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Changes and postharvest regulation of activity and gene expression of enzymes related to cell wall degradation in ripening apple fruit
Postharvest Biology and Technology 56 (2010) 147–154
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Changes and postharvest regulation of activity and gene expression of enzymes related to cell wall degradation in ripening apple fruit Jianmei Wei a,b , Fengwang Ma a,∗ , Shouguo Shi a , Xiudong Qi c , Xiangqiu Zhu b , Junwei Yuan b a b c
College of Horticulture, Northwest A & F University, Yangling, Shaanxi 712100, China Changli Fruit Institute, Hebei A & F Science Academy, Changli, Hebei 066600, China Hebei Normal University of Science and Technology, Changli, Hebei 066600, China
a r t i c l e
i n f o
Article history: Received 30 August 2009 Accepted 6 December 2009 Keywords: Cell wall enzyme activity Fruit softening Gene expression Malus domestica Borkh. Regulation
a b s t r a c t To elucidate the roles of cell wall-modifying enzymes in apple fruit, we investigated the activity and gene expression of -galactosidase (-Gal), ␣-l-arabinofuranosidase (␣-l-Af), polygalacturonase (PG), and pectin methylesterase (PME). Their regulation by ethylene and cold storage (0 ◦ C) was also assessed. ‘Golden Delicious’ and ‘Fuji’ fruit showed differences in their rates of respiration and decline of firmness, as well as demonstrating unique regulated effects. Activities of -Gal and ␣-l-Af were higher in ‘Golden Delicious’ than in ‘Fuji’ fruit, although both had similar patterns of change. They were dramatically inhibited by 1-methylcyclopropene (1-MCP) and 0 ◦ C, and enhanced by exposure to ethephon, with stronger response in ‘Golden Delicious’ fruit. Gene expression of cell wall enzymes also was significantly affected by 0 ◦ C, 1-MCP, and ethephon. The difference in ␣-l-Af expression among treatments in ‘Golden Delicious’ was more significant than in ‘Fuji’ fruit, especially early in storage. In contrast, expression of -Gal was inhibited by 1-MCP at early stages in ‘Golden Delicious’ fruit and over the entire storage period in ‘Fuji’ fruit, and was significantly enhanced by ethephon treatment in the former but only slightly in the latter. At 0 ◦ C, -Gal mRNA accumulation was inhibited in both cultivars. PG and PME activities increased during softening, and differed at different stages for each cultivar, and were obviously regulated by ethylene and 0 ◦ C. PME expression was higher in ‘Golden Delicious’ fruit, with far greater differences between cultivars than that detected for PG. Both PME and PG mRNA were more intensively influenced by ethylene and cold storage in ‘Golden Delicious’ fruit. The results confirm that cell wall enzymes play an important role in fruit softening. Of these, -Gal and ␣-l-Af may be more closely related to the storability of apples than PG and PME, especially when fruit ripening and softening begin. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The softening of fruit texture is a major process of ripening, leading to a loss of firmness, facilitating pathogen infection, increasing postharvest decay, and reducing shelf-life and fruit quality. Changes in cell wall structure and composition are the main cause of this softening (Brummell and Harpster, 2001). This complex biochemical process is correlated with the action of a number of cell wall-modifying enzymes (Brummell and Harpster, 2001; Giovannoni, 2001). During ripening, water-soluble polyuronides increase while insoluble and covalently bound pectin decreases. Polygalacturonase (PG) and pectin methylesterase (PME) are two major enzymes that act on the pectin fraction of the cell wall. PME catalyzes pectin demethylation, making the walls susceptible to further degradation by PG (Brummell and Harpster, 2001).
∗ Corresponding author. Tel.: +86 29 87082648; fax: +86 29 87082648. E-mail addresses: [email protected], [email protected] (F. Ma). 0925-5214/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2009.12.003
As is common at the initiation of fruit ripening, pectins generally undergo the earliest modification that involves the loss of neutral sugars (NS), particularly galactose and arabinose (Redgwell et al., 1997). Both NS usually exist as side chains attached to rhamnosyl residues of the rhamnogalacturonan backbone; these are the most dynamic cell wall glycosyl residues (Gross and Sams, 1984; Redgwell et al., 1997), and are degraded by the action of -galactosidase (-Gal) and ␣-l-arabinofuranosidase (␣-l-Af) (Fry, 1995; Beldman et al., 1997). -Gal and ␣-l-Af are the two major glycosidases that may remove galactosyl and arabinosyl residues from cell wall polysaccharides. They are thought to be responsible for the decrease in such residues in many ripening fruit (Ross et al., 1994; Smith and Gross, 2000; Tateishi et al., 2001; Sozzi et al., 2002; Chávez Montes et al., 2008). Among these hydrolases, PG has been widely studied, but its role has not been well defined. Results from transgenic trials with tomato have suggested that this enzyme alone is neither necessary nor sufficient for fruit softening (Giovannoni et al., 1989). Many other reports have also implied that any individual enzyme is not
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sufficient, but that it is part of an overall process incorporating several isoforms and post-transcriptional regulatory events (Goulao et al., 2007). The loss of firmness during apple ripening is associated with the activity of several cell wall-modifying enzymes (Brummell, 2006). However, little study has been made about how these cell wall hydrolase activities and related gene expression influence fruit textural attributes. Generally, ‘Fuji’ is a crisp and juicy apple that stores well whereas ‘Golden Delicious’ apples easily become mealy after harvest. Therefore, the objective of our research was to examine alterations in cytohydrolytic activities and gene expression. By understanding how these are regulated by low temperature and ethylene in apple, we hoped to gain insight into the possible roles of cell wall-modifying enzymes in two cultivars with different softening and ripening mechanisms. 2. Materials and methods 2.1. Plant materials and treatments Fruit of two apple (Malus domestica Borkh.) cultivars, ‘Fuji’ and ‘Golden Delicious’, were harvested at the commercial maturity stage from the Yanshan mountain region at Changli, Province of Hebei (39◦ 45 N, 119◦ 12 E) (China). After their immediate transfer to the laboratory, fruit that were free of mechanical injury, insects, and diseases were selected and divided into four groups. Our treatments included: (1) control, fruit placed directly into plastic bags (15 m thick) and stored at room temperature (RT, 20 ± 1 ◦ C); (2) cold storage, fruit pre-cooled to remove field heat followed by storage at 0 ◦ C; (3) 1-methylcyclopropene (1-MCP), fruit placed inside a sealed container with 500 nL L−1 1-MCP (Ansip® , 0.18%), fumigated for 24 h at RT, then ventilated, and held under ambient conditions at RT; (4) ethephon, fruit treated with 2 mL L−1 ethephon solution (40%), soaked 5 min, air-dried, then stored at RT. Fruit were sampled at appropriate intervals based on their storability. For subsequent analysis, the flesh tissue of each sample was cut into approximately 2 cm2 pieces, frozen in liquid nitrogen, and stored at −70 ◦ C. 2.2. Determination of firmness and respiration rate Using a hand penetrometer (model GY-1; China) equipped with a flat probe, we determined firmness at four equatorial regions on the flesh of three apples (ten replications per sampling time). The respiration rate was measured via the air-stream method (three replications per treatment). 2.3. Enzyme extraction and analysis of activity Cell wall enzymes were extracted according to the method described by Zhou et al. (2000), with slight modifications. The frozen flesh (3.0 g) was powdered with a pestle and mortar, then stirred into 6 mL of cold 12% polyethyleneglycol containing 0.2% sodium bisulphite. After the homogenate was centrifuged for 10 min at 12,000 × g, the pellet was washed with 4 ◦ C aqueous
0.2% sodium bisulfite. Pellets were collected and extracted for PG (EC3.2.1.15), PME (EC3.1.1.11), ␣-l-Af (EC3.2.1.55), and -Gal (EC 3.2.1.23). The extraction conditions included 6 mL of cold extraction buffer [0.1 M sodium acetate (pH 5.2), 100 mM NaCl, 2% (v/v) -mercaptoethanol, and 5% (w/v) polyvinylpyrrolidone (PVP)] at 4 ◦ C for 1 h. Following centrifugation as above, the supernatant was used to assay for enzyme activity. All of these steps were performed at 4 ◦ C. PG activity was determined as described by Gross (1982). Enzyme extract (0.2 mL) was mixed with 0.8 mL of 0.5% polygalacturonic acid (Sigma Chemical Co., St. Louis, MO, USA) in 50 mM sodium acetate buffer (pH 5.2), and incubated at 37 ◦ C for 2 h. To measure the amount of galacturonic acid released, we added 2 mL of borate buffer (0.1 M, pH 9.0) and 0.3 mL of cyanoacetamide to the reaction mixture. After boiling for 10 min and then cooling, absorbance was read at 276 nm. Galacturonic acid was used as our standard and the controls for the boiled extract were run in the reaction buffer. One unit of activity was defined as 1 g of galacturonic acid released g−1 fresh weight (FW) min−1 . For PME activity (Lin et al., 1989), 1 mL of crude extract was mixed with 4 mL of 1% (w/v) citrus pectin (Sigma Chemical Co., St. Louis, MO, USA) and titrated with 0.01 M NaOH to maintain pH 7.4 while incubating at 37 ◦ C for 1 h. One unit of activity was calculated as 1 mmol NaOH consumed g−1 FW 10 min−1 . The activities of ␣-l-Af and -Gal were measured by using 3 mM p-nitrophenyl--d-galactopyranoside and p-nitrophenyl-␣d-arabinofuranoside (Sigma Chemical Co., St. Louis, MO, USA) as substrates, respectively. Here, 0.5 mL of 0.1 M sodium acetate (pH 5.2) and 0.5 mL of substrate were pre-incubated at 40 ◦ C for 10 min before 0.5 mL of the enzyme extract was added. After incubation at 37 ◦ C for 30 min, the reaction was stopped by adding 2.0 mL of 0.5 M sodium carbonate, and the p-nitrophenol that was released was then measured spectrophotometrically at 400 nm. A calibration curve was obtained by using free p-nitrophenol (PNP) (Sigma Chemical Co., St. Louis, MO, USA) as our standard. Enzyme activity was expressed as nmol PNP g−1 FW min−1 (Yoshioka et al., 1995; Brummell et al., 2004). In all assays, the experiment with boiled enzyme extract was taken as the control. 2.4. Analysis of cell call enzyme-related gene expression by QRT-PCR The expression of genes involved in cell wall metabolism, including PME, PG, -Gal, and ␣-l-Af, was determined by quantitative reverse transcription-polymerase chain reaction (QRT-PCR). Total RNA was extracted from fruit by the modified CTAB method (Gasic et al., 2004), and DNase was used to remove DNA before reverse transcription. Gene-specific primers (Table 1) were designed (Primer5.0 software) from the coding sequences of apple genes. QRT-PCR was performed with a PrimeScriptTM RT Reagent Kit (TaKaRa, Japan), using oligo(dT)20 and random primers for cDNA synthesis according to the manufacturer’s protocol. The amplified PCR products were quantified on an iQ5 Multicolor RealTime PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA), with the SYBR Premix Ex Taq kit (TaKaRa, Japan). The vari-
Table 1 Primer sets for QRT-PCR amplification of cell wall enzyme-related gene expression. Gene
Primer sequence
PG PME -Gal ␣-l-Af 18S rRNA
S5 -GTAACTGCACCAGAGGACA-3 S5 -GATGCCTTGGAGTGGAGA-3 S5 -AAGAACGGAAAGTCCCCAC-3 S5 -AGAAACGCCTATCCTGAC-3 S5 -CCATTGGAGGGCAAGTCT-3
A5 -TTCTTCACCACCAAGTTATT-3 A5 -TGCTAATGTATTGCGTTC-3 A5 -TCCAATGACCCATACACGG-3 A5 -CACGGCATACTCGCTCAC-3 A5 -GGTTCTCACGCTACACGA-3
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ous cDNA samples were standardized with 18S rRNA transcripts. Our QRT-PCR experiments were repeated three times, based on three separate RNA extracts from three samples, and the expression levels were set at 1.0 for day 0 for each gene.
‘Fuji’ fruit, perhaps related to variations in their softening attributes and regulation mechanisms.
2.5. Experimental design and statistical analysis
For both cultivars, decline in fruit firmness followed the same trend as softening rates, although this decrease was only moderate for ‘Fuji’ and more rapid for ‘Golden Delicious’ fruit (Fig. 2). However, significant differences were found among 1-MCP, ethephon, and 0 ◦ C treatments in the latter (Fig. 2B), where flesh firmness did not noticeably decrease with 1-MCP or cold storage, but did decline quickly during the control or ethephon treatments. Although no difference was found between 1-MCP and 0 ◦ C treatments or between ethephon treatment and the control, data for both 1-MCP and 0 ◦ C treatments differed significantly from that for the control. However, ‘Fuji’ fruit firmness was maintained at higher levels than 8.0 kg cm−2 after 70 d of storage, whether fruit had been treated with 1-MCP, 0 ◦ C treatment, or ethephon, and those apples retained their original crisp texture (Fig. 2A). All these results suggest that the two cultivars have distinctive softening characteristics, and that both ethylene and low temperatures play important roles in fruit softening.
Experiments were performed according to a completely randomized design. Numerical data were examined by analysis of variance (ANOVA), and significance was detected with Duncan’s multiple range tests at a level of 5%. 3. Results 3.1. Fruit respiration rate Although their dynamic trends were similar, the respiration rate was dramatically higher in ‘Golden Delicious’ than in ‘Fuji’ apples. Rates peaked at day 7 (‘Golden Delicious’) and day 14 (‘Fuji’) after harvest (Fig. 1). In the latter, this rate was only slightly accelerated by ethephon treatment compared with the controls, whereas storage at 0 ◦ C or exposure to 1-MCP had no apparent affect (Fig. 1A). However, for ‘Golden Delicious’ fruit, both 1-MCP and 0 ◦ C dramatically inhibited respiration and postponed its climatic peak, especially at the early stage of storage. For that cultivar, however, only a slight acceleration in respiration was found in ethephontreated fruit (Fig. 1B). These results demonstrate the important difference in respiratory patterns between ‘Golden Delicious’ and
3.2. Fruit firmness
3.3. Enzyme activity and gene expression 3.3.1. Polygalacturonase (PG) Polygalacturonase activity was significantly different between ‘Fuji’ and ‘Golden Delicious’ fruit, and was sensitive to ethephon
Fig. 1. Effect of ethephon, 1-MCP, and 0 ◦ C treatment on respiration rate in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
Fig. 2. Effect of ethephon, 1-MCP, and 0 ◦ C on firmness in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
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Fig. 3. Effect of ethephon, 1-MCP, and 0 ◦ C on PG activity and PG gene expression in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. The PG expression levels were set at 1.0 for day 0. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
and cold storage. In ‘Fuji’ fruit (Fig. 3A), this activity increased steadily and in a similar manner during storage, regardless of treatment, with values being negatively correlated with flesh firmness (r = −0.834*). PG levels were relatively low at the beginning of the 1-MCP treatment, then rose, and differences from the control diminished after 28 d. The promotive effect by ethephon did not surpass the inhibitory effect from 1-MCP treatment though ethephon treatment could enhance PG activity in ‘Fuji’ fruit. Activity was initially low under 0 ◦ C storage but began to increase later. For ‘Golden Delicious’ fruit (Fig. 3B), PG levels peaked at day 56 before declining rapidly, producing a negative correlation with flesh firmness (r = −0.707*). Ethephon greatly enhanced PG activity and reduced the time to its peak whereas both 1-MCP and 0 ◦ C treatment were strong inhibitors, with PG maintaining relatively low activity during the storage period. Expression of PG changed little in ‘Fuji’ fruit and was similar to the control throughout our experiments. However, it was accelerated by ethephon and inhibited by 1-MCP; 0 ◦ C treatment had only a small influence except on day 14 of storage (Fig. 3C). In ‘Golden Delicious’ fruit, such expression was obviously enhanced after 28 d of ethephon treatment, and was dramatically higher than in the control, especially during late storage. For that cultivar, PG expression was significantly inhibited in 1-MCP-treated fruit, with transcript levels being very low early on and increasing only slightly by the end of the storage period. The inhibitory effect of 0 ◦ C storage for ‘Golden Delicious’ was similar to that for ‘Fuji’ fruit, but was not obviously lower than that observed for the control (Fig. 3D). 3.3.2. Pectin methylesterase (PME) Pectin methylesterase activity increased slightly in the early stage of ‘Fuji’ fruit storage and remained fairly constant from day 14 to 42 before decreasing gradually. This demonstrated a negative correlation with firmness (r = −0.881**). PME levels were enhanced by ethephon and were higher than the control after 42 d. Both 1MCP treatment and storage at 0 ◦ C had inhibitory effects and PME activity did not tend to increase during storage (Fig. 4A). In ‘Golden
Delicious’ fruit, activity rose as fruit ripening and softening ensued; this process was more rapid during the early stage and was negatively correlated with firmness (r = −0.982**). Ethephon treatment did not significantly accelerate this activity, but both 1-MCP and 0 ◦ C treatment slowed it (Fig. 4B). In ‘Fuji’ apples (Fig. 4C), PME expression remained constant up to day 42, and increased only in the late stage of storage. Treatment with either ethephon or 0 ◦ C had little effect on transcripts in the early stage of storage, but differences in expression among treatments were manifest after day 56. By contrast, expression of PME in ‘Golden Delicious’ fruit (Fig. 4D) increased rapidly after harvest and was significantly regulated by ethylene and low temperature. This ethylene-regulated expression exceeded that of the control, but that difference was diminished in later storage. 1-MCP treatment strongly repressed PME transcript levels throughout the experimental period, whereas cold storage inhibited such expression mainly in the first 42 d, after which it did not differ significantly from the control. 3.3.3. ˇ-Galactosidase (ˇ-Gal) For ‘Fuji’ fruit, -galactosidase activity was very low until day 42 (Fig. 5A); for ‘Golden Delicious’ fruit this activity increased rapidly at the beginning of storage and continued to rise throughout the period (Fig. 5B). Our analysis indicated a significant negative correlation between -Gal activity and firmness in ‘Fuji’ (r = −0.726*) and ‘Golden Delicious’ (r = −0.912**) fruit, thereby demonstrating that this increase in enzyme levels was closely associated with the softening process. Treatment with either 1-MCP or cold storage dramatically inhibited this activity in both cultivars, but especially in ‘Fuji’ fruit. However, the ethephon-treated and control apples had noticeably higher levels. These results indicate that -Gal plays an important role during apple softening, and is closely related to the unique storage characteristics of each cultivar. The expression patterns of -Gal differed significantly between ‘Fuji’ and ‘Golden Delicious’ fruit. In the latter, expression was mainly inhibited early in storage by 1-MCP; this effect disappeared
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Fig. 4. Effect of ethephon, 1-MCP, and 0 ◦ C on PME activity and PME gene expression in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. The PME expression levels were set at 1.0 for day 0. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
over time. In contrast, expression in ‘Fuji’ fruit continued to be strongly repressed throughout the entire storage period. Ethephon treatment significantly increased transcript levels in ‘Golden Delicious’ but not in ‘Fuji’ fruit. Under cold storage, expression was significantly reduced in both cultivars (Fig. 5C and D).
3.3.4. ˛-l-Arabinofuranosidase (˛-l-Af) For both cultivars, the trend in activity of ␣-larabinofuranosidase was similar to that seen with fruit ripening and softening. In ‘Fuji’ fruit, ␣-l-Af levels were very low but increased slightly after 14 d of storage (Fig. 6A), while a very steady
Fig. 5. Effect of ethephon, 1-MCP, and 0 ◦ C on -Gal activity and -Gal gene expression in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. The -Gal expression levels were set at 1.0 for day 0. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
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Fig. 6. Effect of ethephon, 1-MCP, and 0 ◦ C on ␣-l-Af activity and ␣-l-Af gene expression in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. The ␣-l-Af expression levels were set at 1.0 for day 0. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
rise was found beginning just after harvest in ‘Golden Delicious’ fruit (Fig. 6B). These increases were significantly and negatively correlated with fruit firmness, with correlation coefficients of −0.842** and −0.778* for ‘Golden Delicious’ and ‘Fuji’ fruit, respectively. Ethephon had different effects on activities – slightly higher than for the control in ‘Fuji’ (Fig. 6A) and noticeably higher than the control in ‘Golden Delicious’ fruit (Fig. 6B). By contrast, the difference between cultivars in response to 1-MCP and 0 ◦ C inhibition was very small. Such activity in ‘Golden Delicious’ fruit was almost completely stopped before rising only in the late storage period following 1-MCP treatment (Fig. 6A and B). In ‘Fuji’ fruit, ␣-l-Af expression was insensitive to ethylene and almost no difference was found among 1-MCP, ethephon, and the control treatments, especially during early storage. However, 0 ◦ C treatment dramatically inhibited such expression, and transcript levels did not increase until day 56 (Fig. 6C). Expression of ␣-l-Af in ‘Golden Delicious’ fruit was more sensitive to ethephon and 1-MCP treatment than that in ‘Fuji’ fruit. In ‘Golden Delicious’ fruit, ␣-l-Af mRNA expression in the former treatment was higher than that in the control after 28 d, and was maintained at this level throughout the remainder of the period. Exposure to 1-MCP significantly inhibited gene expression during storage. Finally, at 0 ◦ C, ␣-l-Af expression was repressed in ‘Golden Delicious’ fruit, which was stronger than in ‘Fuji’ fruit (Fig. 6D). 4. Discussion Cell wall degradation is the main factor involved in fruit softening and texture changes (Bennett and Labavitch, 2008; Billy et al., 2008). It is increasingly apparent that softening and textural changes are brought about by the actions of a multitude of cell wall-localized enzymes acting on specific, potentially highly localized substrates (Brummell et al., 2004). Cell wall enzymes are major contributors to cell wall disassembly, reducing intercellular connections and cell dispersion and playing various roles at different
stages during fruit development (Brummell, 2006; Almeida and ˜ Huber, 2007; Sanudo-Barajas et al., 2009). PME mainly serves to catalyze pectin demethylation, damaging the calcium horizontal links of the acid polysaccharide chain and leading to cell separation, and generating a suitable substrate for PG (Brummell et al., 2004). Also, transgenic suppression of PME activity in tomato decreases pectin de-methylesterification, reducing the depolymerization of pectin by PG during ripening (Harriman et al., 1991; Tieman et al., 1992). This indicated that the fruit remained firmer because of diminished PME activity during ripening (Phan et al., 2007). Goulao et al. (2008) reported that the transcriptional pattern of PME de-emphasises its role in ripening of apple. Our study showed that PME activity and mRNA abundance between two cultivars were obviously different at the early stage of storage and were regulated by ethylene and 0 ◦ C, having more significant effects on PME transcripts in ‘Golden Delicious’ than that in ‘Fuji’ fruit. However, the level of PME mRNA did not changed coincidently with PME activity after being regulated by ethylene and low temperature; there should be further research to confirmi if this phenomenon is in accordance with previous studies (Ray et al., 1988; Harriman et al., 1991). PG is another cell wall-modifying enzyme. It hydrolyzes pectin acid along with the main chain of polygalacturonic acid, causing pectin degradation, cell wall dissolution, and ultimately, fruit softening (Wakabayashi et al., 2000; Brummell et al., 2004). Many reports suggest that PG-mediated pectin depolymerization requires a pectin substrate that is at least partially de-methylesterified (Wakabayashi et al., 2000). Brummell et al. (2004) consider that PG -subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening, suppression of PME activity does not affect firmness during normal ripening, and suppression of -subunit protein accumulation increases softening. This activity affects the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture. Accumulation of PG mRNA is
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regulated by ethylene, with low levels of ethylene being sufficient for induction, and mRNA accumulation increasing with ethylene exposure (Sitrit and Bennett, 1998; Brummell and Harpster, 2001). It is reported that the transcription level of the MdPG must be correlated with rate of firmness loss of fruit through the enzyme activity (Wakasa et al., 2006; Mann et al., 2008). In this work, based on the different responses to ethylene and 0 ◦ C, as well as the cultivar differences in PG activity, we consider that PG might play roles mainly at the mid- and late-stages of storage. Moreover, PG expression was also markedly regulated by ethylene and 0 ◦ C, and was weaker than PME, especially in ‘Golden Delicious’ fruit. However, the promotional effect of ethephon and the inhibitory effect of 1-MCP on PG expression was stronger in ‘Golden Delicious’ fruit, although no difference was found between cultivars when fruit had been chilled. However, in ‘Fuji’ fruit, PG activity was not tightly coupled with the accumulation of PG mRNA, that is, the increase of PG activity in the late stages of storage when specific gene expression does not show any increase. This suggests that PG activity may be manifest in a cultivar-dependent manner, or be regulated by translational or post-translational mechanisms. -Gal has been studied in various fruits, including tomato (Smith and Gross, 2000), pear (Tateishi et al., 2001; Mwaniki et al., 2007), and strawberry (Martínez and Civello, 2008). It has been characterized in association with the removal of galactosyl residues from cell wall polymers during fruit softening (Gross and Sams, 1984). Indeed, de-galactosidation in response to -Gal action has been proposed as an important process in cell wall modification during ripening (Smith and Gross, 2000). Therefore, such modification might involve glycosidases, such as -Gal, acting in cooperation with pectolytic enzymes (e.g., PG) in pectin metabolism during fruit ripening (Giovannoni, 2001). The expression of -Gal mRNA was also investigated using over-ripe apple fruit, and its transcripts could be unambiguously detected by semiquantitative RT-PCR during fruit ripening (Goulao et al., 2008). Here, we demonstrated that an increase in -Gal activity and related gene expression during ripening and softening was concomitant with a decrease in firmness. -Gal activity was rapidly enhanced at the early ripening stage in ‘Golden Delicious’ fruit, and was greater than that measured in ‘Fuji’ fruit. Treatments with 1MCP and 0 ◦ C dramatically postponed fruit softening and inhibited -Gal activity and -Gal mRNA accumulation, whereas the opposite response was found with ethephon. In addition, this change in pattern of activity did not differ from that for the control, indicating that -Gal has an important role in apple fruit softening, particularly the initial process. The glycoside hydrolase ␣-l-Af cleaves the glycosidic bonds between l-arabinofuranoside side chains and various oligosaccharides. Its activity is increased during the ripening of persimmon (Xu et al., 2003), tomato (Sozzi et al., 2002), peach (Brummell et al., 2004), banana (Zhuang et al., 2007), and pear fruit (Tateishi et al., 1996, 2005; Mwaniki et al., 2007). The ␣-l-Af enzyme has been purified from pears, and northern blots have shown that its mRNA is highly expressed only in ripening fruit (Tateishi et al., 2005). Many ␣-l-Af isoforms have been characterized from tomato (Sozzi et al., 2002) and pear (Mwaniki et al., 2007); one of them has been shown to be regulated by ethylene, functioning by releasing arabinosyl residues from the pectic fraction. This indicates that ␣-l-Af plays an important role in Arab metabolism during tomato ripening. In apple, degradation of a branched arabinan occurs prior to softening (P˜ena and Carpita, 2004), and interestingly, ␣-l-Af activity increases dramatically from commercial maturity to the over-ripe stages in ‘Royal Gala’ fruit (Goulao et al., 2007). Expression analysis of ␣-l-Af isolated from other fruit has shown that it has an important role in altering fruit texture during ripening (Itai et al., 2003; Tateishi et al., 2005; Mbéguié-A-Mbéguié et al., 2009). In our experiments, ␣-l-Af activity in both cultivars increased with ripening and
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softening, although the rate was obviously higher in ‘Golden Delicious’ fruit. Expression of its encoding gene followed a similar trend. Both activity and expression were notably inhibited by 1-MCP and 0 ◦ C treatment, but somewhat promoted by ethephon. This indicated that ␣-l-Af is sensitive to ethylene and low temperatures and has an important role in softening the fruit texture. We conclude that cell wall-modifying enzymes are closely correlated with fruit texture and cultivar characteristics. -Gal and ␣-l-Af may be more closely related to the storability of apples than PG and PME, especially when fruit ripening and softening begin. PME activity appeared at the early stage of storage, while PG activity occurred later; perhaps PG operates mainly at the mid- and latestages and is weaker than PME during fruit softening. Nevertheless, although the parameters analyzed here provide some information about fruit softening, our results cannot adequately explain the entire mechanism. This means that other factors such as cell wallneutral sugars, the functioning of genes for related enzymes, or non-enzymatic factors that contribute to texture softening should be taken into account in further research in order to identify apple fruit mechanisms. Acknowledgements This work was supported by the National Science & Technology Pillar program during the eleventh five-year plan period (2007BA79B05). The authors are grateful to Miss Priscilla Anne Licht for her help in writing. References Almeida, D.P.F., Huber, D.J., 2007. Polygalacturonase-mediated dissolution and depolymerization of pectins in solutions mimicking the pH and mineral composition of tomato apoplast. Plant Sci. 172, 1087–1094. Beldman, G., Schols, H.A., Pitson, S.M., Searle-van Leeuwen, M.J.F., Voragen, A.G.J., 1997. Arabinans and arabinan degrading enzymes. In: Advances in Macromolecular Carbohydrate Research. U.S.A. pp. 1–64. Bennett, A.B., Labavitch, J.M., 2008. Ethylene and ripening-regulated expression and function of fruit cell wall modifying proteins. Plant Sci. 175, 130–136. Billy, L., Mehinagic, E., Royer, G., Renard, C.M.G.C., Arvisenet, G., Prost, C., Jourjon, F., 2008. Relationship between texture and pectin composition of two apple cultivars during storage. Postharvest Biol. Technol. 47, 315–324. Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311–340. Brummell, D.A., Dal Cin, V., Crisosto, C.H., Labavitch, J.M., 2004. Cell wall metabolism during maturation, ripening and senescence of peach fruit. J. Exp. Bot. 55, 2029–2039. Brummell, D.A., 2006. Cell wall disassembly in ripening fruit. Funct. Plant Biol. 33, 103–119. Chávez Montes, R.A., Ranocha, P., Martinez, Y., Minic, Z., Jouanin, L., Marquis, M., Saulnier, L., Fulton, L.M., Cobbett, C.S., Bitton, F., Renou, J., Jauneau, A., Goffner, D., 2008. Cell wall modifications in Arabidopsis plants with altered ␣-l-arabinofuranosidase activity. Plant Physiol. 147, 63–77. Fry, S.C., 1995. Polysaccharide-modifying enzymes in the plant cell wall. Ann. Rev. Plant Physiol. Plant Mol. Biol. 46, 497–520. Gasic, K., Hernandez, A., Schuyler, S., Korban, S.S., 2004. RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Mol. Biol. Rep. 22, 437–1437. Giovannoni, J., 2001. Molecular biology of fruit maturation and ripening. Ann. Rev. Plant Physiol. Plant Mol. Biol. 52, 725–749. Giovannoni, J., DellaPenna, D., Bennett, A.B., Fischer, R.L., 1989. Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening. Plant Cell 1, 53–63. Goulao, L.F., Santos, J., Sousa, I., Oliveira, C.M., 2007. Patterns of enzymatic activity of cell wall modifying enzymes during growth and ripening of apples. Postharvest Biol. Technol. 43, 307–318. Goulao, L.F., Cosgrove, D.J., Oliveira, C.M., 2008. Cloning, characterisation and expression analyses of cDNA clones encoding cell wall-modifying enzymes isolated from ripe apples. Postharvest Biol. Technol. 48, 37–51. Gross, K.C., 1982. A rapid and sensitive spectrophotometric method for assaying polygalacturonase using 2-cyanoacetamide. HortScience 17, 933–934. Gross, K.C., Sams, C.E., 1984. Changes in cell wall neutral sugar composition during fruit ripening: a species survey. Phytochemistry 23, 2457–2461. Harriman, R.W., Tieman, D.M., Handa, A.K., 1991. Molecular cloning of tomato pectin methylesterase gene and its expression in Rutgers, ripening inhibitor, nonripening, and never ripe tomato fruit. Plant Physiol. 97, 80–87.
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Itai, A., Ishihara, K., Bewley, J.D., 2003. Characterization of expression, and cloning of beta-d-xylosidase and alpha-l-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit. J. Exp. Bot. 54, 2615–2622. Lin, T.P., Liu, C.C., Chen, S.W., Wang, W.Y., 1989. Purification and characterization of pectinmethylesterase from Ficus awkeotsang Makino achenes. Plant Physiol. 91, 1445–1453. Mann, H.S., Alton, J.J., Kim, S.H., Tong, C.B.S., 2008. Differential expression of cellwall-modifying genes and novel cDNAs in apple fruit during storage. J. Am. Soc. Hort. Sci. 133, 152–157. Martínez, G.A., Civello, P.M., 2008. Effect of heat treatments on gene expression and enzyme activities associated to cell wall degradation in strawberry fruit. Postharvest Biol. Technol. 49, 38–45. Mbéguié-A-Mbéguié, D., Hubert, O., Baurens, F.C., Matsumoto, T., Chillet, M., FilsLycaon, B., Sidibé-Bocs, S., 2009. Expression patterns of cell wall-modifying genes from banana during fruit ripening and in relationship with finger drop. J. Exp. Bot. 60, 2021–2034. Mwaniki, M.W., Mathooko, F.M., Hiwasa, K., Tateishi, A., Yokotani, N., Ushijima, K., Nakano, R., Inaba, A., Kubo, Y., 2007. -Galactosidase and ␣-l-Arabinofuranosidase activities and gene expression in European and Chinese pear fruit during ripening. J. Jpn. Soc. Hortic. Sci. 76, 85–90. P˜ena, M.J., Carpita, N.C., 2004. Loss of highly branched arabinans and debranching of rhamnogalacturonan I accompany loss of firm texture and cell separation during prolonged storage of apple. Plant Physiol. 135, 1305–1313. Phan, T.D., Bo, W., West, G., Lycett, G.W., Tucker, G.A., 2007. Silencing of the major salt-dependent isoform of pectinesterase in tomato alters fruit softening. Plant Physiol. 144, 1960–1967. Ray, J., Knapp, J., Grierson, D., Bird, C., Schuch, W., 1988. Identification and sequence determination of a cDNA clone for tomato pectin esterase. Eur. J. Biochem. 174, 119–124. Redgwell, R.J., Fischer, M., Kendall, E., MacRae, E.A., 1997. Galactose loss and fruit ripening: high-molecular-weight arabinogalactans in the pectic polysaccharides of fruit cell walls. Planta 203, 174–181. Ross, G.S., Wegrzyn, T., MacRae, E.A., Redgwell, R.J., 1994. Apple -galactosidase activity against cell wall polysaccharides and characterization of a related cDNA clone. Plant Physiol. 106, 521–528. ˜ Sanudo-Barajas, J.A., Labavitch, J., Greve, C., Osuna-Enciso, T., Muy-Rangel, D., SillerCepeda, J., 2009. Cell wall disassembly during papaya softening: role of ethylene in changes in composition, pectin-derived (PDOs) production and wall hydrolases. Postharvest Biol. Technol. 51, 158–167.
Sitrit, Y., Bennett, A.B., 1998. Regulation of tomato fruit polygalacturonase mRNA accumulation by ethylene: a reexamination. Plant Physiol. 116, 1145–1150. Smith, D.L., Gross, K.C., 2000. A family of at least seven -galactosidase genes is expressed during tomato fruit development. Plant Physiol. 123, 1173–1183. Sozzi, G.O., Fraschina, A.A., Navarro, A.A., Cascone, O., Greve, L.C., Labavitch, J.M., 2002. ␣-l-Arabinofuranosidase activity during development and ripening of normal and ACC synthase antisense tomato fruit. HortScience 37, 564–566. Tateishi, A., Kanayama, Y., Yamaki, S., 1996. ␣-l-Arabinofuranosidase from the cell wall of Japanese pear fruit. Phytochemistry 42, 295–299. Tateishi, A., Inoue, H., Shiba, H., Yamaki, S., 2001. Molecular cloning of galactosidase from Japanese pear (Pyrus pyrifolia) and its gene expression with fruit ripening. Plant Cell Physiol. 42, 492–498. Tateishi, A., Mori, H., Watari, J., Nagashima, K., Yamaki, S., Inoue, H., 2005. Isolation, characterization, and cloning of ␣-l-arabinofuranosidase expressed during fruit ripening of Japanese pear. Plant Physiol. 138, 1653–1664. Tieman, D.M., Harriman, R.W., Ramamohan, G., Handa, A.K., 1992. An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit. Plant Cell 4, 667–679. Wakabayashi, K., Chun, J.P., Huber, D.J., 2000. Extensive solubilization and depolymerization of cell wall polysaccharides during avocado (Persea americana) ripening involves concerted action of polygalacturonase and pectinmethylesterase. Physiol. Plant 108, 345–352. Wakasa, Y., Kudo, H., Ishikawa, R., Akada, S., Senda, M., Niizeki, M., Harada, T., 2006. Low expression of an endopolygalacturonase gene in apple fruit with long-term storage potential. Postharvest Biol. Technol. 39, 193–198. Xu, C.G., Nakatsuka, A., Kano, H., Itamura, H., 2003. Changes in ethylene production and activities of cell wall degrading enzymes during rapid fruit softening of Japanese persimmon ‘Saijo’. J Jpn. Soc. Hortic. Sci. 72, 460–462. Yoshioka, H., Kashimura, Y., Kaneko, K., 1995. -d-Galactosidase and ␣-larabinofuranosidase activities during the softening of apples. J. Jpn. Soc. Hortic. Sci. 63, 871–878. Zhou, H.W., Sonego, L., Khalchitski, A., Ben-Arie, R., Lers, A., Lurie, S., 2000. Cell wall enzymes and cell wall changes in ‘Flavortop’ nectarines: mRNA abundance, enzyme activity, and changes in pectic and neutral polymers during ripening and in woolly fruit. J. Am. Soc. Hortic. Sci. 125, 630–637. Zhuang, J.P., Su, J., Li, X.P., Chen, W.X., 2007. Changes in ␣-l-arabinofuranosidase activity in peel and pulp of banana (Musa sp.) fruit during ripening and softening. J. Plant Physiol. Mol. Biol. 33, 31–136.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Changes and postharvest regulation of activity and gene expression of enzymes related to cell wall degradation in ripening apple fruit Jianmei Wei a,b , Fengwang Ma a,∗ , Shouguo Shi a , Xiudong Qi c , Xiangqiu Zhu b , Junwei Yuan b a b c
College of Horticulture, Northwest A & F University, Yangling, Shaanxi 712100, China Changli Fruit Institute, Hebei A & F Science Academy, Changli, Hebei 066600, China Hebei Normal University of Science and Technology, Changli, Hebei 066600, China
a r t i c l e
i n f o
Article history: Received 30 August 2009 Accepted 6 December 2009 Keywords: Cell wall enzyme activity Fruit softening Gene expression Malus domestica Borkh. Regulation
a b s t r a c t To elucidate the roles of cell wall-modifying enzymes in apple fruit, we investigated the activity and gene expression of -galactosidase (-Gal), ␣-l-arabinofuranosidase (␣-l-Af), polygalacturonase (PG), and pectin methylesterase (PME). Their regulation by ethylene and cold storage (0 ◦ C) was also assessed. ‘Golden Delicious’ and ‘Fuji’ fruit showed differences in their rates of respiration and decline of firmness, as well as demonstrating unique regulated effects. Activities of -Gal and ␣-l-Af were higher in ‘Golden Delicious’ than in ‘Fuji’ fruit, although both had similar patterns of change. They were dramatically inhibited by 1-methylcyclopropene (1-MCP) and 0 ◦ C, and enhanced by exposure to ethephon, with stronger response in ‘Golden Delicious’ fruit. Gene expression of cell wall enzymes also was significantly affected by 0 ◦ C, 1-MCP, and ethephon. The difference in ␣-l-Af expression among treatments in ‘Golden Delicious’ was more significant than in ‘Fuji’ fruit, especially early in storage. In contrast, expression of -Gal was inhibited by 1-MCP at early stages in ‘Golden Delicious’ fruit and over the entire storage period in ‘Fuji’ fruit, and was significantly enhanced by ethephon treatment in the former but only slightly in the latter. At 0 ◦ C, -Gal mRNA accumulation was inhibited in both cultivars. PG and PME activities increased during softening, and differed at different stages for each cultivar, and were obviously regulated by ethylene and 0 ◦ C. PME expression was higher in ‘Golden Delicious’ fruit, with far greater differences between cultivars than that detected for PG. Both PME and PG mRNA were more intensively influenced by ethylene and cold storage in ‘Golden Delicious’ fruit. The results confirm that cell wall enzymes play an important role in fruit softening. Of these, -Gal and ␣-l-Af may be more closely related to the storability of apples than PG and PME, especially when fruit ripening and softening begin. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The softening of fruit texture is a major process of ripening, leading to a loss of firmness, facilitating pathogen infection, increasing postharvest decay, and reducing shelf-life and fruit quality. Changes in cell wall structure and composition are the main cause of this softening (Brummell and Harpster, 2001). This complex biochemical process is correlated with the action of a number of cell wall-modifying enzymes (Brummell and Harpster, 2001; Giovannoni, 2001). During ripening, water-soluble polyuronides increase while insoluble and covalently bound pectin decreases. Polygalacturonase (PG) and pectin methylesterase (PME) are two major enzymes that act on the pectin fraction of the cell wall. PME catalyzes pectin demethylation, making the walls susceptible to further degradation by PG (Brummell and Harpster, 2001).
∗ Corresponding author. Tel.: +86 29 87082648; fax: +86 29 87082648. E-mail addresses: [email protected], [email protected] (F. Ma). 0925-5214/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2009.12.003
As is common at the initiation of fruit ripening, pectins generally undergo the earliest modification that involves the loss of neutral sugars (NS), particularly galactose and arabinose (Redgwell et al., 1997). Both NS usually exist as side chains attached to rhamnosyl residues of the rhamnogalacturonan backbone; these are the most dynamic cell wall glycosyl residues (Gross and Sams, 1984; Redgwell et al., 1997), and are degraded by the action of -galactosidase (-Gal) and ␣-l-arabinofuranosidase (␣-l-Af) (Fry, 1995; Beldman et al., 1997). -Gal and ␣-l-Af are the two major glycosidases that may remove galactosyl and arabinosyl residues from cell wall polysaccharides. They are thought to be responsible for the decrease in such residues in many ripening fruit (Ross et al., 1994; Smith and Gross, 2000; Tateishi et al., 2001; Sozzi et al., 2002; Chávez Montes et al., 2008). Among these hydrolases, PG has been widely studied, but its role has not been well defined. Results from transgenic trials with tomato have suggested that this enzyme alone is neither necessary nor sufficient for fruit softening (Giovannoni et al., 1989). Many other reports have also implied that any individual enzyme is not
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sufficient, but that it is part of an overall process incorporating several isoforms and post-transcriptional regulatory events (Goulao et al., 2007). The loss of firmness during apple ripening is associated with the activity of several cell wall-modifying enzymes (Brummell, 2006). However, little study has been made about how these cell wall hydrolase activities and related gene expression influence fruit textural attributes. Generally, ‘Fuji’ is a crisp and juicy apple that stores well whereas ‘Golden Delicious’ apples easily become mealy after harvest. Therefore, the objective of our research was to examine alterations in cytohydrolytic activities and gene expression. By understanding how these are regulated by low temperature and ethylene in apple, we hoped to gain insight into the possible roles of cell wall-modifying enzymes in two cultivars with different softening and ripening mechanisms. 2. Materials and methods 2.1. Plant materials and treatments Fruit of two apple (Malus domestica Borkh.) cultivars, ‘Fuji’ and ‘Golden Delicious’, were harvested at the commercial maturity stage from the Yanshan mountain region at Changli, Province of Hebei (39◦ 45 N, 119◦ 12 E) (China). After their immediate transfer to the laboratory, fruit that were free of mechanical injury, insects, and diseases were selected and divided into four groups. Our treatments included: (1) control, fruit placed directly into plastic bags (15 m thick) and stored at room temperature (RT, 20 ± 1 ◦ C); (2) cold storage, fruit pre-cooled to remove field heat followed by storage at 0 ◦ C; (3) 1-methylcyclopropene (1-MCP), fruit placed inside a sealed container with 500 nL L−1 1-MCP (Ansip® , 0.18%), fumigated for 24 h at RT, then ventilated, and held under ambient conditions at RT; (4) ethephon, fruit treated with 2 mL L−1 ethephon solution (40%), soaked 5 min, air-dried, then stored at RT. Fruit were sampled at appropriate intervals based on their storability. For subsequent analysis, the flesh tissue of each sample was cut into approximately 2 cm2 pieces, frozen in liquid nitrogen, and stored at −70 ◦ C. 2.2. Determination of firmness and respiration rate Using a hand penetrometer (model GY-1; China) equipped with a flat probe, we determined firmness at four equatorial regions on the flesh of three apples (ten replications per sampling time). The respiration rate was measured via the air-stream method (three replications per treatment). 2.3. Enzyme extraction and analysis of activity Cell wall enzymes were extracted according to the method described by Zhou et al. (2000), with slight modifications. The frozen flesh (3.0 g) was powdered with a pestle and mortar, then stirred into 6 mL of cold 12% polyethyleneglycol containing 0.2% sodium bisulphite. After the homogenate was centrifuged for 10 min at 12,000 × g, the pellet was washed with 4 ◦ C aqueous
0.2% sodium bisulfite. Pellets were collected and extracted for PG (EC3.2.1.15), PME (EC3.1.1.11), ␣-l-Af (EC3.2.1.55), and -Gal (EC 3.2.1.23). The extraction conditions included 6 mL of cold extraction buffer [0.1 M sodium acetate (pH 5.2), 100 mM NaCl, 2% (v/v) -mercaptoethanol, and 5% (w/v) polyvinylpyrrolidone (PVP)] at 4 ◦ C for 1 h. Following centrifugation as above, the supernatant was used to assay for enzyme activity. All of these steps were performed at 4 ◦ C. PG activity was determined as described by Gross (1982). Enzyme extract (0.2 mL) was mixed with 0.8 mL of 0.5% polygalacturonic acid (Sigma Chemical Co., St. Louis, MO, USA) in 50 mM sodium acetate buffer (pH 5.2), and incubated at 37 ◦ C for 2 h. To measure the amount of galacturonic acid released, we added 2 mL of borate buffer (0.1 M, pH 9.0) and 0.3 mL of cyanoacetamide to the reaction mixture. After boiling for 10 min and then cooling, absorbance was read at 276 nm. Galacturonic acid was used as our standard and the controls for the boiled extract were run in the reaction buffer. One unit of activity was defined as 1 g of galacturonic acid released g−1 fresh weight (FW) min−1 . For PME activity (Lin et al., 1989), 1 mL of crude extract was mixed with 4 mL of 1% (w/v) citrus pectin (Sigma Chemical Co., St. Louis, MO, USA) and titrated with 0.01 M NaOH to maintain pH 7.4 while incubating at 37 ◦ C for 1 h. One unit of activity was calculated as 1 mmol NaOH consumed g−1 FW 10 min−1 . The activities of ␣-l-Af and -Gal were measured by using 3 mM p-nitrophenyl--d-galactopyranoside and p-nitrophenyl-␣d-arabinofuranoside (Sigma Chemical Co., St. Louis, MO, USA) as substrates, respectively. Here, 0.5 mL of 0.1 M sodium acetate (pH 5.2) and 0.5 mL of substrate were pre-incubated at 40 ◦ C for 10 min before 0.5 mL of the enzyme extract was added. After incubation at 37 ◦ C for 30 min, the reaction was stopped by adding 2.0 mL of 0.5 M sodium carbonate, and the p-nitrophenol that was released was then measured spectrophotometrically at 400 nm. A calibration curve was obtained by using free p-nitrophenol (PNP) (Sigma Chemical Co., St. Louis, MO, USA) as our standard. Enzyme activity was expressed as nmol PNP g−1 FW min−1 (Yoshioka et al., 1995; Brummell et al., 2004). In all assays, the experiment with boiled enzyme extract was taken as the control. 2.4. Analysis of cell call enzyme-related gene expression by QRT-PCR The expression of genes involved in cell wall metabolism, including PME, PG, -Gal, and ␣-l-Af, was determined by quantitative reverse transcription-polymerase chain reaction (QRT-PCR). Total RNA was extracted from fruit by the modified CTAB method (Gasic et al., 2004), and DNase was used to remove DNA before reverse transcription. Gene-specific primers (Table 1) were designed (Primer5.0 software) from the coding sequences of apple genes. QRT-PCR was performed with a PrimeScriptTM RT Reagent Kit (TaKaRa, Japan), using oligo(dT)20 and random primers for cDNA synthesis according to the manufacturer’s protocol. The amplified PCR products were quantified on an iQ5 Multicolor RealTime PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA), with the SYBR Premix Ex Taq kit (TaKaRa, Japan). The vari-
Table 1 Primer sets for QRT-PCR amplification of cell wall enzyme-related gene expression. Gene
Primer sequence
PG PME -Gal ␣-l-Af 18S rRNA
S5 -GTAACTGCACCAGAGGACA-3 S5 -GATGCCTTGGAGTGGAGA-3 S5 -AAGAACGGAAAGTCCCCAC-3 S5 -AGAAACGCCTATCCTGAC-3 S5 -CCATTGGAGGGCAAGTCT-3
A5 -TTCTTCACCACCAAGTTATT-3 A5 -TGCTAATGTATTGCGTTC-3 A5 -TCCAATGACCCATACACGG-3 A5 -CACGGCATACTCGCTCAC-3 A5 -GGTTCTCACGCTACACGA-3
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ous cDNA samples were standardized with 18S rRNA transcripts. Our QRT-PCR experiments were repeated three times, based on three separate RNA extracts from three samples, and the expression levels were set at 1.0 for day 0 for each gene.
‘Fuji’ fruit, perhaps related to variations in their softening attributes and regulation mechanisms.
2.5. Experimental design and statistical analysis
For both cultivars, decline in fruit firmness followed the same trend as softening rates, although this decrease was only moderate for ‘Fuji’ and more rapid for ‘Golden Delicious’ fruit (Fig. 2). However, significant differences were found among 1-MCP, ethephon, and 0 ◦ C treatments in the latter (Fig. 2B), where flesh firmness did not noticeably decrease with 1-MCP or cold storage, but did decline quickly during the control or ethephon treatments. Although no difference was found between 1-MCP and 0 ◦ C treatments or between ethephon treatment and the control, data for both 1-MCP and 0 ◦ C treatments differed significantly from that for the control. However, ‘Fuji’ fruit firmness was maintained at higher levels than 8.0 kg cm−2 after 70 d of storage, whether fruit had been treated with 1-MCP, 0 ◦ C treatment, or ethephon, and those apples retained their original crisp texture (Fig. 2A). All these results suggest that the two cultivars have distinctive softening characteristics, and that both ethylene and low temperatures play important roles in fruit softening.
Experiments were performed according to a completely randomized design. Numerical data were examined by analysis of variance (ANOVA), and significance was detected with Duncan’s multiple range tests at a level of 5%. 3. Results 3.1. Fruit respiration rate Although their dynamic trends were similar, the respiration rate was dramatically higher in ‘Golden Delicious’ than in ‘Fuji’ apples. Rates peaked at day 7 (‘Golden Delicious’) and day 14 (‘Fuji’) after harvest (Fig. 1). In the latter, this rate was only slightly accelerated by ethephon treatment compared with the controls, whereas storage at 0 ◦ C or exposure to 1-MCP had no apparent affect (Fig. 1A). However, for ‘Golden Delicious’ fruit, both 1-MCP and 0 ◦ C dramatically inhibited respiration and postponed its climatic peak, especially at the early stage of storage. For that cultivar, however, only a slight acceleration in respiration was found in ethephontreated fruit (Fig. 1B). These results demonstrate the important difference in respiratory patterns between ‘Golden Delicious’ and
3.2. Fruit firmness
3.3. Enzyme activity and gene expression 3.3.1. Polygalacturonase (PG) Polygalacturonase activity was significantly different between ‘Fuji’ and ‘Golden Delicious’ fruit, and was sensitive to ethephon
Fig. 1. Effect of ethephon, 1-MCP, and 0 ◦ C treatment on respiration rate in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
Fig. 2. Effect of ethephon, 1-MCP, and 0 ◦ C on firmness in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
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Fig. 3. Effect of ethephon, 1-MCP, and 0 ◦ C on PG activity and PG gene expression in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. The PG expression levels were set at 1.0 for day 0. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
and cold storage. In ‘Fuji’ fruit (Fig. 3A), this activity increased steadily and in a similar manner during storage, regardless of treatment, with values being negatively correlated with flesh firmness (r = −0.834*). PG levels were relatively low at the beginning of the 1-MCP treatment, then rose, and differences from the control diminished after 28 d. The promotive effect by ethephon did not surpass the inhibitory effect from 1-MCP treatment though ethephon treatment could enhance PG activity in ‘Fuji’ fruit. Activity was initially low under 0 ◦ C storage but began to increase later. For ‘Golden Delicious’ fruit (Fig. 3B), PG levels peaked at day 56 before declining rapidly, producing a negative correlation with flesh firmness (r = −0.707*). Ethephon greatly enhanced PG activity and reduced the time to its peak whereas both 1-MCP and 0 ◦ C treatment were strong inhibitors, with PG maintaining relatively low activity during the storage period. Expression of PG changed little in ‘Fuji’ fruit and was similar to the control throughout our experiments. However, it was accelerated by ethephon and inhibited by 1-MCP; 0 ◦ C treatment had only a small influence except on day 14 of storage (Fig. 3C). In ‘Golden Delicious’ fruit, such expression was obviously enhanced after 28 d of ethephon treatment, and was dramatically higher than in the control, especially during late storage. For that cultivar, PG expression was significantly inhibited in 1-MCP-treated fruit, with transcript levels being very low early on and increasing only slightly by the end of the storage period. The inhibitory effect of 0 ◦ C storage for ‘Golden Delicious’ was similar to that for ‘Fuji’ fruit, but was not obviously lower than that observed for the control (Fig. 3D). 3.3.2. Pectin methylesterase (PME) Pectin methylesterase activity increased slightly in the early stage of ‘Fuji’ fruit storage and remained fairly constant from day 14 to 42 before decreasing gradually. This demonstrated a negative correlation with firmness (r = −0.881**). PME levels were enhanced by ethephon and were higher than the control after 42 d. Both 1MCP treatment and storage at 0 ◦ C had inhibitory effects and PME activity did not tend to increase during storage (Fig. 4A). In ‘Golden
Delicious’ fruit, activity rose as fruit ripening and softening ensued; this process was more rapid during the early stage and was negatively correlated with firmness (r = −0.982**). Ethephon treatment did not significantly accelerate this activity, but both 1-MCP and 0 ◦ C treatment slowed it (Fig. 4B). In ‘Fuji’ apples (Fig. 4C), PME expression remained constant up to day 42, and increased only in the late stage of storage. Treatment with either ethephon or 0 ◦ C had little effect on transcripts in the early stage of storage, but differences in expression among treatments were manifest after day 56. By contrast, expression of PME in ‘Golden Delicious’ fruit (Fig. 4D) increased rapidly after harvest and was significantly regulated by ethylene and low temperature. This ethylene-regulated expression exceeded that of the control, but that difference was diminished in later storage. 1-MCP treatment strongly repressed PME transcript levels throughout the experimental period, whereas cold storage inhibited such expression mainly in the first 42 d, after which it did not differ significantly from the control. 3.3.3. ˇ-Galactosidase (ˇ-Gal) For ‘Fuji’ fruit, -galactosidase activity was very low until day 42 (Fig. 5A); for ‘Golden Delicious’ fruit this activity increased rapidly at the beginning of storage and continued to rise throughout the period (Fig. 5B). Our analysis indicated a significant negative correlation between -Gal activity and firmness in ‘Fuji’ (r = −0.726*) and ‘Golden Delicious’ (r = −0.912**) fruit, thereby demonstrating that this increase in enzyme levels was closely associated with the softening process. Treatment with either 1-MCP or cold storage dramatically inhibited this activity in both cultivars, but especially in ‘Fuji’ fruit. However, the ethephon-treated and control apples had noticeably higher levels. These results indicate that -Gal plays an important role during apple softening, and is closely related to the unique storage characteristics of each cultivar. The expression patterns of -Gal differed significantly between ‘Fuji’ and ‘Golden Delicious’ fruit. In the latter, expression was mainly inhibited early in storage by 1-MCP; this effect disappeared
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Fig. 4. Effect of ethephon, 1-MCP, and 0 ◦ C on PME activity and PME gene expression in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. The PME expression levels were set at 1.0 for day 0. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
over time. In contrast, expression in ‘Fuji’ fruit continued to be strongly repressed throughout the entire storage period. Ethephon treatment significantly increased transcript levels in ‘Golden Delicious’ but not in ‘Fuji’ fruit. Under cold storage, expression was significantly reduced in both cultivars (Fig. 5C and D).
3.3.4. ˛-l-Arabinofuranosidase (˛-l-Af) For both cultivars, the trend in activity of ␣-larabinofuranosidase was similar to that seen with fruit ripening and softening. In ‘Fuji’ fruit, ␣-l-Af levels were very low but increased slightly after 14 d of storage (Fig. 6A), while a very steady
Fig. 5. Effect of ethephon, 1-MCP, and 0 ◦ C on -Gal activity and -Gal gene expression in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. The -Gal expression levels were set at 1.0 for day 0. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
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Fig. 6. Effect of ethephon, 1-MCP, and 0 ◦ C on ␣-l-Af activity and ␣-l-Af gene expression in apple fruit. The concentrations of 1-MCP and ethephon treatments (at RT) were 500 nL L−1 and 2 mL L−1 , respectively; control was stored at RT; cold storage was at 0 ◦ C. The ␣-l-Af expression levels were set at 1.0 for day 0. Values are means of 3 replicates ±S.E. Significance in same sampling day is detected at 0.01 and 0.05 levels using Duncan’s multiple range tests, respectively.
rise was found beginning just after harvest in ‘Golden Delicious’ fruit (Fig. 6B). These increases were significantly and negatively correlated with fruit firmness, with correlation coefficients of −0.842** and −0.778* for ‘Golden Delicious’ and ‘Fuji’ fruit, respectively. Ethephon had different effects on activities – slightly higher than for the control in ‘Fuji’ (Fig. 6A) and noticeably higher than the control in ‘Golden Delicious’ fruit (Fig. 6B). By contrast, the difference between cultivars in response to 1-MCP and 0 ◦ C inhibition was very small. Such activity in ‘Golden Delicious’ fruit was almost completely stopped before rising only in the late storage period following 1-MCP treatment (Fig. 6A and B). In ‘Fuji’ fruit, ␣-l-Af expression was insensitive to ethylene and almost no difference was found among 1-MCP, ethephon, and the control treatments, especially during early storage. However, 0 ◦ C treatment dramatically inhibited such expression, and transcript levels did not increase until day 56 (Fig. 6C). Expression of ␣-l-Af in ‘Golden Delicious’ fruit was more sensitive to ethephon and 1-MCP treatment than that in ‘Fuji’ fruit. In ‘Golden Delicious’ fruit, ␣-l-Af mRNA expression in the former treatment was higher than that in the control after 28 d, and was maintained at this level throughout the remainder of the period. Exposure to 1-MCP significantly inhibited gene expression during storage. Finally, at 0 ◦ C, ␣-l-Af expression was repressed in ‘Golden Delicious’ fruit, which was stronger than in ‘Fuji’ fruit (Fig. 6D). 4. Discussion Cell wall degradation is the main factor involved in fruit softening and texture changes (Bennett and Labavitch, 2008; Billy et al., 2008). It is increasingly apparent that softening and textural changes are brought about by the actions of a multitude of cell wall-localized enzymes acting on specific, potentially highly localized substrates (Brummell et al., 2004). Cell wall enzymes are major contributors to cell wall disassembly, reducing intercellular connections and cell dispersion and playing various roles at different
stages during fruit development (Brummell, 2006; Almeida and ˜ Huber, 2007; Sanudo-Barajas et al., 2009). PME mainly serves to catalyze pectin demethylation, damaging the calcium horizontal links of the acid polysaccharide chain and leading to cell separation, and generating a suitable substrate for PG (Brummell et al., 2004). Also, transgenic suppression of PME activity in tomato decreases pectin de-methylesterification, reducing the depolymerization of pectin by PG during ripening (Harriman et al., 1991; Tieman et al., 1992). This indicated that the fruit remained firmer because of diminished PME activity during ripening (Phan et al., 2007). Goulao et al. (2008) reported that the transcriptional pattern of PME de-emphasises its role in ripening of apple. Our study showed that PME activity and mRNA abundance between two cultivars were obviously different at the early stage of storage and were regulated by ethylene and 0 ◦ C, having more significant effects on PME transcripts in ‘Golden Delicious’ than that in ‘Fuji’ fruit. However, the level of PME mRNA did not changed coincidently with PME activity after being regulated by ethylene and low temperature; there should be further research to confirmi if this phenomenon is in accordance with previous studies (Ray et al., 1988; Harriman et al., 1991). PG is another cell wall-modifying enzyme. It hydrolyzes pectin acid along with the main chain of polygalacturonic acid, causing pectin degradation, cell wall dissolution, and ultimately, fruit softening (Wakabayashi et al., 2000; Brummell et al., 2004). Many reports suggest that PG-mediated pectin depolymerization requires a pectin substrate that is at least partially de-methylesterified (Wakabayashi et al., 2000). Brummell et al. (2004) consider that PG -subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening, suppression of PME activity does not affect firmness during normal ripening, and suppression of -subunit protein accumulation increases softening. This activity affects the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture. Accumulation of PG mRNA is
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regulated by ethylene, with low levels of ethylene being sufficient for induction, and mRNA accumulation increasing with ethylene exposure (Sitrit and Bennett, 1998; Brummell and Harpster, 2001). It is reported that the transcription level of the MdPG must be correlated with rate of firmness loss of fruit through the enzyme activity (Wakasa et al., 2006; Mann et al., 2008). In this work, based on the different responses to ethylene and 0 ◦ C, as well as the cultivar differences in PG activity, we consider that PG might play roles mainly at the mid- and late-stages of storage. Moreover, PG expression was also markedly regulated by ethylene and 0 ◦ C, and was weaker than PME, especially in ‘Golden Delicious’ fruit. However, the promotional effect of ethephon and the inhibitory effect of 1-MCP on PG expression was stronger in ‘Golden Delicious’ fruit, although no difference was found between cultivars when fruit had been chilled. However, in ‘Fuji’ fruit, PG activity was not tightly coupled with the accumulation of PG mRNA, that is, the increase of PG activity in the late stages of storage when specific gene expression does not show any increase. This suggests that PG activity may be manifest in a cultivar-dependent manner, or be regulated by translational or post-translational mechanisms. -Gal has been studied in various fruits, including tomato (Smith and Gross, 2000), pear (Tateishi et al., 2001; Mwaniki et al., 2007), and strawberry (Martínez and Civello, 2008). It has been characterized in association with the removal of galactosyl residues from cell wall polymers during fruit softening (Gross and Sams, 1984). Indeed, de-galactosidation in response to -Gal action has been proposed as an important process in cell wall modification during ripening (Smith and Gross, 2000). Therefore, such modification might involve glycosidases, such as -Gal, acting in cooperation with pectolytic enzymes (e.g., PG) in pectin metabolism during fruit ripening (Giovannoni, 2001). The expression of -Gal mRNA was also investigated using over-ripe apple fruit, and its transcripts could be unambiguously detected by semiquantitative RT-PCR during fruit ripening (Goulao et al., 2008). Here, we demonstrated that an increase in -Gal activity and related gene expression during ripening and softening was concomitant with a decrease in firmness. -Gal activity was rapidly enhanced at the early ripening stage in ‘Golden Delicious’ fruit, and was greater than that measured in ‘Fuji’ fruit. Treatments with 1MCP and 0 ◦ C dramatically postponed fruit softening and inhibited -Gal activity and -Gal mRNA accumulation, whereas the opposite response was found with ethephon. In addition, this change in pattern of activity did not differ from that for the control, indicating that -Gal has an important role in apple fruit softening, particularly the initial process. The glycoside hydrolase ␣-l-Af cleaves the glycosidic bonds between l-arabinofuranoside side chains and various oligosaccharides. Its activity is increased during the ripening of persimmon (Xu et al., 2003), tomato (Sozzi et al., 2002), peach (Brummell et al., 2004), banana (Zhuang et al., 2007), and pear fruit (Tateishi et al., 1996, 2005; Mwaniki et al., 2007). The ␣-l-Af enzyme has been purified from pears, and northern blots have shown that its mRNA is highly expressed only in ripening fruit (Tateishi et al., 2005). Many ␣-l-Af isoforms have been characterized from tomato (Sozzi et al., 2002) and pear (Mwaniki et al., 2007); one of them has been shown to be regulated by ethylene, functioning by releasing arabinosyl residues from the pectic fraction. This indicates that ␣-l-Af plays an important role in Arab metabolism during tomato ripening. In apple, degradation of a branched arabinan occurs prior to softening (P˜ena and Carpita, 2004), and interestingly, ␣-l-Af activity increases dramatically from commercial maturity to the over-ripe stages in ‘Royal Gala’ fruit (Goulao et al., 2007). Expression analysis of ␣-l-Af isolated from other fruit has shown that it has an important role in altering fruit texture during ripening (Itai et al., 2003; Tateishi et al., 2005; Mbéguié-A-Mbéguié et al., 2009). In our experiments, ␣-l-Af activity in both cultivars increased with ripening and
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softening, although the rate was obviously higher in ‘Golden Delicious’ fruit. Expression of its encoding gene followed a similar trend. Both activity and expression were notably inhibited by 1-MCP and 0 ◦ C treatment, but somewhat promoted by ethephon. This indicated that ␣-l-Af is sensitive to ethylene and low temperatures and has an important role in softening the fruit texture. We conclude that cell wall-modifying enzymes are closely correlated with fruit texture and cultivar characteristics. -Gal and ␣-l-Af may be more closely related to the storability of apples than PG and PME, especially when fruit ripening and softening begin. PME activity appeared at the early stage of storage, while PG activity occurred later; perhaps PG operates mainly at the mid- and latestages and is weaker than PME during fruit softening. Nevertheless, although the parameters analyzed here provide some information about fruit softening, our results cannot adequately explain the entire mechanism. This means that other factors such as cell wallneutral sugars, the functioning of genes for related enzymes, or non-enzymatic factors that contribute to texture softening should be taken into account in further research in order to identify apple fruit mechanisms. Acknowledgements This work was supported by the National Science & Technology Pillar program during the eleventh five-year plan period (2007BA79B05). The authors are grateful to Miss Priscilla Anne Licht for her help in writing. References Almeida, D.P.F., Huber, D.J., 2007. Polygalacturonase-mediated dissolution and depolymerization of pectins in solutions mimicking the pH and mineral composition of tomato apoplast. Plant Sci. 172, 1087–1094. Beldman, G., Schols, H.A., Pitson, S.M., Searle-van Leeuwen, M.J.F., Voragen, A.G.J., 1997. Arabinans and arabinan degrading enzymes. In: Advances in Macromolecular Carbohydrate Research. U.S.A. pp. 1–64. Bennett, A.B., Labavitch, J.M., 2008. Ethylene and ripening-regulated expression and function of fruit cell wall modifying proteins. Plant Sci. 175, 130–136. Billy, L., Mehinagic, E., Royer, G., Renard, C.M.G.C., Arvisenet, G., Prost, C., Jourjon, F., 2008. Relationship between texture and pectin composition of two apple cultivars during storage. Postharvest Biol. Technol. 47, 315–324. Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311–340. Brummell, D.A., Dal Cin, V., Crisosto, C.H., Labavitch, J.M., 2004. Cell wall metabolism during maturation, ripening and senescence of peach fruit. J. Exp. Bot. 55, 2029–2039. Brummell, D.A., 2006. Cell wall disassembly in ripening fruit. Funct. Plant Biol. 33, 103–119. Chávez Montes, R.A., Ranocha, P., Martinez, Y., Minic, Z., Jouanin, L., Marquis, M., Saulnier, L., Fulton, L.M., Cobbett, C.S., Bitton, F., Renou, J., Jauneau, A., Goffner, D., 2008. Cell wall modifications in Arabidopsis plants with altered ␣-l-arabinofuranosidase activity. Plant Physiol. 147, 63–77. Fry, S.C., 1995. Polysaccharide-modifying enzymes in the plant cell wall. Ann. Rev. Plant Physiol. Plant Mol. Biol. 46, 497–520. Gasic, K., Hernandez, A., Schuyler, S., Korban, S.S., 2004. RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Mol. Biol. Rep. 22, 437–1437. Giovannoni, J., 2001. Molecular biology of fruit maturation and ripening. Ann. Rev. Plant Physiol. Plant Mol. Biol. 52, 725–749. Giovannoni, J., DellaPenna, D., Bennett, A.B., Fischer, R.L., 1989. Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening. Plant Cell 1, 53–63. Goulao, L.F., Santos, J., Sousa, I., Oliveira, C.M., 2007. Patterns of enzymatic activity of cell wall modifying enzymes during growth and ripening of apples. Postharvest Biol. Technol. 43, 307–318. Goulao, L.F., Cosgrove, D.J., Oliveira, C.M., 2008. Cloning, characterisation and expression analyses of cDNA clones encoding cell wall-modifying enzymes isolated from ripe apples. Postharvest Biol. Technol. 48, 37–51. Gross, K.C., 1982. A rapid and sensitive spectrophotometric method for assaying polygalacturonase using 2-cyanoacetamide. HortScience 17, 933–934. Gross, K.C., Sams, C.E., 1984. Changes in cell wall neutral sugar composition during fruit ripening: a species survey. Phytochemistry 23, 2457–2461. Harriman, R.W., Tieman, D.M., Handa, A.K., 1991. Molecular cloning of tomato pectin methylesterase gene and its expression in Rutgers, ripening inhibitor, nonripening, and never ripe tomato fruit. Plant Physiol. 97, 80–87.
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