Effects of nitric oxide treatment on the cell wall softening related enzymes and several hormones of papaya fruit during storage

Abstract

Papaya fruits (Carica papaya L. cv ‘Sui you 2’) harvested with <5% yellow surface at the blossom end were fumigated with 60 µL/L of nitric oxide for 3 h and then stored at 20 ℃ with 85% relative humility for 20 days. The effects of nitric oxide treatment on ethylene production rate, the activities of cell wall softening related enzymes including polygalacturonase, pectin methyl esterase, pectate lyase and cellulase and the levels of hormones including indole acetic acid, abscisic acid, gibberellin and zeatin riboside were examined. The results showed that papaya fruits treated with nitric oxide had a significantly lower rate of ethylene production and a lesser loss of firmness during storage. A decrease in polygalacturonase, pectin methyl esterase, pectate lyase and cellulase activities was observed in nitric oxide treated fruit. In addition, the contents of indole acetic acid, abscisic acid and zeatin riboside were reduced in nitric oxide treated fruit, but no significant reduction in the level of gibberellin was found. These results indicate that nitric oxide treatment can effectively delay the softening and ripening of papaya fruit, likely via the regulation of cell wall softening related enzymes and certain hormones.

Keywords

Nitric oxide papaya softening cell wall softening related enzyme hormone

Introduction

Papaya (Carica papaya L.) is a typical climacteric fruit, undergoing massive physio-chemical changes in a very short period as a consequence of the activation of biochemical pathways (Fabi et al., 2009). Several fruit quality parameters including appearance, ﬂavor and nutritional value may decline greatly due to the way the fruits are handled and treated after being harvested. The reduction in shelf-life time of papaya fruit can be accelerated by exposure to ethylene.

Papaya softening is one of the most remarkable changes during ripening, signiﬁcantly limiting the market sales due to a short shelf-life. The major changes, resulting in the softening of the fruit, are due to enzyme-mediated alterations of the composition and structure of cell wall. The cell wall softening related enzymes include pectin methyl esterase (PME), polygalacturonase (PG), pectate lyase (PL) and cellulase (C_X) (Goulao et al., 2007; Nunes et al., 2009). The activities of these enzymes are believed to be regulated by ripening related hormones or other signal molecules. Lohani et al. (2004) discovered that abscisic acid (ABA) stimulated the activities of the abovementioned enzymes, especially PL. Indole acetic acid (IAA) suppressed the activities of the cell wall softening related enzymes (Lohani et al., 2004). Gibberellic acid (GA) treatment could retard the peaks of PG and PL, and delay fruit ripening (Payasi et al., 2004).

At present, a number of different methods, such as heat treatment (Parker et al., 2010), plant regulators (Huerta-Ocampo et al., 2012) and chemical additives (Ali et al., 2011) have been developed to slow down the ripening and softening process. A common approach for preventing ethylene-mediated ripening is the use of 1-methylcyclopropene (1-MCP). However, it has been reported that 1-MCP treated papaya fruit cannot normally soften at color-break stage (Hofman et al., 2001). Experiment in our laboratory (data not shown) found that papaya fruit treated with 1-MCP at the color-break stage failed to soften completely and developed a more elastic flesh texture, which was regarded as ‘rubbery’. Therefore, an effective way is needed to control softening of papaya fruit for long-term storage.

As a plant regulator, nitric oxide (NO) is synthesized within the plants and involved in the regulation of plant ripening and senescence, since it was first reported by Leshem and Haramaty (1996). Various reports have demonstrated that NO controls many physiological processes in plants, such as growth (Zhang et al., 2011), ripening (Leshem and Haramaty, 1996) and disease resistance (Zheng et al., 2011). NO has been shown to prevent vegetative stress and softening of horticultural products (Zaharah and Singh, 2011). Earlier studies have suggested that hormones could act as important regulators in fruit development and ripening (Crane, 1964). Indeed, it has been shown that IAA, GA, cytokinin-like substances, ABA and ethylene, alone or in combination with other hormones, could act in different periods of fruit development (Ozga and Reinecke, 2003), regulating plant growth, development and differentiation. However, little research has been done on the changes of hormones in postharvest papaya fruit treated with NO before storage. Also, the effects of hormones on the activities of cell wall softening related enzymes have not been elucidated in detail in papaya fruit. Therefore, we aim to investigate the effect of NO on papaya fruit softening, the cell wall softening related enzymes and certain hormones and to explore their interactions during the storage of papaya fruit.

Materials and methods

Plant material and treatment

Papaya fruits (Carica papaya L. cv ‘Sui you 2’) were harvested in October 2009, from an orchard at Panyu District, Guangzhou, China, with <5% yellow surface at the blossom end. The fruits were selected for uniform size, maturity and appearance free from defects and mechanical damage and dipped in 500 µL/L of prochloraz for 1 min.

Papaya fruits (36 per group) were treated with NO (NO treated fruit) or remained untreated (control fruit). For NO treatment, the fruits were sealed in plastic containers (30.8 L) and flushed with nitrogen gas to displace all oxygen as NO can actively react with oxygen, so it would interfere with the experiment. Then, pure NO gas (provided by Source Gas Co., Ltd., Guangzhou) was injected into the containers, by controlling the released velocity with injector, to attain certain concentrations. In this study, we applied a concentration of NO at 60 µL/L, based on our previous work (Guo et al., 2011). The control fruits were also flushed with nitrogen gas instead of NO to ensure consistent experimental conditions and justifiable comparisons. After 3 h, all fruits were immediately placed in plastic bags, stored at 20 ℃ with 85% relative humility. Each treatment was repeated three times.

Determination of ethylene and firmness

The ethylene level was determined as follows. Each subsample of five fruits was placed in a hermetically sealed container. After 2 h, 1 mL of the internal atmosphere of the container was extracted with a syringe and the ethylene was quantified in a gas chromatograph (GC-2014C, Shimadzu Co., Ltd, Kyoto, Japan). The temperatures of the column, injector and flame ionization detector were 80 ℃, 140 ℃ and 150 ℃, respectively. The ethylene levels were expressed as microliter per hour per kilogram fresh weight.

Fruit firmness was measured at six points on three peeled fruits per repeat, using an Instron Harness Tester 5542 (Instron, Norwood, NJ, USA) equipped with an 8-mm diameter cylindrical tip. Each fruit was compressed at the equatorial zone at a rate of 300 mm/min and the maximum force during the test was recorded. The fruit firmness was expressed in Newton (N).

Assay of cell wall softening related enzymes

Polygalacturonase

The measurement of PG activity was based on the method described by Pathak and Sanwal (1998) with slight modiﬁcations. Pulp tissues (1 g) were homogenized in 2 mL of cold 0.04 M sodium acetate buffer (pH 6.0) at 4 ℃ and centrifuged at 12,000 r/min for 25 min. The supernatant was used as crude enzyme. The reaction mixtures, containing 0.1 mL of crude extract, 0.3 mL of 0.1% (w/v) polygalacturonic acid in 0.2 mL of 0.04 M sodium acetate buffer (pH 4.6) and 0.4 mL H₂O, were incubated at 37 ℃ for 60 min. The reaction was stopped with 1 mL of 3, 5-dinitrosalicylic acid (DNS) and immersed in a boiling water bath for 5 min. After cooling on ice, distilled water was added to 10 mL. The absorbance was measured at 540 nm with UV-2450 spectrophotometer (Shimadzu, Japan). A calibration curve was obtained using D-galacturonic acid as a standard. One unit of PG activity was expressed as 1 µg of galacturonosyl-reducing groups liberated per minute per gram of original fresh weight.

Pectin methylesterase

The activity of PME was assayed according to the method of Hagerman and Austin (1986). Pulp tissues (1 g) were homogenized in 4 mL of 8.8% NaCl (w/v) and centrifuged at 12,000 r/min for 30 min (4 ℃). The supernatant was collected, adjusted to pH 7.5 with NaOH and used for assaying the enzyme activity. The mixture containing 2 mL of 0.5% (w/v) pectin, 0.15 mL of 0.01% bromothymol blue in 0.75 mL H₂O and 0.1 mL enzymatic extract was incubated at 25 ℃ for 30 min. The absorbance at 620 nm was measured. One unit of PME activity was expressed as the absorbance change in 1 min/g of original fresh weight.

Pectate lyase

The activity of PL was measured by a modiﬁed method of Lowry et al. (1951). Pulp tissues (1 g) were added in 9 mL of Tris–HCl (20 mM, pH 7.0, containing 20 mM cysteine–HCl, 20 mM EDTA and 0.05% Triton X-100). The homogenate was centrifuged at 12,000 r/min for 30 min at 4 ℃. The supernatant was used as crude enzyme. The reaction mixture, containing 3 mL of 4 mM sodium acetate buffer (pH 4.5), 1.5 mL polygalacturonic acid (PGA, 1% aqueous solution adjusted to pH 4.5) and 0.5 mL enzymatic extract, was incubated at 37 ℃ for 30 min and then stopped by boiling in a water bath for 2 min. The absorbance of the reaction mixture was measured at 235 nm. The increase in the absorbance against the control with pre-boiled enzyme was taken as a measure of the PL activity. One unit of PL activity was expressed as the amount of enzyme required to liberate 1 µg of aldehyde groups from PGA per minute per gram of original fresh weight.

Cellulase

C_X activity was measured as previously described by Zhang et al. (2010). Pulp tissues (1 g) were homogenized with 4 mL of 50 mM sodium acetate buffer (pH 4.5, containing 7.5% NaCl and 0.5 g polyvinylpyrrolidone) at 4 ℃ and centrifuged at 12,000 r/min for 30 min. The supernatant was used as crude enzyme. C_X was assayed by adding 1 mL crude enzyme extracts to 2 mL of 1% carboxymethylcellulose sodium as substrate and incubating the mixture at 40 ℃ for 60 min. The reaction was stopped with 1 mL DNS and immersed in a boiling water bath for 5 min. After cooling on ice, distilled water was added to 10 mL. The absorbance at 540 nm was determined. A calibration curve was obtained using glucose as a standard. One unit of C_X activity was expressed as 1 µg of glucose-reducing groups liberated per minute per gram of original fresh weight.

Evaluation of endogenous hormones

The hormones of papaya were assayed according to the method of Li et al. (2010). Pulp tissues (1 g) were stirred and extracted in 2 mL of cold 80% methanol containing butylhydroxytoluene (1 mM) as an antioxidant, overnight at 4 ℃. The supernatant was collected after centrifugation at 12,000 r/min for 20 min at 4 ℃. Then, the crude extract was passed through C₁₈ (Waters, Milford, MA, USA). The eluate was evaporated to dryness in vacuum and the residue was dissolved in 2 mL phosphate buffer saline (pH 7.5, containing 0.1% Tween-20 and 0.1% gelatin).

The total endogenous contents of IAA, ABA, GA₃ and ZR were measured by the enzyme-linked immunosorbent assay (Yang et al., 2001). A 96-well microtitration plate was coated with 100 µL coating buffer (pH 9.6, containing 1.5 g/L Na₂CO₃, 2.93 g/L NaHCO₃ and 0.02 g/L NaN₃) containing synthetic ovalbumin conjugates for IAA, ABA, GA₃ and ZR, respectively, incubated for 4 h at 37 ℃ for ABA, GA₃ and ZR and overnight at 4 ℃ for IAA. After washing with phosphate buffer saline containing 0.1% (v/v) Tween-20 for four times, 50 µL of either samples or standard IAA, ABA, GA₃ and ZR (0–2000 ng/mL dilution range) and 50 µL antibodies were added in each well and incubated for 0.5 h at 37 ℃. The antibodies against IAA, ABA, GA₃ and ZR were prepared according to the method described by Weiler et al. (1981). After washing as abovementioned, 100 µL of 1.25 µg/mL horseradish peroxidases-labeled goat anti-rabbit immunoglobulin was added to each well and incubated for 0.5 h at 37 ℃. After washing four times, 100 µL buffered enzyme substrate containing 1.5 mg/mL ortho-phenylenediamine and 0.008% (v/v) H₂O₂ was added, and the enzyme reaction was carried out in the dark at 37 ℃ for 15 min and then stopped by adding 50 µL of 2 mol/L H₂SO₄ per well. The absorbance was measured at 490 nm. Contents of IAA, ABA, GA₃ and ZR were calculated according to Weiler et al. (1981). Three replicates were performed for each measurement.

Statistical analyses

Statistical analyses included analysis of variance and Duncan’s multiple range tests. SPSS (Version 16.0) was applied to analyze the statistical significance for any measurement by comparing the means of three replicates at certain time points from the control and NO treated samples (p < 0.05). Sigmaplot (Version 10.0) was used for data plotting. Error bars show standard errors of the means for three replicates.

Results and discussion

Effect of NO on ethylene production rates and ﬁrmness of papaya fruit

The ethylene production rates in control fruit increased rapidly, reaching a maximum at day 6 (Figure 1(a)). Thereafter, a drop happened and then slight fluctuations occurred frequently. However, ethylene production rates in NO treated fruits were undetected at day 2 and significantly lower than those in control fruits during storage. Firmness declined rapidly during the ﬁrst 10 days, indicating that the fruits softened quickly (Figure 1(b)). Firmness in NO treated fruit was higher compared to the control fruit, being 65.77 N at day 10, about three times that of control fruit (24.48 N).

The softening in papaya is one of the most important features during ripening, directly influencing postharvest storage and commercial value of the fruit. In this work, short-term exposure to NO could reduce the ethylene production rates and firmness of papaya, which was consistent with a previous study by Singh et al. (2009). However, our previous work showed that NO treatment had no effect on papaya fruit with complete yellow surface (data not shown). We observed that the firmness in papaya dropped dramatically with the increase in ethylene production before the sixth day, while after the 10th day there was no marked change in both ethylene production and firmness. These results indicate that NO treatment could slow down fruit ripening and softening via a lowered ethylene production.

Effect of NO on cell wall softening related enzymes of papaya fruit

It has been widely recognized that the cell wall softening related enzymes play a key role in cell wall degradation and softening of fruit (Goulao et al., 2007). In the present work, PG activity in control fruit increased rapidly, while the activity of this enzyme in NO treated fruit was strongly prevented in the first 4 days and remained lower than that in the control fruit till day 10 (Figure 2). However, after that day, although PG activity still rose gradually, no significant difference was observed between control and NO treated fruit. We also found that firmness in control and NO treated fruit began to decrease slowly after the 14th day (Figure 1(b)), indicating that PG, to some extent, is required for the extensive softening of papaya fruit.

The activity of PME in control fruit increased continuously, peaked at day 18, with a 2-fold increase compared to day 2 and then decreased (Figure 3). PME activity in NO treated fruit remained lower than that in the control fruit throughout the storage. PME could hydrolyze the methyl ester bond of pectin to give pectic acid and methanol, whereas PG could cleave the a-1, 4-glycosidic bond between the anhydrogalacturonic acid units (Benen and Visser, 2003). Therefore, it is speculated that NO may prevent these processes through inhibiting PME and PG activities to slow down the softening progress. We also observed a peak for PME activity at day 18, while PG activity remained at a high level after day 18 in both control and NO treated fruit. It is supposed that PME and PG may work in a concerted manner such that PME removes the methyl groups from the C₆ position of galacturonic acid units, which then enables PG to depolymerize the de-esteriﬁed polygalacturonide chain (Koch and Nevins, 1989).

The changes in PL activities were maintained at similarly increased trends between the control and NO treated fruit during storage (Figure 4), but the levels in NO treated fruit was slightly lower than that in control fruit. Furthermore, the changes in PL activity showed a highly positive correlation with those of PME and PG (Table 1). Like PG, PL also catalyzes the cleavage of the α(1-4)-galacturonan linkage, but by β-elimination instead of a hydrolytic cleavage. Bermudez-Jimenez et al. (2002) discovered that the PL gene has been cloned in strawberry and using antisense technology its role in fruit softening has been tangibly demonstrated. The effects of pectate lyase-silencing in strawberry could also extend the softening of fruit while color and soluble solids were not affected (Youssef et al., 2009).

Table 1.

The correlation analysis between the activities of cell wall softening related enzymes and hormones of papaya fruit

	Correlations
	IAA	GA₃	ABA	ZR	PG	PME	PL	C_X
IAA	1.000	0.279	0.498	0.659	0.845	0.718	0.861	0.458
GA₃	0.279	1.000	0.129	0.108	0.410	−0.113	0.175	0.256
ABA	0.498	0.129	1.000	0.429	0.665	0.844	0.737	0.214
ZR	0.659^*	0.108	0.429	1.000	0.609	0.420	0.734	0.187
PG	0.845^**	0.410	0.665^*	0.609^*	1.000	0.812	0.784	0.651
PME	0.718^**	−0.113	0.844^**	0.420	0.812^**	1.000	0.896	0.284
PL	0.861^**	0.175	0.737^**	0.734^**	0.784^**	0.896^**	1.000	0.342
C_X	0.458	0.256	0.214	0.187	0.651^*	0.284	0.342	1.000

IAA: indole acetic acid; GA₃: gibberellins; ABA: abscisic acid; ZR: zeatin riboside; PME: pectin methyl esterase; PG: polygalacturonase; PL: pectate lyase.

Data are the mean ± SE of determinations made for each sample during the 20 days of storage at 20 ℃.

Correlation significant at the 0.05 level (p < 0.05).

Correlation significant at the 0.01 level (**p < 0.01).

C_X activity in control fruit increased sharply and reached a maximum at day 8, about 1.4-fold higher than that of NO treated fruit at day 12 and then decreased continuously (Figure 4). These observations are consistent with those reported by Nunes et al. (2008) who studied the importance of C_X activity of fresh plums. The activity of C_X in NO treated fruit was significantly lower than that in control fruit and the peak of C_X activity in NO treated fruit was retarded for 4 days. This lower C_X activity could be related to the lower demethylesteriﬁcation of pecticpolysaccharides by the action of PME whose activity was inhibited by NO. PME could directly or indirectly assist other enzymes, mainly by promoting accessibility of the enzymes to their substrates. The observed activity of C_X was relevant to PG activity, which degrades cellulose and the β-1, 4-glucan backbone of xyloglucan leading to extensive polysaccharide depolymerization (Wakabayashi et al., 2000).

In general, softening of fruit is associated with solubilization and hydrolysis of pectic substances in the cell wall (Brummell and Labavitch, 1997). Changes in the solubilization of pectic substances involve the action of cell wall softening related enzymes. In the present study, application of NO could inhibit the cell wall softening related enzymes to further extend softening and ripening of postharvest papaya fruit.

Effects of NO treatment on the levels of hormones in papaya fruit

The effects of NO treatment on the levels of hormones, IAA, ABA, GA₃ and ZR, in papaya fruit are shown in Figure 5. Among these hormones, their change trends were similar between control and NO treated fruit. The IAA content in control fruit was higher than that of NO treated fruit (Figure 5(a)). There was no significant difference in GA₃ content between control and NO treatment (Figure 5(b)). The ABA content in control fruit increased gradually, with a dramatic increase on day 18 (Figure 5(c)). The content of ABA in NO treated fruit gradually increased and was lower compared to control fruit. ZR content in the fruits fluctuated frequently, usually gradually increased, but its changing trend in NO treated fruit was lower than the control fruit (Figure 5(d)). These results indicate that NO treatment could affect the levels of these hormones.

Our results also showed that though NO treatment could decrease the levels of ABA and ZR, the extent of this decrease was much less compared to IAA, indicating that IAA is an important hormone in the regulation of papaya fruit. GA₃ level in NO treated fruit was not significantly different from the control fruit. It is presumed that NO may have no remarkable inhibition on GA₃. Lohani et al. (2004) found that ABA alone can cause softening of the banana to a lesser extent in comparison to ethylene treated fruit, while we discovered that NO treatment could significantly suppress the ethylene production and ABA content. Therefore, it is also concluded that NO treatment could restrain IAA, ABA and ZR to delay papaya fruit softening and ripening.

Correlation analysis between cell wall softening related enzymes and hormones in control fruit

The fact that both the inhibition of the activities of cell wall softening related enzymes and reduction of the levels of several hormones are caused by NO treatment suggests that there may be certain correlations between these cell wall softening related enzymes and hormones. Thus, we performed correlation analysis. As shown in Table 1, IAA was, to some extent, correlated with ZR and PME was correlated with PL. PG activity had a remarkable correlation with PME and PL, while it had a marked correlation with C_X.

IAA and ABA had a significantly positive correlation with PG, PME and PL, indicating that both of them could enhance the activities of PG, PME and PL in papaya in comparison to GA₃, which had no remarkable correlation with the cell wall softening related enzymes. ZR was also associated with PG and PL during storage, suggesting that ZR could also enhance the activities of PG and PL. These responses suggest that PG and PL activities are not only regulated by IAA and ABA, but also by ZR. In addition, IAA and ABA could have synergistic effects on PME activity. Therefore, it is likely that NO treatment could directly or indirectly suppress PG, PME and PL to delay the softening.

It was indicated that cell wall softening related enzymes and hormones in papaya had certain relevance and affected each other, together deciding the process of fruit softening and ripening. Jiang et al. (2000) discovered that ABA alone or in combination with other hormones could modulate the activities of cell wall softening related enzymes in banana fruit. Likewise, a delay in ripening and softening of banana fruit treated with IAA was observed by Pathak and Sanwal (1999). Lohani et al. (2004) suggested that ethylene stimulated activities of cell wall softening related enzymes and ABA might enhance this effect of ethylene. IAA inhibited activities of all four hydrolases enzymes irrespective of whether they were treated with ethylene or not. Therefore, it is concluded that treatment of papaya fruit with NO could inhibit cell wall softening related enzymes except C_X by suppressing the IAA, ABA or ZR, and, thus, further retard the softening and ripening.

Regression analysis between cell wall softening related enzymes and hormones in control fruit

The regression analysis results are shown in Table 2, from which we found that the equation was true and credible. Standardized coefficients showed IAA had more remarkable influence on PG than the other three hormones whose effects are not significant. PME activity mainly resulted from the actions of IAA and ABA, while PL activity from the action of ABA. However, NO regression equation was drawn by stepwise regression analysis between C_X and endogenous hormones. It was indicated that endogenous hormones in papaya fruit might have no significant effects in regulation of C_X.

Table 2.

The regression analysis between the activities of cell wall softening related enzymes and contents of hormones of papaya fruit

Correlation factor	Regression equation	Correlation coefficient	Partial correlation coefficient	F-test	Standardized coefficients (β)	Significant level/P
R(Y_PG, X)	Y = 96.456 + 0.015X_IAA	0.714	0.845	22.477	0.845	0.001
R(Y_PME, X)	Y = 7.431 + 0.003X_IAA + 0.056X_ABA	0.889	0.784	28.135	0.475	0.012
R(Y_PME, X)	Y = 7.431 + 0.003X_IAA + 0.056X_ABA	0.889	0.879	28.135	0.665	0.002
R(Y_PL, X)	Y = 1.916 + 0.003X_ABA	0.868	0.700	26.294	0.411	0.024

IAA: indole acetic acid; ABA: abscisic acid; PME: pectin methyl esterase; PG: polygalacturonase; PL: pectate lyase.

Regression analysis further suggested that PG activity was mainly controlled by IAA, PME activity was mainly associated with IAA and ABA and PL activity was mainly affected by ABA. These results suggest that the cell wall softening related enzymes might be directly or indirectly regulated by these hormones in vivo. Therefore, we conclude that NO treatment could mainly inhibit IAA to suppress PG activity, inhibit IAA and ABA to prevent PME activity and inhibit ABA to suppress PL activity. Although Chiwocha et al. (2003) showed that the increased ABA catabolism was concurrent with a marked accumulation of IAA, it is still not well known how the accumulation of endogenous hormones influence the synthesis or catabolism of the cell wall softening related enzymes.

Conclusion

Sixty microliter per liter NO fumigation might be a potentially useful way, to some extent, to delay ‘Sui you 2’ papaya fruit ripening and softening. NO treatment modulates the activities of the cell wall softening related enzymes including PG, PME and PL, possibly by indirectly reducing the levels of IAA, ABA and ZR in vivo. Further work is required to understand the mechanisms of signal transduction pathways involved in NO-mediated regulation of hormone levels and the activities of the cell wall softening related enzymes in papaya fruit. These studies could be achieved by adopting a technique that uses N¹⁵O or NO¹⁸ tracer in the future.

Footnotes

Funding

This work was supported by Natural Science Foundation of Guangdong Province (Project No. 06200670); National Key Technology R &D Program of China (Grant No. 2011BAD024B02) and Special Fund for Agro-scientific Research in the Public Interest of the Ministry of Agriculture of China (No. 201303077).

Conflict of interest

None declared.

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