decreased throughout ontree ripening (Fig. 1E) reaching 60 N at day 147 and to 30 N at the LR stage. No differences in free juice were found between UR and MR fruit. In contrast, significantly less juice was extracted from pears left on the tree until day 161 (Fig. 1F). We also performed a sensory analysis test with a panel of assessors able to discriminate taste and aroma. Results for juiciness are shown in Fig. 1G, whereas a plot for other attributes is depicted in Fig. S1. Fruit harvested early showed lower acidity and sweetness scores than pears from the later harvests (Fig. S1). The most prominent differences detected in the pears held on the tree until the latest ripening stage (161 DAA) were lower juiciness (Fig. 1G) and acidity (Fig. S1). Overall, results on fruit physical and chemical analysis showed that ripening progressed as the fruit was held on the tree. However, LR pears did not develop full juiciness and/or buttery texture upon ripening. Terasaki et al. (2006) by using a non-destructive vibrometer found a loss of elasticity in fruit developing textural disorders. In order to prevent this, pears may be harvested at the mature green stage and ripened off the tree. A number of protocols to induce ripening, including a period of low temperature storage and exposure to ethylene have been developed for the different varieties (Kondo ˜ and Mitcham, 2008). and Takano, 2000; Villalobos-Acuna
3.2. Cell wall solubilization of pectin and cross-linking glycans We evaluated the changes in the composition of cell wall components to identify some uncommon disassembly events that may be associated with the failure to develop normal texture. The AIR yield dropped 33% from the UR to the MR stage (Fig. 1H). A reduction in total AIR is a common feature observed during ripening, resulting from wall turnover and also fruit growth and starch degradation (Raffo et al., 2011). The starch content represented 15% of the AIR and did not change from the UR to the MR stage. Subsequently, there was a decrease in starch which accounted for 5% of the AIR at day 161. The highly lignified thick-walled stone cells may also increase the ambiguity of the AIR (Jermyn and Isherwood, 1956). No further decrease of AIR content was observed when the fruit was maintained in the tree for more than 146 DAA. We then fractionated the cell wall components based on their ease of extraction. The water soluble uronic acids (UA) increased from the UR to the MR stage (Fig. 2A). An accumulation in water soluble polyuronides and a reduction in insoluble pectin were found when pear fruit ripens off the tree (Ben-Arie and Sonego, 1979; Yoshioka et al., 1992). The fruit held on the tree 161 days, developing abnormal texture upon ripening, presented lower water soluble UA than that at the MR and even the UR stage. This is unexpected, since a common feature accompanying firmness loss of many fruit is an increase in water extractable pectin (Brummell, 2006). Pears with altered texture had less water-soluble polyuronides than fruit developing a buttery and juicy texture (Murayama et al., 1998). The UA content of the NSF and 1KSF increased also until day 146, but no changes were observed afterwards. This differs from studies on detached pears, which showed that tightly bound pectins show large reductions as normal textural properties develop (Murayama et al., 1998, 2006b). As expected, the 4KSF fraction represented less than 2% of total UA and showed no changes at any ripening stage analyzed. The solubilization of pectin was not completely arrested during the transition from stages MR to LR. In fact, a marked increase was detected in the CSF, which represented almost 40% of total cell wall UA of LR pears. The increase of CDTA-soluble pectins likely occurred from insolubilization of water soluble polyuronides. The degree of esterification of pectins decreases during pear ripening (Yoshioka et al., 1992). The generation of calcium binding sites by polymethylesterase-mediated demethylation of polyuronides together with the maintenance of a proper molecular size to allow
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Fig. 1. (A) Ethylene, (B) lightness, (C) hue, (D) chroma, (E) firmness, (F) free juice, (G) juiciness profile and (H) yields of the alcohol insoluble residue (AIR) from “Bartlett” pears at unripe (UR, 119 DAA), mid-ripe (MR, 147 DAA) and late-ripe stage (LR, 161 DAA). The standard deviations are shown. Different letters indicate significant differences based on a Tukey test at a level of significance of P < 0.05.
the formation of ionic bridges with calcium, may be involved in the observed shift, but this should be further evaluated. Neutral sugars (NS) at the UR stage were more abundant in the 4KSF (ca. 37%), followed by the 1KSF, NSF and CSF with 24, 20 and 13% of the total each (Fig. 2B). The water soluble NS at this stage were very low. A large reduction in the 4KSF, and an accumulation in the WSF were found in NS from the unripe to the mid-ripe stage. In MR fruit the NS were quite evenly distributed in all the wall fractions. The water soluble-neutral sugars continued increasing throughout development. At the LR stage the WSF contained the
highest content of NS followed by the CSF. The rise in NS at this stage seemed to result from 4 M KOH insoluble material associated with ␣-cellulose. The similar starch contents at the UR and MR stages suggests that the modifications observed in the AIR were mostly related to changes in cell wall polysaccharides. Between MR and LT some starch was degraded and this may be reflected mostly in the changes in the KOH soluble fractions, where this component usually appears (Alayón-Luaces et al., 2012). This should have not affected the reduction in the solubility of water soluble pectins observed in LR pears.
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Fig. 2. (A) Uronic acids (UA) and (B) neutral sugar (NS) distribution of cell wall fractions from “Bartlett” pears at unripe (UR, 119 DAA), mid-ripe (MR, 147 DAA), and late-ripe stage (LR, 161 DAA). Different letters indicate significant differences based on a Tukey test at a level of significance of P < 0.05.
3.3. Monosaccharide composition of pectin and cross-linking glycans At all the developmental stages studied arabinose (Ara) followed by galactose (Gal) and glucose (Glc) were the most abundant NS in the WSF (Fig. 3). The sugar content increased markedly from the
UR to the LR stage. Ara accumulated 8-fold followed by Gal, which rose 5-fold from the initial to the final ripening stage. The accumulation of Ara in the WSF took place mainly between UR and MR stages. In contrast, the level of Gal in the fraction soluble in water increased at a same rate throughout development. An RGI with large and highly branched arabinan side chains has already been identified in “Bartlett” pear (Dick and Labavitch, 1989). RG-I is degraded in ripening pears, with the initial loss of much of its arabinose (Dick and Labavitch, 1989). The CSF showed a similar pattern to that described for the WSF with Ara and Gal as the main NS. The NSF was the fraction containing the highest Ara contents. Before ripening, it accounted for more than 50% of total NS, interestingly (as observed for the water- and CDTA-soluble fractions) its content also increased as ripening progressed, suggesting that some of the Ara-rich polysaccharides were strongly associated to cross-linking glycans or cellulose. The 1KSF showed more prominence of other sugars besides Ara, such as Glc, Gal, Xyl (xylose) and Man (mannose) (Fig. 4). In this case, Ara content also increased from the UR to the MR stages, together with a rapid loss of Glc from UR to MR. Extensive loss of Glc was also detected between these stages in the 4KSF, but later on Glc content increased in both glycan-rich fractions. Overall, results show that Ara is the most dynamic cell wall NS in “Bartlett” pear ripening. Ahmed and Labavitch (1980) found that substantial amounts of galacturonic acid and arabinose were lost during “Bartlett” pear ripening. A more detailed evaluation of all fractions indicated that the solubilization of this sugar occurred more markedly between the UR and MR stages and that it may have been released at least in part from the KOH-insoluble material.
3.4. Depolymerization pectin and cross-linking glycans Ahmed and Labavitch (1980) reported that the molecular weight of pear homogalacturonan (HGA) decreased during ripening. We evaluated the modifications of molecular weight distribution of the different fractions by SEC (Fig. 5). In stored Red “Bartlett” pear the water soluble polyuronides were depolymerized (Yoshioka et al., 1992). In contrast in the present study, no changes in polymer size were observed on the WSF and NSF throughout on tree
Fig. 3. Sugar composition (g kg AIR−1 ) of the water-, CDTA-, and Na2 CO3 -soluble fractions (WSF, CSF and NSF, respectively) from “Bartlett” pears at unripe (UR, 119 DAA), mid-ripe (MR, 147 DAA), and late-ripe stage (LR, 161 DAA).
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Fig. 4. Sugar composition (g kg AIR−1 ) of the 1 M KOH- (1KSF) and 4 M KOH-soluble fraction (4KSF) from “Bartlett” pears at unripe (UR, 119 DAA), mid-ripe (MR, 147 DAA), and late-ripe stage (LR, 161 DAA).
ripening. The CSF showed some depolymerization from the UR to the MR stage, but subsequently no further changes were detected. Pectin depolymerization of stored pears has been positively correlated with polygalacturonase (PG) activity (Yoshioka et al., 1992).
Fonseca et al. (2005) also found that physical or chemical factors reducing the resistance of pears to ripening (ethylene or chilling) induce the expression of several cell wall-degrading proteins including polygalacturonase and expansin. The patterns of
Fig. 5. Size exclusion chromatography profiles of the WSF, CSF, NSF, 1KSF and 4KSF of “Bartlett” pears at unripe (UR, 119 DAA), mid-ripe (MR, 147 DAA), and late-ripe stage (LR, 161 DAA), on Sepharose CL-6B (WSF, 1KSF and 4KSF) or CL-2B (CSF and NSF) columns. V0 , void volume; Vt , total volume.
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and Agencia Nacional de Promoción Científica y Tecnológica (PICT 2006-01267 and PICT 2009-0059) for financial support. N.M.A.P., G.O.S., C.A.S. and A.R.V. are Research Members of the National Research Council of Argentina (CONICET). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.postharvbio.2012.06.002. References
Fig. 6. Sequence of wall changes in tree ripened “Bartlett” pears at unripe (UR, 119 DAA) or mid-ripe (MR, 147 DAA) developing normal texture upon ripening, and at late-ripe stage (LR, 161 DAA) which did not result in buttery and juicy fruit.
transcript accumulation for the pectin-depolymerizing enzymes polygalacturonase (PcPG1 and PcPG3) genes showed good association with fruit softening in pear fruit ripened on and off the tree (Murayama et al., 2006a). Girdling treatments on the branch over the abscission zones resulted in increased expression of the mentioned PG genes, and a partial recovery of wall solubilization. However, the changes in polymer size of the fruit were not directly studied. A shift toward lower molecular weights was also observed in the 4KSF between the UR and MR stages, but no changes were detected afterwards. Our results show that depolymerization of polysaccharides from tree ripened “Bartlett” pears takes place at intermediate stages. The limited pectin depolymerization when the fruit is maintained in the tree is at least unusual since polyuronide hydrolysis has been shown to be a late ripening event in most fruits (Yoshioka et al., 1992; Brummell, 2006). 4. Conclusion “Bartlett” pears ripened on-tree, are less juicy and develop mealy texture upon ripening. The cell wall analysis showed arabinose solubilization as one of the clearest features, occurred at a higher rate from the UR to the MR stage, but proceeded throughout on-tree ripening (Fig. 6). Water soluble pectins increased from the UR to the MR but later on decreased in association with the accumulation of CDTA-soluble polyuronides in LR fruit. Both crosslinking glycans solubilization and depolymerization took place mainly at intermediate stages, and some cellulose-derived glucans appear to solubilize in the last stage. In contrast, pectin depolymerization, which is normally described as a late ripening event, was not observed in any of the fractions if fruit is held on the tree after the MR stage. As for an explanation for the suboptimal ripening of LR fruit, fewer differences than might have been expected have emerged. However, it seems clear that some insolubilization of pectin results in LR fruit as well as a modification of arabinose metabolism. An imbalance in pectin degradation, including decreased pectin backbone depolymerization, together with continuing removal of Ara from RG-I, and/or other modifications increasing ionic interactions, may lead to fruit that losses firmness but yields an abnormal mealy texture upon ripening. Acknowledgements We thank Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 0353), Universidad de Buenos Aires (UBACyT Program),
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