Journal of Applied Botany and Food Quality 91, 109 - 113 (2018), DOI:10.5073/JABFQ.2018.091.015 1 College of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou, Henan, China 2 Department of Horticulture, Oregon State University, Mid-Columbia Agricultural Research and Extension Center, Hood River, Oregon, USA Effect of hydrogen sulfide on surface pitting and related cell wall metabolism in sweet cherry during cold storage Huanhuan Zhi1, Yu Dong2* (Submitted: December 25, 2017 Accepted: March 28, 2018) * Corresponding author Summary This study was to determine whether postharvest hydrogen sulfide (H2S) fumigation effected surface pitting development of ‘Lapins’ and ‘Regina’ cherries after cold storage, and if so, how it minimizes surface pitting injury and its relation to cell wall metabolism. Fruit were exposed to H2S gas released from an aqueous solution of so- dium hydrosulfide (NaHS) at rates of 0.5, 1, 2, or 3 mM, then stored at 0 °C for 4 weeks. Fruit of both cultivars treated with 1 or 2 mM NaHS had a greater firmness and a lower respiration rate compared to control fruit. However, no treatments retarded losses in soluble solids content (SSC) and titratable acidity (TA). Fruit fumigated with H2S displayed significantly suppressed stem browning, decay, and pitting incidences, but not weight loss. The positive effect of H2S on reduced pitting might be due to the lower yields of water-soluble polysaccharides (WSP) and CDTA-soluble polysaccharides (CSP) or/ and due to the lower activities of polygalacturonase (PG), pectate lyase (PL), and β-D-galactosidase (β-GAL). Overall, results demon- strated that H2S applied to sweet cherry as a postharvest vapor has ability to control surface pitting development by stabilizing cell wall structure and regulating cell wall catabolism. Keywords: sweet cherry; hydrogen sulfide; surface pitting; storage quality; cell wall metabolism Abbreviations: H2S, hydrogen sulfide; NaHS, sodium hydrosulfide; SSC, soluble solids content; TA, titratable acidity; WSP, water- soluble polysaccharides; CDTA, cyclohexanetrans-1, 2-diamine tetra acetate; CSP, CDTA-soluble polysaccharides; NSP, Na2CO3-soluble polysaccharides; AIR, alcohol insoluble residue; PG, polygalacturo- nase; PME, petin methylesterase; PL, pectate lyase; β-GAL, β-D- galactosidase. Introduction Fresh sweet cherries (Prunus avium L.) gain popularity among consumers worldwide due to their attractive dark mahogany color, pleasing flavor, desirable taste and high nutrients. However, they are highly perishable, non-climacteric, and available only a few weeks after storage under the optimal conditions (SERRANO et al., 2005; WANI et al., 2014). Changes in fruit appearance (i.e. color, surface pitting), flavor, softening, stem quality, and dehydration are major symptoms of quality deterioration. Under current marketing condi- tions, pitting greatly influences consumer perception of fruit quality (KAPPEL et al., 2006). Fruit pulp temperature had been identified as an important factor implicated in the development of pitting during handling, storage, and shipping of sweet cherry (LIDSTER and TUNG, 1980; ZOFFOLI and RODRIGUEZ, 2009). Rapid cooling of fruit to al- most 0 °C immediately after harvest is an extremely important step in postharvest handing, known to reduce pitting (DRAKE et al., 1988; WADE and BAIN, 1980; YOUNG and KUPFERMAN, 1994). During storage or shipping, relatively stable low-temperature (0 °C) maintain fruit quality and low respiratory activity that retard skin darkening and loss of flavor and acidity; these quality changes, when not re- stricted, increase cherry susceptibility to pitting (WANG and LONG, 2014). However, cherry stored at higher temperatures and lower levels of humidity developed increased pitting, likely due to rapid water loss from the fruit (PATTERSON, 1987; SCHICK and TOIVONEN, 2002). Additionally, pitting development may be related to the length of storage (LIDSTER and TUNG, 1980; DRAKE and ELFVING, 2002). It is clear that surface pitting occurs either during handling or storage and shipping; therefore improved strategies discourage development of pitting during the postharvest period will have economic benefits for growers. Pre-harvest sprays of plant growth regulators such as CaCl2, gib- berellic acid, or benzyladenine increase fruit firmness and improve resistance to pitting disorder (CANLI et al., 2015; EINHORN et al., 2013; WANG et al., 2015). However, sweet cherries’ response to these applications is known to delay on-tree fruit maturation and fruit with lower sugar and acid levels that are likely subject to quality deterioration or mold (KAPPEL and MACDONALD, 2002). Recently, hydrogen sulfide (H2S), an endogenous signaling molecule similar to carbon monoxide and nitric oxide, has been reported to activate defense responses and prolong fruit life in studies on kiwi (ZHU et al., 2014), banana (LI et al., 2016), strawberry (HU et al., 2012), and pear (HU et al., 2014) fruit. It has been hypothesized that H2S could up-regulate antioxidant enzymes while down regulating the expres- sion of chlorophyll degradation-related genes and reduce membrane oxidative damage caused by reactive oxygen species (FOTOPOULOS et al., 2015). However, whether H2S plays a functional role in reduc- ing susceptibility to surface pitting injury remains unclear. The objective of this work was to investigate (1) the effect of H2S on fruit quality and pitting development of ‘Lapins’ and ‘Regina’ sweet cherries during cold storage and (2) to evaluate the possible mecha- nism of H2S in reducing development of pitting. Materials and methods Plant materials ‘Lapins’ and ‘Regina’ cherries were harvested from the orchard of the Mid-Columbia Agriculture Research and Extension Center in Hood River, OR, USA (latitude 45.7°N, longitude 121.545.7°W, elevation 150 m, average annual rainfall ~800 mm). For both culti- vars, trees were 15-years old and on Mazzard rootstock. They were maintained with standard cultural, fertilizer, herbicide and pesticide practices. Twenty-five boxes (~8 kg/box) of each cultivar, selected to be free of any visible damage or fungal infection and of uniformity size were picked at commercial maturity. Commercial maturity was determined as color grade 5 according to the Pacific Northwest Dark Sweet Cherry Development Index Chart developed by Oregon State University, in which 1 = Blush, 2 = Rose, 3 = Ruby, 4 = Crimson, 5 = Currant, 6 = Merlot, and 7 = Mahogany. Fruit of each cultivars with pedicels intact were divided into 5 treatments × 3 replications × 1 lot per replication = 15 lots of 10 kg/lot. Each lot was loaded 110 H. Zhi, Y. Dong into a 70-L latching box (650 × 430 × 340 mm) and fumigated with 0.5, 1.0, 1.5, or 3.0 mM of sodium hydrosulfide (NaHS) dissolved in 500 mL distilled water. Control fruit were fumigated with the same volume of distilled water. After fumigating, H2S gas concentration in each lot was monitored using an H2S gas detector (Ventis MX4, Industrial Scientific, Pasadena, TX, USA). The H2S concentration reached approximately 0.1, 0.6 2.4, and 4.8 × 10-4 μM after 30 min when the fruit were exposed to 0.5, 1.0, 1.5, or 3.0 mM NaHS, res- pectively, then maintained at a constant concentration for 24 h during the fumigation treatment. All latching boxes were held at 0 °C for 24 h. After treatment, fruit were packed into commercial poly- ethylene bags (~1 kg), then stored at 0 °C and >90% relative humi- dity for 4 weeks. Respiration rate, storage quality, weight loss, stem browning, decay and pitting incidence, cell wall compositions, and related wall-modifying enzymes were evaluated. Methods Respiration rate Thirty fruit per replicate from each treatment were equilibrated in air at 0 °C for 5 h before being place into hermetically sealed glass containers (960 mL). After 1 h incubation, the headspace CO2 were determined using CO2 analyzer (900161, Bridge Analyzers Inc., Ala- meda, CA, USA). Respiration rate was expressed as μg CO2 kg-1 s-1. Fruit firmness, SSC, and TA Before quality evaluations, fifty fruit from each treatment per re- plicate were randomly selected and placed in the laboratory at 20 °C for 4 h, until condensation on the surface of the fruit was dried. Fruit firmness was measured on opposite sides of each fruit, mid- way between the pedicel and calyx using a FirmTech 2 fruit firmness instrument (BioWorks Inc., Stillwater, OK, USA) and expressed as g mm-1. Juice was prepared for SSC and TA measurements using a juicer (Acme Model 6001, Acme Juicer Manufacturing Co., Sierra Madre, CA, USA) equipped with a uniform milk filter strip (Schwartz Manufacturing Co., Two Rivers, WI). SSC was determined using a refractometer (PAL-1, Atago, Tokyo, Japan). TA was determined by titrating 10 mL of cherry juice, diluted with 40 mL distilled water to pH 8.1 with 0.1 N NaOH using a titrator (DL-15, Mettler-Toledo, Zurich, Switzerland) and expressed as the equivalent percentage of malic acid. Weight loss, stem browning, decay, and surface pitting incidence The bags of fruit were weighed initially and after 4 weeks of sto- rage. Weight loss was expressed as percentage loss of original weight. Stem browning was recorded for those pedicels exhibiting >30% of the entire surface browned. Fruit decay was assessed as a percent of fruit from one hundred fruit samples showing any type of decay, however, decay organisms were not identified. Surface pitting was evaluated and recorded as a percentage of one hundred fruit sam- ples showing visible pitting. One hundred fruit from each treatment of each replicate were randomly selected and were scored for stem browning and surface pitting. Isolation and measurement of cell wall polysaccharides Cell wall polysaccharides were isolated and measured as described by SALATO et al. (2013). Flesh tissue samples (10 g) were homogenized in 20 mL of 80% ethanol, then boiled for 30 min, and centrifuged at 9,000 g for 10 min at 20 °C. The supernatant was decanted and the residue twice suspended with ethanol, then twice with acetone, and dried at 75 °C for 6 h. The samples were AIR. AIR (100 mg) was treated with distilled water twice, centrifuged, and two supernatants were collected as WSP. Next, the residue was treated with 50 mM sodium acetate buffer (pH 6.5) containing 50 mM CDTA twice, cen- trifuged, and two supernatants were collected as CSP. Finally, the residue was treated with 50 mM Na2CO3 containing 10 mM NaBH4 twice, centrifuged, and two supernatants were collected as NSP. Extract polysaccharides solution (0.2 mL) was added to 1 mL of sul- furic acid containing 75 mM sodium borate. After boiling for 10 min, 20 μL of 0.5% NaOH containing 0.15% m-phenylphenol was added to the mixture and loaded in a 96-well plate. Absorbance at 520 nm was monitored in a plate reader (Elx800, Bio-Tek Instruments Co., Winooski, VT, USA). A standard calibration curve was constructed using galacturonic acid and the data were expressed as AIR mg g-1. Cell wall-modifying enzymes PG activity was performed as described by GROSS (1982). Flesh tis- sue samples (0.25 g) were homogenized in 5 mL of 0.3 M NaCl. After centrifugation at 12,000 g for 15 min at 4 °C, the supernatant (50 μL) was added to 0.6 mL of 0.1% polygalacturonic acid, 0.6 mL of 50 mM sodium acetate buffer (pH 4.5), and 0.15 mL of 0.3 M NaCl, then incubated at 37 °C for 2 h. After adding 4 mL of 0.1 M borate buffer (pH 9.0) and 0.6 mL of 1% 2-cyanoacetamide, the mix- ture was boiled in a water bath for 10 min. One unit of PG activity was defined as the release of 1 μg of galacturonic acid at 276 nm absorbance per min. PME activity was performed as described by BASAK and RAMAS- WAMY (1996). All solutions, including distilled water, NaCl, pectin, and supernatant were adjusted to pH 7.5 with 0.1 M NaOH. Flesh tis- sue samples (4 g) were homogenized in 4 mL of 0.3 M NaCl. After centrifugation, the supernatant (2 mL) was added to 25 mL of 0.5% pectin and then incubated for 1 h at 37 °C. The mixture was titrated to pH 8.1 with 0.1 M NaOH and the volume of NaOH solution was recorded. One unit of PME activity was defined as the release of 1 mM of ester hydrolyzed per min. PL activity was performed as described by BELGE et al. (2015). Flesh tissue samples (0.25 g) were homogenized in 5 mL of 50 mM Tris- HCl (pH 8.5) containing 0.6 mM CaCl2, 5 mM EDTA, and 0.05% Triton X-100. After centrifugation, the supernatant (0.4 mL) was added to 3.1 mL of 50 mM Tris-HCl (pH 8.5) containing 0.6 mM CaCl2 and 0.24% polygalacturonic acid, then incubated at 37 °C for 30 min. After boiling in a water bath for 10 min, one unit of PL activity was defined as the formation of 1 μM of 4,5-unsaturated product at 232 nm absorbance per min. β-GAL was performed as described by BELGE et al. (2015). Flesh tissue samples (0.5 g) were homogenized in 5 mL of 0.3 M NaCl. After centrifugation, the supernatant (0.1 mL) was added to 2 mL of 0.04% p-nitrophenyl-β-D-galactoside and 0.3 mL of 0.3 M NaCl, then incubated at 37 °C for 30 min. After adding 2 mL of 0.2 M Na2CO3, one unit of β-GAL activity was defined as the release of 1 mg of p-nitrophenol at 400 nm absorbance per min. Statistical Analysis The experiment design was completely randomized with three repli- cates for each treatment since the trees were uniform in the orchard. Homogeneity tests were performed for two continuous years and the data were homogeneous for each parameter tested. The data were combined and subjected to analysis of variance using SPSS software (SPSS Inc., Chicago, IL, USA). When appropriate, means were sepa- rated by Fisher’s protected least significant difference test at P < 0.05. Results and discussion Effect of H2S on fruit respiration Postharvest application of H2S has been shown to reduce fruit qua- lity deterioration, including appearance and texture, to resist patho- Effect of H2S on pitting in sweet cherry 111 gen infection, and to extend fruit storability (HU et al., 2012; HU et al., 2014; LI et al., 2016; ZHU et al., 2014). Respiration rate is a major indicator of physiological and biochemical activity, and it in- creases as fruit deterioration. In this study, respiration rates were inhibited by H2S in both cultivars, especially in 1 and 2 mM NaHS treatments, compared to the control (Tab. 1). This demonstrated that H2S may lower respiration and lower the loss of quality for stored fruit. decay, and surface pitting (Tab. 2). HU et al. (2002) reported reduced physiological disorders as a response to greater antioxidant enzymes (i.e. catalase, guaiacol peroxidase, ascorbate peroxidase, and gluta- thione reductase) activity which might be activated by H2S. Other reports indicated that the effect of H2S on quality deterioration dis- orders was probably due to fruit higher energy status (LI et al., 2016). However, susceptibility to pitting is an intrinsic characteristic of cherry skin. Therefore, cell wall metabolism seems to be implicated in the development of pitting. Tab. 1: Effect of H2S on respiration rate, fruit firmness, soluble solids content (SSC), and titratable acidity (TA) of ‘Lapins’ and ‘Regina’ cherries at harvest and after 4 weeks of storage at 0 °C. Treatments Respiration rate Fruit firmness SSC (%) TA (%) (μg CO2 kg-1 s-1) (g mm-1) ‘Lapins’ At harvest 3.21 da 315.01 e 17.01 a 0.59 a Control 4.55 a 335.12 c 16.00 b 0.47 b 0.5 mM NaHS 4.39 a 328.49 d 16.01 b 0.45 b 1 mM NaHS 3.51 c 347.30 a 15.93 b 0.46 b 2 mM NaHS 3.44 c 346.07 a 15.87 b 0.46 b 3 mM NaHS 3.87 b 342.10 b 15.64 b 0.47 b ‘Regina’ At harvest 2.98 d 361.14 d 18.10 a 0.45 a Control 3.98 a 370.11 c 18.01 a 0.38 b 0.5 mM NaHS 3.85 ab 371.08 c 17.83 a 0.41 b 1 mM NaHS 3.34 c 391.10 a 17.93 a 0.41 b 2 mM NaHS 3.31 c 390.08 a 18.06 a 0.41 b 3 mM NaHS 3.76 b 379.33 b 18.03 a 0.38 b aMeans within a column in each cultivar followed by different letters indicate significant differences according to the Fisher’s protected least significant dif- ference test P < 0.05. Effect of H2S on fruit quality ‘Lapins’ and ‘Regina’ cherries were picked at typical commercial harvest maturity; fruit firmness was 315.01 and 361.14 g mm-1, SSC was 17.01% and 18.10%, and TA was 0.59% and 0.45%, respectively. After 4 weeks of storage at 0 °C, firmness increased in all treatments. This increased firmness may be related to moisture loss (DRAKE and ELFVING, 2002), although no significant weight losses were detected between the control and H2S-treated fruit after 4 weeks (Tab. 2). In both cultivars, H2S significantly increased fruit firmness, while losses in SSC and TA were not delayed by the treatment. Both 1 and 2 mM NaHS treatments in both cultivars led to significant- ly higher firmness than control and other NaHS treatments. The 0.5 mM NaHS treatment did not affect storage quality. The effect of the 3 mM NaHS treatment were similar to the 1 or 2 mM NaHS treatment for firmness, but fruit firmness was lower than that in 1 or 2 mM NaHS treatment (Tab. 1), indicating that high H2S concen- tration might increase the levels of malondialdahyde, and oxidative damage to cell membrane systems (ZHANG et al., 2011). These results indicated that the effect of H2S in both cherry cultivars was dose- dependent, the greatest positive effect exerted by the 1 to 2 mM dose. FACTEAU (1982) reported that fruit firmness was negatively related to pitting development in ‘Bing’ cherries; our finding in H2S-treated fruit confirmed his observations. As expected, consumers favor sweet cherries with less surface pit- ting (CHAUVIN et al., 2009). In both cultivars, fruit treated with H2S expressed delayed deterioration of fruit indicators stem browning, Effects of H2S on cell wall composition and metabolism Pitting appears as one or more irregular depressions around the fruit surface and can occur anywhere without particular location (PORRITT et al., 1971). Examinations of the ultrastructure of fruit skin tissue has shown that a single layer of epidermal cells covers the surface of fruit, 4-5 layers of hypodermal cells with thicker walls are found below the epidermal layer, underneath the hypodermal layers are composed of radially elongated and rounded parenchy- matic mesocarp cells (LANE et al., 2000; PARAM and ZOFFOLI, 2016). Pitting originating from mechanical damage is caused by the col- laps of injured cells in the epidermis and hypodermis or/and paren- chymatic mesocarp. Anatomical differences among epidermal, hy- podermal and mesocarp cells across cultivars affects cell layout, cell number, cell size, and susceptibility to pitting (PORRITT et al., 1971; PARAM and ZOFFOLI, 2016). ‘Regina’ had a great uniformity in cell size and arrangement with less susceptibility to pitting, while ‘Lapins’ had smaller and less-uniform cells and greater susceptible to pitting (PARAM and ZOFFOLI, 2016). In this study, even though H2S did not affect the initiation of mechanical damage in either cultivars, it postponed the development of macroscopically visible dents that might alter the structural composition of the cell wall in fruit skin tissue. Compared to the control, H2S-treated cherries of both culti- vars had lower WSP and CSP and higher NSP (Tab. 3), especially in 1 mM NaHS treatments. Previous work failed to show a relationship between the depolymerization of cell wall polysaccharides and pit- ting development (FACTEAU et al., 1982; KAPPEL et al., 2006; SALATO et al., 2013). Our results indicated that H2S has the ability to rein- force the connection between cell wall polysaccharides and the cell Tab. 2: Effect of H2S on weight loss, stem browning, decay, and surface pit- ting of ‘Lapins’ and ‘Regina’ cherries after 4 weeks of storage at 0 °C. Treatments Weight loss Stem browning Decay Surface pitting (%) (%) (%) (%) ‘Lapins’ Control 0.87 aa 34.51 a 6.71 a 13.34 a 0.5 mM NaHS 0.85 a 33.93 a 5.11 b 12.19 a 1 mM NaHS 0.87 a 23.66 c 3.52 d 8.76 c 2 mM NaHS 0.86 a 23.71 c 3.30 e 8.97 c 3 mM NaHS 0.86 a 26.87 b 4.01 b 10.34 b ‘Regina’ Control 0.84 a 23.51 a 5.87 a 9.97 a 0.5 mM NaHS 0.83 a 23.32 a 5.57 ab 9.35 ab 1 mM NaHS 0.83 a 13.21 b 3.41 b 6.13 d 2 mM NaHS 0.84 a 13.17 b 3.67 b 7.07 c 3 mM NaHS 0.83 a 23.50 a 5.14 b 8.64 b aMeans within a column in each cultivar followed by different letters indicate significant differences according to the Fisher’s protected least significant dif- ference test P < 0.05. 112 H. Zhi, Y. Dong wall. Firmer fruit was more resistant to mechanical damage, and displayed reduced susceptibility to surface pitting (TOIVONEN et al., 2004; KAPPEL et al., 2006). ANDREWS and LI (1995) reported that the cell wall-modifying enzymes including PG, PME, and carboxymethylcellulase increased in sweet cherry during development and reached to maximum acti- vity when the fruit skin color turned to dark-red. After fruit were harvested and subjected to cold storage, the activity of PG and β-GAL continued to increase, but PME activity was not affected by cold temperatures (BELGE et al., 2015). In this study, the activity of cell wall-modifying enzymes in ‘Lapins’ and ‘Regina’ cherries at harvest were PG of 118.96 and 84.79 U g-1, PME of 0.44 and 0.35 U g-1, PL of 229.20 and 187.63 U g-1, and β-GAL of 437.64 and 227.67 U g-1, respectively. After 4 weeks of storage, the activity of PG and β-GAL in control fruit of both cultivars increased, while no signifi- cant differences were observed in PME activity (Tab. 4). Fruit treated with 1 mM NaHS in both cultivars displayed a significant reduction in PG activity over the control as reported in strawberry (HU et al., 2002). Furthermore, PG and β-GAL activity were delayed by the 1 mM NaHS treatment, while PME activity was not effected by H2S (Tab. 4). These results indicate that PG and β-GAL may play impor- tant roles in the development of surface pitting, and that H2S could inhibit their activity, and result in reduced cell wall disassembly and improved resistance to pitting injury. BELGE et al. (2015) reported that PL did not show any distinct changes in ‘Celeste’ or ‘Somerset’ cherries when fruit were held at 0 °C for 14 days. By contrast, PL in the control of both varieties studies here increased after 4 weeks of storage; the H2S-treated fruit displayed significantly inhibited PL activity. Further studies should be conducted to see if the low acti- vity of β-GAL in cherries is protects fruit against the development of surface pitting. Conclusion This study demonstrated that postharvest application of H2S gas (1-2 mM NaHS) increased fruit firmness and reduced respiration rate, stem browning, decay, and surface pitting in ‘Lapins’ and ‘Regina’ cherries after 4 weeks of cold storage. The potential benefit of H2S in minimizing pitting development was associated closely with reduc- tion of the degradation of cell wall polysaccharides and suppression of cell wall-modifying enzymes (PG, PL, and β-GAL) activities. These results are the first report on the effect of H2S on surface pit- ting development in ‘Lapins’ and ‘Regina’ cherries. Although ap- proval of the use of H2S gas on food has not yet been granted, an alternative reducing agent gas based on H2S tended to be effective in controlling physiological disorders and improving fruit quality in sweet cherry. Acknowledgement We are grateful to the Columbia Gorge Fruit Growers Association and the NW Fresh Pear Research Committees for financial support. 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DOI: 10.5073/JABFQ.2015.088.037 CHAUVIN, M.A., WHITING, M., ROSS, C.F., 2009: The influence of harvest Tab. 3: Effect of H2S on water-soluble polysaccharides (WSP), CDTA- soluble polysaccharides (CSP), and Na2CO3-soluble polysaccharides (NSP) of ‘Lapins’ and ‘Regina’ cherries after 4 weeks of storage at 0 °C. Treatments WSP CSP NSP (AIRa mg g-1) (AIR mg g-1) (AIR mg g-1) ‘Lapins’ Control 11.72 ab 8.96 a 13.44 c 0.5 mM NaHS 10.86 b 8.89 a 14.47 b 1 mM NaHS 7.00 e 6.17 d 17.71 a 2 mM NaHS 7.72 d 6.64 c 17.33 a 3 mM NaHS 8.53 c 7.16 b 17.04 a ‘Regina’ Control 8.89 a 6.44 a 15.41 c 0.5 mM NaHS 7.96 b 5.98 a 15.39 c 1 mM NaHS 5.43 c 3.98 b 18.46 a 2 mM NaHS 5.19 c 4.17 b 17.41 b 3 mM NaHS 7.79 b 6.35 a 14.97 c aAIR = alcohol insoluble residue bMeans within a column in each cultivar followed by different letters indicate significant differences according to the Fisher’s protected least significant dif- ference test P < 0.05. Tab. 4: Effect of H2S on activities of polygalacturonase (PG), petin methyl- esterase (PME), pectin lyase (PL), and β-D-galactosidase (β-GAL) at harvest and after 4 weeks of storage at 0 °C. Treatments PG PME PL β-GAL (U g-1) (U g-1) (U g-1) (U g-1) ‘Lapins’ At harvest 118.96 ba 0.44 a 229.20 e 437.64 e Control 125.69 a 0.43 a 420.59 a 950.22 a 0.5 mM NaHS 123.34 a 0.44 a 367.33 c 828.87 b 1 mM NaHS 117.50 b 0.45 a 289.76 d 685.89 d 2 mM NaHS 121.36 a 0.43 a 294.20 d 722.88 c 3 mM NaHS 127.62 a 0.44 a 409.60 b 841.30 b ‘Regina’ At harvest 84.79 c 0.35 a 187.63 e 227.67 e Control 99.87 a 0.36 a 338.46 a 556.34 a 0.5 mM NaHS 102.23 a 0.34 a 348.54 a 413.60 b 1 mM NaHS 88.46 b 0.35 a 223.39 d 399.85 b 2 mM NaHS 85.35 bc 0.36 a 249.98 c 427.66 b 3 mM NaHS 96.98 a 0.38 a 290.30 b 405.57 b aMeans within a column in each cultivar followed by different letters indicate significant differences according to the Fisher’s protected least significant dif- ference test P < 0.05. 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